Aging is one of the major risk factors for OA, precipitating onset predominantly among adults 60 years of age or older (Lotz and Loeser, 2012). The low turnover of ECM and articular cartilage likely increases the sensitivity to accumulate age-related changes, including a disturbed structural organization of ECM due to formation and accumulation of AGEs, cartilage calcification, and fibrosis, which alter cartilage mechanical properties (Roberts et al., 2016). Cellular age-related changes, those include senescence, reduction in the number of chondrocytes, mitochondrial dysfunction, and altered growth factors responsiveness. Alterations in protective mechanisms such as decreased antioxidant defense and reduced autophagy also occur with aging, disturbing the anaboliccatabolic equilibrium and reducing the remodeling and repairing ability of cartilage (Lotz and Loeser, 2012; Hui et al., 2016). Autophagy can regulate age-related changes in articular cartilage. Both aging human and mice chondrocytes exhibit a reduction in constitutive autophagy. Moreover, compromised autophagy as a consequence of aging precedes the decrease in cartilage cellularity and the onset of structural damage (Carames et al., 2010, 2015; Hui et al., 2016). Chondrocyte-specific ablation of autophagy gene Atg5 in mice leads to age-related OA but no alteration of injury-induced OA (Bouderlique et al., 2016). Altered tissue repair, misbalanced homeostasis and defective autophagy are important hallmarks of aged tissues in general (Cuervo et al., 2005; Cuervo, 2008). Consistently, the molecular mechanisms occurring during aging can explain the impairment in autophagy, including defects in induction and inefficient lysosomal clearance. For instance, the altered hormonal regulation of autophagy occurring in aged tissues can be a possible explanation for the compromised autophagy observed in old organisms caused by glucagon upregulation (Cuervo et al., 2005). Signaling pathways involving pro-longevity factors such as Sirtuin 1 (SIRT1), transcription factor forkheadbox O3 (FOXO3), and pro-senescence factors NF-kB and p53 are also known autophagy regulators and thus can be involved in cartilage aging (Salminen and Kaarniranta, 2009). Decreased autophagy can be explained by the impaired capacity of the lysosomes to fuse with autophagosomes and the failure of lysosome hydrolases that decrease the proteolysis efficiency of lysosomes (Cuervo, 2008). In fact, the accumulation in the lysosomes of undegradable materials that occurs with aging hampers the degradative capacity and results in decreased autophagy (Brunk and Terman, 2002a,b; Terman and Brunk, 2004a).

In cardiac and skeletal muscle of mice and humans with progeria, autophagy decreases with aging, while mTOR activity is elevated (Ramos et al., 2012; Sandri et al., 2013). Pharmacological and genetic inhibition of mTOR extends lifespan by unknown mechanisms (Lamming, 2016). mTOR inhibition represses autophagy promoting the degradation of aberrant proteins and damaged organelles, thus protecting from toxicity, and consequently slowing aging. Other processes including the regulation of protein synthesis, regulation of mitochondrial function, anti-inflammatory effects, increased stress resistance and preservation of stem-cells might also contribute to the prolongevity effects of mTOR inhibition (Johnson et al., 2013). Aging and OA are clearly intertwined, and autophagy might be a common mechanism involved in cartilage degradation in both conditions.

Kashin-Beck is a rare disease of the bone occurring in children, with high prevalence in certain areas of Asia. Functional genetic studies identified ATG4C as a novel susceptibility gene in Kashin-Beck (Wu et al., 2014). In normal human chondrocytes stimulated with IL-1β, Parkin, a component of the multiprotein proteasome complex, eliminates dysfunctional mitochondria promoting survival and protecting from apoptosis (Ansari et al., 2017). In age-related and surgical OA mice, Bach1 deficiency reduces the severity of articular cartilage damage probably by antioxidant effects of HO-1 and downregulation of ECM catabolic enzymes (Takada et al., 2015). Chondrocyte-derived extracellular organelles from adult porcine and osteoarthritic patients evaluated by light scatter nanoparticle counting have constitutive autophagy that is increased with rapamycin and suppressed by autophagy inhibitors and genetic silencing of ATG5, supporting the role of autophagy in cartilage disease and repair (Rosenthal et al., 2015).

### microRNA

Small non-coding RNA molecules have been extensively used as tools to study the post-transcriptional regulation of gene expression. miRNA have been manipulated in mice, articular cartilage and chondrocytes to test different hypothesis related to musculoskeletal system physiology and disease. In OA model mice and SW1353 human chondrosarcoma cells treated with interleukin-1β, miR-17-5p was decreased (Li et al., 2018). Autophagy was found suppressed in knee joints mainly through suppressing p62/SQSTM1, an autophagosome cargo protein that targets other proteins that bind to it for selective autophagy. In an hypoxic environment as occurs in aged articular cartilage, miR-146a targets related to inflammation Traf6 and IRAK1, as well as chondrocyte-specific Smad4 were down-regulated (Chen et al., 2017). Traf6 and IRAK1 were identified as regulators of autophagy. Previous reports found that miR-146a promote chondrocytes autophagy via depressing Bcl-2 (Zhang F. et al., 2015). In primary human and TC28a2 chondrocytes, miR-155 inhibited autophagy and contributed to the autophagy defects in OA (D'Adamo et al., 2016). In rats with OA, miR-4262 regulates chondrocyte fate by influencing PI3K/AKT/mTOR signaling (Sun et al., 2018). In OA chondrocytes and in zebra fish, Fis1 suppression induces accumulation and inhibition of lysosomes by altering miRNAs and energy signals (Kim et al., 2016). These results indicate that post-transcriptional targeting of autophagy with specific miRNAs might be considered as a promising preclinical model to study musculoskeletal physiology and disease.

These pathways span a broad range of biological processes, and therefore targeting specific pathways with small molecules should be taken into consideration regarding the wide endogenous functions of these pathways in non-target tissues. **Table 1** summarizes relevant advances in the autophagy-related molecular mechanisms in OA.

# CURRENT DEVELOPMENT IN INNOVATIVE THERAPEUTIC APPROACHES

Although many advances to understand the pathophysiological processes have been made, no effective treatments to stop or prevent OA exist. Few symptomatic treatments are available, mainly focusing on pain relief and improving joint function. Current clinical management relies in a combination of pharmacological approaches with surgical procedures. Also, decisions on treatment greatly depend on different patient-related factors, such as the occurrence of other co-morbidities and the presence of one or multiple affected joints. Pharmacological options include analgesic and anti-inflammatory agents primarily. When the goals are not achieved with conservative treatment and the disease progresses to diminished quality of life, surgical options are considered. However, the burden, risks and lack of long-term efficacy that come with surgical interventions, ultimately may affect the quality of life of patients and impact the health care system. Thus, efforts focused on generating strong preclinical evidence that can lead to the development of novel targeted therapies are necessary.

# Approaches Targeting Autophagy as OA Therapeutics

Pharmacological activation of autophagy with small molecules has been the subject of several studies showing in some cases some preclinical efficacy in OA (**Table 2**). Targeting PI3K/AKT/mTOR pathway to modulate autophagy stands out as a major focus of pharmacological interventions in preclinical studies. Rapamycin induces autophagy activation through the inhibition of mTORC1 in many cell and tissues (Galluzzi et al., 2017). It is used as an immunosuppressive in transplantation and autoimmune disorders, and in cancer due to its activity at inhibiting growth and proliferation of tumor cells

#### TABLE 2 | Preclinical treatments targeting autophagy in OA.

(Lamming et al., 2013). In articular cartilage, mTOR inhibition by Rapamycin protects from oxidative stress, and cell death (Carames et al., 2012b; Sasaki et al., 2012; Lopez de Figueroa et al., 2015), increases the expression of aggrecan and type II collagen, while decreases MMP-13 in OA chondrocytes (Zhang Y. et al., 2015). C57Bl/6J mice with experimentally induced OA treated with Rapamycin show autophagy activation, including increased LC3-II levels and inhibited rpS6 phosphorylation in articular cartilage. Treatment also protects against OA-like alterations, including the maintenance of cartilage cellularity and prevention of ECM damage. Furthermore, the severity of synovitis was attenuated and inflammatory cytokine IL-1β was decreased. These results suggest that induction of autophagy can significantly reduce the severity of experimental OA (Carames et al., 2012a). It is interesting to note that beneficial effects are also observed in meniscus, a specific type of cartilage not present in all human joints (Meckes et al., 2017). However, systemic long-term administration of Rapamycin is associated with side effects including edema, mucositis, hair and nail disorders, and dermatological effects, among others, making its utility problematic for chronic conditions (Lamming et al., 2013). In order to avoid these side effects, it has been suggested the intra-articular injection as a more appropriate route for clinical use. Indeed, intra-articular injection reduces articular degeneration and preserves hyaline cartilage, by downregulating cartilage degrading enzymes (MMP9 and MMP13), hypertrophyrelated genes (vascular endothelial growth factor-VEGF- and COL10A1), inflammation-related genes (IL-1β and IL-6), and stress-responsive genes (CCAAT/enhancer binding protein beta-C EBPβ and mTOR) and by upregulating Col2a1 (Matsuzaki et al., 2014a; Takayama et al., 2014). These studies show that direct administration of autophagy activators in the joint have protective effects, while potentially avoiding the side effects observed with systemic administration. Selective ATP-competitive inhibition of mTOR with Torin1 blocks phosphorylation of both mTORC1 and mTORC2 and induces autophagy by blocking signals required for cell growth and proliferation (Thoreen et al., 2009). Torin 1 treatment reduces severity of OA pathology in rabbits (Cheng et al., 2016a,b), highlighting again the important role of PI3K/AKT/mTOR in articular cartilage degeneration. However, it is important to consider the negative feedback loop of the PI3K/Akt pathway. mTOR signaling regulates protein synthesis and autophagy mediated, at least in part, through changes in the phosphorylation of downstream molecules such as p70S6K, 4E-BP1, and ULK1. Ultimately, the cascade leads to the inhibition of the insulin receptor substrate (IRS1), an upstream regulator of PI3K/Akt (Laplante and Sabatini, 2012). On the other hand, mTOR inhibition leads to an enhancement of PI3K/Akt signaling and increased MMP production by chondrocytes. Based on this, Chen et al. proposed the dual inhibition of both pathways as a more promising approach for OA to abolish the negative feedback mechanism and its possible side effects (Chen et al., 2013). On the other hand, excessive inhibition of mTOR gene expression could be harmful for tissues. OA outpatients with lower mTOR gene expression in PBMCs compared to healthy controls, exhibited more pain while standing and upon joint function, and have increased total joint stiffness than OA outpatients with higher mTOR gene expression (Tchetina et al., 2013). Inhibition of PI3K/AKT/mTOR promotes autophagy and attenuates inflammation in rat articular chondrocytes (Xue et al., 2017). Chronic upregulated autophagy has been associated with compromised lifespan and pathology. For instance, mouse models of premature aging show systemic degeneration and weakening of the musculoskeletal system partly due to DNA damage defects (Marino et al., 2008). Therefore, continuous autophagy activation might not likely be the best therapeutic approach. Instead, repairing autophagy defects, or restoring the normal levels of autophagy, could be a more efficient therapeutic strategy for age-related diseases. Sugars, as precursors of the synthesis of glycosylated proteins and lipids, have also been tested as therapeutic agents for OA. Glucosamine is a dietary supplement marketed to support the structure and function of joints. In chondrocytes and in articular cartilage, it protects nucleus pulposus cells through the inhibition of mTOR and consequent activation of autophagy (Carames et al., 2013; Jiang et al., 2014). Primary human chondrocytes treated with sucrose are protected from degradation, chondrocyte inflammation and death via PI3K/Akt/mTOR (Khan et al., 2017), and trehalose reduces ER and oxidative stress-mediated by autophagy stimulation (Tang et al., 2017). Although these results are promising, the time-dependent dual role of glucosamine (shortterm exposure increases autophagy, while long-term exposure have inhibitory effects (Kang Y. H. et al., 2015), making these data difficult to interpret. Interestingly, drugs widely used in the management of OA such as glucocorticoid dexamethasone have also shown a similar dual profile of action in chondrocytes via FOXO3 and in meniscus via inositol 1,4,5-trisphosphate receptor (Liu et al., 2014; Shen et al., 2015a,b). Perhaps, autophagy activation is an early protective response, whereas prolonged treatments eventually lead to permanent autophagy defect. These dual effects might be due to the particular metabolism and pharmacokinetics of these molecules and the wide interference with diverse metabolic and inflammatory pathways. Whether this dual effect is generally observed with such molecules potentially interfering with autophagy, remains to be clarified. Glucose metabolism might play a relevant role in cartilage as diabetic mice with experimental OA have is degenerated joint tissue and Rapamycin treatment decreases both pathology and inflammation (Ribeiro et al., 2016b). Indeed, insulin might exert catabolic effects in chondrocytes, inducing loss of proteoglycans and inflammation by downregulating autophagy, similarly to what is observed in chondrocytes from diabetic-OA patients (Ribeiro et al., 2016a). On the other hand, sirtuins have arisen as potential targets of phenol-like molecules such as Hydroxytyrosol to prevent inflammation by promoting cell autophagy (Cetrullo et al., 2016; Zhi et al., 2018).

Preclinical studies targeting autophagy and related molecular mechanisms in cartilage with advanced therapies medical products such as gene and cell therapy have also received some attention. Intra-articular treatment with Parathyroid hormone (1–34) alleviates cartilage degeneration by activating autophagy (Chen et al., 2018). Adiponectin protects chondrocytes from apoptosis related to oxidative stress by inducing autophagy possibly via PI3K/Akt/mTOR (Hu et al., 2017). Other biological products such as Platelet rich plasma (PRP), adipose-stem cells, and serum have also shown efficacy at protecting from degeneration and inflammation through autophagy-related mechanisms in cartilage disease (Jiang et al., 2016; Moussa et al., 2017; Zhang et al., 2018).

Gene therapy and genome editing by nucleases has dramatically expanded the toolbox for the identification and therapeutic validation of targets. Agents interfering with transcription to achieve therapeutic benefit have also received special attention. Nanoparticles targeting NF-κB not only are bioavailable, but reduce early apoptosis, synovitis, and

maintain cartilage homeostasis by enhancing AMPK signaling that ultimately attenuate inflammation (Yan et al., 2016). Other examples include miRNA targets (miR-20, miR-30b) that maintain cartilage homeostasis by enhancing autophagy in chondrocytes (Chen et al., 2016; He and Cheng, 2018). One would expect a dramatic transformation of drug discovery pipelines by the generation of engineered cell-based models with disease-relevant genetic modifications in the upcoming years.

# Current and Future Therapeutic Approaches of Inflammation in OA

OA is now considered an inflammation-associated multifactorial disorder. As compared to rheumatoid arthritis, the inflammation in OA is chronic, comparatively low grade and mainly mediated by the innate immune pathways including complement and pattern-recognition receptors such as damage associated molecular patterns (Robinson et al., 2016). The recent identification of novel regulators (Liu-Bryan and Terkeltaub, 2015) of inflammation in OA holds promise for the development of transformative therapies. Among these emerging regulators, the bioenergy sensors serine/threonine kinase AMPK and sirtuin-1 (SIRT1) have received particular attention. The role of AMPK and SIRT1 is actually not restricted to energy metabolism, but includes resistance to stress and inflammation. It is noteworthy their role in the tuning of mitochondrial activity to adapt energy consumption, in the resolving of inflammation via the down-regulation of NF-κB, in the control of matrix catabolic responses and last but not least in promoting autophagy. These wide range activities of AMPK and SIRT1 make it a promising entry point for the development of novel therapeutics. A large variety of AMPK activators such as aspirin (Hui et al., 2016), metformin or methotrexate are clinically used in inflammatory disorders (O'Neill and Hardie, 2013). Their use in OA is conceptually attractive and sound, and the results of randomized placebo-controlled trials will certainly bring conclusive data.

The positioning of such sensors at the crossroad of inflammation and autophagy paves the way of research avenues to identify the missing links between chronic inflammation and metabolism alterations related to aging, exercise, nutrition or even circadian rhythm (CR) (Berenbaum and Meng, 2016). CR not only interferes with several physiological processes ranging from behavioral and physiological activities such as sleep/wake cycles, blood pressure, body temperature and metabolism, but also contributes to the severity of aging-related disorders (Longo and Panda, 2016). CR is centrally regulated by a core pacemaker located in the hypothalamus and by an integrated network of peripheral oscillators in tissues and cell types. The coordinated activity of this network controls diurnal pattern of rest and activity in mammals. The pacemaker consists in a group of genes coding for transcriptional activators/repressors that interact in a 24 hourly-tuned feedback loop. The rhythmic activity of the positive regulators brain and muscle aryl hydrocarbon molecular pattern.

receptor nuclear translocator-like protein 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) drives the expression of E-box promoter containing genes that are pivotal in cell differentiation. Among these clock-controlled genes, the negative regulators cryptochrome (CRY) and mammalian period (PER) in turn repress the expression of BMAL1 and CLOCK, thereby completing the feedback loop that cycles every 24 h. While it remained poorly understood, pain and stiffness in OA patients intriguingly follows a daily pattern notably for hand, knee and hip OA. On the other hand, it has been recently identified a circadian clock in chondrocytes (Gossan et al., 2013) that controls the expression of genes involved in cartilage homeostasis and can be altered during aging. Together these data provide a rationale for questioning the role of CR alteration in OA. Following the pioneering description of BMAL1/CLOCK in chondrocytes, it has been subsequently demonstrated that inflammatory cytokines such as IL-1β, conversely to TNF-alpha, severely affects the expression of BMAL1/CLOCK through functional interference with NF-kB (Guo et al., 2017). More recently, it has also been demonstrated that BMAL1, which is essential to maintain the differentiated phenotype of chondrocytes through its interaction with a large number of TGF-β signaling members (Akagi et al., 2017), was repressed in OA and associated with a switch of catabolic phenotype (Snelling et al., 2016). These converging reports have culminated in the description that genetic ablation of BMAL-1 specifically in mouse chondrocytes deeply affects intrinsic CR,

contribute to induce a catabolic profile in chondrocytes and leads to cartilage damage (Dudek et al., 2016). Interestingly, it has also been described that BMAL1 predisposes to a marked degeneration of the fibrocartilaginous intervertebral disc (Dudek et al., 2017). Altogether, these data support the notion that disruption of chondrocyte CR may predispose individuals to the onset of cartilage disorders. In search of novel therapeutic interventions in OA, it seems thus reasonable to speculate that the development of drugs interfering with the peripheral clock in cartilaginous tissues may open new therapeutic spaces in the management of OA.

Consistent with this idea, the recent description of a molecular dialog between clock gene BMAL1 and the energy/nutrient sensor SIRT1 has shed further light on the clinical translatability of this therapeutic concept (Dvir-Ginzberg et al., 2016). SIRT1 is well-known to form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression notably in muscle and heart. Such a crosstalk has also been described in human OA cartilage (Yang et al., 2016), where a direct SIRT1-mediated circadian regulation of BMAL1 occurs. Strikingly and of particular clinical applicability, the sirtuin-activating molecule resveratrol, also known as an interacting agent with the aryl hydrocarbon receptor, is well-known to exert beneficial effects on cartilage anabolism. To address the clinical efficacy of such a molecule, the effects of oral resveratrol are currently being assessed in a phase 3 double blind placebo-controlled and randomized clinical trial (NCT02905799-Arthrol) in OA patients.

Also in line with the concept that cartilage intrinsic CR may be a pragmatic entry point for the development of integrative therapeutics in OA, is the intriguing connection between circadian genes and autophagy. As extensively described above, autophagy is a key process in the onset of OA and is thus considered a promising therapeutic target. Of interest, BMAL1 has recently been shown to positively regulate autophagy through mTOR in cardiomyocytes (Qiao et al., 2017). While it remains to be determined whether such a relationship also operates in cartilage, it raises the possibility that restoring the cartilage CR may contribute to increase autophagy processes which in turn contribute to limit cell stress, inflammation and catabolism. Among the molecules that could interfere with central and peripheral CR, melatonin has been widely studied in the context of rheumatologic disorders (Jahanban-Esfahlan et al., 2017). Melatonin is a circadian multi-tasking hormone mainly produced in the pineal gland but also in a wide range of other tissues. Melatonin exhibits diverse biological activities such as anti-oxidation, anti-inflammation or antiapoptosis through its interaction with specific plasma membrane or nuclear receptors and intracellular targets. In the context of OA, intraarticular injection of melatonin protected against cartilage damages induced by partial meniscectomy in rabbits (Lim et al., 2012). Deciphering the underlying mechanisms has revealed that the anti-inflammatory effect of melatonin could be mediated by a SIRT1-dependant inhibition of the NF-kB pathway (Lim et al., 2012). Consistent with a role of cartilage clock in OA, low doses of oral melatonin, in association with exercise, was found to concomitantly restore the OA-associated reduced levels of clock-controlled genes and reduce the severity of cartilage damages in a murine model of collagenase-induced OA (Hong et al., 2017). This converging body of evidence suggesting that melatonin exerts a protective effect in OA remains however to be consolidated through well-conducted in vitro and in vivo experiments in animal model of OA or in clinical trial, notably with respect to the ambiguous role of SIRT1 in mediating the pro-inflammatory effects of IL-1β or H2O<sup>2</sup> in chondrocytes (Guo et al., 2017; Hong et al., 2017).

Despite some areas of darkness still subsist notably regarding the molecular links between CR, SIRT1/AMPK energy sensors, and autophagy, these data reinforce the notion that local clock genes may be considered as clinically relevant targets for the development of novel therapeutic intervention. See **Figure 1** summarizing hypothetical mechanisms underlying the role of the circadian rhythm in OA.

# CONCLUSIONS

Failures of autophagy homeostasis lead to cell dysfunction senescence and death and initiate the musculoskeletal degeneration and inflammation characteristic of rheumatic

# REFERENCES

diseases. In this sense, preclinical models of such fundamental homeostasis mechanisms might create opportunities to build biobanks with patient derived material that can be used to perform drug screens and facilitate drug development. Clinical and preclinical models include blood, tissues from human joints and experiments in rodents showing that aging and OA of articular cartilage are invariably associated with defective autophagy and inflammation. Studying these dimensions of cartilage aging, chondrocyte senescence, inflammation and autophagy could facilitate to open the pharmacological space to novel disease-modifying therapies for OA. In this review, we highlighted what is currently known about preclinical models of OA homeostasis, the major targets and pathways identified, and its potential value for translation. See **Figure 2** illustrating the interrelation between autophagy, senescence and inflammation in OA.

# REVIEW CRITERIA

Data for this review were collected by searching in PubMed database for articles published from 1965 to 2018 using the following keywords "osteoarthritis," "inflammation," "aging," "senescence," "SASP," "chondrocyte," "homeostasis," "autophagy," "therapeutics," "metabolism," "circadian clock" alone or in combination. English-language original publications and Review articles were selected on the basis of their relevance for the inclusion in the bibliography.

# AUTHOR CONTRIBUTIONS

All authors approved the final version to be published. CV, ED, JG, BC: manuscript design and drafting and revising the manuscript.

# FUNDING

This study was supported by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain and Fondo Europeo de Desarrollo Regional (FEDER), Una manera de hacer Europa, PI14/01324. BC was supported by Miguel Servet Type II Program- CPII16/00045-A, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain and thank the FOREUM Foundation for Research in Rheumatology for their support. JG and CV would like to thank the Fondation arthritis, the French Society of Rheumatology, the ROAD Network and the FOREUM-Foundation for Research in Rheumatology for their supports. ED would like to thank to Ministerio de Economía y Competitividad Spain and Fondo Europeo de Desarrollo Regional (FEDER) (Project Code: RTC-2015-4207-1) and the Kaertor Foundation for their support.

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patients and disease activity. Arthritis 2013:461486. doi: 10.1155/2013/ 461486

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a past co-authorship with one of the authors BC.

Copyright © 2018 Vinatier, Domínguez, Guicheux and Caramés. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Obesity, Metabolic Syndrome, and Musculoskeletal Disease: Common Inflammatory Pathways Suggest a Central Role for Loss of Muscle Integrity

Kelsey H. Collins 1,2, Walter Herzog1,2, Graham Z. MacDonald<sup>1</sup> , Raylene A. Reimer 1,3 , Jaqueline L. Rios 1,2,4, Ian C. Smith<sup>1</sup> , Ronald F. Zernicke1,5,6 and David A. Hart 1,2,7,8 \*

<sup>1</sup> Human Performance Laboratory, Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada, <sup>2</sup> McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, AB, Canada, <sup>3</sup> Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada, <sup>4</sup> CAPES Foundation, Brasilia, Brazil, <sup>5</sup> Departments of Orthopaedic Surgery and Biomedical Engineering, School of Kinesiology, University of Michigan, Ann Arbor, MI, United States, <sup>6</sup> Department of Surgery, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada, <sup>7</sup> Department of Family Practice, The Centre for Hip Health and Mobility, University of British Columbia, Vancouver, BC, Canada, <sup>8</sup> Alberta Health Services Bone and Joint Health Strategic Clinical Network, Calgary, AB, Canada

#### Edited by:

Alexandrina Ferreira Mendes, University of Coimbra, Portugal

#### Reviewed by:

Oreste Gualillo, Servicio Gallego de Salud, Spain Francis Berenbaum, Université Pierre et Marie Curie, France Tom Appleton, University of Western Ontario, Canada

> \*Correspondence: David A. Hart<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 30 October 2017 Accepted: 05 February 2018 Published: 23 February 2018

#### Citation:

Collins KH, Herzog W, MacDonald GZ, Reimer RA, Rios JL, Smith IC, Zernicke RF and Hart DA (2018) Obesity, Metabolic Syndrome, and Musculoskeletal Disease: Common Inflammatory Pathways Suggest a Central Role for Loss of Muscle Integrity. Front. Physiol. 9:112. doi: 10.3389/fphys.2018.00112

Inflammation can arise in response to a variety of stimuli, including infectious agents, tissue injury, autoimmune diseases, and obesity. Some of these responses are acute and resolve, while others become chronic and exert a sustained impact on the host, systemically, or locally. Obesity is now recognized as a chronic low-grade, systemic inflammatory state that predisposes to other chronic conditions including metabolic syndrome (MetS). Although obesity has received considerable attention regarding its pathophysiological link to chronic cardiovascular conditions and type 2 diabetes, the musculoskeletal (MSK) complications (i.e., muscle, bone, tendon, and joints) that result from obesity-associated metabolic disturbances are less frequently interrogated. As musculoskeletal diseases can lead to the worsening of MetS, this underscores the imminent need to understand the cause and effect relations between the two, and the convergence between inflammatory pathways that contribute to MSK damage. Muscle mass is a key predictor of longevity in older adults, and obesity-induced sarcopenia is a significant risk factor for adverse health outcomes. Muscle is highly plastic, undergoes regular remodeling, and is responsible for the majority of total body glucose utilization, which when impaired leads to insulin resistance. Furthermore, impaired muscle integrity, defined as persistent muscle loss, intramuscular lipid accumulation, or connective tissue deposition, is a hallmark of metabolic dysfunction. In fact, many common inflammatory pathways have been implicated in the pathogenesis of the interrelated tissues of the musculoskeletal system (e.g., tendinopathy, osteoporosis, and osteoarthritis). Despite these similarities, these diseases are rarely evaluated in a comprehensive manner. The aim of this review is to summarize the common pathways that lead to musculoskeletal damage and disease that result from and contribute to MetS. We propose the

overarching hypothesis that there is a central role for muscle damage with chronic

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exposure to an obesity-inducing diet. The inflammatory consequence of diet and muscle dysregulation can result in dysregulated tissue repair and an imbalance toward negative adaptation, resulting in regulatory failure and other musculoskeletal tissue damage. The commonalities support the conclusion that musculoskeletal pathology with MetS should be evaluated in a comprehensive and integrated manner to understand risk for other MSK-related conditions. Implications for conservative management strategies to regulate MetS are discussed, as are future research opportunities.

Keywords: joint diseases, muscle, bone, tendon, NFkB, MAPK

# METHODOLOGY

The studies presented in this review were identified through PubMed Searches and the review of relevant papers in the area of diet-induced obesity, musculoskeletal health, musculoskeletal disease, and inflammation.

# INTRODUCTION TO METABOLIC SYNDROME AND OBESITY

Metabolic syndrome (MetS) is a cluster of conditions visceral obesity, hypertension, dyslipidemia, and elevated fasting glucose—that increase an individual's risk for diabetes and cardiovascular complications (Alberti and Zimmet, 1998; Manuel et al., 2014). Human metabolism has evolved to efficiently convert chemical energy obtained through the consumption of food into thermal and chemical energy. Our body's metabolic pathways have developed to provide energy to tissues in times of physical threat and survival, or to efficiently conserve energy in times of food deprivation. Today, westernized societies have an abundance of food (food security) and many individuals have little need to perform physical activity. This combination has led to excessive nutrient storage, placing significant stress on our metabolic pathways, and leading to an increase in the prevalence of disease stemming from metabolic dysfunction (Miranda et al., 2005).

Concordant with the rise in MetS prevalence, there is also a global increase in the prevalence of musculoskeletal (MSK) diseases and disorders (Wearing et al., 2006). Recent evidence demonstrates that metabolic complications also increase the risk for the most prominent MSK diseases, such as sarcopenic obesity (muscle loss in obesity), osteoporosis, tendinopathy, and osteoarthritis, conditions which contribute significantly to disability and time lost from work. The resultant damage and pain associated with these conditions likely develops through low-level systemic inflammation (Hoy et al., 2014; Smith et al., 2014; **Figure 1**) in addition to loading due to obesity (Felson et al., 1988), and reduced ability to withstand loading due to sarcopenia.

MSK disease is of particular concern, for example, as osteoarthritis-related walking disability significantly increases risk for all-cause mortality and cardiovascular events, when controlling for other cofounders (Hawker et al., 2014). This suggests that MSK disability associated with MetS can contribute to the worsening of MetS through sedentary behavior. Although obesity has received considerable attention regarding chronic cardiovascular conditions and diseases, as well as diabetes, the MSK complications that result from obesity associated MetS are less frequently discussed and rarely evaluated comprehensively.

Muscle mass is a key predictor of longevity in older adults (Srikanthan and Karlamangla, 2014), and since muscle is highly plastic and undergoes regular remodeling, it is a vulnerable tissue in a chronic low-level inflammatory environment, such as that seen with metabolic dysfunction (Tidball, 2005; Fink et al., 2014; D'Souza et al., 2015; Collins et al., 2016a). For example, intramuscular lipid deposits increase with obesity and are also positively correlated with insulin resistance (Akhmedov and Berdeaux, 2013; Addison et al., 2014; Fellner et al., 2014), linking structural alterations with altered capacity for glucose homeostasis. Functionally, impaired muscle integrity, persistent atrophy, and lipid accumulation in muscle are risk factors for tendinopathy (Meyer and Ward, 2016), osteoporosis (Ormsbee et al., 2014), osteoarthritis (Lee et al., 2012), and integrity of a motion segment, such as the leg (**Figure 2**).

# A ROLE FOR METABOLIC DISTURBANCE IN MOTION SEGMENT TISSUE DAMAGE

A hypothesis has emerged proposing that metabolically mediated damage to MSK tissues may be one additional component of the MetS, as adipose-based inflammation links obesity, MetS, and MSK tissue damage (Hart and Scott, 2012; Zhuo et al., 2012). Increased visceral adiposity is linked to induction of increased levels of catabolic mediators (Fontana et al., 2007) and ultimately tissue damage (**Figures 1** – **3**). Additionally, the presence of hypertension may be linked to tissue damage through vasoconstriction and ultimately depriving tissues of appropriate nutrient exchange (McMaster et al., 2015). Moreover, high cholesterol is speculated to link dysregulated lipid metabolism and endothelial dysfunction and has been directly associated with tissue damage in tendons (Tilley et al., 2015), decreased bone mineral density (Makovey et al., 2009), and osteoarthritis damage (Farnaghi et al., 2017). In parallel, investigations into the impact of low-level systemic inflammation from metabolic disturbance on the onset of sarcopenic obesity tendinopathy, osteoporosis, and osteoarthritis have been conducted. Many of these authors report associations between low-level systemic inflammation from diet-induced obesity (DIO) with MSK disease outcomes (**Table 1**), however these diseases are seldom evaluated

comprehensively to evaluate a common inflammatory pathway to disease induction and progression.

As such, we suggest a hypothesis linking altered muscle integrity to direct and indirect consequences on the motion segment (**Figures 2**, **3**). An obesogenic diet, resulting in over nutrition and development of a low-level systemic inflammation, acutely challenges associated tissues of the motion segment and can result in positive adaptation (i.e., through dynamic compensation that helps the tissue accommodate metabolic challenge), as well as negative adaptation (i.e., vulnerability to deleterious changes in tissue integrity). With these initial challenges, such tissue adaptive responses are balanced and help preserve the associated tissues and motion segment integrity. However, with chronic exposure to obesogenic diet and its inflammatory consequences, tissues demonstrate dysregulated repair, an imbalance toward negative adaptation resulting in regulatory failure, tissue damage (i.e., ectopic lipid storage and tissue fibrosis), and failure of motion segment integrity progressing to loss of function and ultimately, disease.

# AIM AND SCOPE OF REVIEW

The aim of this review is to summarize the links between induction of local and systemic inflammation, DIO, and a central role for muscle integrity in the inflammation-based pathogenesis of these MSK diseases. Early loss of muscle integrity can have both direct and indirect "ripple" effects downstream on tendons and bones, as well as the functioning of multi-tissue complex joints (e.g., the joint as an organ, **Figure 2**) (Frank et al., 2004; Loeser et al., 2012). As muscle plays a critical role in the mechanical and biological homeostasis of bones (through the muscle tendon unit indirectly and directly through the muscle/bone interface), tendons (directly through the muscle tendon unit), and joints (through loading and stabilization), we suggest that with the influence of dysregulated muscle loading, inflammation, and altered muscle integrity, failure of motion segment integrity is induced and exacerbated.

This review is focused on outcomes from studies using preclinical DIO models, as the gradual and progressive pathway toward MetS afforded by DIO provides critical insight into the short- and long-term pathophysiology, in addition to the phenotype of MSK diseases (Buettner et al., 2007; Nilsson et al., 2012). Evaluating diet-induced alterations allows for linking results across systems from food, through the gut and the associated microbiome, to early and late tissue-based changes, as well as the inclusion of potential epigenetic outcomes regarding temporal relations for the onset and progression of MSK disease.

# OBESITY AND IMPACT ON MUSCLE INTEGRITY

# Sarcopenic Obesity

Sarcopenic obesity, or low muscle mass and quality with increased fat mass, is not only associated with poor physical function (Zamboni et al., 2008), but also results in additional weight gain and an 8 to 11-fold increase in the risk for three or more additional physical disabilities (Baumgartner, 2000).

Sarcopenic obesity can predict disability and loss of activity in elderly adults (Baumgartner et al., 2004), and sarcopenic

obesity is more closely associated with MetS than either obesity or sarcopenia alone. This suggests important roles for both fat accumulation and muscle loss in the etiology of MetS (Lim et al., 2010). Although the age-dependent declines in muscle structure and strength are well-documented, the mechanism by which MetS results in sarcopenic obesity remains to be clarified (Kob et al., 2014). As sarcopenic obesity results in disability, loss of activity, altered mechanical loading, and altered biological function in the muscle due to lipid deposition and its sequelae (Ormsbee et al., 2014), it is likely that sarcopenic obesity is central to the development of other musculoskeletal pathologies. In fact, data from our laboratory in a rat model revealed changes in the integrity of specific muscles as early as 3-days on a high-fat/high-sucrose (HFS) diet (Collins et al., 2016c), and correspond to long-term changes in systemic inflammation and gut microbiota (Collins et al., 2015a). These data support the notion that muscle may be among the first MSK tissues affected by DIO, and inflammation likely plays a substantial role in this loss of integrity.

# Dysregulated Tissue Regeneration of Muscle—Primary Tissue Damaged by MetS

Muscle fiber damage happens on a daily basis and is generally considered to be a beneficial stimulus, leading to growth and adaptation through muscle regenerative processes (Karalaki et al., 2009). In muscle, monocyte and macrophage recruitment, as well as phagocytosis of necrotic material, occurs within the first 24 hours. Muscle is repaired through a series of tightlycontrolled inflammatory processes (Akhmedov and Berdeaux, 2013). Specifically, muscle regeneration is a multistep process involving degeneration, regeneration, and remodeling, ultimately restoring structure and function (Laumonier and Menetrey, 2016).

The three most active cells in the regeneration of skeletal muscle are macrophages, satellite cells, and fibroblasts

(Akhmedov and Berdeaux, 2013). The metabolic complications associated with obesity can result in an inappropriate temporal recruitment of these cells, which in turn leads to impaired angiogenesis and myocyte formation, while promoting the deposition of fibrotic and adipose tissue, ultimately leading to a reduction in structural integrity and functional capacity of a muscle (Karalaki et al., 2009).

With DIO, metabolic dysfunction and the presence of chronic low level inflammation can impair the "normal" inflammatory response and the regenerative capacities of skeletal muscles, resulting in a pseudo-injury (Collins et al., 2016c; **Figure 3**). For example, elevated levels of leptin, a satiety hormone now appreciated to have a role in low-level systemic inflammation, can impair angiogenesis, leading to tissue ischemia (Brown et al., 2015). IL-6 expression at the muscle level is a key mediator of macrophage infiltration and muscle repair (Zhang et al., 2013), while elevated expression of TGF-β promotes an increased fibrotic tissue deposition (Laumonier and Menetrey, 2016).

Efficient muscle regeneration can be attributed to satellite cells being readily available, and the cells' ability to re-establish residual pools to support multiple rounds of regeneration (Karalaki et al., 2009). Satellite cells are limited by the complex physiological environment in which they interact, an environment that can be significantly altered in individuals with obesity (D'Souza et al., 2015). A pathological host environment can limit a satellite cell's ability to be activated, proliferate, and differentiate into a muscle fiber (D'Souza et al., 2015; Meng et al., 2015). This was elegantly shown by Boldrin and colleagues through the transplantation of satellite cells from mdx mice, a genetic mouse model of muscular dystrophy, into a neutral environment (Boldrin et al., 2015). Despite the impairment of mdx satellite cells as a result of being in the pathogenic environment of an mdx mouse, following transplantation, mdx satellite cells were fully capable of being activated, and could proliferate and differentiate into a fully functional muscle fiber (Boldrin et al., 2015). Macrophages may also inhibit satellite cell activity, suggesting another mechanism by which low-level systemic inflammation may inhibit muscle repair (Tidball and Villalta, 2010).

Impairments in satellite cell activity have been reported to promote fibro/adipogenic progenitor cells (FAP) that normally aid in muscle regeneration, to differentiate into fibroblasts and/or adipocytes (Chapman et al., 2016). FAPs have also been demonstrated to be a source of intramuscular lipid deposition with rotator cuff/supraspinatus tendon injury (Liu et al., 2016). FAPs are thought to be vulnerable to reprogramming in TABLE 1 | Examples of MSK damage resulting from DIO.

the presence of low-level inflammation, which may result in increased lipid and fibrosis deposition, and may limit reversibility of fibrosis (Mann et al., 2011). Thus, chronic obesity and associated MetS, with inflammation and fatty infiltration of muscles, may lead to a compromise in the regeneration of muscle integrity.

In addition to the environmental challenges to the muscle regeneration posed by metabolic disturbance, skeletal muscle from individuals with obesity displays a greater number of glycolytic-fibers vs. oxidative-fibers when compared to a healthy individual (Pattanakuhar et al., 2017). Since oxidativefibers generally contain a greater number of satellite cells relative to glycolytic fibers (Karalaki et al., 2009), individuals with obesity may also have fewer satellite cells. Based on this information, impairments in satellite cell function as a result of alterations in cell metabolism, a reduction in cell number, suppression of cell activation, depleted cell reserves, and impaired cell proliferation and differentiation may lead to impairments in muscle fiber regeneration (Akhmedov and Berdeaux, 2013).

Work from our laboratory has demonstrated that the oxidative soleus muscle is protected against HFS-induced damage over short-term and long-term exposures in a rat model (Collins et al., 2017b; **Figure 4**). By 3-days on HFS, dynamic increases in mRNA levels for superoxide dismutase (SOD2) in HFS animals implicate compensatory oxidative stress scavenging in the soleus muscle compared to control animals. By 2 weeks on HFS, increased mRNA levels for oxidative capacity [succinate dehydrogenase (SDH)] were detected compared to chow-fed controls, suggesting one adaptation strategy that the soleus muscle may employ with HFS metabolic challenge. Although the precise mechanism(s) by which the soleus is protected from metabolic disturbance-induced muscle damage remains to be clarified, it appears that increasing the oxidative capacity and the oxidative stress scavenging ability of muscles (i.e., with aerobic exercise) may be a beneficial strategy for mitigating obesity-induced muscle damage and its consequences.

Inflammation related to obesity can also impair myocyte remodeling as a result of a reduction in protein synthesis due to elevated TNF-α levels (Brown et al., 2015). Furthermore, there is evidence for adipocyte-muscle cross talk in vitro, whereby adipocyte-derived inflammation can contribute to inflammation and atrophy in muscle cells subjected to a metabolically dysfunctional environment, possibly through IGF-1 (Pellegrinelli et al., 2015). Impaired protein synthesis can also prevent muscle from properly adapting to mechanical stimuli. Brown et al. (2015) demonstrated this inability, showing that following muscle damage, obese muscle displayed no adaptations, while lean mice displayed an increase in muscle wet weight and muscle fiber hypertrophy (Brown et al., 2015). Potential contributing factors to a reduction in protein synthesis may be elevated lipid metabolites resulting from impaired mitochondrial function contributing to elevated TNF-α levels, which are known to have inhibitory effects on IGF-1 (Akhmedov and Berdeaux, 2013). Decreases in IGF-1 result in the inhibition of the muscle growth signaling pathway (IGF-1 → P13K → Akt → mTOR), effectively blunting muscle protein synthesis (Brown et al., 2015).

Furthermore, increased myostatin levels can also contribute to impaired growth in obese muscle. Myostatin is not only significantly up-regulated in obese skeletal muscle, but in adipose tissue as well, further inhibiting myogenesis, providing another avenue through which potential muscle-adipose cross talk may occur (Karalaki et al., 2009).

# Functional Muscle Damage with Metabolic Derangement

Generally speaking, adults with obesity are reported to have significantly higher absolute strength in lower limb muscles, but lower strength when normalized to body mass (Tomlinson et al., 2016). When the upper limb muscles are evaluated, there are no statistical significant differences between individuals with obesity and normal-weight controls (Tomlinson et al., 2016). Potentially, the characterization of obesity by body mass, which is common in these studies, may be inappropriately representing body composition. We have shown, in a healthy and overweight population cohort, that body mass index (BMI) inappropriately estimates body composition in 30% of the population, with a specific disparity between BMI and body composition measurements in healthy females (Collins et al., 2017c). Also, there is a lack of data describing the effect of obesity on muscle integrity, and a lack of consistent protocols to assess muscle strength (Tomlinson et al., 2016). However, data from our lab (Collins et al., 2016a,c, 2017b) and others, in rodents (Ciapaite et al., 2015) and large mammals (Clark et al., 2011), have demonstrated deleterious alterations in muscle structural integrity with metabolic disturbance. Computational approaches modeling the gastrocnemius muscle have demonstrated that whole-muscle force is dependent on muscle integrity, specifically regarding reductions of muscle force due to intramuscular lipid (Rahemi et al., 2015). A zebrafish model of diet-induced obesity further demonstrated that obesity induces decreases in locomotor performance, isolated muscle isometric stress, workloop power output, and muscle relaxation rates (Seebacher et al., 2017). Of note, these decrements in performance and function were not reversed with weight loss, generating interesting questions about the potential reversibility of impaired muscle function with obesity.

Additional sources of muscle damage and altered repair are advanced glycation end products (AGEs), which accumulate over time due to increased availability of glucose and hyperglycemia (**Figure 5**). Dietary AGEs can interfere with muscle healing and impair contractile function in a mouse model of obesity (Egawa et al., 2017). HFS diets can induce hyperglycemia in rodents, further linking diet-induced obesity to AGEs (Sumiyoshi et al., 2006). The receptor for AGEs on macrophages, called RAGE, is associated with a pro-inflammatory state, and RAGE/AGEs are reported to be involved in the onset and progression of metabolic disturbance, insulin resistance, and adipokine expression (Leuner et al., 2012; Hofmann et al., 2014).  Reactive AGEs can also cross-link with collagen fibers, which subsequently can affect the fiber's mechanical and biological properties (Abate et al., 2013). Although AGE collagen cross-linking can be reversed (Asif et al., 2000), it is unclear whether glycation itself can be reversed. Of note, endurance exercise has been shown to attenuate AGEs in cardiac muscle of rats (Wright et al., 2014).With weight loss, reversal of metabolic dysfunction may not be fully achievable, and weight re-gain is common (Fothergill et al., 2016). As mentioned above, to what degree reversibility of skeletal muscle damage may be achieved in this context remains to be determined (**Figures 2** – **4**), and several factors could contribute to irreversibility (Mann et al., 2011). Likely, clarifying relations between the time of exposure to low-level systemic inflammation and muscle damage should be determined, as impaired muscle integrity has been observed following short-term exposure to a metabolic challenge, likely before "full" metabolic derangement has been achieved (Fink et al., 2014; Collins et al., 2016c). Also, some alterations in satellite cell function can be irreversible (Sacco and Puri, 2015). Generally, DNA methylation, or epigenetic changes, is an actively researched area being explored in the context of muscle (Carrió and Suelves, 2015), and DNA methylation is an important step in muscle cell differentiation (Brunk et al., 1996). However, it is likely that epigenetic changes, which are dynamic and induced by environmental changes, can induce cellular reprogramming, and thus could potentially limit the reversibility of muscle damage, even if the MetS-related inflammation is controlled (Carrió and Suelves, 2015). Investigations to clarify the role of methylation in physiological and pathophysiological muscle changes may provide valuable insight into the reversibility potential of muscle damage.

# Dysregulated Homeostasis of Associated Motion Segment Tissues (Tendon, Bone, Cartilage, and Joint) with Obesity: Acute and Chronic

Muscle, tendon, cartilage, and bone tissues repair and regenerate over different timelines, and these different repair timelines may dictate vulnerability or resistance to damage with metabolic disturbance involving inflammatory processes. Both biological and mechanical stimuli are critical to homeostatic tissue regulatory mechanisms. Across tissues, remodeling rates slow

with age. As discussed above, muscle tissue is likely the most vulnerable MSK tissue to perturbation with metabolic challenge. Repair of such tissues in the face of an active inflammatory environment is likely compromised, and resolution of repair is particularly challenging (Hart et al., 2004).

Altered muscle integrity can challenge the motion segment in a dynamic manner, until the associated tissues ultimately begin to fail (**Figures 2**, **3**). Specifically, tendon healing is slower compared to muscle. In tendon, tenocytes migrate to the wound, initiating type III collagen synthesis. After several weeks to months, remodeling of tendinous tissue occurs such that collagen fibers are aligned in the direction of stress, and maturation occurs over the course of a year. Bone mechanotransduction, initiating the maintenance/remodeling processes (Scott et al., 2008), requires a series of events that can require almost 24-h to ultimately result in the synthesis of bone matrix proteins (Robling and Turner, 2009). Cartilage is considered to have limited intrinsic repair properties, although with the facilitation of autologous stem cells, biologics, and other transplants, some repair may be achievable (Chu et al., 2010). The impaired regenerative capacity of tendon, bone and cartilage likely indicate a lack of reversibility with altered integrity and subsequent compromise in function, underscoring a need for strategies aimed at primary prevention (before it occurs), secondary prevention (limiting the amount of damage as it occurs), tertiary prevention (prevention of progression of damage to disease), or reversal of damage.

# DIET-INDUCED OBESITY AND ELEMENTS OF THE DYSREGULATED INFLAMMATORY RESPONSE

# Inflammatory Mediator Alterations in Obesity

#### Cytokines and Chemokines

Activation of the innate immune system is critical in the pathogenesis of type 2 diabetes and tissue damage. Some of the key signaling pathways involved in this process are nuclear factor –κB (NF-κB), c-Jun N-terminal Kinase (JNK), and the NLRP3 inflammasome, all resulting in transcription of proinflammatory cytokines (Lackey and Olefsky, 2015). NF-κB can be activated by elevated levels of pro-inflammatory cytokines such as TNF-α, which is increased in the adipose tissue of obese and diabetic animals. Neutralizing TNF-α has been shown to reduce insulin resistance (Hotamisligil et al., 1993). Additionally, JNK activity increases in tissues that are sensitive to insulin, is activated by ER stress, and directly inhibits insulin signaling (Gual et al., 2005; Lackey and Olefsky, 2015). The inflammasome is a protein complex that matures and secretes inflammatory cytokines, such as IL-1β and IL-18. Similar to NFκB, the inflammasome can be activated by pro-inflammatory cytokines, lipopolysaccharide (LPS), and some forms of low density lipoproteins (LDLs). LPS, low density lipoproteins, and the AGE-product of LDLs can signal through Toll-like Receptor-4 (TLR-4) (Hodgkinson et al., 2008), activating IL-1β and IL-18 signaling.

TLR-4 recognition of saturated fatty acids is necessary to enable NF-κB signaling and induce expression of proinflammatory cytokines (TNF-α, IL-6, and MCP-1) (Jialal et al., 2014; Lackey and Olefsky, 2015). TLRs sense pathogen-associated molecular patterns and damage-associated molecular patterns (PAMPs), and regulate the inflammatory responses to mitigate tissue repair (Lee et al., 2013). TLR-4 gene expression and protein content are increased in muscle from patients with obesity and diabetes, potentially contributing to insulin resistance, as well as compromised muscle integrity observed with metabolic disturbance (Reyna et al., 2008). In the context of OA, TLRs can modulate the catabolic pathways and maintain joint homeostasis (Houard et al., 2013). Of interest, the lubricating molecule proteoglycan-4 (PRG-4) has been shown to modulate the inflammatory response through competitive inhibition of TLR-4 receptors, suggesting that as PRG-4 concentration is reduced with OA severity, inflammatory modulation may also be reduced, contributing to the progression of OA (Iqbal et al., 2016). In tendinopathy, there may not be a clear role for TLRs, as catabolic processes in Achilles tendinopathy seem to occur independently of TLR4-induced gene expression from IL-1β or TNF-α (de Mos et al., 2009). In bone, LPS-induced activation of TLR-4 in neutrophils is reported to upregulate the catabolic RANKL osteoclast cascade, linking TLR based inflammation to increased bone resorption (Chakravarti et al., 2009).

Appropriate levels of pro-inflammatory cytokines are necessary for tissue homeostasis. However, exogenous exposure to pro-inflammatory cytokines, or endogenous high levels of pro-inflammatory cytokines, are associated with damage across all musculoskeletal tissues. In muscle, TNF-α, IL-1β, and IL-6 activate transcription of MuRF-1 and MAFBx/atrogin-1, the key muscle atrophy pathway, through IGF/Akt-1 (Akhmedov and Berdeaux, 2013). For example, IL-6 and TNF-α inhibit bone-forming osteoblast cells, and NF-κb, RANKL, TNF-α, IL-6, M-CSF, and MCP-1 can contribute to osteoclast recruitment, maturation, and inhibit osteoclast apoptosis (Roy et al., 2016). In tendon, pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) can disrupt tissue homeostasis, by inducing extracellular matrix degradation, induce other pro-inflammatory cytokines, which result in necrotic and apoptotic cell changes, and affect collagen and elastin expression (Schulze-Tanzil et al., 2011). Cytokine effects in tendon are modulated by mechanical loading in this complex mechano-biological environment (Killian et al., 2012).

The knee joint is a complex organ system with many resident inflammatory cells, particularly in the synovium and infrapatellar fat pad. These tissues secrete pro-inflammatory cytokines, like IL1-α and IL-1β (Sanchez-Adams et al., 2014). In humans, systemic and synovial fluid inflammatory profiles can differentiate between patients based on OA severity, suggesting that cytokine levels may help define OA pathogenesis (Heard et al., 2013). In addition to the infrapatellar fat pad, there is also a synovial adipose depot, and these two depots differ significantly from each other in lean individuals with OA (Harasymowicz et al., 2017). Moreover, infrapatellar fat pads and synovium adipose depots differed in adipocyte size, fibrosis, and macrophage infiltration in OA patients with obesity compared to lean OA patients (Harasymowicz et al.,

2017). High glucose concentrations can contribute to increased chondrocyte responsiveness to cytokines, increased levels of reactive oxygen species, leading to an overproduction of IL-6 and PGE2 (Laiguillon et al., 2015). These cytokines can also contribute to mitochondrial dysfunction within the joint, and, in turn, mitochondrial dysfunction can amplify chondrocyte responsiveness to cytokines (Vaamonde-García et al., 2012).

for intramuscular fat; Picro, Picrosirius stain for collagen).

Unpublished data from the author's laboratories suggest that intramuscular lipid deposits and fibrotic material in the vastus lateralis muscle of the knee are associated with the presence of metabolic-induced knee joint damage after a 12-week obesity induction period (**Figure 6**). Based on the inflammatory mediator profile of joints undergoing damage (Collins et al., 2015a,b, 2016b), inflammation is likely playing a significant role in this process and in the relations between muscle damage and joint damage. Ongoing efforts will probe the role of associated inflammation on muscle integrity in the onset and progression of such metabolic joint damage and whether it leads to overt OA.

Given the systemic-to-local hypothesis in metabolic OA (Zhuo et al., 2012; Collins et al., 2016b; Berenbaum et al., 2017), we have evaluated hip, knee, and shoulder joints using the HFS rat model system for 12-weeks (Collins et al., 2017a). A HFS diet, in the absence of trauma, resulted in significant increases in joint damage/OA-like changes in the shoulder and knee joints of rats after a standard 12-week obesity induction period. The hip joint, however, was not significantly affected by DIO, which is consistent with findings from human epidemiological studies. Total joint damage, assessed by adding the individual Modified Mankin Scores across all three joints, was increased in DIO animals compared to chow-fed animals, and was associated with the percentage of body fat. Positive significant predictive relations for total joint score were found between body fat and two serum mediators (IL-1α and VEGF). These data suggest that systemic inflammatory alterations from DIO in this model system may result in a higher incidence of knee, shoulder, and multi-joint OA-like/joint damage with the HFS diet over the long term (Collins et al., 2017a). Due to the preliminary nature of these studies, longitudinal experiments with multiple time points are needed to validate these proposed relations. If these relations are supported in future studies, then not all joints are affected equally by obesity-associated inflammation in MetS. Studying these relations are an ongoing direction of current research.

### Adipokines

Adipocytes release adipokines such as leptin, adiponectin, visfatin, and resistin as a signaling mechanism in addition to passively storing energy, which can cause and exacerbate chronic low-level systemic inflammation (Gomez et al., 2009). Adipokines have been shown to induce pro-inflammatory mediators in activated CD4+ T cells from osteoarthritis patients, demonstrating that systemic mediators may play a role in osteoarthritis (Scotece et al., 2017). After interaction with such activated CD4+ T cells, chondrocytes demonstrate increased expression of MMP-13 and decreased expression of collagen-2 and aggrecan.

Leptin is a satiety hormone that increases in a nearlinear fashion to body fat (Friedman and Halaas, 1998). It inhibits appetite and regulates body weight, energy expenditure, and maintains glucose homeostasis. However, with metabolic disturbance, individuals become leptin resistant. Leptin is a class-1 cytokine secreted from adipose tissue, and high concentrations of leptin are associated with musculoskeletal tissue damage (Zabeau et al., 2003; Collins et al., 2015a,b, 2016b,c). Leptin activates downstream pro-inflammatory pathways (IL-2, IFNγ) and inhibits the anti-inflammatory pathway (IL-4) (Lechler et al., 1998). Aside from being produced by adipose tissue, leptin levels can be increased by TNF-α, IL-1, and LPS, creating a positivefeedback loop with low-level chronic inflammation (Grunfeld et al., 1996).

Leptin signaling is critical to conventional muscle maintenance (Akhmedov and Berdeaux, 2013), and basal levels of satellite cells are reduced in animals with impaired leptin

Data are shown as fold change ± standard error. DIO n = 12–14; chow n = 5–7.\* Indicates p < 0.05 vs. control; \*\*Indicates p < 0.01 vs. control, # Indicates different in SF between DIO and chow.

signaling (Peterson et al., 2008). However, hyperphysiological levels of leptin also stimulate the proliferation and activation of macrophages, which may be a mechanism by which leptin concentration influences tissue damage (Santos-Alvarez et al., 1999). Leptin may also positively upregulate myostatin (Rodríguez et al., 2015), a member of the TGF-β superfamily that negatively regulates muscle mass and growth, and a molecule which is up-regulated in muscle from individuals with metabolic disturbance (Hittel et al., 2009). Upregulation of follistatin has been an effective treatment for muscle degenerative diseases by increasing muscle growth due to its ability to inhibit myostatin, a negative regulator of muscle mass. Follistatin also alleviates synovitis and mitigates OA-like changes from inflammatory arthritis in mice (Yamada et al., 2014). As muscle has a role in OA pathogenesis, protecting both muscles and joints with follistatin represents an attractive therapeutic opportunity for MetS-induced musculoskeletal damage.

Leptin also may be involved in mediating the pathogenesis of osteoarthritis in humans (Fowler-Brown et al., 2014) and other animals (Griffin et al., 2009, 2010, 2012; Collins et al., 2015a,b, 2016b). Leptin is found in synovial fluid of humans (Lübbeke et al., 2013) and other animals (Collins et al., 2015a,b, 2016b). Unpublished mRNA data (**Table 2**); from our laboratory indicate increased mRNA expression levels for leptin in the fat pad and synovium of rats with DIO compared to those on a chow diet, after a 12-week obesity induction period.

Additionally, leptin may be involved in mediating the pathogenesis of osteoarthritis in humans (Fowler-Brown et al., 2014) and other animals (Griffin et al., 2009, 2010, 2012; Collins et al., 2015a,b, 2016b). Leptin is found in synovial fluid of humans (Lübbeke et al., 2013) and other animals (Collins et al., 2015a,b, 2016b). Increased mRNA levels for leptin were accompanied by increased leptin in the serum and synovial fluid (Collins et al., 2016b). After 28-weeks of DIO, the fat pad and synovium demonstrated disparate up-regulation of IL-1β, although IL-1β was detected in the synovial fluid of these animals (Collins et al., 2015a). These findings suggest that the fat pad and synovium contribute to increased pro-inflammatory synovial fluid profiles (Collins et al., 2016b). However, cartilage explants are not substantially damaged when exposed to physiological levels of leptin (Griffin et al., 2010), despite reports of associations between leptin and MMPs (Koskinen et al., 2011), calling into question the direct involvement of leptin in eliciting cartilage damage.

In the context of bone, leptin influences the formtion and resorption of mineralized tissue by increasing the activity of osteoclasts through RANKL (Ducy et al., 2000). However, there are also reports that leptin supports bone growth and bone MSC differentiation into osteoblasts (Thomas et al., 1999), so the precise mechanism for leptin's effects on bone is unclear, but likely is dependent on exposure and dose (Kawai et al., 2009). Low-level systemic inflammation and leptin can negatively influence tendon structural integrity (Abate, 2014; Abate et al., 2016), can result in accelerated heterotopic ossification in tendon tissues (Jiang et al., 2017) and may be associated with increases in tendon ruptures (Ji et al., 2010). Taken together, increases in systemic leptin—or mediators downstream of leptin—appear to have deleterious effects on all major musculoskeletal tissues. These data support the notion that catabolic activity as a result of increased leptin is an important pathway to clarify in the context of global musculoskeletal integrity (Griffin et al., 2009). More details regarding the interface among leptin signaling, inflammation, metabolism, and musculoskeletal disorders are detailed elsewhere (Abella et al., 2017b).

Resistin is an adipokine involved in insulin resistance, inflammation, and energy homeostasis. Serum resistin levels are reported to be derived from visceral adipose tissue (Milan et al., 2002). There is conflicting evidence as to whether resistin is associated with bone mineral density (Mohiti-Ardekani et al., 2014). Within the joint, synovial fluid levels of resistin are associated with inflammatory and catabolic molecules in the joints of human osteoarthritis patients (Koskinen et al., 2014). However, resistin and visfatin, demonstrated positive predictive relations with recovery from upper extremity soft tissue disorders such as tendinopathy, and are thought to be related to antiinflammatory response mechanisms (Rechardt et al., 2014).

Adiponectin, another adipokine, is derived from visceral fat, and is thought to serve a protective role on cardiovascular health and glucose homeostasis (Milan et al., 2002). As body fat decreases, adiponectin levels generally increase, and adiponectin may modulate adipose tissue regulation via NF-κB (Ajuwon and Spurlock, 2005). Specifically, adiponectin treatment in obese mice increases bacterial clearance and hematopoietic progenitor proliferation in bone marrow (Masamoto et al., 2016). Adiponectin was shown to be significantly correlated with bone mineral density in a group of osteoporotic and healthy patients (Mohiti-Ardekani et al., 2014). Adiponectin has also been proposed as a systemic biomarker of OA. Plasma adiponectin was significantly higher in a population of OA patients and was also higher in women with erosive hand OA compared to patients with non-erosive OA (Filková et al., 2009). In a separate cohort of hand OA patients, the individuals with the highest levels of adiponectin demonstrated a decreased risk for hand OA progression (Yusuf et al., 2011). However, the study populations were not the same in these two reports (i.e., total numbers, males and females vs. females alone, and use of European populations potentially differing genetically) and therefore, additional research needs to be performed to better evaluate this relationship. Additionally, in a study evaluating 76 males in Thailand with knee OA revealed that levels of adiponectin in synovial fluid correlated with disease severity (Honsawek and Chayanupatkul, 2010). In muscle, adiponectin has been shown to increase fatty acid oxidation and glucose uptake, and to attenuate local inflammation (Nigro et al., 2015). Adiponectin has been proposed as a treatment for diabetic tendinopathy (Rothan et al., 2013), suggesting it may be protective and have favorable anti-inflammatory effects across MSK tissues.

Progranulin is a more recently identified adipokine that may also have anti-inflammatory characteristics. mRNA levels for progranulin are increased in cartilage, synovium, and infrapatellar fat pads from OA patients, and mRNA levels are increased in response to pro-inflammatory stimulation (Abella et al., 2016). Specifically, progranulin has been shown to counteract pro-inflammatory molecule expression (i.e., NOS, MMP-13) induced by IL-1β and the LPS-TLR-4 axis (Abella et al., 2016). Attstrin, a progranulin-derived peptide, is a promising therapeutic candidate for osteoarthritis (Abella et al., 2017a) given that progranulin can counteract IL-1 driven inflammation through TNFR1 in human chondrocytes, and intraarticular injection of progranulin-derived attstrin prevented OA-progression in a surgical model of murine OA (Xia et al., 2015).

# Obesity and Involvement of Cells of the Inflammatory System Macrophages

Generally speaking, macrophages are dichotomized into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes based on their activity during tissue repair processes (Novak and Koh, 2013). However, it is understood that this dichotomy represents an oversimplification based on in vitro data which may not accurately represent in vivo states (Martinez and Gordon, 2014). Macrophages are described to demonstrate a high degree of functional plasticity and their phenotypes can change based on environmental stimuli (Stout and Suttles, 2004). It is likely useful to consider macrophage activation as a spectrum rather than a binary categorization (Mosser and Edwards, 2008), but for the purposes of this review, and concordant with the current musculoskeletal literature, macrophage activity and these relations are generalized using the M1/M2 paradigm.

Macrophages are derived from monocyte precursor cells. Tissues have resident macrophages, which are responsible for general tissue maintenance. These macrophages are described as alternatively activated, or M2 macrophages, and are induced by TGF-β, IL-4, and IL-13 (Gordon and Martinez, 2010). M1 macrophages (classically activated macrophages) are key phagocytes within tissues, and are induced through IFN-γ activation and LPS-induced TLR signaling or from detection of pathogen-associated molecular patterns (Lampiasi et al., 2016). With obesity, stress, loading, or tissue and niche specific control mechanisms, M2-type macrophages may experience a phenotypic shift. This shift may be influenced by exposure to certain cytokines through their general signaling mechanisms or in the presence of other conditions, including IL-6 (Braune et al., 2017) or TNF-α (Wu et al., 2015). AGEs can also play a role in M2 to M1 polarization (Jin et al., 2015) (**Figure 5**). Typically, an imbalance in the ratio of M1:M2 macrophages is considered maladaptive, creating an imbalance toward tissue degradation and an absence of adequate repair. MAPK is a key pathway in macrophage-mediated inflammatory responses and may play a significant role in diseases mediated by macrophages (Yang et al., 2014).

Within 24 hours following muscle damage, thousands of macrophages have infiltrated the damaged tissue (Tidball, 2005; Grounds, 2011). Pro-inflammatory M1-macrophages first perform cell lysis, removing necrotic muscle tissue debris (Laumonier and Menetrey, 2016). Anti-inflammatory M2 macrophages infiltrate the damaged site once M1-macrophages have removed necrotic debris (∼48 hours following injury), helping resolve the inflammatory response while promoting myogenesis (Akhmedov and Berdeaux, 2013). Through the secretion of a number of growth factors, macrophages promote angiogenesis (FGF, TGF-β), synthesis of ECM proteins (TGFβ), and activation of satellite cells, all promoting myogenesis (Grounds, 2011).

It is possible that an increased presence of M1 macrophage cells in a tissue is an early sign of disrupted tissue homeostasis and repair. With diet-induced obesity, M1-type macrophages are present in the quadriceps muscle of mice (Fink et al., 2014), rats (Collins et al., 2016c), and humans (Fink et al., 2014; Khan et al., 2015). In bone, osteoclasts are considered M1-type macrophages. With a high-fat diet, bone loss is accelerated in young mice due to increased osteoclastogenesis (Shu et al., 2015), and as noted, pro-inflammatory cytokines increase osteoclast activity in humans with obesity through activation of the NFκB/RANKL pathway (Cao, 2011). In the context of the joint as an organ (Frank et al., 2004), the infrapatellar fat pad does not demonstrate inflammation or M1 polarization prior to knee OA in mice (Barboza et al., 2017). Synovial membrane samples from OA patients reveal markers for M1 macrophages (IL-1α, IL-1β, and TNF-α) and decreased levels of the M2 marker IL-1RA (Smith et al., 1997). As such, it is challenging to determine if levels of M1/M2 macrophages are a cause or consequence of disease. In rabbits, intra-articular injections of the proinflammatory mediators IL-1 and TNF-α stimulate M1 activation and degrade cartilage, suggesting macrophage polarization may occur in synovial cells (Pettipher et al., 1986; Henderson and Pettipher, 1989). Conditional macrophage depletion in obese mice with a knee injury does not mitigate OA severity (Wu et al., 2017b). Moreover, in mice where macrophages were depleted, there was an increase in neutrophils and CD3+ T cells compared to control animals (Wu et al., 2017b). These data suggest that a potential redundancy may exist between macrophages and other inflammatory cells. Macrophages may modulate inflammatory homeostasis within the joint OA and obesity, and that a basal level of macrophages are likely needed to maintain joint health (Wu et al., 2017b). While this study is not conclusive, it demonstrates that the relations among macrophage polarization and presence in obesity and OA are complex. It is important to note that what constitutes a healthy or maladaptive balance in macrophage phenotype in one tissue or disease may differ from another tissue or disease process. As such, these findings are likely context- and tissue-dependent. More studies are needed to better understand whether synovial macrophage polarization, mitigation/modulation, or ablation is a promising therapeutic target in OA pathogenesis (Sun et al., 2016).

# SPECIFIC CELL INVOLVEMENT IN METABOLIC OVERLOAD WITH OBESITY

Many cells (e.g., adipocytes, hepatocytes, and myocytes) contribute to the conversion of food into chemical energy and can be placed under significant metabolic stress when exposed to a typical western-style diet. Significant increases in the postprandial flux of metabolic substrates results in drastic nutrient spikes characterized by elevated glucose, free fatty acid (FFA), and triglyceride (TG) levels (Weiss et al., 2013). Chronic nutrient overload places metabolic pathways under significant stress, overwhelming subcutaneous adipose depots, leading to adipocyte mitochondrial dysfunction and impaired insulin sensitivity (Miranda et al., 2005). Normally, insulin inhibits lipolysis and promotes glucose transport. However, during chronic metabolic overload, mitochondrial dysfunction can disrupt adipocyte insulin signaling pathways, leading to impaired glucose transport into adipocytes and the inability to suppress cell lipolysis, further increasing blood glucose and free fatty acid (FFA) levels in circulation (Miranda et al., 2005). As a result of increased circulating blood glucose and FFA levels, hepatocytes and myocytes are placed under great metabolic stress.

# Mitochondrial Dysfunction and Adipose Adaptations with Metabolic Challenge

With the onset of metabolic challenge, the imbalance of redox states and mitochondrial dysfunction may be instrumental to the development of insulin resistance and MetS (Long et al., 2015). With nutrient overload, mitochondrial dysfunction in adipose tissue will give rise to an increase in inflammatory mediators (adipokines) resulting in tissue remodeling, and potentially cell death (Trayhurn, 2005; Vernochet et al., 2014). Adipokine dysregulation can result in the fibrosis, or inadequate healing response, of adipose tissue (Kwon et al., 2012), as well as liver (Chiang et al., 2011). As adipose depots become inflamed and fibrotic, they become limited in their energy storing and endocrine function, resulting in the deposition of ectopic lipid in other tissues coupled with the presences of insulin resistance (i.e., muscle and liver) (Sun et al., 2013). Altered mitochondrial function, ectopic lipid formation, and incomplete or inadequate healing responses are hallmarks of MSK diseases, and are likely initiated by low-level systemic inflammation.

# Mechanisms of Tissue Metabolic Dysfunction with Obesity

Once subcutaneous adipose depots become overwhelmed, lipid spill-over leads to ectopic lipid accumulation in visceral adipose tissue depots and other insulin sensitive tissues, such as muscle (Miranda et al., 2005). In muscle, the nutrient overload placed on myocytes, similar to adipocytes, can lead to metabolically dysfunctional cells, further contributing to the already dysregulated inflammatory environment. The complexity and interconnectedness between these processes and mitochondrial dysfunction are illustrated in **Figure 7**.

# DIETARY CONSIDERATIONS, NUTRIENT CONTENT, AND OBESITY PHENOTYPES

While discussing inflammation from DIO, it is important to consider the composition of the experimental diets, as the dietary profile and energy density of a given diet likely will impact the resultant inflammatory profile in each preclinical model (reviewed in Hariri and Thibault, 2010). For example, diets rich in fat elicit different types of glucose intolerance compared to diets rich in sugar, whereby dietary fat largely compromises muscular glucose tolerance, and sugar challenges liver lipogenic and gluconeogenic enzyme activities (Sumiyoshi et al., 2006). As such, different dietary composition may yield different findings or differences in the severity of disease outcomes.

Fat and sugar dietary constituents can contribute to different animal phenotypes, whereby animals on a high sugar diet do not necessarily gain weight, but demonstrate a conversion of lean body mass to fat body mass (Sumiyoshi et al., 2006). Diets high in saturated fat, however, generally result in increased body fat and body mass, although the type of fat employed (i.e., omega-3 fats) can also have anti-inflammatory effects, and can modulate body fat and body mass (Schemmel et al., 1969; Sumiyoshi et al., 2006; Hariri and Thibault, 2010; Wu et al., 2014, 2017a). To add to the complexity of this matter, many animal species, like humans, demonstrate obesity prone and obesity resistant phenotypes (Schemmel et al., 1969).  This feature allows for the experimental evaluation of animals that are exposed to the same amounts of an obesogenic diet, but develop disparate increases in body mass. With MSK disease, this is an important consideration, as tissue damage can be evaluated between animals with similar mass, but increased body fat and lowlevel inflammation (Hariri and Thibault, 2010). Epidemiological studies reveal MetS can also occur in individuals with typical body weight or that are underweight (e.g., HIV-infected patients), with increases in body fat and metabolic derangement, which underscores the relevance of evaluating obesity resistant animals as a surrogate for this phenomenon to understand MSK diseases in the context of MetS (Drelichowska et al., 2015).

# Nutrient Sensing, Mitochondrial Dysfunction, and Metabolic Dysfunction

Mitochondria are the primary cellular organelles that generate adenosine triphosphate (ATP). Mitochondrial dysfunction is attributed to excessive nutrient processing, resulting in the uncoupling of oxidative phosphorylation, effectively increasing reactive oxygen species (ROS) generation and decreasing ATP production (Weiss et al., 2013). The free radical theory states that an imbalance between the generation of ROS and antioxidants produced by peroxisomes can result in ROS stealing electrons from other cellular sources, resulting in cellular damage (Ashok and Ali, 1999). Under nutrient overload, as seen in obesity, peroxisomal numbers and/or activity can be impaired as a result of micronutrient deficiencies and/or excessive inflammatory cytokine (e.g., TNF-α) accumulation resulting from elevated oxidative stress and the accumulation of metabolic by-products.

When exposed to nutrient overload, nutrient sensors [i.e., AMP-activated protein kinase (AMPK), sirtuins (SIRT), and mechanistic target of rapamycin, (mTOR)] become impaired, impair mitochondrial function, and can contribute to systemic inflammation, adiposity, and lipotoxicity. AMPK activity also plays a role in bone homeostasis, as an essential mediator of fat and glucose metabolism on bone remodeling (Jeyabalan et al., 2012; reviewed in Finkel, 2015). In skeletal muscle, leptin has been shown to activate AMPK (Salminen and Kaarniranta, 2012), inhibiting FA synthesis, increasing FA oxidation and glucose uptake (Minokoshi et al., 2002). Moreover, limiting nutrient supply and the associated increase in SIRT-1 activity are hypothesized to enhance muscle cell proliferation, while nutrient overload, or age-related loss of SIRT-1, is expected to provide an unfavorable environment for cell proliferation (Akhmedov and Berdeaux, 2013). Likely, reduced cell proliferation occurs, in part, through age-related loss of SIRT, potentially as a consequence of reduced AMPK activity, which may result in loss or dysfunction of mitochondrial activity (Wang et al., 2015; Berenbaum et al., 2017).

In chondrocytes from human OA patients, mitochondrial activity is deficient, leading to a catabolic cascade of responses. Activating AMPK/SIRT-1, mitochondrial master regulator PGC-1a, and FOXO3A mediates chondroprotection, suggesting these pathways may also be critical to maintaining mitochondrial function in chondrocytes, which is critical to preserving cartilage tissue structure (Zhao et al., 2014; Wang et al., 2015; Berenbaum et al., 2017). Mitigating age-related changes in oxidative stress in cartilage (i.e., through SIRT-3) protects against OA-progression and osteoclastogenesis in bone, suggesting that improving the resistance of cartilage to oxidative stress may be one therapeutic target for OA, or for maintaining bone homeostasis (Fu et al., 2016; Huh et al., 2016).

# Reactive Oxygen Species (ROS) and Oxidative Stress Alterations with Obesity

As a by-product of mitochondrial metabolism and homeostasis, ROS are produced and are highly regulated. With cell damage, mitochondrial dysfunction, or oxidative stress, ROS levels accumulate and are associated with the onset of metabolic dysfunction (Serra et al., 2013). ROS accumulation can lead to: lipid peroxidation and disruption of the cellular membrane; ER stress, resulting in protein mis-folding and unfolding, and a decrease in protein synthesis, ultimately rendering cells incapable of clearing misfolded proteins; and activation of Caspase-3 and cell apoptosis (Weiss et al., 2013). Furthermore, oxidative stress in tissues is reported to be an important pathogenic mechanism of MetS onset (Furukawa et al., 2004).

Increased fat mass has been linked to increased systemic markers of oxidative stress in humans and mice (**Figure 7**) (Furukawa et al., 2004). For example, increased peroxide and reduced endothelial nitric oxide synthase have been associated with cancellous bone loss in an obesity model (Ohnishi et al., 2009). In bone, reactive oxygen species and oxidative stress are critical to osteoclast differentiation. As such, ROS may contribute to osteoporosis and bone catabolism, through activation of RANKL—or receptor activator of NF-κB ligand- influencing osteoclast activity, as well as other pathways downstream such as NF-κB (Callaway and Jiang, 2015). With increased systemic inflammation associated with obesity, bone marrow macrophages and their progenitors can be increased (Singer et al., 2014), with related stimulation of osteoclastogenesis and reduced osteoblast development (Kyung et al., 2009; Halade et al., 2011). Yue and co-workers also reported that leptin produced from obese adipose tissue can bind to leptin receptors on mesenchymal stem cells promoting differentiation to adipocytes and inhibiting osteoblast formation (Yue et al., 2016). The central effects of leptin can also promote cancellous bone loss via the sympathetic nervous system (Ducy et al., 2000; Takeda et al., 2002).

In skeletal muscle, although low-levels of ROS are necessary for force production, high levels of ROS can result in great susceptibility to fatigue and contractile dysfunction (Powers et al., 2011) and are associated with reduced muscle repair capacity (Kozakowska et al., 2015). There may be a link between dysfunctional repair and overuse tendinopathies through ROS production, but this idea remains to be tested experimentally (Bestwick and Maffulli, 2004; Longo et al., 2008). Although the signaling pathway between ROS and osteoarthritis is not clear, there are examples of antioxidant therapy (e.g., dietary polyphenols and hyaluronic acid) that are useful in human studies (reviewed in Lepetsos and Papavassiliou, 2016).

# Lipotoxicity and Insulin Resistance with Obesity

Accumulation of lipids and lipid by-products can result in dysfunction in myocyte metabolic pathways. Lipid concentration is considered a strong indicator of the sensitivity of myocytes insulin-mediated pathways, as well as adipocyte and hepatocyte insulin sensitivity, significantly affecting substrate metabolism and circulating metabolite concentrations (Weiss et al., 2013). The adverse metabolic effects associated with ectopic lipid storage are supported by lipodystrophy studies, showing that muscle lipid accumulation can result in severe insulin resistance and diabetes (Weiss et al., 2013). In addition to the liver, primarily utilized for short term energy storage when circulating nutrient levels are elevated, skeletal muscle is a primary site of glucose uptake and utilization (Yu et al., 2012). Skeletal muscle insulin resistance largely contributes to whole body glucose levels and the presence of a pro-inflammatory environment, as the body is comprised of 40–50% muscle by weight (DeFronzo and Tripathy, 2009; Weiss et al., 2013). In response to low-level inflammation from DIO, extracellular matrix remodeling may contribute to the onset of insulin resistance, thereby inducing collagen synthesis and muscle fibrosis, and contributing to decreased insulin signaling (Williams et al., 2015).

Insulin resistance can also impact tissues of the joint organ system, because insulin resistance has been linked to chondrocyte dysfunction, and insulin appears to have a protective role for synoviocytes, whereby insulin blunts TNF-induced matrix metalloproteinase release (Hamada et al., 2015). However, patients with diabetes exhibit increased synovial levels of TNF-α and macrophages, suggesting that insulin resistance may impair the protective effect of insulin in the joint (Hamada et al., 2016), and greater insulin resistance is related to lower femoral neck strength (Srikanthan et al., 2014).

# Accumulation of Metabolic By-Products and Toxic Lipid Metabolites in Obesity

Elevated levels of oxidative stress can result in incomplete substrate oxidation. These impairments can lead to the excessive accumulation of toxic lipid metabolites (e.g., diacylglycerol, fatty acetyl CoA, and ceramides) and ROS, all natural by-products of cellular metabolism (Broussard and Devkota, 2016). Fatty acid trafficking in muscle may be one of the key factors involved in the onset of insulin resistance, by changing the availability of substrates involved in formation and clearance of harmful lipid intermediates (diacylglycerides and ceramide) (Mittendorfer, 2011). The balance between synthesis and breakdown of these intermediates may influence the balance between the formation and breakdown of FA and TGs, resulting in the storage and development of lipid depots in non-adipose tissues (Mittendorfer, 2011) from alterations in clearance mechanisms.

# Hyperglycemia and Advanced Glycation End Products with Obesity

Advanced glycation end products (AGEs) have a pronounced effect on many proteins, particularly collagen. Generally, AGEs accumulate in tissues and form cross-links with targeted proteins, alter cell structure, and interact with receptors that induce oxidative stress and inflammation, resulting in tissue damage (Sanguineti et al., 2014), **Figure 5**. Specifically, AGEs can alter the physiological failure behavior of some tissues, including tendons (Fessel et al., 2014), by increasing lateral spacing, accumulating cross-links between collagen fibrils, and reducing mechanical properties of tissues by reducing sliding behavior (Gautieri et al., 2017). Moreover, AGEs affect growth and modulate the physiological processes of OA (Franke et al., 2009), and damageassociated molecular patterns (DAMPS) can bind to RAGE and contribute to the pathogenesis of OA (Rosenberg et al., 2017). AGEs can induce muscle atrophy in a RAGE-mediated AMPK down-regulated manner (Chiu et al., 2016) and may affect skeletal muscle growth and contractile function in mice fed a diet high in AGEs (Egawa et al., 2017). In bone, AGEs are associated with decreased bone mineral density and impaired bone quality, eliciting oxidative stress and inflammatory responses in bone cells, while altering material properties of bone collagen fibers via cross-linking (Yamamoto and Sugimoto, 2016).

# OBESITY, BACTERIAL LIPOPOLYSACCHARIDE (LPS), AND GUT MICROBIOTA

It is well-established that the gut microbiota is altered with DIO in a manner that promotes a pro-inflammatory environment (Cani et al., 2009; Cândido et al., 2017). Through changes in tight junction proteins and intestinal barrier integrity, components of gram-negative bacteria may leak into the systemic circulation, resulting in increased systemic LPS concentrations in obese animals including humans (**Figure 8**) (Cani et al., 2008). Systemic LPS, inflammation, and metabolic disturbance resulting from altered gut microbiota and a leaky gut are believed to be key initiating factors leading to insulin resistance (Cani et al., 2007). In the context of musculoskeletal disease, an impaired gut barrier function has been implicated in rheumatoid arthritis and OA, and may present a viable therapeutic approach for disease management (Scher and Abramson, 2011). However, to date, cause and effect relations between rheumatoid arthritis and the gut microbiota are still being explored (Bravo-Blas et al., 2016).

We were among the first to identify a significant linkage between constituents of the gut microbiota (Methanobrevibacter and Lactobacillis spp.) and musculoskeletal changes in metabolic OA/joint damage severity (Collins et al., 2015a) and the onset of compromised muscle integrity (Collins et al., 2016c). These findings link systemic (serum) and local (synovial fluid) to LPS and musculoskeletal changes with diet-induced obesity (Huang et al., 2016) (**Figure 9**). Using a fecal transplant intervention, lipid profiles can be transferred from donors to germ-free hosts, and altered muscle integrity can be recapitulated, suggesting that the gut microbiota may have a direct effect on muscle development and integrity (Yan et al., 2016). The gut-OA pathophysiological link indicates that dietary and microbiotabased interventions may be important therapeutic opportunities that should be evaluated in the context of MSK health (Collins et al., 2015a; Huang et al., 2016; Berenbaum et al., 2017). There is a need for studies detailing the causal mechanisms of interactions among the gut-microbiota, LPS, and MSK changes. However, given the short-term changes in gut microbiota composition, as well as the decrease in variance in relative abundance across species, studies promoting gut microbiota diversity could be useful in this context. One such candidate is evaluating the impact of prebiotic fiber, which may positively modulate the gut microbiota to promote improved host interactions (Bomhof et al., 2014; Paul et al., 2016; Nicolucci et al., 2017; Parnell et al., 2017).

# IMPLICATIONS FOR CONSERVATIVE CARE STRATEGIES RELATED TO METS AND INFLAMMATION

Information regarding pharmacologic management for obesity and metabolic syndrome can be found elsewhere (Apovian et al., 2015), and promising therapeutic targets are discussed throughout this review (and highlighted in **Table 3**).

The inflammatory interface between damage to MSK components and MetS should be a critical consideration, as protecting and preserving MSK structural integrity could be a key approach to conservative management of MetS. Presently, clinical guidelines indicate that weight loss and exercise are good evidence-based conservative approaches for MSK conditions (McAlindon et al., 2014). In the context of osteoarthritis specifically, the combined effect of 18-months of dietary modulation and a combination program involving both aerobic and strength training-based exercise was found to induce weight loss, reduce knee compressive forces, reduce serum IL-6 levels, decrease infrapatellar fat pad size, decrease pain, and increase function in overweight or obese adults with knee OA (Messier et al., 2013; Beavers et al., 2015; Pogacnik Murillo et al., 2017). As such, careful characterization of exercise and dietary interventions in human and other animal models is needed to implement these strategies.

Although there are several candidates for dietary intervention, prebiotic fiber and probiotic supplementation targeting the microbiome (Nicolucci et al., 2017; Parnell et al., 2017), and decreased intake of dietary sugar (Te Morenga et al., 2013) are three safe, readily available, and translatable dietary interventions to protect against MSK damage due to metabolic disturbance that warrant further investigation. As the onset of MSK damage with metabolic disturbance does not have a readily identified starting point, studies are needed to evaluate the efficacy and utility of these dietary modulations in the patient population of interest (e.g., in prevention vs. reversibility and acute vs. chronic), as well as unintended consequences of these modifications. It would be critical to monitor muscle, tendon, bone, and cartilage tissues simultaneously while evaluating these dietary interventions, to facilitate an understanding of the benefits and interconnectedness of these MSK tissue changes and diseases.

# OPPORTUNITIES AND FUTURE RESEARCH DIRECTIONS

There are several gaps in the current understanding of the effects of low-level systemic inflammation vs. metabolic disturbance on musculoskeletal health. Many of these gaps are summarized in **Table 3**. Whether muscle is a primary target tissue in the motion segment, and whether muscular changes directly result TABLE 3 | Remaining questions, research agenda, and evidence-supported candidate pathways, targets, and conservative care opportunities.

#### Remaining Questions:

#### Research Agenda:

#### Evidence-Supported Candidate Pathways:

#### Evidence-Supported Candidate Targets:

in subsequent changes in bone, tendon, and joints, remains to be assessed and clarified. The studies presented here underscore the need for evaluating multiple musculoskeletal tissues and diseases in concert with MetS (as well as defining which elements of the MetS are responsible for which consequences of obesity). This approach will facilitate an understanding of how damage in each tissue develops with respect to the other tissues comprising a motion segment, how the different tissues may contribute to homeostasis or damage of the motion segment, and what role that MetS/inflammation plays in manifesting the risks of tissue damage. At present, it is unclear if: MetSrelated inflammation increases in severity with chronicity; systemic inflammation is constant with continued metabolic disturbance; or MetS-related inflammation is dynamic and fluctuating. It may be useful to evaluate tissues as "sources" or "targets" of low-level systemic inflammation (**Figure 9**). This conceptual framework is demonstrated in the data from our previous studies, which suggest a systemic-to-local timecourse shift from serum to synovial fluid, and serum to muscle changes (Collins et al., 2016a,b). Briefly, metabolic dysregulation initiates an inflammatory cascade, whereby an early perturbation in a presumptive inflammatory source (i.e., visceral fat and gut microbiota) results in target tissue (i.e., muscle) adaptation and subsequent damage. Although the source of the inflammation is unclear, as are the specific pathways involved, target tissue inflammation and damage may affect subsequent associated motion segment tissues (i.e., bone, tendon, and joints), and these relations are affected by many factors (i.e., the dynamic make-up/profile of the inflammatory components, time of exposure, and severity of the metabolic disturbance).

Furthermore, adaptation and impact of sources of inflammation on target tissues are important knowledge gaps that need to be addressed. It would be of value to determine if metabolic dysfunction and regulation are integrated and interdependent, or if source tissues and target tissues of the motion segment are regulated and affected differently and independently, at least in a first approximation. Identifying the potential for reversibility of tissue damage with dietinduced obesity is another critical area of research that should be addressed. The mechanism(s) by which target tissue or motion segment tissue changes may contribute to MetS and inflammation are incompletely understood at this time. Time-course studies are needed to answer these questions.

Discussion of the impact of metabolic disturbance on neuroregulatory systems, and a role for neuroinflammationmediated influence on loss of musculoskeletal integrity was limited in this review, but this may be a potentially important area for future research. Nearly all of the tissues of a motion segment discussed are innervated except for adult cartilage, and thus, neuro-regulation and how it is affected primarily and secondarily by MetS associated inflammation, may be critical in understanding the underlying mechanistic considerations. In particular, how these changes in neuroregulation and neuroinflammation contribute to a loss of muscle integrity is an interesting area for future research.

# SUMMARY

Studies presented in this review implicate several pathways that may be critical for the onset and progression of systemic inflammation due to metabolic disturbance and musculoskeletal damage. Some of the pathways that could be involved are mitogen-activated protein kinases (MAPK, p38 MAPK, JNK), myeloid differentiation primary response gene 88 (MyD88), NFκB, and the NLRP3 inflammasome. Likely, some or all of these pathways may be activated in parallel creating a potentially positive-feedback based redundant system. Investigations targeting specific pathways in the context of metabolic disturbance and MSK disease will provide much needed mechanistic insights to better understand the consequences of diet-induced obesity.

A motion segment, such as a leg is comprised of a number of interdependent components (i.e., muscles, tendons, bones, and articular joints) that rely on the integrated biological and mechanical integrity of all tissues for optimal function. Thus, the direct and indirect impact of dysregulation via MetS and associated inflammation on any one element may have a "ripple" effect that may be compounded over time. There are many important and relevant gaps in this research area, and addressing them will provide valuable insights into the relations among the motion segment, common inflammatory pathways, and resulting MSK disease.

# AUTHOR CONTRIBUTIONS

KC was responsible for conception and design, collection, and assembly of data analysis and interpretation of the data, drafting of the article, critical revision of the article for important intellectual content and final approval of the article. WH, GM, RR, JR, IS, and RZ were responsible for conception and design, collection and assembly of data analysis, interpretation of the data, critical revision of the article for important intellectual

# REFERENCES

content and final approval of the article. DH was responsible for conception and design, analysis and interpretation of the data, drafting of the article, critical revision of the article for important intellectual content and final approval of the article.

# ACKNOWLEDGMENTS

This work was supported by the Canadian Institutes of Health Research # RT736475 and MOP 115076, the Canada Research Chair Programme, the Alberta Innovates Health Solutions Osteoarthritis Team Grant, Alberta Innovates Health Solutions, Alberta Health Services Strategic Clinical Network Program, Canadian Institutes of Health Research Banting and Best Canada Graduate Scholarship, and the Killam Foundation. Due to space and scope limitations, we apologize to authors in this area whose work we were unable to include in the present review.

infrapatellar adipose tissue depots and their response to class II and III obesity in patients with OA. Arthritis Rheumatol. 69, 1396–1406. doi: 10.1002/art.40102

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Collins, Herzog, MacDonald, Reimer, Rios, Smith, Zernicke and Hart. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Human Milk and Donkey Milk, Compared to Cow Milk, Reduce Inflammatory Mediators and Modulate Glucose and Lipid Metabolism, Acting on Mitochondrial Function and Oleylethanolamide Levels in Rat Skeletal Muscle

#### Edited by:

Oreste Gualillo, Servicio Gallego de Salud, Spain

#### Reviewed by:

Luciana Lopes Guimaraes, Universidade Santa Cecilia, Spain Vicente Lahera, Complutense University of Madrid, Spain Antonio Longo, Università degli Studi di Catania, Italy

# \*Correspondence:

Maria P. Mollica<EMAIL_ADDRESS>
† These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology

Received: 19 June 2017 Accepted: 10 January 2018 Published: 30 January 2018

#### Citation:

Trinchese G, Cavaliere G, De Filippo C, Aceto S, Prisco M, Chun JT, Penna E, Negri R, Muredda L, Demurtas A, Banni S, Berni-Canani R, Mattace Raso G, Calignano A, Meli R, Greco L, Crispino M and Mollica MP (2018) Human Milk and Donkey Milk, Compared to Cow Milk, Reduce Inflammatory Mediators and Modulate Glucose and Lipid Metabolism, Acting on Mitochondrial Function and Oleylethanolamide Levels in Rat Skeletal Muscle. Front. Physiol. 9:32. doi: 10.3389/fphys.2018.00032 Giovanna Trinchese1†, Gina Cavaliere1†, Chiara De Filippo<sup>1</sup> , Serena Aceto<sup>1</sup> , Marina Prisco<sup>1</sup> , Jong Tai Chun<sup>2</sup> , Eduardo Penna<sup>1</sup> , Rossella Negri <sup>3</sup> , Laura Muredda<sup>4</sup> , Andrea Demurtas <sup>4</sup> , Sebastiano Banni <sup>4</sup> , Roberto Berni-Canani <sup>3</sup> , Giuseppina Mattace Raso<sup>5</sup> , Antonio Calignano<sup>5</sup> , Rosaria Meli <sup>5</sup> , Luigi Greco<sup>3</sup> , Marianna Crispino<sup>1</sup> and Maria P. Mollica<sup>1</sup> \*

<sup>1</sup> Department of Biology, University of Naples Federico II, Naples, Italy, <sup>2</sup> Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Naples, Italy, <sup>3</sup> European Laboratory for Food Induced Diseases, Department of Translational Medical Sciences, University of Naples Federico II, Naples, Italy, <sup>4</sup> Dipartimento di Scienze Biomediche, Università degli Studi di Cagliari, Cagliari, Italy, <sup>5</sup> Department of Pharmacy, University of Naples Federico II, Naples, Italy

Scope: Milk from various species differs in nutrient composition. In particular, human milk (HM) and donkey milk (DM) are characterized by a relative high level of triacylglycerol enriched in palmitic acid in sn-2 position. These dietary fats seem to exert beneficial nutritional properties through N-acylethanolamine tissue modulation. The aim of this study is to compare the effects of cow milk (CM), DM, and HM on inflammation and glucose and lipid metabolism, focusing on mitochondrial function, efficiency, and dynamics in skeletal muscle, which is the major determinant of resting metabolic rate. Moreover, we also evaluated the levels of endocannabinoids and N-acylethanolamines in liver and skeletal muscle, since tissue fatty acid profiles can be modulated by nutrient intervention.

Procedures: To this aim, rats were fed with CM, DM, or HM for 4 weeks. Then, glucose tolerance and insulin resistance were analyzed. Pro-inflammatory and anti-inflammatory cytokines were evaluated in serum and skeletal muscle. Skeletal muscle was also processed to estimate mitochondrial function, efficiency, and dynamics, oxidative stress, and antioxidant/detoxifying enzyme activities. Fatty acid profiles, endocannabinoids, and N-acylethanolamine congeners were determined in liver and skeletal muscle tissue.

Results: We demonstrated that DM or HM administration reducing inflammation status, improves glucose disposal and insulin resistance and reduces lipid accumulation in skeletal muscle. Moreover, HM or DM administration increases redox status, and mitochondrial uncoupling, affecting mitochondrial dynamics in the skeletal muscle. Interestingly, HM and DM supplementation increase liver and muscle levels of the N-oleoylethanolamine (OEA), a key regulator of lipid metabolism and inflammation.

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Conclusions: HM and DM have a healthy nutritional effect, acting on inflammatory factors and glucose and lipid metabolism. This beneficial effect is associated to a modulation of mitochondrial function, efficiency, and dynamics and to an increase of OEA levels in skeletal muscle.

Keywords: human milk, donkey milk, mitochondrial functions, mitochondrial dynamics, oxidative stress, inflammation

# INTRODUCTION

Human milk (HM), the natural food of infants, provides an adequate supply of all nutrients necessary to support postnatal growth and development. In addition, it provides immunomodulant components and plays a key role in preventing inflammation and metabolic diseases throughout life (Agostoni et al., 2013; Cacho and Lawrence, 2017). Interestingly, it has been demonstrated that breastfed individuals have lower rate of obesity and type 2 diabetes than those fed with infant formula (Owen et al., 2006). Such effects have been attributed to appetite regulation and reduced weight gain in breast-fed children and/or to the presence of unique nutrients or bioactive constituents in HM. In fact, metabolism-regulating hormones, such as leptin, adiponectin, resistin and ghrelin, detected in HM (Lönnerdal, 2013), may be involved in the regulation of growth in infancy and might influence the programming of energy balance with long-term consequences on health (Agostoni et al., 2013). In addition, the peculiar high concentration in HM of palmitic acid in the sn-2 position of triacylglycerol backbone seems to play a crucial role in the regulation of energy and lipid metabolism (Innis, 2016). Interestingly, also donkey milk (DM) is characterized by a high concentration of palmitic acid in sn-2 position of triacylglycerol, and is recognized as the best potential substitute for HM due to its remarkable nutritional value coupled with good palatability and reduced allergenicity (Tafaro et al., 2007; Fiocchi et al., 2010). Recently, we have demonstrated that palmitic acid in sn-2 position of triacylglycerol is able to modulate N-acylethanolamine levels in rat tissues, which may possibly explain its effects on lipid and energy metabolism (Carta et al., 2015). Among N-acylethanolamine, palmitoylethanolamide (PEA) and oleylethanolamide (OEA), are PPARα endogenous agonists. PPARs are a family of nuclear receptors that regulate fatty acid metabolism in different tissues. In particular, PPARα activation increases peroxisomal and mitochondrial oxidation and leads to anti-inflammatory and antioxidant effects (Fu et al., 2005; Lo Verme et al., 2005; Contreras et al., 2013).

Recently, we have demonstrated that HM or DM administration improves liver inflammatory state, enhancing hepatic mitochondrial functions and uncoupling (Trinchese et al., 2015). Mitochondrial uncoupling, dissipating the proton gradient across the inner membrane, creates a futile cycle of glucose and fatty acid oxidation without generating ATP (Nedergaard et al., 2005; Tseng et al., 2010), and thereby increases lipid oxidation and reduces intracellular lipid content (Harper et al., 2008). Promoting this inefficient metabolism that generates heat instead of ATP, mitochondrial uncoupling can serve as a potential treatment for obesity. Therefore, drugs, nutrients, or natural molecules modulating the mitochondrial function and efficiency can be considered as potential treatment for obesity and insulin resistance promoting an inefficient metabolism (Li et al., 2000). To ensure a healthy mitochondrial population, cells are equipped with various quality control systems, that regulate mitochondrial shape, function, and mass. These regulatory systems include mitochondrial biogenesis and dynamics (Liesa and Shirihai, 2013). In particular, mitochondrial dynamic is a tightly regulated process controlled by fusion and fission events (Detmer and Chan, 2007). During mitochondrial fusion, optic atrophy (OPA)1 is responsible for mitochondrial inner membrane fusion, and two other proteins mitofusins 1 and 2 (Mfn1 and Mfn2) mediate outer membrane fusion. On the other hand, dynamin-related protein (DRP)1 and fission protein (Fis)1 are involved in mitochondrial fission (Detmer and Chan, 2007). Usually, a shift toward fusion optimizes mitochondrial function and plays a beneficial role in the maintenance of long-term bioenergetic capacity. In contrast, a shift toward fission results in numerous mitochondrial fragments contributing to elimination of irreversibly damaged mitochondria through autophagy.  Skeletal muscle is one of the major targets of insulin accounting for high percentage of the hormone-dependent glucose uptake. Indeed, metabolic impairment in skeletal muscle strongly affects glucose disposal in the whole-body. Moreover, skeletal muscle is a chief determinant of resting metabolic rate, whose reduction is associated with weight gain (Ravussin et al., 1988). Recent data have demonstrated that the insulin-resistant condition in skeletal muscle is characterized by alterations of mitochondrial function and efficiency (Cavaliere et al., 2016).

Here, we examined the effects of supplementation of cow milk (CM), HM, and DM on systemic and skeletal muscle inflammation, insulin sensitivity, endogenous lipids, and endocannabinoids levels in skeletal muscle and liver, and mitochondrial functions in skeletal muscle.

# MATERIALS AND METHODS

# Animals and Chemicals

All chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless specified otherwise. Male Wistar rats (60 days old; 345 ± 7 g; Charles River, Calco, Lecco, Italy) were caged in a temperature-controlled room and exposed to a daily light–dark cycle (12/12 h) with free access to chow diet and drinking water. The rats were divided into four experimental groups (n=7), three of them were supplemented with equicaloric intake (82 kJ) of raw CM, DM, or HM drinking 21, 48, or 22 ml/day, respectively. The animals were treated for 4 weeks. The last group did not receive milk supplement and was used as control. Despite the different volumes used, the energy density provided by the different milk supplements was kept virtually the same (**Table 1**). DM from the Ragusana breed and CM were obtained from "Azienda Agricola Insalata" (Oliveto Citra, SA, Italy). Human milk was kindly provided by the milk bank "Banca del Latte Materno" Ospedale Colle dell'Ara—Chieti-Italy. At the end of the treatments, the animals were anesthetized by intra-peritoneal injection of chloral hydrate (40 mg/100 g body weight), and blood was taken from the inferior cava. Skeletal muscle was removed from the hind leg for analysis, and the samples not immediately used for mitochondrial preparation were stored at −80◦C. All experiments were conducted in compliance with national guidelines for the care and use of research animals (D.L. 116/92, implementation of EEC directive 609/86).

# Serum and Tissue Parameters

Plasma concentrations of triglycerides and cholesterol were measured by colorimetric enzyme reaction by use of commercial kits (SGM Italia, Italy and Randox Laboratories ltd., United Kingdom). Instead, specific ELISA kits were used to measure the serum and tissue levels of IL-1α, IL-10 (Thermo Scientific, Rockford, IL, USA), TNF-α, and monocyte chemoattractant protein-1 (MCP-1) (Biovendor R&D, Brno, Czech Republic), adiponectin and leptin (B-Bridge International Mountain View, CA).

# Oral Glucose Tolerance Test and Insulin Tolerance Test

For oral glucose tolerance test, rats were allowed to fast overnight and then orally dosed with glucose (3 g/kg body weight) dissolved in water. For insulin tolerance test, fasting rats (5 h)

Values represent means ± SEM from n = 7 animals/group. Labeled means without a common letter differ (P < 0.05).

were intraperitoneally injected with insulin (homolog-rapidacting, 10 unit/kg body weight in sterile saline; Novartis, Basel, Switzerland). Blood was collected by direct flow from a small tail cut, before and after the treatments at certain intervals, and the glucose levels were determined by glucose monitor calibrated for rats (BRIO, Ascensia, NY), and the insulin levels by ELISA (Mercodia rat insulin; Mercodia, Uppsala, Sweden). Basal fasting values of serum glucose and insulin were used to calculate Homoeostatic Model Assessment (HOMA) index as [Glucose (mg/dL) <sup>∗</sup> Insulin (mU/L)]/405 (Cacho et al., 2008).

# Mitochondrial Parameters and Basal and Inducible Proton Leak

Mitochondrial isolation, oxygen consumption, and proton leakage measurements were performed as previously reported (Cavaliere et al., 2016). Oxygen consumption (polarographically measured using a Clark-type electrode) was measured in the presence of substrates and ADP (state 3) or with substrates alone (state 4), and respiratory control ratio (RCR) was calculated. Mitochondrial proton leakage was assessed by a titration of the steady-state respiration rate as a function of the mitochondrial membrane potential in skeletal muscle mitochondria. Carnitine palmitoyl-transferase (CPT) system, aconitase, and superoxide dismutase (SOD) specific activity were measured spectrophotometrically as previously reported (Flohe and Otting, 1984; Alexson and Nedergaard, 1988; Hausladen and Fridovich, 1996). Rate of mitochondrial H2O<sup>2</sup> release was assayed by following the linear increase in fluorescence (ex 312 nm and em 420 nm) due to the oxidation of homovanillic acid in the presence of horseradish peroxidase (Barja, 1998).

# Lipid Content, Redox Status, and Nrf2-Activated Enzyme Activities in Skeletal Muscles

Total lipid content in the skeletal muscle was estimated by using the Folch method. Briefly, the skeletal muscle was weighed, chopped into small pieces, thoroughly mixed, and finally homogenized with water (volumes equal to twice the skeletal muscle weight) in a Polytron homogenizer. On appropriate aliquots of the homogenate, lipid content was determined gravimetrically after extraction in chloroform–methanol and evaporation to constant weight by a rotating evaporator (Folch et al., 1957).

TABLE 2 | The sequences (5′ -3′ ) of the primers used in Real Time PCR.

Reduced (GSH) and oxidized (GSSG) glutathione concentrations in skeletal muscle were measured with the dithionitrobenzoic acid (DTNB)-GSSG reductase recycling assay (Bergamo et al., 2007), and the GSH/GSSG ratio was used as an oxidative stress marker. To investigate the possible involvement of NF-E2-related factor 2 (Nrf2) in the diet-induced stress, cytoplasmic extracts were prepared from rat skeletal muscle. The enzymatic activities of glutathione S-transferases (GSTs) and quinone-oxidoreductase (NQO1) were evaluated spectrophotometrically in cytoplasmic extracts (Benson et al., 1980; Habig and Jakoby, 1981; Levine et al., 1990).

# Lipid Analysis

Aliquots of total lipid extract from tissues were mildly saponified as previously described (Banni et al., 1996) in order to obtain free fatty acids for HPLC analysis. Separation of unsaturated fatty acids was carried out with an Agilent 1100 HPLC system (Agilent, Palo Alto, Calif., USA) equipped with a diode

common letter differ (P < 0.05).

array detector as previously reported. Spectra (195–315 nm) of the eluate were obtained every 1.28 s and were electronically stored. These spectra were acquired to confirm the identified HPLC peaks (Melis et al., 2001)**.** Quantification of anandamide (AEA), 2-arachidonoyl glycerol (2-AG), PEA, and OEA was

conducted by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) using as internal standards their deuterated homologs as previously described (Piscitelli et al., 2011).

# Real Time PCR Analysis

Total RNA was extracted from skeletal muscle using the TRIzol reagent (Ambion). After DNase treatment, RNA was quantified and reverse-transcribed (1 µg) using the Advantage RT-PCR kit (Clontech). For the evaluation of mitochondrial fission and fusion gene transcription, we used murine primers, whose sequences are shown in **Table 2**. qPCR was performed as previously described (Trinchese et al., 2015).

# Western Blot Analysis

Tissues were homogenized in lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, Tris-HCl 20 mM, pH 7.5) containing protease and phosphatase inhibitors. Proteins (15– 30 µg/lane) were separated on 10–12% SDS-polyacrilamide gel and transferred to PVDF membranes (GE Healthcare). Blocking (5% non-fat milk, 30 min) and immunoreaction (o/n) were performed in TBST buffer (0.1% Tween, 150mM NaCl, 10mM Tris-HCl, pH 7.5) at room temperature. The primary antibodies were anti-Mitofusin1, anti-Mitofusin2, anti-DRP1 (all Santa Cruz), and anti-β-actin (BD Biosciences). Signals were visualized by horseradish peroxidase-linked secondary antibodies in enzyme-linked chemiluminescence (ECL, Millipore). Relative protein levels were normalized with β-actin levels on the same membrane.

# Transmission Electron Microscopy

Samples of soleus muscle were cut into ∼1 mm<sup>3</sup> fragments and fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, (pH 7.4) for 4 h at 4◦C. After washing in phosphate buffer, tissues were post-fixed with 1% osmium tetroxide in phosphate buffer for 2 h at 4◦C. Samples were dehydrated through graded alcohols (50, 70, 90, and 100%) and propylene oxide, and subsequently embedded in EMbed 812 resin (Electron Microscopy Science) (48 h at 60◦C). Ultrathin (70 nm thick) sections were cut at a Sorvall Porter-Blum ultramicrotome, contrasted with uranyl acetate and lead citrate. The ultrastructure of the sample was visualized on a transmission electron microscope Philips 208S at Centro

PEA, N-palmitoylethanolamine; AEA, anandamide; 2AG, 2-arachidonoyl glycerol. Values represent means ± SE from n = 7 animals/group.

TABLE 3 | Tissue levels of AEA, PEA, and 2AG.

(P < 0.05).

di Servizi Metrologici Avanzati (CeSMA, University of Naples Federico II); micrographs were acquired with a Mega View II Soft Imaging System camera. Mitochondrial compartment volume density was measured using the stereological point-sampling technique (Weibel et al., 1966; Altunkaynak et al., 2012). For each experimental group, 10 random images were taken at 25,000 x magnification. A grid with equally and symmetrically spaced intersection points was overlaid on each micrograph using the Image J1.50 software (NIMH). Mitochondrial volume density for each micrograph was calculated as the ratio of points overlapping the cellular compartment of interest and the total number of grid points overlapping the cytoplasm area. To measure mitochondrial fusion and fission percentage, the number of mitochondria undergoing fusion and fission on total mitochondrial number in the visual field was counted in 10 images for each experimental group.

# Statistical Analysis

Data were analyzed by one-way ANOVA followed by the Bonferroni post-hoc test. Labeled means without a common letter

differ (P < 0.05). Analyses were performed and plotted using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

# RESULTS

# Body Weight and Food Intake

As expected, although different milk treatments provided similar total food intake, CM-treated animals exhibited a significant increase in body weight and body weight gain when compared with control, DM- and HM-treated rats (**Table 1**).

# Serum Metabolites and Inflammatory Parameters

Serum parameters were reported in **Figure 1**. Triglycerides significantly decreased in the DM-fed animals (**Figure 1A**), while cholesterol and MCP1 serum levels were not affected by different

presented as means ± SEM from n = 7 animals/group. Labeled means without a common letter differ (P < 0.05).