# ARE RODENT MODELS FIT FOR INVESTIGATION OF HUMAN OBESITY AND RELATED DISEASES?

EDITED BY: Patrick C. Even, Sam Virtue, Nicholas M. Morton, Gilles Fromentin and Robert K. Semple PUBLISHED IN: Frontiers in Nutrition and Frontiers in Endocronology

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ISSN 1664-8714 ISBN 978-2-88945-425-9 DOI 10.3389/978-2-88945-425-9

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# **ARE RODENT MODELS FIT FOR INVESTIGATION OF HUMAN OBESITY AND RELATED DISEASES?**

#### Topic Editors:

**Patrick C. Even,** UMR Physiologie de la Nutrition et du Comportement Alimentaire, AgroParisTech, INRA, Université Paris Saclay, France **Sam Virtue,** Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, United Kingdom

**Nicholas M. Morton,** University/BHF Centre for Cardiovascular Sciences, Queens Medical Research Institute, University of Edinburgh, United Kingdom

**Gilles Fromentin,** UMR Physiologie de la Nutrition et du Comportement Alimentaire, AgroParisTech, INRA, Université Paris Saclay, France

**Robert K. Semple,** Centre for Cardiovascular Sciences, Queens Medical Research Institute, University of Edinburgh, United Kingdom

Not only developed countries, but also most developing areas of the world, have experienced a surge in obesity prevalence over recent decades. Obesity complications are now among the leading causes of premature mortality, encompassing conditions such as coronary heart disease, stroke, and type 2 diabetes. This places a heavy burden on contemporary healthcare systems. While rodent models have limitations as experimental models of human obesity-related disease, study of rats and mice either spontaneously prone - or resistant - to obesity, or genetically engineered to illuminate underlying mechanisms has yielded key information about the metabolic defects linked to obesity, and their associated diseases. This topic includes both original research studies and reviews of the use of animal studies in specific areas of obesity-related disease. Various methodological approaches are discussed, with Cover image: Emilia Stasiak/Shutterstock.com evaluation of the extent to which use of animal models has facilitated progress, or, conversely, has proved a cul de sac in investigation of human disease mechanisms. Consideration is also given to future strategies to use such rodent models optimally to enhance comprehension and treatment of pandemic human obesity-related diseases.

**Citation:** Even, P. C., Virtue, S., Morton, N. M., Fromentin, G., Semple, R. K., eds. (2018). Are Rodent Models Fit for Investigation of Human Obesity and Related Diseases? Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-425-9

# Table of Contents

*06 Editorial: Are Rodent Models Fit for Investigation of Human Obesity and Related Diseases?* Patrick C. Even, Sam Virtue, Nicholas M. Morton, Gilles Fromentin and Robert K. Semple *08 Knowledge Gaps in Rodent Pancreas Biology: Taking Human Pluripotent Stem Cell-Derived Pancreatic Beta Cells into Our Own Hands* Munirah Mohamad Santosa, Blaise Su Jun Low, Nicole Min Qian Pek and Adrian Kee Keong Teo *17 Mouse Models of Human GWAS Hits for Obesity and Diabetes in the Post Genomic Era: Time for Reevaluation* Samantha Laber and Roger D. Cox *22 The Importance of Context: Uncovering Species- and Tissue-Specific Effects of Genetic Risk Variants for Type 2 Diabetes* Soren K. Thomsen, Mark I. McCarthy and Anna L. Gloyn *28 How Useful Are Monogenic Rodent Models for the Study of Human Non-Alcoholic Fatty Liver Disease?* Jake P. Mann, Robert K. Semple and Matthew J. Armstrong *39 Genetic Rodent Models of Obesity-Associated Ovarian Dysfunction and Subfertility: Insights into Polycystic Ovary Syndrome* Isabel Huang-Doran and Stephen Franks *49 Relationships between Rodent White Adipose Fat Pads and Human White Adipose Fat Depots* Daniella E. Chusyd, Donghai Wang, Derek M. Huffman and Tim R. Nagy *61 Mechanisms of Comorbidities Associated With the Metabolic Syndrome: Insights from the JCR:LA-cp Corpulent Rat Strain* Abdoulaye Diane, W. David Pierce, Sandra E. Kelly, Sharon Sokolik, Faye Borthwick, Miriam Jacome-Sosa, Rabban Mangat, Jesus Miguel Pradillo, Stuart McRae Allan, Megan R. Ruth, Catherine J. Field, Rebecca Hutcheson, Petra Rocic, James C. Russell, Donna F. Vine and Spencer D. Proctor *73 Obesity in the Otsuka Long Evans Tokushima Fatty Rat: Mechanisms and Discoveries* Sheng Bi and Timothy H. Moran *78 Modeling Diet-Induced Obesity with Obesity-Prone Rats: Implications for Studies in Females*

Erin D. Giles, Matthew R. Jackman and Paul S. MacLean

*91 Animal Models for the Study of the Relationships between Diet and Obesity: A Focus on Dietary Protein and Estrogen Deficiency*

Tristan Chalvon-Demersay, François Blachier, Daniel Tomé and Anne Blais

*104 Consumption of Substances of Abuse during Pregnancy Increases Consumption in Offspring: Possible Underlying Mechanisms*

Kinning Poon and Sarah F. Leibowitz

*117 Hedonics Act in Unison with the Homeostatic System to Unconsciously Control Body Weight*

Heike Münzberg, Emily Qualls-Creekmore, Sangho Yu, Christopher D. Morrison and Hans-Rudolf Berthoud

Patrick C. Even and Anne Blais

*153 Normocaloric Diet Restores Weight Gain and Insulin Sensitivity in Obese Mice* Giovanni Enrico Lombardo, Biagio Arcidiacono, Roberta Francesca De Rose, Saverio Massimo Lepore, Nicola Costa, Tiziana Montalcini, Antonio Brunetti, Diego Russo, Giovambattista De Sarro and Marilena Celano

# Editorial: Are Rodent Models Fit for Investigation of Human Obesity and Related Diseases?

*Patrick C. Even1 \*, Sam Virtue2 , Nicholas M. Morton3 , Gilles Fromentin1 and Robert K. Semple2,3*

*1UMR Physiologie de la Nutrition et du Comportement Alimentaire, AgroParisTech, INRA, Université Paris Saclay, Paris, France, 2Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, United Kingdom, 3University/BHF Centre for Cardiovascular Sciences, Queens Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom*

Keywords: obesity, disease, rodent models, non-alcoholic fatty liver disease, type 2 diabetes mellitus

#### **Editorial on the Research Topic**

#### **Are Rodent Models Fit for Investigation of Human Obesity and Related Diseases?**

#### *Edited by:*

*Maurizio Muscaritoli, Sapienza Università di Roma, Italy*

#### *Reviewed by:*

*Maurizio Crestani, Universita` degli Studi di Milano, Italy*

> *\*Correspondence: Patrick C. Even<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Clinical Nutrition, a section of the journal Frontiers in Nutrition*

*Received: 09 October 2017 Accepted: 13 November 2017 Published: 01 December 2017*

#### *Citation:*

*Even PC, Virtue S, Morton NM, Fromentin G and Semple RK (2017) Editorial: Are Rodent Models Fit for Investigation of Human Obesity and Related Diseases? Front. Nutr. 4:58. doi: 10.3389/fnut.2017.00058*

Mice and rats provide the primary model systems in which the pathophysiology of obesity and its associated pandemic diseases are investigated. Rodent models have been instrumental in the garnering of numerous insights into fundamental pathophysiological mechanisms that are conserved across species. Nevertheless, in key respects rodent physiology is distinct from that of humans, and uncritical overreliance on rodent findings risks impeding translational progress toward improving human health. This research topic aimed to assess whether current use of mouse and rat models is appropriate to maximize generalizable insights and to minimize erroneous conclusions being drawn about human disease. It solicited 16 publications, encompassing 14 opinions or reviews and 2 original research articles, describing or evaluating many aspects of current research performed using rats or mice, covering both causes and consequences of obesity.

Type 2 diabetes is the main focus of three articles. Santosa et al. discuss the use of the rodent pancreatic beta cell to understand the signaling pathways involved in human beta cell differentiation. Laber and Cox and Thomsen et al. consider the successes for rodent modeling of human genome wide association studies for adiposity and type 2 diabetes mellitus (T2DM), encompassing similarities and differences between mouse and human genomes. They consider how animal models can be developed as more precise disease models of T2DM by targeted gene manipulation in the correct developmental and tissue context. Significant limitations of this strategy are also discussed.

Rodent modeling of two major obesity associated diseases, namely non-alcoholic fatty liver disease (NAFLD) and polycystic ovary syndrome (PCOS), is the focus of two articles. The use of monogenic mouse models to interrogate the pathophysiology and genetic predisposition to NAFLD is discussed by Mann et al. This points to interspecies differences and variability in experimental protocols as a limit for the extent to which results from rodent models can currently be extrapolated to humans. Huang-Doran and Franks reviewed the characteristics of the PCOS and assessed the adequacy of rodent models for investigation of this complex pathology. They highlight the variable recapitulation of the PCOS phenotype in rodents, and the relative lack of insulin resistance-related PCOS, quite unlike humans.

Rat models have been used for three decades to study facets of nutrition, endocrinology, the metabolic syndrome, obesity, lipid metabolism, vascular myocardial pathophysiology and pharmacology, and are the primary focus of several articles in the topic.  The authors also elaborate on sexual dimorphism in

adipose tissue depots and attempt to explain why premenopausal women generally have a healthier metabolic risk profile than men. Diane et al. discuss how the JCR:LA-cp rat has contributed to understanding of the gut's contribution to increased production of chylomicron particles, and how ruminant-derived trans fatty acids participate in regulating lipid metabolism. The authors also consider modeling of PCOS in this strain of rat. Bi and Moran focused on the insights into the neural basis of food intake and body weight control that have been yielded by studies of the obese OLEF-T rat, a CCK-1 receptor knockout model. The OLEF-T rat has also been valuable in dissection of interactions among exercise and food intake, and in investigating the role of the DMH in energy balance.

Giles et al. report lessons learned from study of the OP/OR rat, including the observation that OP rats have disturbances in hypothalamic signaling pathways involved in energy homeostasis before obesity develops, and while still on a low-fat diet. They also highlight the sexual dimorphism of obesity and point out that this is often ignored because most preclinical studies are performed on male rodent models. The authors have extended the study of the obese rat model to address the menopause and breast cancer using surgical ovariectomy in OP/OR female rats to mimic loss of ovarian function. Surgical ovariectomy was also used in mice by Chalvon-Demersay et al. to address the consequences of estrogen deficiency, however they suggest that differences observed even between rats and mice imply that extrapolations to humans must be made with caution. In the same article, the authors also discuss the "protein leverage hypothesis," which proposes that insufficient protein intake may be a key factor in obesity development. The authors discuss in detail mechanisms whereby dietary protein levels may affect food intake.

The brain is the focus of two further articles. Poon and Leibowitz discuss the various techniques used for the administration of substances to rodents in studies of the neuronal and molecular mechanisms determining the behavioral outcomes of gestational exposure to non-illicit substances of abuse, such as excessive dietary fat, ethanol, and nicotine. Münzberg et al. suggest that environmental factors and genetic predisposition, rather than personal choices, are at the root of the obesity pandemic, and critically evaluate how rodent models can help to understand the contribution of hedonic neural processes to body weight regulation. They summarize how these models helped to solidify the new view that homeostatic and hedonic controls are closely interrelated, often acting in unison at the unconscious level to affect biologically adaptive responses.

**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 © 2017 Even, Virtue, Morton, Fromentin and Semple. This is an open-access article distributed under the terms of the Creative Commons*

Lutz and Bueter review the advantages and limitations of studying mechanisms underlying the benefits of bariatric surgical approaches to obesity treatment (Roux-en-Y gastric bypass and vertical sleeve gastrectomy) in rats and mice. They conclude that most animal models recapitulate remarkably well findings in humans, but indicate that animals larger than rats and mice may offer additional specific advantages.

Thornton reviews experiments in favor of the specific hypothesis that increased hydration leads to body weight and fat loss through a decrease in feeding. The hypothesis derives from a broad association between chronic dehydration, raised levels of angiotensin II and chronic diseases such as obesity, diabetes, cancer, and cardiovascular diseases. Proposed mechanisms involve an increase in metabolism due to expansion of cell volume by hydration.

Two original studies were published in this topic. Even and Blais sound a cautionary methodological note by demonstrating that the cost of thermoregulation in mice housed at room temperature strongly affects attempts to estimate thermogenic responses to feeding accurately, due to heat transfer between diet-induced thermogenesis and non-shivering thermogenesis. They suggest that these observations undermine the use of mice housed below thermoneutrality to model human disorders of energy balance. Finally, Lombardo et al. show that use of a simple standard diet, without additive agents and without caloric restriction, is sufficient to rescue high-fat feeding-induced insulin resistance and prevent the evolution of diabetes without the need for a hypocaloric diet.

The articles in this topic offer a cross section of current approaches to studying obesity and its associated diseases in rodents. They highlight successes and failures, as well as illustrating important methodological issues and some emerging hypotheses in the field. Collectively, they attest to the enduring utility of highly tractable rodent models of complex human metabolic disease and to the importance of continued and careful adjustment of rodents to the disease being modeled. They further raise awareness of areas where species-specific pathophysiology appears irrevocably distinct. Appropriately critical use of rodent models will remain vital to interrogation of pandemic human metabolic disease.

# AUTHOR CONTRIBUTIONS

All authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

*Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor 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.*

# Knowledge Gaps in Rodent Pancreas Biology: Taking Human Pluripotent Stem Cell-Derived Pancreatic Beta Cells into Our Own Hands

*Munirah Mohamad Santosa1,2 , Blaise Su Jun Low1 , Nicole Min Qian Pek1,3 and Adrian Kee Keong Teo1,2,3,4\**

*1 Stem Cells and Diabetes Laboratory, Discovery Research Division, Institute of Molecular and Cell Biology, Singapore, 2 School of Biological Sciences, Nanyang Technological University, Singapore, 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 4 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore*

#### *Edited by:*

*Robert Kenneth Semple, University of Cambridge, UK*

#### *Reviewed by:*

*Eusebio Chiefari, University of Catanzaro, Italy Neil Hanley, University of Manchester, UK*

#### *\*Correspondence:*

*Adrian Kee Keong Teo<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Genomic Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 30 October 2015 Accepted: 25 December 2015 Published: 14 January 2016*

#### *Citation:*

*Santosa MM, Low BSJ, Pek NMQ and Teo AKK (2016) Knowledge Gaps in Rodent Pancreas Biology: Taking Human Pluripotent Stem Cell-Derived Pancreatic Beta Cells into Our Own Hands. Front. Endocrinol. 6:194. doi: 10.3389/fendo.2015.00194*

In the field of stem cell biology and diabetes, we and others seek to derive mature and functional human pancreatic β cells for disease modeling and cell replacement therapy. Traditionally, knowledge gathered from rodents is extended to human pancreas developmental biology research involving human pluripotent stem cells (hPSCs). While much has been learnt from rodent pancreas biology in the early steps toward Pdx1<sup>+</sup> pancreatic progenitors, much less is known about the transition toward Ngn3+ pancreatic endocrine progenitors. Essentially, the later steps of pancreatic β cell development and maturation remain elusive to date. As a result, the most recent advances in the stem cell and diabetes field have relied upon combinatorial testing of numerous growth factors and chemical compounds in an arbitrary trial-and-error fashion to derive mature and functional human pancreatic β cells from hPSCs. Although this hit-or-miss approach appears to have made some headway in maturing human pancreatic β cells *in vitro*, its underlying biology is vaguely understood. Therefore, in this mini-review, we discuss some of these late-stage signaling pathways that are involved in human pancreatic β cell differentiation and highlight our current understanding of their relevance in rodent pancreas biology. Our efforts here unravel several novel signaling pathways that can be further studied to shed light on unexplored aspects of rodent pancreas biology. New investigations into these signaling pathways are expected to advance our knowledge in human pancreas developmental biology and to aid in the translation of stem cell biology in the context of diabetes treatments.

Keywords: pancreas, islet, beta cell, human, pluripotent stem cell

# INTRODUCTION

In the field of stem cells and diabetes, many scientists are actively pursuing the generation of insulinsecreting pancreatic β cells from human pluripotent stem cells (hPSCs) for β cell transplantation/ replacement and treatment of diabetes (1–3). While insights from rodent pancreas developmental biology has guided the generation of PDX1+ pancreatic progenitors from hPSCs, the specific developmental principles thereafter remain murky. Henceforth, research groups have relied upon the transplantation of pancreatic progenitors, derived *in vitro*, into rodents for *in vivo* maturation (4–6). However, there has been considerable progress toward the generation of mature and functional human pancreatic β cells *in vitro* in the recent years. These β cells purportedly co-express cardinal β cell markers, such as PDX1, NKX6.1, musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), prohormoneprocessing enzymes, insulin, and C-peptide. Importantly, they are also monohormonal and glucose responsive.

Developmental biologists believe that there is much to be learnt from rodent developmental biology to guide hPSC-based generation of clinically useful cell types, such as pancreatic β cells. Owing to such efforts, the progression of definitive endoderm (DE) germ layer to PDX1+ pancreatic progenitors has been wellexplored. However, the investigations on the later steps of pancreatic endocrine development and β cell maturation have not been quite fruitful. The most substantial advances in stem cell biology have relied upon an arbitrary approach of iterative trial-and-error testing to achieve mature and functional pancreatic β cells *in vitro* (7). Therefore, several pertinent questions remain: why were we not able to extrapolate rodent developmental principles and apply them on hPSCs to derive mature and functional pancreatic β cells? Are there differences between rodent and human pancreas development that prevent such an application? In this review, we look at signaling pathways that have been activated or repressed in stem cell biology and retrospectively revisit existing knowledge about rodent pancreas biology. Our efforts highlight novel aspects of signaling pathways that can be further investigated in our translational efforts for diabetes.

#### INHIBITION OF TRANSFORMING GROWTH FACTOR-**β** SIGNALING IN THE LATER STAGES OF PANCREATIC DIFFERENTIATION

The transforming growth factor-β (TGF-β) superfamily of proteins regulates pancreas development and function (8). TGF-β1, TGF-β2, and TGF-β3 are expressed in pancreatic epithelial cells at E12.5 in mice. Thereafter, they become localized in the acinar cells (9). TGF-β1 can promote the development of mouse pancreatic β cells from pancreatic buds (10). Perplexingly, it also indirectly inhibits the formation of mouse pancreatic epithelial cells (11). In tandem, TGF-β2 has been demonstrated to inhibit *Hnf1*β and *Pdx1* gene expression. Hence, TGF-β can purportedly restrain the specification of pancreatic cell fate (12). TGF-β signaling effector SMAD3 can bind the *Ins* gene promoter to suppress its expression. In agreement, *Smad3*-deficient islets exhibit an active insulin signaling pathway (13). Collectively, these evidences suggest the requirement to inhibit TGF-β signaling for the derivation of mature and functional pancreatic β cells (**Figure 1A**).

In 2011, Nostro et al. used small molecule SB431542 (14), an Activin/TGF-β receptor antagonist, in their pancreatic differentiation protocol. SB431542 inhibits activin receptor-like kinases (ALK) 4/5/7 and the downstream TGF-β/Activin/Nodal signaling. SB431542 treatment was demonstrated to increase *INS* gene expression and the development of C-peptide<sup>+</sup> cells (15). Similarly, Cho et al. also utilized SB431542, in the presence of retinoic acid (RA), for pancreatic differentiation (16). Alternatively, Schulz et al. used TGF-βRI kinase inhibitor IV to obtain pancreatic progenitors from CyT49 hPSCs (17). Rezania et al. identified that the use of 2-(3-[6-Methylpyridin-2-yl]-1*H*pyrazol-4-yl)-1,5-naphthyridine (ALK5iII) can effectively induce the expression of *NGN3*, *NEUROD1*, *INS,* and *GCG* transcripts to promote pancreatic endocrine specification (18). Rezania et al. further demonstrated that 1 μM ALK5iII is necessary for the induction of NEUROD1<sup>+</sup> cells, but it suppressed the proportion of NKX6.1<sup>+</sup> cells (4), a hallmark of functional β cells (19). Most recently, Rezania et al. compared the effects of several ALK5 inhibitors at a later phase of differentiation of hPSCs and found that only ALK5iII downregulated *NGN3* while increasing *INS*, *GCG,* and *SST* transcripts (6). Furthermore, 10 μM ALK5iII induced the expression of nuclear v-maf MAFA transcript, a critical mature β cell transcription factor, in diabetic rodents (20–22). Rezania et al. (6) concluded that ALK5iII was the most effective and specific inhibitor as it inhibited ALK5 but had minimal inhibition of other kinases. Similarly, Pagliuca et al. also employed 10 μM Alk5iII to derive mature and functional human pancreatic β cells from hPSCs (7) (**Figure 1B**; **Table 1**).

Overall, the inhibition of ALK5/TGF-βRI with ALK5iII appears to be more desirable as compared to the general inhibition of TGF-β signaling via the use of SB431542. Further studies are certainly required to investigate the intricacies of TGF-β signaling during pancreas development and β cell maturation.

### PROTEIN KINASE C SIGNALING ENHANCEMENT

Protein kinase C (PKC) is a family of serine/threonine kinases that are involved in diverse cellular processes, including survival, apoptosis, cell cycle regulation, proliferation, migration, and differentiation (23). In maturing neonatal rat islets, PKCα was only found in β cells, PKCγ in α cells, and PKCϵ in δ cells (24). This differential expression of PKC isoenzymes (25) hints that PKC signaling may play a role in the functional maturation of pancreatic endocrine progenitors (**Figure 1A**).

Chen et al. (26) was the first to demonstrate that 300 nM (−)-indolactam V (ILV) or PKC agonists {500 nM [(2*S*,5*S*)- (*E*,*E*)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam (TPB)] or 14 nM phorbol-12-myristate-13-acetate (PMA)} can efficiently increase the formation of PDX1+ pancreatic progenitors from hPSCs via the activation of PKC signaling. ILV treatment resulted in an increased gene expression of several pancreatic progenitor markers, including *SOX9*, *PDX1*, *PTF1A*, *HNF6*, and *PROX1*, and endocrine progenitor markers, including *NGN3*, *NKX2.2,* and *NKX6.1*. The protein expression of pancreatic progenitor markers FOXA2, PTF1A, HNF6, and NKX6.1 were increased, whereas the expression of intestinal marker CDX2 and liver marker AFP were suppressed (26). In addition, they also found that ILV and PKC agonists, TPB or PMA, can synergize with FGF10 signaling to promote the proliferation of PDX1<sup>+</sup> cells derived from hPSCs. Interestingly, ILV treatment also works on

differentiation process [as described by Rezania et al. (6) and Pagliuca et al. (7)] are represented by the decreasing shades of green. The differing colors co-existing in an aggregate illustrates the heterogeneity of cells prevalent in such a differentiation scheme. While some of the cells will transit from being endocrine progenitors (light orange) to Pdx1+ insulin-producing β-like cells (brown), the end-product will include an assortment of maturing endocrine cell types (represented by orange and light orange).

mouse embryonic stem cells, suggesting a conservation of signaling pathway in both mouse and human cells (26) (**Table 1**).

Subsequently, Rezania et al. (4) employed two PKC activators in the mid-late stage of hPSC differentiation; 50 nM TPB (safer profile) and phorbol 12,13-dibutyrate (PDBu) (27), demonstrating that the activation of PKC signaling induces the gene expression of pancreatic lineage markers *PTF1A*, *NGN3*, *NEUROD1*, and *NKX6.1* while suppressing the expression of intestinal (*CDX2*) and liver (*ALB*) markers (4). This demonstrated that PKC signaling enriches the development of pancreatic progenitors while inhibiting intestinal and hepatic lineages. Similarly, Pagliuca et al. used 500 nM PDBu in their pancreatic differentiation protocol (7) (**Figure 1B**; **Table 1**). Although these data are encouraging, more studies remain to be done to thoroughly clarify the role of PKC signaling and the specific mechanisms in the maturation of pancreatic endocrine progenitors.

# LOWER LEVEL OF RETINOIC ACID SIGNALING AS PANCREATIC DIFFERENTIATION PROGRESSES

It is well-established that RA signaling plays critical roles in the early and late stages of pancreas development (28). RA is a

*BTC, Betacellulin; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; GPx-1, glutathione peroxidase-1; GSiXX,* γ *secretase inhibitor XX; ILV, (*−*)-indolactam V; NAC, N-acetylcysteine; PDBu, phorbol 12,13-dibutyrate; PMA, phorbol-12-myristate-13-acetate; T3, l-3,3*′*,5-Triiodothyronine; TPB, [(2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam].*

Novel Pathways for

β Cell Maturation

lipid-soluble vitamin A derivative synthesized from the oxidation of retinaldehyde via enzymes retinaldehyde dehydrogenase 1 (RALDH1), RALDH2, and RALDH3. RA produced at the splanchnic lateral plate mesoderm and Raldh2 expressed in the dorsal pancreatic mesenchyme promote Pdx1 induction in the dorsal foregut endoderm (**Figure 1A**). Raldh2 mutant mice exhibit dorsal pancreatic bud agenesis (29) as they fail to form pancreatic progenitors, indicated by the loss of Pdx1, Prox1, altered Isl1, and reduced Hlxb9 expression (29, 30).

Many existing protocols differentiating hPSCs into pancreatic cells utilize 1–3 μM RA. As an alternative, Schulz et al. replaced RA with 3 nM TTNPB/arotinoid acid – a more stable retinoid analog that can selectively activate RA receptors (RARs) (17). Interestingly, Rezania et al. started out using 1 μM RA during the early posterior foregut differentiation, subsequently reducing to 100 nM RA during pancreatic endoderm phase and further reducing to 50 nM during the pancreatic endocrine phase (6). Pagliuca et al. also reported a similar pancreatic differentiation protocol in which a decreasing dose of RA was used, starting with 2 μM followed by 100 nM RA at later stages, which was eventually reduced to 25 nM (7) (**Figure 1B**; **Table 1**).

While it is widely accepted that RA is crucial for pancreatic specification, using progressively lower doses of RA as practiced by Rezania et al. (6) and Pagliuca et al. (7) raises questions about the importance of RA concentrations during pancreas development both *in vitro* and *in vivo*. RA signals by binding RARs and retinoid X receptors (RXRs); so it is postulated that a decrease in RA signaling may be more conducive for subsequent pancreatic endocrine specification since retinoid receptors are upregulated in the pancreatic exocrine (31). However, given the lack of understanding in this regard, the significance of the dose of RA during human pancreas development warrants further studies.

#### INHIBITING **γ**-SECRETASE/NOTCH FOR ACCELERATED PANCREATIC ENDOCRINE DIFFERENTIATION

Notch signaling is essential for the proper development of pancreatic endocrine progenitors as it regulates their decision between differentiation and proliferation (32). The reduction of Notch signaling is known to promote accelerated pancreatic endocrine differentiation (33). Similarly, the inhibition of Notch signaling via γ-secretase (an intra-membrane protease) inhibitor can downregulate the expression of Notch target Hes-1, an inhibitor of pro-endocrine gene Ngn3 (34) (**Figure 1A**). Conversely, the activation of Notch in PDX1<sup>+</sup> pancreatic progenitors prevents pancreatic differentiation (35).

*N*-[*N*-(3,5-difluorophenacetyl-l-alanyl)]-*S*-phenylglycine t-butyl ester (DAPT) is a commonly used γ-secretase inhibitor with an IC50 in the nM range. The inhibition of Notch signaling with DAPT can increase Ngn3 mRNA and protein expression in adult islets (36). Likewise, DAPT increases *NGN3* and *NEUROD1* gene expression in hPSC-derived pancreatic progenitors (18). In recent protocols developed by Pagliuca et al. (7) and Rezania et al. (6), other γ-secretase inhibitors have been employed to retard Notch signaling. Rezania et al. used γ-secretase inhibitor XX (GSiXX) that has an IC50 in the low nM range. They showed that GSiXX can induce the expression of β cell maturation genes but inhibit the expression of *PTF1A*, a marker of pancreatic exocrine lineage (6). GSiXX can also act in concert with triiodothyronine (T3) to increase the percentage of NKX6.1<sup>+</sup>INS<sup>+</sup>GCG<sup>−</sup> β-like cells (6). Alternatively, Pagliuca et al. employed the use of XXI at 1 μM (7), which has an IC50 in the picomolar range, and demonstrated that XXI worked with other factors to improve β cell gene expression (**Figure 1B**; **Table 1**). However, it remains unclear whether there are differences between DAPT, GSIXX, or XXI in the induction or suppression of key pancreatic transcription factors for the eventual promotion of pancreatic β cell formation.

# TRIIODOTHYRONINE COULD PROMOTE PANCREATIC **β** CELL MATURATION

Studies in the 1980s suggest that thyroid hormones regulate insulin secretion, possibly via control over glucose oxidation and calcium uptake rates (37). T3, a thyroid hormone, can potentiate insulin signaling and increase insulin synthesis in diabetic rodents (38), in rodent islets (39), and in a rodent pancreatic β cell line (40) (**Figure 1A**). Mechanistically, T3 phosphorylates and activates AKT in pancreatic β cells, improving their survival (39, 41); 0.1 μM of T3 can increase rodent pancreatic β cell proliferation via the MAPK/extracellular signal-regulated kinase (ERK) signaling pathway (42). Interestingly, T3 apparently induces the transdifferentiation of human pancreatic ductal cell line (hPANC-1) into β-like cells, with an increased expression of *INS* transcripts (43). T3 can also increase both the mRNA expression of pro-endocrine gene Ngn3 and the number of β cells, indirectly inducing endocrine differentiation from exocrine cells; possibly via Akt signaling (44).

Of late, T3 has been shown to promote pancreatic β cell maturation and proliferation in rats (45). Based on these findings, Rezania et al. went on to demonstrate that 1 μM of T3 can actually induce the expression of *INS* and mature β cell markers, and enhance the co-expression of NKX6.1 and INSULIN protein (6). Similarly, Pagliuca et al. employed the same dose of 1 μM T3 in the later stages of their pancreatic differentiation protocol to generate human pancreatic β cells from hPSCs (7) (**Figure 1B**; **Table 1**). While the biology and role of T3 in pancreatic β cell maturation remains to be explored further, its inclusion in pancreatic differentiation protocols appears to serve a positive function.

# INHIBITION OF TYROSINE KINASE RECEPTOR AXL INDUCES MATURE PANCREATIC **β** CELL MARKER (MAFA) EXPRESSION

AXL is a member of the Tyro3-Axl-Mer (TAM) transmembrane receptor tyrosine kinase (RTK) family that plays an important role in essential cellular processes, such as cell survival, growth, proliferation, and differentiation. Its ligand, growth arrest specific 6 (Gas6), binds AXL to activate downstream signaling, including the phosphoinositide 3-kinase (PI3K), ERK, and signal transducer and activator of transcription 3 (STAT3) signaling (46). Interestingly, Rezania et al. performed small molecule and growth factor library screening to identify compounds that can induce mature β cell marker MAFA from hPSC-derived pancreatic progenitors and found that 2 μM BGB324 (R428), an inhibitor of AXL, can induce MAFA protein expression (6) (**Figure 1B**; **Table 1**). However, there is little information linking AXL to pancreas development and β cell maturation.

In 1999, Augustine et al. reported that the overexpression of AXL results in diabetes in mice. Furthermore, the administration of exogenous Gas6 exacerbated the condition (47). Haase et al. recently confirmed that GAS6 is expressed in pancreatic tissues and found that GAS6 reduced *Mafa* gene expression in rodents (48), likely due to the activation of AXL (**Figure 1A**). This corresponds with the increase in MAFA expression observed by Rezania et al. after the inhibition of AXL signaling (6). While there seems to be a *bona fide* association between AXL signaling and pancreas development, the cellular mechanism(s) remain a mystery.

### ANTIOXIDANTS MAY BENEFIT PANCREATIC DIFFERENTIATION

Excessive levels of reactive oxygen species (ROS) have been implicated in glucotoxicity-induced pancreatic β cell destruction and dysfunction. In this regard, antioxidants play important defensive roles against ROS. In the endocrine pancreas, the antioxidant vitamin C is known to be an effective co-factor for the peptidyl α-amidation of several biologically active peptides and is necessary for optimal insulin secretion from pancreatic β cells (**Figure 1A**). In fact, high concentrations of ascorbic acid (vitamin C) were found in neonatal rat endocrine pancreas (49). Mechanistic studies involving other antioxidants, such as glutathione peroxidase-1 (GPx-1), *N*-acetylcysteine (NAC), and ebselen, were reported to maintain the protein expression of mature β cell marker MAFA (20–22) (**Table 1**).

Interestingly, Rezania et al. used 1–2 mM NAC during their pancreatic differentiation and found that it also increased nuclear MAFA protein expression (6). However, this was not replicated with another antioxidant, vitamin E. Pagliuca et al. also relied upon the use of 0.25 mM of vitamin C throughout S1–S5 phase of their pancreatic differentiation protocol to generate mature and functional human pancreatic β cells (7) (**Figure 1B**; **Table 1**). While the metabolism of vitamins C and E are altered before the onset of diabetes in rats, their contribution to the pancreas is unclear (50). Antioxidant treatments may preserve β cell function, exerting positive effects in diabetes (51), but their role in pancreas development and β cell maturation certainly remains elusive.

# BETACELLULIN DIRECTS A PANCREATIC **β** CELL FATE

Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family that plays a role in the differentiation of pancreatic β cells (52) (**Figure 1A**). It is largely expressed in the liver, kidney, small intestine, and pancreas (53), and is specifically expressed in 9- to 24-week-old human fetal pancreas (54). BTC binds to ErbB-1 and ErbB-4 receptors (55) to initiate downstream signaling pathways involving PI3K/Akt, MAPK, STAT, and mTOR signaling pathways (56).

Betacellulin appears to direct a pancreatic β cell fate. It can convert exocrine cells (57) and α cells (58) into insulin-secreting cells. It can also induce β cell neogenesis from ductal cells in diabetic mice (59). Li et al. demonstrated that exogenous BTC can promote β cell regeneration in 90% pancreatectomized rats (60) and convert δ to β cells in STZ-induced diabetic mice (61). Also, Yamamoto et al. observed that long-term administration of BTC reverses STZ-induced hyperglycemia in mice (62). Surprisingly, the loss of BTC in mice yielded no overt defect (63) despite their active roles in the pancreas. This could be explained by compensatory effects exhibited by the other EGFR ligands (64). The overexpression of BTC in transgenic islets does not affect islet structure, endocrine cell ratio, or β cell mass but enhances glucose-stimulated insulin secretion (65). However, ubiquitous overexpression of BTC in mice results in various pathologies (66). Intriguingly, gene variants and polymorphisms in the *BTC* gene have also been found to be associated with types 1 and 2 diabetes (54, 67).

The induction of β cell development/differentiation by BTC (52) could be an outcome of the downstream increase in insulin receptor substrate-2 (IRS-2) expression (68), an important mediator of β cell function (69). BTC can induce *Pax4* gene expression in rat islets, promoting β cell functionality (70). It can also sustain *PDX1* expression and induce β cell differentiation from hPSCs (71). Ten ng/ml BTC are sufficient to increase *Pdx1* gene expression, insulin production, and to inhibit amylase and glucagon production in mouse embryonic pancreas explants (72). Lately, Pagliuca et al. employed 20 ng/ml BTC in the later stages of their pancreatic differentiation protocol (7). Similarly, 50 ng/ml EGF was added to preserve cell mass (17) (**Figure 1B**; **Table 1**). These data strongly indicate that BTC and/or other EGF ligands are of importance in pancreas development and β cell maturation. Nonetheless, more detailed molecular mechanisms remain to be uncovered.

# CONCLUDING REMARKS

Relating to the current most advanced human pancreatic β cell differentiation protocols, the biological mechanisms involved in the later stages of β cell development and maturation still remain elusive. The fact that the postnatal stage in rodents is grossly equivalent to the third trimester in human pancreas development (73) invites new approaches to study this aspect of β cell biology. In this review, we revisited some of the least-understood developmental signaling pathways in rodent pancreas biology. Our efforts unraveled interesting aspects of these signaling pathways that demand to be thoroughly elucidated at the mechanistic level. Future studies should seek to highlight how immature β cells transit into mature and functional β cells. This would certainly advance our knowledge of human pancreas developmental biology and boost translational efforts in the use of stem cells for diabetes treatment.

## AUTHOR CONTRIBUTIONS

MS: reviewed the literature, wrote and edited the paper. BL: edited the paper. NP: edited the paper and prepared the figure. AT: conceptualized the review topic and contents, wrote, edited, and approved the paper.

#### REFERENCES

#### ACKNOWLEDGMENTS

We thank members of the Teo laboratory, including Chang Siang Lim, Hwee Hui Lau, and Larry Sai Weng Loo for their comments on the manuscript.

#### FUNDING

AT is supported by the Institute of Molecular and Cell Biology (IMCB), A\*STAR, NHG-KTPH SIG/14033, the NUHS-CG Metabolic *In Vitro* Core Seed Funding and the JCO Career Development Award (CDA) 15302FG148, A\*STAR.

patterning and pancreatic specification of human pluripotent stem cells. *Development* (2011) **138**:861–71. doi:10.1242/dev.065904

**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 © 2016 Santosa, Low, Pek and Teo. 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) or licensor 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.*

# Mouse Models of Human GWAS Hits for Obesity and Diabetes in the Post Genomic Era: Time for Reevaluation

*Samantha Laber1,2\* and Roger D. Cox1 \**

*1Mammalian Genetics Unit, Medical Research Council Harwell Institute, Oxfordshire, UK, 2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK*

Keywords: GWAS, epigenetics, obesity, cross-species conservation, genetic association

In recent years, genome-wide association studies (GWAS) have identified hundreds of loci and thousands of single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes mellitus (T2DM) and obesity traits [such as body mass index (BMI) and waist–hip ratio (WHR)] in the human population (1–4). The vast majority of these SNPs are in non-coding regions of the genome and distal to promoters, suggesting they act through gene regulation which makes their functional interpretation challenging (5). Collectively, comparing the epigenetic landscape between mouse and human has established new pathways involved in obesity and diabetes, and in fact, inter-species conservation has successfully been used as criteria in finding functional and disease-relevant elements (6–8). By contrast, genome-wide comparative analysis of the mouse and human epigenome across tissues has highlighted the presence of cis-regulatory divergence (9, 10). New mouse engineering approaches together with bioinformatics dissection of trait-associated regions, for example, epigenetic modifications and genome interactions hold great promise to fully understand the underlying mechanisms of human disease-associated non-coding variants in T2DM and obesity.

#### *Edited by:*

*Nicholas Michael Morton, University of Edinburgh, UK*

#### *Reviewed by:*

*Mirela Delibegovic, University of Aberdeen, UK David Hodson, University of Birmingham, UK*

#### *\*Correspondence:*

*Samantha Laber<EMAIL_ADDRESS>Roger D. Cox<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Genomic Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 04 November 2016 Accepted: 13 January 2017 Published: 07 February 2017*

#### *Citation:*

*Laber S and Cox RD (2017) Mouse Models of Human GWAS Hits for Obesity and Diabetes in the Post Genomic Era: Time for Reevaluation. Front. Endocrinol. 8:11. doi: 10.3389/fendo.2017.00011*

THE CONTEXT-SPECIFIC NATURE OF HUMAN GWA SIGNALS IN HUMAN

Over 80% of loci identified by GWAS are in intergenic and intronic regions and many of these genetic risk regions are enriched for histone modifications (5), suggesting they act as regulatory elements which appear to function in a highly cell-selective manner. Due to the tissue specificity as well as the developmental and epigenetic complexity of gene regulation, functional approaches require the study of the relevant tissue and cell type as well as genetic and bioinformatics approaches that reliably assess the regulatory role of non-coding variants (7, 11, 12). Ongoing progress in high-throughput sequencing and the development of new experimental tools are greatly advancing our capacity to study chromatin biology and genome function. In particular, ChIP-seq allows identification of transcription factor binding sites and chromatin states; chromosome conformation capture-based techniques (including 3C, 4C, 5C, CaptureC, and HiC) allow the study of chromatin interactions; and DNase hypersensitivity or ATAC-seq can identify accessible chromatin (13–16). Additionally, tools like HaploReg (17), Enlight (18), RegulomeDB (19), and The Islet Regulome Browser (11) are emerging that allow the integration of GWAS results with genetic and epigenetic annotations that can be used to dissect the gene regulatory networks that underpin genomic association signals.

By integrating the information gained from functional genomics efforts such as the ENCODE (5) and Roadmap Epigenomics projects (20) together with expression quantitative trait loci (eQTL) results and functional studies, it becomes increasingly clear that adipose tissue is one of several key effectors of genetic risk loci for T2DM and obesity trait associations, particularly for WHR signals (2, 7, 21, 22). However, there is currently still a lack of comprehensive maps linking distal elements that harbor disease-associated variants with their target genes in relevant tissues and developmental stages. Furthermore, extensive fine-mapping of risk associations is crucial in order to narrow down the association signal to the likely causative variants which can then be functionally investigated (23). This has resulted in many studies being performed assuming that the closest gene to a given disease-associated signal is the causative one. Traditionally, target genes based on proximity to a signal were selected to model in the mouse using global or tissue-specific gene knockout or overexpression alleles to characterize gene function (24). However, with this approach, many target genes for GWA signals have potentially been overlooked, for example, in the case of the BMI-associated variants in *Fto* (25, 26). An additional level of complexity comes with the possibility for an association signal—that usually harbors dozens of SNPs—to potentially contain a number of disease-causing variants that might act in different tissues and/or at different times, affecting different genes. For example, there is currently evidence for intronic *FTO* risk variants to alter the expression of nearby genes in both adipose tissue and brain. An eQTL in human cerebellum links re9930509 to altered *IRX3* expression (25), rs1421085 has very convincingly been shown to be located within an enhancer for *IRX3* and *IRX5* in adipocyte precursors (7, 26) and rs1421085 and rs8050136 have been proposed to selectively alter *FTO* and *RPGRIP1L* expression in human-induced pluripotent stem cell-derived neurons (27). Therefore, mouse models which could help pinpoint variants, target genes, and relevant tissues would prove invaluable in the mechanistic dissection of human disease-associated sequence variants. However, whether it is possible to use the mouse for modeling regulatory variants (which is essential to capture the relevant spatiotemporal effects) will depend on the functional conservation of the regulatory circuitry of a given signal in human and mouse.

#### CONSERVATION BETWEEN HUMAN AND MOUSE

It is estimated that our last common ancestor with the mouse was about 90 million years ago (28). At this point, many of the core physiological regulatory mechanisms had evolved, for example, mouse and human share the same basic mechanisms for controlling food intake *via* leptin and hypothalamic anorexigenic and orexigeneic neurons, and similarly insulin and glucagon are core effectors in glucose homeostasis. However, there have clearly been many evolutionary changes over this long period of time. At the level of the genome, chromosome number and organization have changed, although it is striking how large tracts of DNA have conserved their order of genes and show high coding sequence conservation (29). Thus, if we wish to use the mouse as model of human metabolic disease we can rely on much of the core conservation of ancient metabolic pathways and their regulation but cannot ignore the fact of their continued evolution that adapts and changes these mechanisms for the survival of two very different organisms. The mouse ENCODE Consortium reported that comparative gene expression data from human and mouse reveals that some sets of genes tend to cluster more by species than by tissue and *vice versa* (29). More recently, it has been suggested that gene clustering by tissue rather than species is much stronger than originally thought (30). Interestingly, single-cell sequencing of human and mouse pancreatic alpha and beta cells showed good cross-species correlation of transcriptomes although with some important species differences (31). Finally, Breschi et al. (32) describe how transcriptomes show a continuum of variation from species dominated clustering to organ dominated clustering. Importantly, for modeling GWA signals in other species, genes that varied little between species (and are more organ-specific) are more likely to overlap with human risk variants (32).

#### EPIGENOMIC CONSERVATION BETWEEN HUMAN AND MOUSE—INSIGHTS FROM THE MOUSE ENCODE CONSORTIUM

Some of the other key findings of ENCODE in the mouse genome were that human and mouse trans-regulatory networks (transcription factor networks) are considerably more conserved than the cis-regulatory landscape, which in fact accounts for the majority of regulatory plasticity between human and mouse (28, 29). At the same time, the degree of divergence of regulatory elements varies widely between different types of elements that are active in different tissue contexts (9, 28). The Mouse ENCODE Consortium (29) demonstrated that 79.3% of mouse candidate enhancers (predicted by patterns of histone modifications) and 66.7% of transcription factor binding sites have sequence orthologs in humans. Further, 61.5% of tested candidate mouse-specific enhancers also show enhancer activity in human embryonic stem cells in a reporter assay (29), suggesting a degree of functional conservation between human and mouse gene regulation. Based on this level of conservation, it is intriguing to ask the question whether mouse chromatin states could be used to identify potential sites for functional characterization in mouse for human GWAS hits. Mapping 4,265 SNPs from human GWAS studies onto the mouse genome using 15 mouse samples revealed that human GWAS hits are associated with specific chromatin states in relevant mouse tissues (29). For example, in mouse kidney, H3K4me1 is enriched in specific GWAS hits associated with urate levels and metabolites. For mouse liverspecific H3K36me3, GWAS hits related to HDL cholesterol and triglyceride levels are enriched. Together, 55% of mapped SNPs overlapped with at least one histone mark in mouse (29). These results suggest that histone modification marks can be used to inform about human risk variants and for the identification of candidate functional sequences for characterization of human GWAS hits in mouse.

Furthermore, SNPs with high regulatory potential are enriched in conserved transcription factor binding sites (19). Cheng et al. (33) show that conserved sequences occupied by orthologous transcription factors in human and mouse are enriched for GWAS variants. When investigating whether this is true for individual phenotypes, they found that SNPs associated with type I diabetes and several other traits are significantly enriched in conserved transcription factor binding sites, with 13 out of 20 type 1 diabetes SNPs being in conserved binding sites. By contrast, all of the SNPs associated with pulmonary function were found to be human-specific, suggesting that besides GWAS SNPs generally being enriched in conserved regulatory elements, that this enrichment is dependent on the trait (33). Whether this is the case for T2DM and obesity traits association is yet to be investigated. With continuous efforts and the increase in available mouse genome data sets, it will become possible to draw conclusions about the human–mouse conservation of transcription factor occupancy and enrichment of GWAS SNPs in adipose tissue. Indeed, on a cellular level, a systematic comparison between the human and mouse epigenome during adipocyte development and in different fat depots is largely missing, and Mouse ENCODE has currently only limited adipose tissue datasets that could be matched to human. Though, Mikkelsen et al. (34) generated a comparative analysis of chromatin state maps together with gene expression profiles from human adipose tissue and mouse 3T3- L1 at four time points during differentiation. They showed that although a significant amount of open chromatin in orthologous regions were shared between the two models (15–30%), most of them were species-specific. While we are not proposing that this affected the key findings of this study, it is worth pointing out that

comparing a mouse cell line and primary human tissue-derived pre-adipocytes with their accompanying ontogenetic differences can potentially hinder the interpretation when using these data sets for dissecting specific GWAS loci with the aim to establish relevant functional sites.

Taken together, although the cis-regulatory landscape has substantially diverged between human and mouse on a global level, human trait-associated SNPs are enriched in sites that are conserved between the two species for the majority of traits investigated.

# THE POTENTIAL FOR NEW STRATEGIES IN MOUSE MODEL ENGINEERING

With our current knowledge of the context-specificity of gene regulation and consequently the many layers of complexity of most GWAS signals, it becomes increasingly clear that it is necessary to study and understand the underlying regulatory network in the relevant human tissue (**Figure 1**). In the past,

conservation of a regulatory site (or the lack of data sets that can determine the latter) a classical tissue-specific target gene manipulation can prove valuable; in the case of a high functional conservation (based on genomics and bioinformatics dissection of the loci), a model that selectively manipulates the regulatory region can in principle be useful. Comparing the human–mouse epigenome can be expected to become increasingly powerful with the improvement of quality as well as the comprehensiveness of genomic data sets and tools. The translational utility will depend on the mouse model chosen and the information gained can feedback and help interpret human GWAS signals.

a successful approach to studying the function of individual candidate genes *in vivo* has been achieved by generating global knockout and overexpression models (24). However, these models do not resemble the tissue-specific nature of alterations in regulatory elements. Tissue-specific target gene manipulation using CRE drivers can be a powerful tool to overcome this problem. However, another challenge comes with the current lack of reliable pre-adipocyte-specific CRE lines that can be used to assess the tissue-specific effect of identified target genes in cases of pre-adipocyte-specific signals. Recent advances in genome engineering, namely CRISPR/Cas9, opened the opportunity to conveniently alter any regulatory sequence of interest (35). In other words, it is now possible to genome edit transcription factor binding sites and enhancer elements in the mouse which in principle has the potential to create mouse models of human risk variants that (i) are cell type-specific; (ii) alter all target genes; (iii) alter target genes at the relevant level and direction; and (iv) alter target genes at the relevant time of development (**Figure 1**).

#### CONCLUSION AND FUTURE DIRECTIONS

The majority of human genetic variants associated with common metabolic disease traits are located within distal regulatory elements. With our current knowledge of gene regulation and the context-specificity of the signal, it is necessary to understand the signal in human. Identifying targets and context is crucial in engineering a *relevant* mouse model. A comprehensive human-tomouse epigenomic comparison can be informative about human risk variants. Although intriguingly, whether manipulation of regulatory elements will become a tool to dissect human obesity/ T2DM risk variants in the mouse will depend on the functional conservation of a given signal. This is yet to be established and offers an exciting avenue to explore.

#### GLOSSARY

**ATAC-seq**—assay for transposase-accessible chromatin followed by high-throughput sequencing. This technique allows the identification of open chromatin*.*

**BMI**—body mass index. A measure of body weight that takes account of an individual's size and calculated by dividing body weight by height squared.

**ChIP-seq**—chromatin immunoprecipitation followed by high-throughput sequencing. This technique allows the identification of DNA fragments that are bound by a specific antibody.

**Cis-regulatory**—non-coding DNA sequences in or near a gene required for its spatiotemporal expression that characteristically contain transcription factor binding sites.

**CRE**—Cre recombinase recognizes DNA sequences known as LoxP sites and when a pair of sites is provided in the same

#### REFERENCES

1. Locke AE, Kahali B, Berndt SI, Justice AE, Pers TH, Day FR, et al. Genetic studies of body mass index yield new insights for obesity biology. *Nature* (2015) 518:197–206. doi:10.1038/nature14177

orientation this leads to deletion of the intervening sequence. In this way, a segment of DNA such as a key exon (said to be floxed) can be deleted resulting in, for example, a null mutation. This can be done *in vivo* by gene editing to place LoxP sites in the required location and then crossing animals that carry this modification to Cre recombinase strains, which then results in recombination. The expression of Cre recombinase can be driven by a promoter of choice either as a transgene or knocked into an endogenous gene promoter. Thus, the recombinase can be expressed in specific tissues as required allowing cell- or tissue-specific recombination, i.e., for the generation of a conditional knockout.

**Epigenome**—a network of chemical compounds (for example, DNA methylation or histone modifications) surrounding DNA that modify the genome without altering the DNA sequence itself. These modifying elements play a role in determining which genes are active in a particular cell at a particular time.

**eQTL**—expression quantitative trait loci are genomic loci that contribute to variation in the expression levels of mRNAs. For example, in individuals in a population inheriting SNP allele A, the expression of gene Y is found to be quantitatively increased or decreased on average relative to the other SNP alleles inherited across the population assayed. This is a correlated trait rather than a direct functional link between a SNP and the expression of a gene. Further, any particular SNP marks a haplotype (a linked co-inherited group) of SNPs and as such represents a locus.

**GWAS**—Genome-Wide Association Study.

**iPSC**—induced pluripotent stem cell.

**SNP**—single-nucleotide polymorphism.

**T2DM**—type 2 diabetes mellitus.

**Transcriptome**—the entire mRNA expressed from the genes of a cell.

**Trans-regulatory**—in the context of transcriptional regulation, a trans-acting element is usually a DNA sequence that contains a gene. This gene encodes for a protein (or other molecules such as microRNA) that will regulate another target gene.

**WHR**—waist-to-hip ratio.

#### AUTHOR CONTRIBUTIONS

SL and RC wrote, edited, and approved the manuscript.

#### ACKNOWLEDGMENTS

The authors thank Thomas Agnew and Cecilia Lindgren for their valuable comments on the manuscript.

#### FUNDING

SL was supported by an MRC PhD studentship and with RC MRC funding MC\_142661184.

architecture and pathophysiology of type 2 diabetes. *Nat Genet* (2012) 44:981–90. doi:10.1038/ng.2383

**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 © 2017 Laber and Cox. 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) or licensor 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.*

# The Importance of Context: Uncovering Species- and Tissue-Specific Effects of Genetic Risk Variants for Type 2 Diabetes

*Soren K. Thomsen1 , Mark I. McCarthy1,2,3 and Anna L. Gloyn1,2,3\**

*1Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, UK, 2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK, 3Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford, UK*

Keywords: type 2 diabetes, genome-wide association study, genetic association, genetic mechanism, genomic annotation, gene regulation, epigenetics

Genome-wide association studies (GWAS) have been highly successful in identifying genetic variation associated with type 2 diabetes (T2D) risk and related quantitative traits (1–3). The vast majority of association signals are located in non-coding regions of the genome, influencing nearby genes through regulation of transcriptional, translational, or splicing activity (4). Due to the highly context-dependent nature of gene expression, the effects of many risk variants are restricted to specific cell types and produce more subtle effects than those observed in organism-wide (or "global") knockouts. In addition, identification of the underlying causal genes and target tissues is often a major challenge, hindering translation into disease mechanisms. Recent studies have shown that the intersection of genetic data and genomic annotations can be used to produce a cellular atlas with which to understand the phenotypes of GWAS signals. Through the generation of directed hypotheses, this integrated framework has the potential to bridge the gap between association signals and disease biology.

#### *Edited by:*

*Robert Kenneth Semple, University of Cambridge, UK*

*Reviewed by: Luca Andrea Lotta, University of Cambridge, UK*

> *\*Correspondence: Anna L. Gloyn<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Genomic Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 24 April 2016 Accepted: 04 August 2016 Published: 31 August 2016*

#### *Citation:*

*Thomsen SK, McCarthy MI and Gloyn AL (2016) The Importance of Context: Uncovering Species- and Tissue-Specific Effects of Genetic Risk Variants for Type 2 Diabetes. Front. Endocrinol. 7:112. doi: 10.3389/fendo.2016.00112*

THE BASIS FOR TISSUE SPECIFICITY

Across the human population, differences in complex traits, such as height and disease susceptibility, are influenced by the presence of single-nucleotide polymorphisms (SNPs). Some of these genetic variants modulate binding of transcription factors (TFs), which in turn drive differences in gene expression (5, 6). TF binding is also influenced by co-factors and chromatin state, which are highly dependent on cell type and developmental stage. To establish and maintain cellular identity, cell-type-specific TFs tend to bind in clusters, often referred to as cis-regulatory modules (7–9). Intriguingly, association signals for T2D have been found to show a significant overlap with islet-selective enhancer clusters (9, 10). Although other tissues have also been implicated in T2D susceptibility, this is consistent with physiological studies establishing islet dysfunction as a central mechanism of disease-associated variants (1, 11–13).

The overlap between T2D signals and enhancer clusters suggests that the effect of risk variants could be subject to the same tissue specificity observed at the level of regulatory activity (11). In other words, a motif-altering allele would only be expected to produce a molecular phenotype in those contexts (cell type or developmental stage) where the binding site has the potential to be occupied. In support of this notion, T2D risk variants were found to be enriched for nearby binding sites of the pioneer TF FOXA2 in islet and liver (14). T2D-association signals also show a significant overlap with SNPs affecting islet expression of regional transcripts, so-called cis-expression quantitative trait loci (cis-eQTL), most of which are not found to be eQTLs in other tissues (15). Together, these general observations outline some of the context-dependent effects that T2D risk variants can be subject to.

### CONTEXT SPECIFICITY OF CAUSAL MECHANISMS FOR T2D SUSCEPTIBILITY

Recent studies have provided more specific evidence to support the notion that context specificity is a key aspect of GWAS causal mechanisms. The following cases collectively provide examples of mechanisms where studying the right tissue, species, and developmental stage proved critical to uncovering the relevant phenotypes.

At the *MTNR1B* locus, a convergence of evidence has pointed to effects of a non-coding T2D-association signal on the pancreatic β cell. Physiological studies have revealed a phenotype indicative of β-cell dysfunction in risk-variant carriers, with some evidence for additional effects on insulin action (12, 16, 17). Fine-mapping efforts identified a single likely causal variant that overlaps active islet and liver enhancers, and a cis-eQTL for *MTNR1B* in islets (14, 15, 18, 19). The risk allele, which increases islet *MTNR1B* expression, was predicted to create a NEUROD1 binding site and shown to selectively bind this key TF in human β cells. These results establish a likely causal mechanism for the non-coding risk allele, and illustrate how motif-altering alleles can generate highly tissue-specific effects. Surprisingly, exon re-sequencing of the *MTNR1B* gene has also shown coding loss-of-function (LOF) mutations to be associated with increased risk of T2D (20). The reason for the opposite directions of effect observed for coding and non-coding risk variants is unclear but may reflect differences between global and islet-specific roles of *MTNR1B.*

In the case of *PTF1A,* studying the right tissue proved necessary but not sufficient to elucidating the underlying mechanism for non-coding mutations in the region. Previous work had identified a group of patients suffering from unexplained isolated pancreatic agenesis, which includes neonatal diabetes as a clinical feature (21). To filter causal mutations from incidental variation, one study used pancreatic endoderm to define regulatory regions that are active during pancreatic development (22). Their strategy identified a distal enhancer that harbors mutations abolishing enhancer activity toward *PTF1A* (23). Coding LOF variants in *PTF1A* had previously implicated the gene in syndromic pancreatic agenesis, characterized by severe neurological features in affected individuals. The observation that the identified enhancer region is not active in any cell type other than pancreatic endoderm provides a plausible explanation for the absence of any cerebral defects (22). Remarkably, even adult pancreatic tissue did not show active chromatin marks in the region, highlighting that studying the right developmental stage was critical to the success of the approach.

The mechanisms underlying GWAS signals are sometimes studied using individuals that carry LOF mutations in positional candidate genes. The observed phenotypes will be a function of global effects across all the tissues where the gene is expressed, which may confound or mask the more context-dependent actions of regulatory risk alleles. At the *CDKN2A* locus, noncoding T2D signals have been robustly associated with measures of islet dysfunction, and a number of studies have established effects of *CDKN2A* on insulin secretion and cellular senescence in β-cells (12, 24, 25). By contrast, coding LOF mutations in *CDKN2A*, which are a cause of familial melanoma, were recently shown to result in a metabolic phenotype consistent with effects on both liver and β-cells (26). This discrepancy was proposed to arise from islet-specific TF binding of the enhancer region containing the T2D signals.

In animal studies, context-dependent knockouts can provide improved spatial and temporal resolution for targeting candidate causal genes. Even so, the disease relevance of the observed phenotypes is determined by the confidence with which the target tissue of the risk allele is known. One example is provided by an intronic T2D signal at the *TCF7L2* locus, which has been the focus of conflicting observations. Tissue-specific knockout studies have demonstrated primary roles of *TCF7L2* in a number of different tissues, including liver and islets, whereas the noncoding GWAS signal has been consistently associated with a relatively narrow insulin secretion defect. Genomic annotations provide a clue as to the underlying reason, with the risk variants being located in an islet-specific region of open chromatin (8, 10, 27). Furthermore, the region has chromatin marks indicative of regulatory activity in islets, but not in a wide range of other tissues (10). The annotations can, thus, be applied as a filter to exclude non-disease-relevant tissues, and guide efforts to study the effect of the risk allele in the most appropriate context.

Coding GWAS variants can also produce context-dependent mechanisms through the restricted expression of gene isoforms. A striking example of this is provided by a coding variant identified in the *TBC1D4* gene in a small founder population of Greenlandic Inuit (28). The risk allele, which produces a truncated transcript that results in nonsense-mediated decay, is positioned in an exon excluded from the short isoform of the transcript. Unlike the widely expressed short isoform, the long form is predominantly expressed in skeletal muscle (29). The decreased insulin sensitivity resulting from reduced expression of *TBC1D4* is, therefore, selectively imposed on muscle tissue. As a result, the risk variant has a different effect on fasting glucose from that observed in individuals carrying LOF mutations affecting both isoforms (28).

Similar to alternative spliceforms, gene homologs can contribute to concealing the primary effect of a GWAS signal in a specific context. At the *ADCY5* locus, T2D risk alleles have been linked to both decreased islet expression of the *ADCY5* gene and β-cell dysfunction, though the underlying molecular mechanism remains unclear (12, 15). Expression studies in rodents have shown *Adcy5* to be nearly undetectable compared with the closely related homolog *Adcy6* (30, 31). By contrast, human islets show roughly equal expression of these orthologous genes, hinting at a non-conserved function of *ADCY5* between the species (30, 32). It also highlights an underlying species-specificity that makes rodents less well suited as models for mechanistic studies.

### LIMITATIONS OF TRADITIONAL APPROACHES

The examples above demonstrate that the molecular phenotypes of GWAS signals can be modulated by a multitude of context-dependent factors. In the case of non-coding variants, tissue- or developmentally restricted activity of the surrounding chromatin can limit effects on gene expression. For coding variants, alternative splicing or expression of homologs can mask a broader phenotype to produce context-dependent effects. These insights have important implications for how we design studies to translate genetic signals into molecular mechanisms.

Traditionally, particular genes have been selected for followup studies based on a combination of known candidate-gene biology and proximity to the GWAS signal (**Figure 1**, *left*). For whole-organism gene knockouts, this could involve engineering

FIGURE 1 | Translating GWAS signals into disease mechanisms using traditional and integrative approaches. The top panel shows a schematic representation of a GWAS locus with a non-coding association signal for T2D risk located near two genes, *A* and *B*. The left-hand side represents the traditional approach followed for elucidating the causal mechanisms leading to a T2D phenotype. This approach can produce a range of phenotypes that are difficult to translate into causal GWAS mechanisms, because the selected genes are often knocked out globally (bottom left panel), or in a tissue-specific context that is irrelevant to understanding the effects of the T2D susceptibility phenotype (bottom center panel). Sequencing approaches can also be used to identify gain-offunction mutations (not shown), which are subject to the same limitations. The right-hand side outlines an alternative approach using integration of emerging datasets to produce directed hypotheses. For optimal resolution, several types of genetic and genomic datasets can be integrated, including TF binding, enhancer regions (defined based on chromatin state), and cis-regulatory relationships (e.g., identified by cis-eQTL or chromatin-conformation capture studies). In this example, the GWAS signal is observed to disrupt an enhancer cluster with cis-regulatory activity toward gene *B*. Importantly, the disruption exclusively affects enhancer activity in disease-relevant tissues. This proposes a follow-up experiment for manipulation of gene *B* in a specific context (bottom right panel), producing a phenotype that is likely to be directly relevant to understanding the molecular basis of T2D susceptibility.

of animal models or using genetic testing to identify individuals carrying LOF variants. As discussed, the relevance of these approaches for delineating disease-relevant mechanisms is limited by the potential for unspecific global phenotypes. Tissuespecific knockouts provide higher spatiotemporal resolution but require the target tissue(s) of the GWAS signal to be known. Human physiological associations can narrow down the list of likely relevant tissues, but these measures are often too crude to pinpoint specific cell types.

In principle, using animal models to target non-coding regions could produce more precise disease models, but this strategy is constrained by the low conservation at the level of regulatory architecture. In a study of the *Cdkn2a* locus in mice, targeted deletion of a 70 kb non-coding interval established enhancer activity toward nearby genes, but the relevance to humans has been questioned by subsequent findings (33, 34). The region encodes a long non-coding RNA that has no clear ortholog in rodents, highlighting the possibility of divergent cisregulatory mechanisms. More generally, TF binding sites have been shown to diverge even faster than the underlying sequence itself (35). For two key liver TFs, the majority of binding events were shown to be species specific, while only 10–30% of hepatic enhancer clusters have corresponding rodent orthologs (36, 37). Although subsets of conserved clusters may aid in the prioritization of causal variants, these observations suggest that a different approach is required to delineate GWAS mechanisms (37, 38).

Even in those cases where animal models do provide targeted gene manipulation in an appropriate context (whether through tissue-specific knockout or transcriptional dysregulation), the resulting phenotypes may not be directly relevant for understanding human disease. Though rodent models continue to be an important tool for studying type 2 diabetes pathogenesis, it has become increasingly clear that murine pancreatic islets differ in a number of ways from their human counterparts (39–41). Certain monogenic forms of diabetes are, therefore, not well recapitulated in rodents, and molecular mechanisms elucidated in animal models should be interpreted with caution (42).

### TOWARD AN INTEGRATED UNDERSTANDING OF GWAS SIGNALS

To successfully study disease-relevant phenotypes, experimental designs can be guided by the integration of genetic association data and genomic annotations (**Figure 1**, *right*). At the core of this framework is the overlaying of a static dataset – a list of variants linked to disease susceptibility and/or physiological traits – with layers of highly dynamic functional information that provide spatial and temporal dimensions. These layers encompass diverse datasets, and include information centered on single variants, such as histone marks and TF binding sites, and higher-order information that signifies relationships between distinct elements, such as chromatin interactions and cis-eQTL data.

#### REFERENCES

1. Voight BF, Scott LJ, Steinthorsdottir V, Morris AP, Dina C, Welch RP, et al. Twelve type 2 diabetes susceptibility loci identified through

Genomic annotations provide a cellular atlas with which to navigate and interpret genetic data in the context of specific cell types and developmental stages. For instance, if a set of likely causal variants has been identified from GWAS or fine-mapping studies, the tissue of action may be inferred from comparing chromatin states across cell types (8–11, 43–46). Conversely, if the target tissue is known from physiological associations, the causal mechanism can be pinpointed by overlaying with relevant functional annotations (14, 22, 47–51). This process generates a plethora of directed hypotheses that can be followed up with specific functional experiments. Increasingly, such studies are likely to be focused on differentiated cells derived from human stem cells, which can provide disease models and chromatin maps that are both functionally and developmentally relevant.

The broader applicability of this approach is, in part, determined by the tractability of individual loci. For regions with extensive linkage disequilibrium, the arising complexity can hinder experimental follow-up. Starting from a limited set of credible variants is, thus, essential. More generally, the value in taking an integrative approach is dictated by the extent to which genomic annotations for disease-relevant tissues have been made available (or can be obtained). The construction of a truly integrated framework is an incremental and monumental effort, facilitated by the Encyclopedia of DNA Elements (ENCODE) and the NIH Roadmap Epigenomics project, which together cover hundreds of tissues and epigenetic annotations. Since each dataset is merely a snapshot of a given cell type in a particular metabolic and developmental state, this on-going process will continue to produce an atlas with ever-finer spatiotemporal resolution. For many hard-to-obtain organs, such as pancreatic islets, power to detect relevant genomic features is still limiting.

Even so, chromatin landscapes for tissues relevant to T2D have begun to emerge in recent years, enabling biological inferences to be made. As we have seen, this has successfully uncovered tissue- and species-specific effects of T2D risk variants. The insights have also provided compelling evidence to demonstrate that context-dependent phenotypes are not the exception but, in fact, a fundamental aspect of GWAS biology. In the coming years, we need to build on this paradigm to accelerate the translation of genetic findings into molecular mechanisms.

# AUTHOR CONTRIBUTIONS

ST, MM, and AG wrote, edited, and approved the manuscript.

#### FUNDING

ST is a Radcliffe Department of Medicine scholar, MM is a Wellcome Trust Senior Investigator, and AG is a Wellcome Trust Senior fellow in Basic Biomedical Science (095101).

large-scale association analysis. *Nat Genet* (2010) 42:579–89. doi:10.1038/ ng.609

2. Morris AP, Voight BF, Teslovich TM, Ferreira T, Segrè AV, Steinthorsdottir V, et al. Large-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes. *Nat Genet* (2012) 44:981–90. doi:10.1038/ng.2383

**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 reviewer LL declared a past co-authorship with one of the authors MM to the handling Editor, who ensured that the process met the standards of a fair and objective review.

*Copyright © 2016 Thomsen, McCarthy and Gloyn. 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) or licensor 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.*

# How Useful Are Monogenic Rodent Models for the Study of Human Non-Alcoholic Fatty Liver Disease?

*Jake P. Mann1 , Robert K. Semple2,3\* and Matthew J. Armstrong4,5*

*1Department of Paediatrics, University of Cambridge, Cambridge, UK, 2 The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK, 3 The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK, 4Centre for Liver Research, National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, UK, 5 Liver Unit, Queen Elizabeth University Hospital Birmingham, Birmingham, UK*

*Edited by:*

*Nils P. Krone, University of Sheffield, UK*

#### *Reviewed by:*

*Rodolfo A. Rey, Consejo de Investigaciones Científicas y Técnicas (CONICET), Argentina Mark Cleasby, Royal Veterinary College, UK*

*\*Correspondence:*

*Robert K. Semple<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Genomic Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 24 July 2016 Accepted: 01 November 2016 Published: 16 November 2016*

#### *Citation:*

*Mann JP, Semple RK and Armstrong MJ (2016) How Useful Are Monogenic Rodent Models for the Study of Human Non-Alcoholic Fatty Liver Disease? Front. Endocrinol. 7:145. doi: 10.3389/fendo.2016.00145*

Improving understanding of the genetic basis of human non-alcoholic fatty liver disease (NAFLD) has the potential to facilitate risk stratification of affected patients, permit personalized treatment, and inform development of new therapeutic strategies. Animal models have been widely used to interrogate the pathophysiology of, and genetic predisposition to, NAFLD. Nevertheless, considerable interspecies differences in intermediary metabolism potentially limit the extent to which results can be extrapolated to humans. For example, human genome-wide association studies have identified polymorphisms in PNPLA3 and TM6SF2 as the two most prevalent determinants of susceptibility to NAFLD and its inflammatory component (NASH), but animal models of these mutations have had only variable success in recapitulating this link. In this review, we critically appraise selected murine monogenic models of NAFLD, NASH, and hepatocellular carcinoma (HCC) with a focus on how closely they mirror human disease.

Keywords: steatosis, animal model, metabolic syndrome, steatohepatitis, genetic models

#### INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is a pandemic disorder associated with premature morbidity and mortality. Its etiology is multifactorial, with both genetic predisposition and environmental factors playing important parts (1). Animal models have been central to recent major translational research efforts, serving as discovery tools to identify new candidate pathogenic mechanisms, as a means of testing hypotheses arising from human studies, and as pre-clinical models in which to assess potential therapeutic strategies (2). There are multiple different experimental perturbations known to trigger NAFLD in animal models, which have been used to generate all components of the NAFLD spectrum (3–10). Such perturbations include genetic manipulation (e.g., ob/ob mouse), pro-steatotic or pro-inflammatory diets [e.g., methionine–choline deficient (MCD) diet], and toxic insults (e.g., streptozotocin injection). Combinations of these may be used to reflect the multifactorial nature of the human disease (e.g., ob/ob mice fed a MCD diet); however, a detailed discussion of rodent diets is beyond the scope of this review.

While animal models do potentially give key insights into the disease process that cannot be obtained through any other means, it is necessary to temper appreciation of this utility with understanding of the limitations of each of the models used.

We, now, briefly review the current understanding of the genetic architecture of human NAFLD, and the major approaches adopted to modeling it in animals, while critically appraising the utility of these models.

#### OVERVIEW OF NON-ALCOHOLIC FATTY LIVER DISEASE PATHOGENESIS

Human NAFLD is defined by the presence of macrovesicular steatosis in more than 5% of hepatocytes in individuals with a history of less than 20 g/day ethanol intake (11). The differentiation of macrovesicular from microvesicular steatosis is a qualitative histological assessment, and the two may coexist in more advanced disease (12). NAFLD exists as a pathological spectrum ranging from non-alcoholic fatty liver (NAFL, also known as "simple" steatosis), which denotes hepatic steatosis in the absence of steatohepatitis, through to non-alcoholic steatohepatitis (NASH), in which there is histological evidence of inflammatory infiltrates, hepatocyte ballooning (featuring Mallory-Denk bodies), and commonly fibrosis of varying severity. Cirrhosis can develop at the most severe end of the spectrum, conferring increased risk of hepatocellular carcinoma (HCC), portal hypertension, and liver failure (13). Longitudinal studies have shown 10–20% of patients with each stage of disease to progress to the following stage in, while there is also an element of reversibility, particularly in NAFL and NASH (14, 15).

A "two-hit hypothesis" has been suggested to account for the pathogenesis of advanced NAFLD, whereby the first "hit" drives development of steatosis and the second triggers inflammation and its sequelae, critically including fibrosis (16).

In principle, hepatic steatosis may result from pre-hepatic derangements, intra-hepatic derangements, or both, as illustrated in simplified form in **Figure 1**. A comprehensive review of pathways leading to hepatic steatosis is beyond the scope of this article and is provided elsewhere (17–19). In brief, pre-hepatic pathogenic factors encompass increased substrate flux to the liver (e.g., non-esterified free fatty acids (NEFA), monosaccharides, or amino acids) and dysregulation of hormones that act directly on hepatocyte metabolism (e.g., insulin, glucagon and related peptides, and adipokines). Increased "preload" in the form of NEFA delivery generally results from failure of adipose tissue adequately to buffer positive energy balance. This may be a consequence of hyperphagic obesity, in which even normal adipose buffering capacity is overwhelmed, or lipodystrophy, in which adipose energy buffering capacity is pathologically constrained. In some situations, a mixture of these is at play, as in generalized lipodystrophy, where the harmful results of absent adipose tissue are potentiated by concomitant lack of leptin.

Many pre-hepatic hormonal factors also influence propensity to NAFLD by acting on adipocytes to modulate lipolysis and/or through direct actions on hepatocytes (e.g., insulin, glucagon, glucagon-like peptides). There has been a particular focus on the ability of high levels of insulin, secondary to peripheral insulin resistance, to drive hepatic *de novo* lipogenesis. Another emerging influence on liver metabolism is the gut microbiome, which may affect gut hormone release and also signal directly through flux of bacterial metabolites such as acetate (20, 21).

Hepatocyte-autonomous (intra-hepatic) defects may also lead to triglyceride accumulation. Such defects may broadly be classified into: those increasing *de novo* synthesis of triglyceride; those perturbing lipid droplet dynamics, triglyceride mobilization and lipoprotein assembly or secretion; and those impairing catabolism of fatty acids by beta-oxidation. Although reduced ability to catabolize fatty acids *via* beta-oxidation (e.g., due to Mendelian disorders in key catabolic enzymes, or mitochondrial dysfunction) does result in hepatic steatosis, however, this is usually microvesicular in appearance and has a distinct clinical profile that often includes hypoglycemia, liver failure, and encephalopathy (22). These disorders will, thus, not be discussed further here.

Development of NASH is multifactorial; a comprehensive review of the inflammatory and fibrotic sequelae of hepatic lipid accumulation can be found elsewhere (23–26). Key elements of pathogenesis include oxidative stress (from lipid peroxidation and mitochondrial dysfunction) and activation of pro-inflammatory pathways (e.g., NF-κB) in hepatocytes, but other cellular pathways, including the endoplasmic reticulum stress response, have also been implicated (27). Coactivation of Kupffer cells, sinusoidal endothelium, and hepatic stellate cells gives rise to cytokines that augment inflammation [e.g., tumor necrosis factor alpha (TNFα), interleukin-1/-6] and drive fibrosis [e.g., transforming growth factor beta (TGFβ)] (19, 28, 29). These processes are also exacerbated by pre-hepatic factors, such as adipose inflammation/lipotoxicity, gut bacterial translocation, and endogenous alcohol production.

#### HUMAN GENETICS OF NAFLD

In the vast majority of patients, NAFLD is a multifactorial condition rooted in obesity and insulin resistance, based on strong clinical association and natural history studies in humans. Pandemic, idiopathic NAFLD is often referred to as "primary" NAFLD (30). Genetics can play a role in each stage of the pathophysiology of NAFLD, as illustrated both by rare monogenic conditions that feature severe NAFLD, and by the association of much more frequent single nucleotide polymorphisms (SNPs) with "common" NAFLD (31–33). The proliferation of recent human genetic findings puts their detailed treatment beyond the scope of this discussion; however, we select a series of mechanistically informative sentinel examples to appraise against rodent models.

#### "PRE-HEPATIC" NAFLD

#### Monogenic Hyperphagic Obesity

Flux of substrates, such as free fatty acids, amino acids, and lactate, provide the building blocks for hepatocyte triglyceride accumulation as well as the energy required for activation of anabolic pathways (see **Figure 1**). Excess flux can thus be a potent driver for NAFLD. Most attention has been paid to flux of free fatty acids, the product either of lipolysis of triglyceride

in adipose tissue or lipolysis of triglyceride in triglyceride-rich lipoproteins such as chylomicron remnants. Key determinants of free fatty acid flux to the liver are thus the dietary intake of fat and the efficiency of fatty acid trapping and storage in adipose tissue. Correspondingly, in states of hyperphagia or adipose tissue insufficiency, free fatty acid flux may be pathologically increased. Of note, "adipose tissue insufficiency" may arise from overload of adipose tissue that is normal or increased in amount, or may arise from frank anatomical deficiency of adipose tissue, or lipodystrophy.

Several human examples of monogenic hyperphagia exist. The first to be discovered was congenital leptin deficiency (34), with loss-of-function mutations in the leptin receptor gene discovered later (35). Leptin is an adipose-derived peptide adipokine that acts on the hypothalamus to signal replete energy stores and suppress appetite (36). It is expressed and secreted by white adipose tissue (WAT) in direct proportion to WAT volume, and serum levels fall commensurately with depletion of adipose stores during starvation. Primary leptin deficiency produces severe, hyperphagic obesity from a young age; however, liver fat accumulation has only rarely been commented on in published studies. In one study, severe NAFLD was identified in an obese leptin-deficient child that resolved quickly on introduction of recombinant leptin therapy (37). The long-term hepatic outcome of untreated human leptin deficiency or leptin receptor mutation is not known; however, leptin receptor polymorphisms have been associated with NASH and insulin resistance in patients with NAFLD (38, 39).

Several other forms of human monogenic obesity are now known. In contrast to genetic leptin or leptin receptor loss-offunction, which are extremely rare, heterozygous mutations in the melanocortin-4-receptor (MC4R) are the most common monogenic cause of obesity, accounting for around 6% of severe, early-onset obesity (40). There are no published reports of the impact of heterozygous MC4R mutations on hepatic steatosis in monogenic obesity, while polymorphisms in MC4R have been associated with alanine aminotransferase and BMI (41), but not with hepatic fat content in population-wide studies (42). Several other genes have been implicated in human monogenic obesity; however, in aggregate, these affect only a very small number of patients, and there is little information on the liver phenotype. Moreover, some of the gene products involved, such as prohormone convertase 1 and pro-opiomelanocortin have pleiotropic roles, not just in appetite control but also in the action of key peripheral hormones (peptide and steroid hormones respectively) which have independent effects on liver fat accumulation, so shall not be considered here.

The sparse attention paid in published human reports to the natural history of NAFLD in human monogenic obesity is likely to be attributable to the rarity of the diseases, to the fact that most patients identified are children in whom clinically overt liver NAFLD has not yet had time to develop and to that fact that, in leptin deficiency, curative therapy with recombinant human leptin is the standard of care. This means that the long-term natural history of NAFLD in leptin deficiency will be extremely difficult to document in future, although determining the long-term liver outcome of MC4R loss-of-function should be both tractable and informative.

#### Monogenic Lipodystrophy

In the face of severe hyperphagia, the ability even of "normal" adipose tissue to buffer chronic positive energy balance by trapping and sequestering lipid is finite and is eventually overwhelmed, leaving the liver exposed to excess free fatty acid flux. However, some rare humans have a congenital deficiency of adipose tissue, which we shall consider only in its most severe, generalized form. In this situation, there is effectively absolute adipose failure, and complete lack of energy buffering is compounded by secondary lack of leptin and consequent hyperphagia, as the hypothalamus erroneously interprets very low or absent leptin as an indication of starvation. Around 95% of cases in humans congenital generalized lipodystrophy (CGL) are accounted for by biallelic mutations in either AGPAT2, encoding an enzyme involved in triglyceride synthesis, or in BSCL2, encoding an endoplasmic reticulum protein involved in lipid droplet regulation and adipocyte differentiation (43, 44). A clinical hallmark of CGL is very severe NAFLD, with early development of inflammation, fibrosis, and HCC. Indeed, clinical experience and published case series suggest that complications of advanced liver disease are among the major sources of mortality in CGL (45, 46). Observations in CGL and other acquired and genetic lipodystrophies establish unequivocally that primary disorders of adipose tissue are sufficient to cause the full spectrum of NAFLD in humans (47). This does not prove that adipose dysfunction is the primary mechanism at play in pandemic NAFLD; however, current population-wide data do not rule this out.

#### Endocrine Drivers

Many hormones exert a major influence on lipid accumulation within hepatocytes, although in each case this is only possible in the context of adequate energy charge and substrate flux to the liver, so these cannot easily be teased apart. Hormones of proven importance in altering liver lipid accumulation include insulin, glucagon, and the incretin gut peptides (e.g., GLP-1). In humans, insulin has attracted particular attention as several lines of evidence suggest that insulin, acting through the insulin receptor, stimulates *de novo* lipogenesis sufficiently to make a major contribution to liver fat accumulation in NAFLD (48–50). Some negative evidence in support of this model comes from the observation that humans with loss-of-function mutations in the insulin receptor itself, although showing very severe insulin resistance, are protected from dyslipidemia and liver fat accumulation (51). Preliminary evidence suggests that this also holds for patients with lipodystrophy and loss-of-function mutations in *PIK3R1*, a component of phosphatidylinositol-3-kinase involved in insulin signaling (52).

# HEPATOCYTE-AUTONOMOUS NAFLD

# Lipid Trafficking Abnormalities

Some genetic abnormalities can influence development of steatosis by hepatocyte-autonomous mechanisms. One of these mechanisms is impaired assembly and export of lipoproteins from hepatocytes, leading to intracellular accumulation of triglycerides. The best examples of this are heterozygous truncating mutations in APOB [encoding apolipoprotein B (ApoB)], causing hypobetalipoproteinaemia and biallelic mutations in MTTP (encoding microsomal triglyceride transfer protein), which cause abetalipoproteinaemia (53). These two conditions have a similar phenotype including NASH, low serum triglycerides, low LDL cholesterol, and neuropathy. Patients often develop progressive NASH with fibrosis even in the absence of diabetes, obesity, or other oxidative stresses (53, 54).

Further evidence in support of altered lipoprotein assembly and secretion as a significant player in NAFLD comes from GWAS of the commoner form of the disease. These have implicated a SNP in TM6SF2 (encoding transmembrane 6 superfamily member 2), which is needed for secretion of very-low density lipoproteins (VLDL), in NAFLD. The risk allele is found in around 1% patients with NAFLD (55) and correlates positively with serum aminotransferase levels (33), hepatic steatosis (31), NASH activity, and fibrosis (but not HCC) (56). Importantly, patients with this SNP have lower circulating LDL cholesterol, lower triglycerides, and reduced incidence of atherosclerotic disease, providing an example of dissociation of NAFLD from dyslipidemia and cardiovascular disease (57). Data relating to the association with insulin resistance and type 2 diabetes are currently inconclusive (58).

Population-wide human genetics has also implicated fundamental abnormalities in lipid droplets in NAFLD: for example, the p.Ile148Met SNP in PNPLA3, whose product is patatin-like phospholipase domain-containing 3, is associated with all stages of NAFLD, from simple steatosis, NASH, fibrosis, and development of HCC (59, 60). PNPLA3 (also known as adiponutrin) is a membrane-bound enzyme expressed at the surface of lipid droplets and on the smooth endoplasmic reticulum (61) and plays a role in lipid droplet dynamics. There has been conflicting evidence on whether this polymorphism is associated with insulin resistance, however.

#### Inflammation/Fibrosis

Inflammation and fibrosis are the hallmarks of complicated NAFLD, and a genetic basis for the susceptibility to these complications is often mooted as the "second hit" needed to drive progression from simple steatosis to inflammatory and fibrotic sequelae. Unsurprisingly, no single gene examples of a primary inflammatory or fibrotic disorders leading to end stage NAFLD have been reported (62, 63); however, the GWAS approach has identified common genetic variants associated with progression of fibrosis (e.g., near the gene for platelet-derived growth factor alpha) or with the development of lobular inflammation (e.g., near genes encoding interleukin-6, or collagen type XIII alpha 1).

#### RODENT GENETIC MODELS OF NAFLD

The ideal "one stop" animal model of NAFLD would recapitulate the full progression from simple steatosis through NASH to fibrosis, cirrhosis, and HCC. Ideally, the disease process would progress rapidly (in contrast to the human condition) to maximize experimental tractability of the model. However, a valid alternative would be to have a series of different models for different stages of the disease sequence, which may be primarily metabolic, inflammatory, or fibrotic. It has been suggested that the primary goal of rodent models should be to mimic the pathology of NAFLD, including the underlying histopathology (3). On this narrow basis, it has been argued that rodents do not accurately model human NAFLD because of differences in ballooning degeneration and distribution of inflammatory infiltrate in NASH (4).

Many animal models have been described in which different degrees of NAFLD are seen, and we shall consider selected models only as examples, classifying them into two groups according to the broad pathogenic mechanism at play: (1) models of "extrahepatic" NAFLD (**Table 1**) and (2) models of "intrahepatic" NAFLD (**Table 2**).

## MURINE "PRE-HEPATIC" NAFLD

### Hyperphagic Models

The severely obese *ob/ob* mouse strain arose spontaneously in 1949, and was eventually discovered in 1994 to harbor a loss-offunction mutation in the gene encoding leptin, thereby ushering in the modern era of investigation of the neuroscience of appetite control. Given an *ad libitum* diet, *ob/ob* mice develop obesity, insulin resistance, hyperglycemia, and hepatic steatosis (74), and they have been become a highly popular model for many aspects of obesity and related disorders, either being studied in isolation, or crossed with other strains of interest in order to determine the effect of severe hyperphagia on mice with different genetic perturbations. The severe obesity of *ob/ob* mice is attributable to hypothalamic hyperphagia; however, several studies have provided evidence for additional peripheral effects of leptin to modulate insulin sensitivity and metabolism directly in tissues, such as muscle and liver. Whether such peripheral effects are also seen in humans remains to be established beyond doubt (75–77).

#### TABLE 2 | Examples of murine genetic models relevant to "intrahepatic" NAFLD.

*Features are based on ad libitum standard chow diet. Apob*−*/*− *mouse is unable to secrete VLDL from hepatocytes; fatter liver Shionogi mice are thought to also have impaired VLDL secretion; FLS-ob/ob are FLS mice crossed with leptin-deficient mice; Transmembrane 6 superfamily 2 (Tm6sf2) knockdown mice have reduced triglyceride secretion; hepaticspecific expression of the human I148M allele of patatin-like phospholipase domain-containing 3 (PNPLA3) causes altered lipid droplet composition and storage. NASH, non-alcoholic steatohepatitis; HCC, hepatocellular carcinoma; Y, yes; N, no.*

The *ob/ob* mouse rapidly develops NAFL and at 20 weeks old early features of NASH are present (77, 78). NASH may been accelerated by a second "hit" of inflammatory/oxidative stress, using, for example, intraperitoneal injection of lipopolysaccharide (LPS) (79), dietary stressors such as a MCD diet (80), or crossing with another NASH-prone strain, such as the fatty liver Shionogi (FLS) mice, which are discussed later (81). Notably, however, leptin is required for normal immune function, and its deficiency dampens down both innate and acquired immune responses in humans (82, 83), though it seems to play a proinflammatory and pro-fibrogenic role in mice (78). This may explain why the inflammatory components of NAFLD appear relatively indolent in *ob/ob* mice than in more common NAFLD. On a related note, it has also be shown that the *ob/ob* mouse is relatively resistant to fibrosis (78, 80), partly due to reduced release of TNFα, which is necessary for activation of TGFβ, a key pro-fibrogenic molecule (84–86).

Collectively, these findings caution that, while the *ob/ob* mouse is a valuable model of primary hyperphagia-driven NAFLD, loss of specific actions of leptin on peripheral metabolism, coupled to some degree of immunosuppression, means that it is likely to deviate from pandemic NAFLD in key respects, especially related to the inflammatory and fibrotic end of the disease spectrum. Similar arguments may apply to leptin-resistant *db/ db* mice, which harbor a splice site mutation abolishing expression of the long form of the leptin receptor; however, *db/db* mice are reported to exhibit more severe NAFLD than *ob/ob* mice (64, 80). In practice, a secondary stressor is also usually applied when using *db/db* mice to study NAFLD, with the MCD diet being widely used (87).

Mice in which *Mc4r* is genetically ablated have also been widely studied. Their primary defect lies within hypothalamic appetite control pathways, as the melanocortin 4 receptor responds to the neuropeptide α-MSH, which is generated in response to leptin action. *Mc4r*-null mice feature hyperphagic obesity without pathologically suppressed leptin levels, suggesting that they have the potential to model the extended spectrum of NAFLD more faithfully than *ob/ob* mice. In keeping with this, it has been reported that *Mc4r*-knockout mice, when exposed to a high-fat diet, develop not only steatosis but also exuberant NASH with established fibrosis after 20 weeks, progressing to HCC in all mice studied by 1 year (65).

#### Lipodystrophic Models

Many different murine genetic models of lipodystrophy have been described. Where direct comparison is possible between mice and humans, it has been found that the common forms of CGL are well modeled in mice, while the situation for human genetic forms of partial lipodystrophy is more complex, and will not be discussed further here (45).

Both *Agpat2*- and *Bscl2*-knockout mice have been described (88, 89). *Agpat2* encodes 1-acylglycerol-3-phosphate O-acyltransferase 2 and is needed for synthesis of triacylglycerols and glycerolphospholipids in white adipose. Many Agpat2<sup>−</sup>/<sup>−</sup> mice die in the first few weeks of life; however, the survivors accurately recapitulate the human condition of lipodystrophy with severe insulin resistance (66). Hepatic steatosis has also been reported to be a major feature of the mice before 16 weeks old, with liver weights twice normal, and modest inflammatory changes seen in addition to pronounced steatosis. More detailed time course studies of the liver disease in this model have not been reported, however.

Bscl2 encodes seipin, which is needed for both differentiation of white adipocytes and normal lipid droplet regulation, and deficiency has been suggested to shift the balance toward release of free fatty acids from adipocytes (89). Bscl2<sup>−</sup>/<sup>−</sup> mice display a lipodystrophic phenotype with near complete absence of WAT. Massive hepatic steatosis is a feature of all three Bscl2<sup>−</sup>/<sup>−</sup> models described. As in Agpat2 null mice, however, detailed natural history studies of liver pathology have not been reported: at 12 weeks, there is no evidence of NASH but there are no reported data beyond this age (90).

Although not directly modeling human disease, some other murine models have provided powerful evidence for the importance of adipose tissue in protection from NAFLD. One impactful model is the A-ZIP/F-1 (or "AZIP") mouse, which is an adiposedeficient mouse generated by transgenic overexpression of an artificially engineered dominant negative protein that interferes with critical adipogenic transcription factors. This causes deficiency of white adipose, severe insulin resistance, and hepatic steatosis, although it was said not to feature inflammation at the relatively young age studied (67). It is not known whether these mice develop NAFLD-related fibrosis, in part because of the reduced survival of the mice, which are severely diabetic. A much more recent model featured knockout of the insulin receptor selectively in adipose tissue, knockout being mediated by *cre* recombinase driven by the adipose-specific adiponectin promoter (68). These mice developed severe lipodystrophy and fatty liver disease very early, and by 12 weeks old, livers demonstrated not only steatosis but also increased ROS, lipid peroxidation, ballooning degeneration of hepatocytes, and elevated serum transaminase levels. By 1 year old the liver accounted for 25% of body weight and showed highly dysplastic liver nodules in addition to worsened inflammation and fibrosis. High fat feeding worsened the liver injury.

#### Liver Insulin Action

Although it is impossible to mimic a primary increase in insulin without inducing complex and potentially confounding changes to systemic metabolism, liver-specific knockout of the insulin receptor does afford the opportunity to test the proposition that liver insulin action is necessary for the development of hepatic steatosis. Indeed, in 2008, it was reported that ablation of the insulin receptor in mouse liver, although inducing severe systemic insulin resistance, did not increase liver triglyceride or liver weight (91).

Conversely, liver-specific knockout of the phosphatase and tensin homolog (PTEN), which is a phosphatidylinositol-3,4,5 triphosphate (PIP3) phosphatase that serves to antagonize insulin's metabolic actions, produces macrovesicular steatosis, NASH (including Mallory-Denk bodies), fibrosis, and HCC (69). Similarly, other modes of genetic activation of phosphatidylinositol-3-kinase pathway signaling also produce steatosis that progresses to HCC, but without such marked steatohepatitis (92, 93).

# HEPATOCYTE-AUTONOMOUS MURINE NAFLD

#### Impaired Lipoprotein Synthesis/Secretion

Assembly and secretion of VLDL represents a major route for the final disposition of intrahepatocyte triglyceride. Apolipoprotein B (ApoB) is a core component of VLDL particles, and genetic partial deficiency in humans causes familial hypobetalipoproteinaemia. Homozygous loss-of-function mutations of ApoB in mice cause embryonic lethality due to exencephalus. However, several different lines of mice heterozygous for mutated ApoB have been described, and these accurately recapitulate the hepatic steatosis, low serum triglycerides, and low HDL cholesterol of the human condition, although little fibrosis has been reported in the absence of additional pro-inflammatory stimuli (70, 94). Nevertheless, an important caveat is that natural history of liver inflammation and fibrosis has not been reported beyond 12 weeks of age. Similar to humans with hypobetalipoproteinaemia, mice with reduced ApoB function do not show marked insulin resistance, which differentiates them from most patients with NAFLD.

The FLS mouse is a further NAFLD model, which arose spontaneously as a result of inbreeding. FLS mice are non-obese and only mildly insulin resistant, but show marked accumulation of macrovesicular triglyceride with mononuclear inflammatory infiltrate and fibrosis, which eventually results in development of HCC by 13–16 months even without additional carcinogenic stimuli (95, 96). The precise underlying genetic defect is not yet known; however, a defect in microsomal triglyceride transfer protein (MTTP) (97) has been suggested. Precisely, why this model is so susceptible to HCC is not known. Crossing of FLS mice with *ob/ob* mice results in a model with the full metabolic syndrome and progressive fibrosis (81, 98); this model most closely recapitulates the whole human spectrum of NAFLD in a practical, experimental timeframe.

As discussed, human GWAS have shown a SNP in TM6SF2 to be associated with NAFLD (99). Reminiscent of hypobetalipoproteinaemia, however, carriers of the risk-conferring T-allele seem relatively protected from atherosclerotic disease despite increased lipid fat content. TM6SF2 is a membrane-bound protein located on the endoplasmic reticulum involved in the secretion of VLDL from hepatocytes (100). Germline knockout of *Tm6sf*− has not yet been described in mice; however, adenoassociated virus-mediated knockdown selectively in mouse liver resulted in increased hepatic triglyceride content and reduced VLDL secretion (32). Moreover transgenic hepatocyte-specific expression of human wild-type TM6SF2 in mice caused an increase in serum LDL cholesterol and lower HDL cholesterol (101); however, the full spectrum of NAFLD has not been demonstrated in this model.

# ABNORMAL LIPID DROPLET DYNAMICS

The p.Ile148Met variant in human PNPLA3, found in around 20% of the population, is associated with NAFLD progression (55). Therefore, efforts have been made to generate a rodent model that recapitulates this genetic predisposing factor and to elucidate the role of PNPLA3 in NAFLD. PNPLA3 is expressed in many tissues in mice, including WAT and liver (102). Expression is suppressed upon fasting (103) and upregulated following a carbohydrate load (104), which is believed to be mediated by steroid regulatory element-binding protein-1c (SREBP-1c) and carbohydrate-response element-binding protein (ChREBP). PNPLA3 was initially thought to function as a lipase and be involved in the release of triglycerides from intracellular lipid droplets (105–107), so it was hypothesized that reduced activity would increase hepatocellular triglyceride content (108).

If reduced PNPLA3 activity was to accelerate NAFLD, *Pnpla3* knockout mice would be expected to have severely fatty livers; however, in fact, they have no evidence of NAFLD (109). Indeed, there is no discernible difference between *Pnpla3*−*/*− and wildtype mice even when fed high-fat diet or crossed onto the *Lepob/ob* background. It should be noted, however, that mice show the highest Pnpla3 expression in WAT, unlike humans, in whom expression is highest in the liver.

In contrast to the knockout mice, mice with hepatic overexpression of human PNPLA3 p.Ile148Met show increased hepatic triglyceride content and fatty acid synthesis (73, 110). They develop steatosis only on a normal chow or sucrose diet, but not on a high-fat diet, which suggests that the mutant PNPLA3 acts upon *de novo* fatty acids, rather than re-absorbed circulating nonesterified fatty acids (111). This is consistent with epidemiological data that suggests fructose-rich diets are more harmful than highfat diets (112).

The cumulative data from these mouse models suggest that PNPLA3 functions as a lysophosphatidic acid acyltransferase, and that the p.Ile148Met polymorphism increases this activity, stimulating development of hepatic steatosis (113, 114). This would have not been identified without the initially surprising finding in the knockout model. Although mice with hepatic expression of human PNPLA3 p.Ile148Met develop NAFLD, it is not known whether these mice develop HCC with age as reported studies extend to only 12 weeks, however.

# CONCLUSION

The spectrum of human NAFLD disease arises from complex environmental influences allied to genetic predisposition. Human monogenic diseases demonstrate unequivocally that adipose tissue failure and primary genetic defects in intrahepatocyte lipid handling can give rise to NAFLD and its sequelae. Genetic perturbation in rodents may be combined with a pro-inflammatory or pro-steatotic diet to accelerate liver injury and mimic the multifactorial nature of human NAFLD. In general, murine genetic models closely mimic monogenic forms of NAFLD, where mouse and human liver phenotypes have been described in sufficient detail to draw conclusions; however, many gaps exist in descriptions of the natural history of NAFLD associated with several of the monogenic diseases in mice or humans. Findings in both species indicate that there is more than one possible pathogenic route to NAFLD, meaning that study of several different human conditions and their models will be important to tease out common mechanisms of liver damage. A critical advantage of mouse models over humans is that the tremendously powerful technologies available to perturb genes conditionally or in an organ-specific way permits isolation of only some parts of a highly interconnected system. So, while alignment of humans and their murine models could be further refined, murine models are a highly valuable tool in the study of NAFLD.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

JM, RS, and MA were all involved in conceptualizing and writing the manuscript, in its critical review, and in approval of the final version.

# FUNDING

RS is supported by the Wellcome Trust (grant number WT098498).

non-alcoholic fatty liver disease. *Nat Commun* (2014) 5:4309. doi:10.1038/ ncomms5309

fatty liver disease. *J Mol Endocrinol* (2013) 51:167–79. doi:10.1530/JME-13-0072

**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 © 2016 Mann, Semple and Armstrong. 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) or licensor 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.*

# Genetic Rodent Models of Obesity-Associated Ovarian Dysfunction and Subfertility: Insights into Polycystic Ovary Syndrome

#### *Edited by:*

*Nicholas Michael Morton, The University of Edinburgh, UK*

#### *Reviewed by:*

*Eusebio Chiefari, University "Magna Græcia" of Catanzaro, Italy Philippa Saunders, The Edinburgh Cancer Research Centre, UK*

#### *\*Correspondence: Isabel Huang-Doran*

<EMAIL_ADDRESS>
#### *†Present address:*

*Isabel Huang-Doran, University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, UK*

#### *Specialty section:*

*This article was submitted to Genomic Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 01 April 2016 Accepted: 17 May 2016 Published: 07 June 2016*

#### *Citation:*

*Huang-Doran I and Franks S (2016) Genetic Rodent Models of Obesity-Associated Ovarian Dysfunction and Subfertility: Insights into Polycystic Ovary Syndrome. Front. Endocrinol. 7:53. doi: 10.3389/fendo.2016.00053*

#### *Isabel Huang-Doran\*† and Stephen Franks*

*Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital, London, UK*

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy affecting women and a leading cause of female infertility worldwide. Defined clinically by the presence of hyperandrogenemia and oligomenorrhoea, PCOS represents a state of hormonal dysregulation, disrupted ovarian follicle dynamics, and subsequent oligo- or anovulation. The syndrome's prevalence is attributed, at least partly, to a well-established association with obesity and insulin resistance (IR). Indeed, the presence of severe PCOS in human genetic obesity and IR syndromes supports a causal role for IR in the pathogenesis of PCOS. However, the molecular mechanisms underlying this causality, as well as the important role of hyperandrogenemia, remain poorly elucidated. As such, treatment of PCOS is necessarily empirical, focusing on symptom alleviation. The generation of knockout and transgenic rodent models of obesity and IR offers a promising platform in which to address mechanistic questions about reproductive dysfunction in the context of metabolic disease. Similarly, the impact of primary perturbations in rodent gonadotrophin or androgen signaling has been interrogated. However, the insights gained from such models have been limited by the relatively poor fidelity of rodent models to human PCOS. In this mini review, we evaluate the ovarian phenotypes associated with rodent models of obesity and IR, including the extent of endocrine disturbance, ovarian dysmorphology, and subfertility. We compare them to both human PCOS and other animal models of the syndrome (genetic and hormonal), explore reasons for their discordance, and consider the new opportunities that are emerging to better understand and treat this important condition.

#### Keywords: androgen, fertility, insulin resistance, mouse models, obesity, PCOS

**Abbreviations:** FSH, follicle-stimulating hormone; GnRH, gonadotrophin-releasing hormone; IR, insulin resistance; LH, luteinizing hormone; PCOS, polycystic ovary syndrome; T2DM, type 2 diabetes mellitus.

# INTRODUCTION