Our data showed PC/PE among total PLs increased in Ob/Ob, but on the other hand, decreased in MCD livers. Thus, PC/PE ratios are changed in a U-shape curve from genetic obesity to non-obese NAFLD/NASH [102]. In addition to PC/PE, ratios among other PLs were also altered with fatty liver. A weak decrease in PE/PI was observed in Ob/Ob livers (Figure 7A). A decrease in PE/PI was observed in HFD-fed WT mice (Figure 7B). A weak decrease of PC/PS and PC/PI ratios was observed in MCD-induced NAFLD (Figure 7C). Such changes during NAFLD can support the changes in electrostatics of PL bilayers and the existence of asymmetric lipid membranes due to charged anionic PLs, such as PS and PE relative to PC [112]. This may correlate with the least extent of hepatic inflammatory status in Ob/Ob mice as compared with HFD- and MCD-fed mice [39–41]. in MCD livers. Thus, PC/PE ratios are changed in a U-shape curve from genetic obesity to non-obese NAFLD/NASH [102]. In addition to PC/PE, ratios among other PLs were also altered with fatty liver. A weak decrease in PE/PI was observed in Ob/Ob livers (Figure 7A). A decrease in PE/PI was observed in HFD-fed WT mice (Figure 7B). A weak decrease of PC/PS and PC/PI ratios was observed in MCD-induced NAFLD (Figure 7C). Such changes during NAFLD can support the changes in electrostatics of PL bilayers and the existence of asymmetric lipid membranes due to charged anionic PLs, such as PS and PE relative to PC [112]. This may correlate with the least extent of hepatic

Our data showed PC/PE among total PLs increased in Ob/Ob, but on the other hand, decreased

iPLA2β deficiency did not alter PC/PE ratio in Ob/Ob (Figure 7A) and MCD-fed mice (Figure 7C). This deficiency however reversed the suppression of PC/PE and PE/PI in HFD-fed mice (Figure 7B). Since marked exacerbation of cholesterol metabolism is reported in Ob/Ob [113] and diabetic mice [114], the elevation of CEs caused a decrease of PC/CE and PE/CE in all three NAFLD models. iPLA2β deficiency reversed the suppression of PC/CE and PE/CE ratios in Ob/Ob and HFD-fed mice, but not in MCD-fed mice (Figure 7A–C). This may suggest that the remodeling with a shuttling of PUFA and MUFA could occur between PC, PE, and CEs via acylation and transacylation in choline/methionine-rich Ob/Ob and HFD livers [115]. Taken together, we have demonstrated a difference between genetic and diet (HFD and MCD) NAFLD regarding the ratios among PL subclasses, a contribution of PS and PI relative to PC and PE, as well as CE metabolism. inflammatory status in Ob/Ob mice as compared with HFD- and MCD-fed mice [39–41]. iPLA2β deficiency did not alter PC/PE ratio in Ob/Ob (Figure 7A) and MCD-fed mice (Figure 7C). This deficiency however reversed the suppression of PC/PE and PE/PI in HFD-fed mice (Figure 7B). Since marked exacerbation of cholesterol metabolism is reported in Ob/Ob [113] and diabetic mice [114], the elevation of CEs caused a decrease of PC/CE and PE/CE in all three NAFLD models. iPLA2β deficiency reversed the suppression of PC/CE and PE/CE ratios in Ob/Ob and HFD-fed mice, but not in MCD-fed mice (Figure 7A–C). This may suggest that the remodeling with a shuttling of PUFA and MUFA could occur between PC, PE, and CEs via acylation and transacylation in choline/methionine-rich Ob/Ob and HFD livers [115]. Taken together, we have demonstrated a difference between genetic and diet (HFD and MCD) NAFLD regarding the ratios among PL subclasses, a contribution of PS and PI relative to PC and PE, as well as CE metabolism.

#### **11. iPLA2**β **and De Novo Lipogenesis Gene Expression in Livers of Mice in 3 NAFLD Models 11. iPLA2β and** *de novo* **Lipogenesis Gene Expression in Livers of Mice in 3 NAFLD Models**

Associated with hepatic steatosis protection in obese but in non-obese NAFLD models [39–41], we further compared expression of iPLA2β protein and de novo lipogenesis in livers of Ob/Ob, HFDand MCD-fed WT mice. Rather than an increase, a slight decrease in iPLA2β protein expression was observed in fatty livers of obese models (Figure 8A,B) and a strong decrease in MCD model (Figure 8C). iPLA2β mRNA expression was not markedly altered (not shown) [39–41]. These data imply that iPLA2β protein may be subjected to degradation at post translational levels during NASH. It is shown that iPLA2 expression is decreased in rat cirrhotic livers [116], and may support iPLA2β as a target for degradation during severe liver injury. Currently, no published data on iPLA2β or PLA2G6 expression in livers of NAFLD/NASH patients are available. We could not correlate iPLA2β protein expression observed in our results with human data. Associated with hepatic steatosis protection in obese but in non-obese NAFLD models [39–41], we further compared expression of iPLA2β protein and *de novo* lipogenesis in livers of Ob/Ob, HFDand MCD-fed WT mice. Rather than an increase, a slight decrease in iPLA2β protein expression was observed in fatty livers of obese models (Figure 8A,B) and a strong decrease in MCD model (Figure 8C). iPLA2β mRNA expression was not markedly altered (not shown) [39–41]. These data imply that iPLA2β protein may be subjected to degradation at post translational levels during NASH. It is shown that iPLA2 expression is decreased in rat cirrhotic livers [116], and may support iPLA2β as a target for degradation during severe liver injury. Currently, no published data on iPLA2β or PLA2G6 expression in livers of NAFLD/NASH patients are available. We could not correlate iPLA2β protein expression observed in our results with human data.

**Figure 8.** Expression of iPLA2β protein and *de novo* lipogenesis mRNA in livers of WT, Ob/Ob mice, HFD-, and MCD-fed mice. Ob/Ob mice, HFD-, and MCD-fed mice are described in Figures 1, 3 and 5, respectively. Expression of (**A**) iPLA2β protein, (**B**) HFD-fed mice, and (**C**) MCD-fed mice. Data are mean ± SEM, N = 5–7 for PCR data; #, *p* < 0.05; \*, *p* < 0.05 between indicated groups. **Figure 8.** Expression of iPLA2β protein and de novo lipogenesis mRNA in livers of WT, Ob/Ob mice, HFD-, and MCD-fed mice. Ob/Ob mice, HFD-, and MCD-fed mice are described in Figures 1, 3 and 5, respectively. Expression of (**A**) iPLA2β protein, (**B**) HFD-fed mice, and (**C**) MCD-fed mice. Data are mean ± SEM, N = 5–7 for PCR data; #, *p* < 0.05; §, *p* < 0.05, KO *versus* KO/MCD; \*, *p* < 0.05 between indicated groups.

With obesity and fatty liver, livers of Ob/Ob and HFD-fed mice showed marked elevation of de novo lipogenesis genes including fatty acid synthase (FAS) and transcription factor SREBP1c (Figure 8A,B). It was shown that the transcription of iPLA2β is regulated by SREBP-1 [117], consistently attenuated expression of FAS and SREBP1c was observed in livers of iPLA2β-deficient obese mice. This attenuation was in indeed correlated with hepatic steatosis protection. On the other hand, MCD feeding of WT mice caused suppressed expression of these genes, which was not altered by iPLA2β deficiency (Figure 8C). This was associated with no steatosis protection by iPLA2β deficiency in this non-obese model. attenuated expression of FAS and SREBP1c was observed in livers of iPLA2β-deficient obese mice. This attenuation was in indeed correlated with hepatic steatosis protection. On the other hand, MCD feeding of WT mice caused suppressed expression of these genes, which was not altered by iPLA2β deficiency (Figure 8C). This was associated with no steatosis protection by iPLA2β deficiency in this non-obese model. **12. Summarized PL Characteristics in Ob/Ob, HFD-, and MCD-Fed Mice and Effects of iPLA2β**

*Biomolecules* **2020**, *10*, x FOR PEER REVIEW 13 of 26

8A,B). It was shown that the transcription of iPLA2β is regulated by SREBP-1 [117], consistently

With obesity and fatty liver, livers of Ob/Ob and HFD-fed mice showed marked elevation of *de*

#### **12. Summarized PL Characteristics in Ob**/**Ob, HFD-, and MCD-Fed Mice and E**ff**ects of iPLA2**β **Deficiency Deficiency**  Because PC and PE are the two major zwitterionic PLs in cells and their metabolism has been a

Because PC and PE are the two major zwitterionic PLs in cells and their metabolism has been a focus as a key mechanistic base for healthy liver, the alterations in PC and PE are critical in liver disease development and NAFLD [53,54,102,108]. iPLA2β deficiency protects obesity and NAFLD with an order of - Ob/Ob [39] › HFD [40] » MCD [41]; and the latter showed no steatosis protection (Figure 9). focus as a key mechanistic base for healthy liver, the alterations in PC and PE are critical in liver disease development and NAFLD [53,54,102,108]. iPLA2β deficiency protects obesity and NAFLD with an order of - Ob/Ob [39] › HFD [40] » MCD [41]; and the latter showed no steatosis protection (Figure 9).

**Figure 9.** Role of iPLA2β deficiency in obese and non-obese NAFLD/NASH mouse models. (**A**) Livers of genetic Ob/Ob and chronic HFD-fed mice exhibited a defect in PL remodeling with suppressed contents of PUFA PLs. PC/PE ratio was increased in Ob/Ob mice while that of HFD-fed mice was decreased. iPLA2β inactivation replenished PLs associated with fatty liver protection. (**B**) Livers of MCD-fed mice exhibited a defect in PL remodeling with suppressed PUFA PLs as well as PC/PE ratio. iPLA2β inactivation in MCD-fed mice did not rescue this defect with no protection. We propose that **Figure 9.** Role of iPLA2β deficiency in obese and non-obese NAFLD/NASH mouse models. (**A**) Livers of genetic Ob/Ob and chronic HFD-fed mice exhibited a defect in PL remodeling with suppressed contents of PUFA PLs. PC/PE ratio was increased in Ob/Ob mice while that of HFD-fed mice was decreased. iPLA2β inactivation replenished PLs associated with fatty liver protection. (**B**) Livers of MCD-fed mice exhibited a defect in PL remodeling with suppressed PUFA PLs as well as PC/PE ratio. iPLA2β inactivation in MCD-fed mice did not rescue this defect with no protection. We propose that iPLA2β deficiency in specific cell types may lead to no protection in liver inflammation and liver fibrosis in HFD and MCD NAFLD model, respectively (marked in red).

Human NAFLD and NASH are associated with numerous changes in the lipid composition of the liver. A decrease of the total PC and a decrease of arachidonic acid (20:4n-6) in FFA, TGs, and PC are reported in both NAFLD and NASH [55]. The contents of eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3) are decreased in NASH livers. In another study, PUFA-PLs are decreased in NASH livers compared to normal livers, and liver CEs are increased in NAFLD and NASH livers compared to normal livers [118]. Thus, a defect in PL remodeling and increased CEs could be observed in livers of human NAFLD and NASH. It is reported that the activity of the desaturase FADS1 is decreased in NAFLD liver biopsies [119]. This decrease in desaturation of FFA would likely lead to a depletion of MUFA and PUFA lipids, particularly PLs in NAFLD/NASH. It is shown that the hepatic PC/PE ratio is decreased in human NASH livers [53]. In another study, this ratio is lower in simple steatosis and NASH patients compared with controls, but it was not different between SS and NASH [120]. PC was lower and PE higher in the liver of simple patients compared with controls, whereas in NASH patients, only PE was higher [120]. Thus, the decrease of hepatic PC/PE ratio is a key parameter for human NAFLD and progression to NASH.

Livers of C57b/S129J mice fed a high-fat/high-cholesterol diet show an increase of hepatic CEs while hepatic PC, PE, and PS contents are decreased in NAFLD and a further decrease in PC and PE are observed in NASH [121]. Not only MUFA-PLs, hepatic PUFA-PC, PUFA-PE and PUFA-PI are reported to be decreased in MCD-fed mice compared with chow-fed or HFD-fed mice [122]. Hepatic 16:0 CEs are also increased in MCD-fed mice compared to chow-fed mice.

Results on liver lipids in our studies overall are consistent with those reported in human [55,118–120] and mouse [121,122] NAFLD/NASH. In our studies, a decrease of PUFA-PLs and MUFA-PLs was observed in livers of Ob/Ob and HFD-fed mice (Figure 9A) and MCD-fed mice (Figure 9B). The elevation of CEs led to a decrease of PC/CE and PE/CE ratios in obese and non-obese lives. iPLA2β deficiency rescued not only the defect of liver PL remodeling, but also reversed the suppression of PC/CE and PE/CE in Ob/Ob and HFD-fed mice (Figure 9A). iPLA2β deficiency in MCD-fed mice did not alter these parameters (Figure 9B). In our models, iPLA2β deficiency did not interfere with liver sphingolipids. Our work suggests the functions of iPLA2β on the hepatocyte PL remodeling in obese NAFLD models. Interestingly, hepatic PC/PE ratio is increased in Ob/Ob mice but decreased in HFD- and MCD-fed mice (Figure 9). Thus, this ratio is a marker in discriminating genetic *versus* diet-induced NASH, which is in a similar manner to human NASH [53,120]. iPLA2β deficiency reversed PC/PE ratio in obese but not non-obese model, rendering this ratio as a marker for phenotypic changes in NASH.

Besides PC and PE, our current data present an additional evidence for changes in other PLs including anionic PS and PI in Ob/Ob mice (Figure 9A). iPLA2β deficiency elicited full protection against fatty liver, obesity, and elevation of liver enzymes in Ob/Ob and HFD-fed mice [39,40]. In these obese models, we propose that iPLA2β specifically hydrolyzes PUFA-PLs and MUFA-PLs for generation of FAs subsequently utilized for TG and CE syntheses for hepatic steatosis. This process may be coordinated with other PLA2 enzymes. By this way, PUFA-PL and MUFA-PL contents in Ob/Ob and HFD livers are suppressed, and this suppression is rescued or replenished by iPLA2β deficiency. In line with this, administration of n-3 essential FAs [123] and PUFAs [124] have been shown to ameliorate hepatic steatosis in obese mice likely by increasing membrane fluidity [125]. Unlike Ob/Ob mice [39], iPLA2β deficiency during HFD feeding did not protect mice from liver inflammation [40]. Consistently, PLs, such as PC, are shown to elicit protection of hepatic steatosis in NAFLD without attenuating liver inflammation in NASH [54]. Alternatively, iPLA2β deletion by birth under the background of leptin deficiency may render a complete protection [39]; possibly due to adaptation in different cell types upon gene deletions throughout the mouse lifetime. Chronic HFD feeding on the other hand would represent an external stress to mice [40]. As iPLA2β KO mice were global deletion, we therefore proposed that iPLA2β deletion may affect specific cell types, such as hepatocytes, immune cells, and adipocytes, differently in response to HFD feeding (Figure 9A).

Due to the lack of choline and methionine in the diet, MCD feeding limits PC synthesis thus resulting in a defect of PL remodeling (Figure 9B). iPLA2β deficiency did not protect mice from MCD-induced fatty liver, but attenuated elevation of liver enzymes (Figure 9B). This attenuation could be due to an inhibition of the uptake of FA as determined by Cd36 expression and FA contents, as well as an inhibition of HDL reverse transport as determined by SR-B1 expression and CE contents [41]. The latter may indicate an involvement of iPLA2β in cholesterol esterification to CEs [114,115]. Despite attenuation of liver enzymes, iPLA2β inactivation during MCD however showed an increase of α-smooth muscle actin and vimentin expression [41]. Such increased liver fibrosis could be due to iPLA2β inactivation in specific cell types such as macrophages or hepatocytes associated with stress induced by MCD feeding (Figure 9B).

Taken together, our results highlight the significance of cross-talk between the metabolism of PLs and neutral lipids, i.e., CEs [115,126] and TGs in lipid droplets [43,127], which can be modulated by iPLA2β deficiency. In the latter case, iPLA2β may co-function with other PLA2s such as group IVA PLA2 in the shuttling of FAs toward TG synthesis [127]. iPLA2β inactivation was effective in attenuating obese (Ob/Ob and chronic-HFD) NAFLD indicating specific involvement of iPLA2β in obesity pathogenesis. As iPLA2β inactivation was ineffective to treat non-obese MCD NAFLD, this indicates that choline and methionine in hepatic PC and PL synthesis and metabolism were necessary for protection in obese models. Our data also emphasize the contributions and involvement of hepatic PL, TG, and CE metabolism in the development of NAFLD.

#### **13. Perspectives**

#### *13.1. Consideration of Cell-Type Specificity of iPLA2*β

The phenotypes of iPLA2β-null mice have been recently reviewed [92,93]. On one hand, iPLA2β is detrimental in mediating ER stress and cell death of pancreatic β-cells. On the other hand, iPLA2β plays a homeostatic role and the loss of iPLA2β in mice leads to ageing-related diseases, such as male infertility, bone-density loss, and neurological disorders. Accordingly, PLA2G6 mutations lead to the pathogenesis of infantile neuroaxonal dystrophy and PARK14-linked Parkinson's disease [92,93]. These data have highlighted various and often opposing functions of iPLA2β in a cell-type specific manner.

In our investigations of obese and non-obese NAFLD/NASH, global deletion iPLA2β-null mice were used. Thus, we could not identify whether the observed effects of iPLA2β inactivation were due to altered functions in adipocytes [99], immune cells such as macrophages [128,129], and Kupffer cells [130], as well as hepatocytes as shown by our work [39–41]. Concurrently, we have also reported that global deletion of iPLA2β was able to sensitize hepatocellular damage induced by concanavalin A [131] or during ageing [132]. It is thought that such epithelial damage caused by iPLA2β deficiency may secrete mediators that in turn activate inflammatory macrophages leading to sensitized injury. The observed sensitization of liver injury [131,132] could therefore be the combined effects of iPLA2β deficiency in hepatocytes and macrophages.

The effects of iPLA2β inactivation in macrophages have been reported [128,129]. On one hand, macrophages from iPLA2β-null mice exhibited suppressed pro-inflammatory M1 response [128,130] and showed enhanced IL-4-induced M2 polarization in vitro [128]. Consistently, iPLA2β-null mice treated with anti-CD95 antibody primed Kupffer cells for attenuated release of TNF-α but enhanced release of interleukin-6 in vitro [130]. iPLA2β-null mice showed the inability to phagocytose infected parasites in vivo [133]; thus iPLA2βdeficiency could lead to a defect in innate immunity. As iPLA2β-null mice showed propensity for increased liver inflammation and fibrosis during HFD and MCD NAFLD (Figure 9), this could be due to the ability of iPLA2β-deficient macrophages to differentially regulate M1 and M2 cytokines during NAFLD.

Our work has demonstrated iPLA2β activity in the hepatocytes by a decrease of products lysoPL and an accumulation of substrates PC and PE observed in livers of iPLA2β-null mice fed with chow [39,40]. Such accumulation could therefore lead to replenishment of PLs and steatosis protection in obese Ob/Ob and HFD NAFLD (Figure 9A). On the other hand, the alterations of hepatocellular PL composition and a decrease of PC/PE in chow-fed iPLA2β-null mice may render altered PL membranes leading to susceptibility for previously observed liver injury [131,132]. Further studies are warranted

to determine whether iPLA2β inactivation specifically in hepatocytes could affect hepatic steatosis and inflammation/fibrosis in HFD (Figure 9A) and MCD (Figure 9B) models.

While knockdown of iPLA2β inhibits hormone-induced differentiation of adipocytes in vitro [99], iPLA2β inactivation in adipocytes may inhibit adipocyte expansion, thus attenuating adipogenesis and obesity observed in our obese NAFLD models [39,40]. It has recently been shown that protection of hepatic steatosis by an ablation of adipocyte PLA2 is mediated by adipocyte hormone leptin [134]. By unknown mechanisms, iPLA2β inactivation may lead to an increase secretion of leptin, and may thus elicit protection in a similar way as adipocyte PLA2. Accordingly, leptin has biological activity in depleting liver TG [135] as well as activating Kupffer cells and thereby altering hepatic lipid metabolism [136]. To figure out the contribution of adipocytes, macrophages, and hepatocytes on the NAFLD/NASH pathogenesis, the generation of tissue-specific iPLA2β-deficient mice is therefore warranted. These results will help us understand that iPLA2β deficiency in which cell type is responsible for steatosis protection in HFD NAFLD and for increased liver fibrosis in MCD NAFLD. Results from tissue-specific iPLA2β KO mice will also help with the designs of iPLA2β inhibitors and formulations for specific-tissue delivery for effective treatment of obese and non-obese NAFLD.

## *13.2. Use of PLs or iPLA2*β *Antagonists for Steatosis Protection in Obese Versus Non-Obese NAFLD*

Because there are no approved drugs for treatment of NAFLD/NASH, our research results may provide some insights for further development toward NAFLD treatment. Here, our data support the idea for repletion of PL loss by use of PLs themselves or use of iPLA2β inhibitors for treatment of obese NAFLD. Some iPLA2β inhibitors have been found to be effective for treatment of diabetes at least in experimental animals [137]. Potent and selective inhibitors of iPLA2β have been developed [138]. Pending the results on the phenotypes of hepatocyte-, macrophage-, and adipose-specific iPLA2β KO mice in obese NAFLD models, investigators may design iPLA2β inhibitors in combination with specific delivery to hepatocytes [139], macrophages [140], or adipocytes [141] for better treatment of the common disease obese NAFLD.

For non-obese MCD NAFLD, iPLA2β inhibitors may be found to be partially effective in attenuating liver enzymes but not hepatic steatosis (Figure 9B). Choline supplementation in patients under parenteral nutrition reverses hepatic steatosis [142], and the strategy for non-obese NAFLD treatment may involve the use of choline and methyl donors as co-treatment with iPLA2β inhibitors. Choline deficiency in humans has been shown to induce hepatic steatosis [143,144] causing liver dysfunctions [145]. Hence, supplementation of choline [142] and methyl donors [146] may be effective in attenuating hepatic steatosis under non-obese NAFLD/NASH. Further investigations in experimental animals are necessary to evaluate the long-term use of iPLA2β inhibitors alone for obese NAFLD and in combination with choline/methyl donor supplementation for non-obese NAFLD models. Nonetheless, the hierarchical mode of action by iPLA2β deficiency indicates that an iPLA2β inhibitor may be designed perhaps with tissue-specific delivery for therapeutic development to treat metabolic syndromes due to obese NAFLD, and may not be suitable for non-obese NASH.

#### **14. Conclusions**

We demonstrated a pivotal role of iPLA2βin the development of hepatic steatosis and inflammation in obese and non-obese NAFLD models. iPLA2β inactivation rescued the defect in PL remodeling and elicited steatosis protection in obese NAFLD models, but not in non-obese MCD model. While our study suggests the use of iPLA2β inhibitors for therapy of obese NAFLD due to genetics or chronic HFD intake, further investigations using tissue-specific iPLA2β-deficient mice are still warranted. Here, the usefulness of the lipidomics methodology is shown in deciphering the alterations in hepatic PL pools and ratios among three NAFLD models.

**Funding:** This research was funded by Deutsche Forschungsgemeinschaft (CH288/6-1 and CH288/6-2).

**Acknowledgments:** We thank John Turk who provided us with iPLA2β-null mice, and previous lab members who were involved in this project. We thank Wolfgang Stremmel and Uta Merle for research insights.

**Conflicts of Interest:** Authors declare no conflict of interest.

### **Abbreviations**

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Metabolic E**ff**ects of Selective Deletion of Group VIA Phospholipase A2 from Macrophages or Pancreatic Islet Beta-Cells**

**John Turk 1,\* ,**† **, Haowei Song 1,2,**† **, Mary Wohltmann 1,**† **, Cheryl Frankfater 1 , Xiaoyong Lei 3 and Sasanka Ramanadham 3,\***

Received: 30 August 2020; Accepted: 13 October 2020; Published: 17 October 2020

**Abstract:** To examine the role of group VIA phospholipase A2 (iPLA2β) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2β deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2β-KO), or in insulin-secreting β-cells (β-Cell-iPLA2β-KO), respectively. MØ-iPLA2β-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2βcontrol mice after consuming a high-fat diet (HFD). MØ-iPLA2β-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2β-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, β-cell-iPLA2β-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. β-Cell-iPLA2β-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. β-Cell-iPLA2β-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2β participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, β-cell iPLA2β plays a role in GSIS and also appears to confer some protection against deterioration in β-cell functions induced by a HFD.

**Keywords:** pancreatic islets; β-cells; insulin secretion; glucose tolerance; insulin resistance; group VIA phospholipase A2

#### **1. Introduction**

Glycerophospholipids are the most abundant molecular components of biological membrane bilayers and are both critical determinants of membrane structure and the source of signaling molecules produced from their hydrolysis by phospholipase enzymes. Glycerophospholipids consist of a glycerol backbone with a phosphate ester in the *sn*-3 position that may form a phosphodiester linkage to a polar head-group, such as choline, ethanolamine, serine, inositol, or glycerol, inter alia. A fatty acid is esterified to the glycerol backbone in the *sn*-2 position of phospholipids, and in the *sn*-1 position there is an ester, ether, or vinyl ether linkage to a fatty acid, fatty alcohol, or fatty aldehyde residue, respectively. Phospholipase A2 (PLA2) enzymes hydrolyze the phospholipid *sn*-2 ester bond to yield a free fatty acid and a 2-lysophospholipid as products [1,2]. The PLA2 superfamily consists of at least 16 groups of structurally and functionally diverse enzymes that include secreted (sPLA2), cytosolic (cPLA2), calcium-independent (iPLA2), lipoprotein-associated (Lp-PLA2), and adipose-PLA2 (AdPLA). These enzymes play central roles in cellular lipid metabolism and signaling [1].

The enzyme that is now designated as Group VIA PLA2 was the first recognized mammalian member of the Ca2+-independent PLA2 enzymes [3–5], and its abbreviated designation is iPLA2β [6,7]. Akin to the plant lipase patatin, iPLA2β contains a GXSXG lipase consensus sequence in the enzyme catalytic center, in which the central Ser is a component of a Ser-Asp catalytic dyad [8]. Enzymes with patatin-like phospholipase domains comprise the PNPLA family, and the human genome expresses nine members of this family, of which iPLA2β is PNPLA9 [9,10]. The phenotypic properties of experimental mouse models with induced mutations in the genes that encode PNPLA family members and the clinical phenotypes of patients with corresponding mutations indicate that several of these lipases play important metabolic roles in mammalian lipid and energy homeostasis [9,10].

Global iPLA2β-null mice produced by homologous recombination exhibit several phenotypic abnormalities, including greatly impaired male fertility [11] and the development of a neurodegenerative condition [12] that is similar to the human genetic disease Infantile Neuroaxonal Dystrophy (INAD), which arises from mutations in the Group VIA PLA2 gene [13,14]. Consistent with previous evidence that iPLA2β participates in signaling events leading to glucose-stimulated insulin secretion (GSIS) from pancreatic islet β-cells [15–22], islets isolated from male [23] or female [24] iPLA2β-null mice exhibit impaired GSIS ex vivo, and male iPLA2β-null mice exhibit impaired glucose tolerance in vivo [23]. In contrast, female iPLA2β-null mice exhibit normal glucose tolerance in the unstressed state [24] but develop a more severe glucose intolerance than wild-type littermates after exposure to the β-cell toxin streptozotocin or after the introduction of a high-fat diet (HFD) [24]. Surprisingly, female iPLA2β-null mice experienced less deterioration in insulin sensitivity than did wild-type littermates after being introduced to a HFD, although pancreatic islets isolated from HFD-fed female iPLA2β-null mice exhibited a much more severe impairment of GSIS than did islets isolated from HFD-fed wild-type littermates [24].

This discordance of the effects of a HFD on insulin secretion and insulin sensitivity in global iPLA2β-null mice suggests that iPLA2β plays distinct roles in the molecular mechanisms underlying insulin secretion and insulin action and in the impact of a HFD on these processes. Evidence indicates that iPLA2β amplifies glucose-induced Ca2+ entry into β-cells [23], suggesting that this is one component of the mechanism(s) through which iPLA2β participates in signaling events underlying GSIS, and iPLA2β also participates in the repair of β-cell mitochondrial membranes that are oxidized upon exposure to high concentrations of palmitic acid [25]. This might represent a mechanism whereby iPLA2β mitigates β-cell injury in HFD-fed mice and accounts for the fact that loss of iPLA2β results in a greater impairment of insulin secretion in HFD-fed iPLA2β-null mice compared to HFD-fed wild-type littermates [24].

Macrophages and their precursor monocytes also express iPLA2β [26–29], and its expression level affects the macrophage phenotype [30]. Migration of monocytes into extravascular sites, including adipose tissue, and their differentiation into macrophages that elaborate cytokines, including TNFα and IL-6, which impair insulin sensitivity, are thought to represent a critical series of events in the development of diet-induced insulin resistance in diabetes and obesity and to involve tissue elaboration of the cytokine Monocyte Chemoattractant-1 (MCP-1) and its interaction with the monocyte MCP-1 receptor CCR2 [31–36]. Genetic and pharmacologic evidence indicates that the monocyte chemotactic response to MCP-1 requires the action of iPLA2β [37–39] to generate the lipid mediator 2-lysophosphatidic acid (LPA) [29,37,39]. These observations suggest the possibility that the relative insensitivity of iPLA2β-null mice to high-fat diet (HFD)-induced insulin resistance might reflect the failure of iPLA2β-null monocytes to migrate into adipose tissue and other extravascular sites in response to HFD-induced tissue elaboration of MCP-1.

A plausible hypothesis is thus that the net metabolic effects of global deletion of iPLA2β might reflect opposing effects on β-cells and monocyte-macrophages. A loss of iPLA2β in β-cells would result in impaired insulin secretion and increased sensitivity to lipid-induced injury, but loss of iPLA2β in macrophages might provide relative protection against the HFD-induced deterioration of insulin sensitivity because of an impaired migration of monocytes into extravascular tissues, differentiation into macrophages, and elaboration of cytokines that result in insulin resistance. To test this hypothesis, we generated mice with floxed-iPLA2β alleles and mated them with mice that express Cre recombinase under control of LysM or RIP2 promoters to produce mice with selective iPLA2β deficiency in macrophages (MØ-iPLA2β-KO) or insulin-secreting β-cells (β-cell-iPLA2β-KO), respectively. The metabolic phenotypes of these mice were then characterized and are described in this report.

#### **2. Materials and Methods**

#### *2.1. Materials*

Enhanced chemiluminescence reagents were obtained from Amersham Biosciences (Piscataway, NJ, USA); SDS-PAGE supplies from Bio-Rad (Richmond, CA, USA); ATP, common reagents, and salts from Sigma (St. Louis, MO, USA); culture media, penicillin, streptomycin, Hanks' balanced salt solution, L-glutamine, agarose, and RT-PCR reagents from Invitrogen (Carlsbad, CA, USA); fetal bovine serum from Hyclone (Logan, UT); Pentex bovine serum albumin (BSA, fatty acid-free, fraction V) from ICN Biomedical (Aurora, OH, USA); forskolin from Calbiochem (La Jolla, CA, USA). Krebs–Ringer bicarbonate (KRB) buffer contained (in mM) 25 HEPES (pH 7.4), 115 NaCl, 24 NaHCO3, 5 KCl, 1 MgCl2, and 2.5 CaCl2.

#### *2.2. Preparation of Mice with Selective Deletion of iPLA2*β *from Restricted Cell Lineages*

The Washington University Animal Studies Committee approved all animal studies. Mice with floxed-iPLA2β alleles were prepared and mated with mice expressing Cre recombinase under control of cell type-restricted promoters to generate conditionally iPLA2β-deficient mice. European Conditional Mouse Mutagenesis (EUCOMM) embryonic stem (ES) cells with an iPLA2β-targeting construct incorporated by homologous recombination [40] were purchased (Figure 1A). In this construct, LoxP sites L2 and L3 were recognized by Cre recombinase [41,42] flank critical iPLA2β gene exons 6–8 (Figure 1A). Removing this "floxed" segment results in a truncated mRNA species eliminated by nonsense mediated decay. The 50 fragment with the neo cassette is flanked by FRT (flippase recognition target) sites F1 and F2 (Figure 1A) that are recognized by FLP (flippase) recombinase [43,44]. Correct integration of 50 and 30 arms was confirmed by PCR (Figure 1B) using primer sets that recognize sequences external to the construct and within the neo cassette, respectively. Primer set Raf 5 and GR3 for the 30 arm yielded a 9.2 kb product (Figure 1A). Two clones (G05, G07) with normal karyotypes were injected into blastocysts that were then implanted into pseudo-pregnant females. Both yielded chimeric mice that transmitted the targeted allele in the germ line to yield heterozygotes for wild-type (WT) and EUCOMM iPLA2β alleles [45], as verified by PCR with primers within the neo cassette and the intron between L2 and exon 6, respectively, that yield a 795 bp product (Figure 1C).

(Figure 1G and H).

recombinase (Figure 1F) and produce no iPLA2β mRNA in macrophages but do so in other tissues

**Figure 1.** Cre-Lox Preparation of Mutant Mice with Tissue-Restricted Deletion of iPLA2β. (**A**) is a schematic illustration of the structure of the gene-targeting construct that was incorporated by homologous recombination into mouse embryonic stem (ES) cells from European Conditional Mouse Mutagenesis (EUCOMM). (**B**) demonstrates correct integration of the 5' and 3' arms of the construct in the ES cell lines by long-range PCR using primer sets that recognize sequences external to the construct and within the neo cassette, respectively, to yield a 9.2 kb product. (**C**) illustrates that injection of the ES cells into blastocysts that were then implanted into pseudo-pregnant female mice resulted in production of chimeric mice with germline transmission of the targeted gene to yield heterozygotes for the wild-type (WT) and EUCOMM iPLA2β alleles. (**D**) illustrates that mating of these heterozygotes with FLP deleter mice resulted in removal of the region between F1 and F2 sites in the targeting construct that contained the neo cassette to yield an iPLA2β allele that contained Lox2 **Figure 1.** Cre-Lox Preparation of Mutant Mice with Tissue-Restricted Deletion of iPLA2β. (**A**) is a schematic illustration of the structure of the gene-targeting construct that was incorporated by homologous recombination into mouse embryonic stem (ES) cells from European Conditional Mouse Mutagenesis (EUCOMM). (**B**) demonstrates correct integration of the 50 and 30 arms of the construct in the ES cell lines by long-range PCR using primer sets that recognize sequences external to the construct and within the neo cassette, respectively, to yield a 9.2 kb product. (**C**) illustrates that injection of the ES cells into blastocysts that were then implanted into pseudo-pregnant female mice resulted in production of chimeric mice with germline transmission of the targeted gene to yield heterozygotes for the wild-type (WT) and EUCOMM iPLA2β alleles. (**D**) illustrates that mating of these heterozygotes with FLP deleter mice resulted in removal of the region between F1 and F2 sites in the targeting construct that contained the neo cassette to yield an iPLA2β allele that contained Lox2 and Lox3 sites flanking iPLA2β exons 6–8 and that this allele is distinguishable from the WT allele upon PCR analyses. (**E**) is a Southern blot of gene fragments after Bam H1 endonuclease digestion and illustrates production of fragments characteristic of the WT and conditional alleles that establish the genotype of individual mice. WT and conditional iPLA2β heterozygotes were mated to yield conditional allele homozygotes, heterozygotes, and WT allele homozygotes in a 1:2:1 ratio. Those mice were then mated with mice that express Cre recombinase under control of LysM or RIP2 promoters to drive Cre expression selectively in macrophages or pancreatic islet β-cells, respectively. (**F**) illustrates that with conditional allele homozygotes these matings result in selective iPLA2β deletion in only the targeted cell line and not in other tissues. (**G**,**H**) illustrate that iPLA2β mRNA is not produced in the Cre-expressing cell type in mice that are iPLA2β conditional allele homozygotes but that iPLA2β mRNA is produced by non-targeted tissues.

These mice were mated with FLP deleter mice to excise the F1-F2 region that contained the neo cassette [43–45] to yield a conditional iPLA2β allele in which loxP sites L2 and L3 flank exons 6–8 (Figure 1A). Progeny included heterozygotes for conditional and WT iPLA2β alleles, as verified by PCR genotyping with primers in introns between exon 5 and F2 and between L2 and exon 6, respectively, which yielded products of 546 and 364 bp for conditional and WT alleles, respectively (Figure 1D). Genotypes were confirmed by Southern blotting after digestion with restriction endonuclease Bam H1, which cleaves at sites B1, B2 (conditional allele), and B3 (WT allele) to yield fragments of 2544 and 2256 bp for WT and conditional alleles (Figure 1E), respectively, recognized by a probe to the 30 end of exon 4 and the following intron. Heterozygous (conditional/WT) mice were mated with mice that express Cre recombinase under control of RIP2 or LysM promoters to direct Cre expression in β-cells or macrophages [46–53], respectively. FLP-negative offspring were mated with Cre mice to produce mice homozygous for iPLA2β conditional alleles that express Cre recombinase (Figure 1F) and produce no iPLA2β mRNA in macrophages but do so in other tissues (Figure 1G,H).

Mice were housed in a specific pathogen-free barrier facility with unrestricted access to water and standard mouse chow containing 6% fat. For mice with a β-cell-specific inactivation of iPLA2β (β-iPLA2β-KO), RIP2-Cre mice (The Jackson Laboratory, number 003573) were crossed with mice carrying iPLA2β alleles with exons 6–8 flanked by loxP recombination sites (iPLA2β lox/lox) [46]. First, generation animals hemizygous for the RIP-Cre gene and bearing one "floxed" LPL allele (iPLA2β lox/wt Cre+) were crossed with iPLA2β lox/lox animals to generate β-cell iPLA2β-deficient (iPLA2β lox/lox Cre+) and β-cell iPLA2β wild-type (iPLA2β lox/lox Cre- ) littermates that were at least N5 in the C57BL/6 background with a conditional deletion of iPLA2β in β-cells. The following primers were used to document iPLA2β gene rearrangement: primer A, 50 -CCCAGCTCTGTGTCTTAGTATG-30 ; primer B, 50 -TTCTTGGCCCAATGGAGTG-30 . Amplification of WT DNA yields a product of 673 bp; amplification of non-rearranged floxed DNA allele yields a product of 855 bp, whereas the amplification of appropriately rearranged DNA will not show a band since the exon 5 is deleted. For mice with a myelomonocytic cell-specific inactivation of iPLA2β (MØ-iPLA2β-KO), mice with loxP-flanked iPLA2β alleles were mated with lysozyme M-Cre mice [51] and crossbred to yield iPLA2β knock-out in macrophage (MØ-iPLA2β-KO) mice that were at least N5 in the C57BL/6 background with conditional deletion of iPLA2βin the myelomonocytic lineage. Mice were genotyped using iPLA2β- and Cre-specific primer sets [54], weaned to chow providing 6% calories as fat, and subsequently fed a high-fat diet (HFD), as described below.

### *2.3. Analyses of iPLA2*β *mRNA in Mouse Tissues*

Northern blots of iPLA2β mRNA were performed as described in [11]. For RT-PCR, total RNA was isolated with an RNeasy kit (Qiagen Inc.). A SuperScript First Strand Synthesis System (Invitrogen) was used to synthesize cDNA in 20 µL reactions that contained DNase I-treated total RNA (2 µg). The cDNA product was treated (20 min, 37 ◦C) with RNase H (2 units, Invitrogen), and was heat inactivated (70 ◦C for 15 min). A reaction without reverse transcriptase was performed to verify the absence of genomic DNA. The PCR performed with the pair of primers 1 and 2 was designed to amplify a fragment that spans the neomycin cassette insertion site. The PCR performed with the pair of primers 3 and 2 was designed to amplify a fragment downstream from the neomycin cassette insertion site. The sequence of primer 1 is tgtgacgtggacagcactagc; that of primer 2 is ccccagagaaacgactatgga; that of primer 3 is tatgcgtggtgtgtacttccg.

#### *2.4. High-Fat Dietary Intervention Studies*

Mice were housed in a pathogen-free barrier facility with unrestricted access to water and standard mouse chow (Purina Mills Rodent Chow 5053) with a caloric content of 13.025% fat, 62.144% carbohydrate, and 24.651% protein. For dietary intervention studies, mice were fed standard chow until 8 weeks of age and thereafter were randomized into groups that were fed either standard chow or a HFD continuously until they reached the age of three or six months, respectively, as described in [55]. The HFD (Harlan Teklad catalog TD88137) had a caloric content of 42% fat, 42.7% carbohydrate, and 15.2% protein.

#### *2.5. Blood Glucose and Insulin Concentrations*

As described previously [56], blood samples were obtained from the lateral saphenous vein in heparinized capillary tubes, and glucose concentrations were measured in whole blood with a blood-glucose monitor (Becton Dickenson) or an Ascensia ELITE XL blood-glucose meter. Plasma was prepared from heparinized blood by centrifugation, and insulin levels were determined in aliquots (5 µL) with a rat insulin ELISA kit (Crystal Chem). Fasting blood samples were obtained after an overnight fast, and fed blood samples were obtained between 9:00 and 10:00 a.m.

#### *2.6. Glucose and Insulin Tolerance Tests*

As described in [23], intraperitoneal glucose tolerance tests (IPGTTs) were performed on mice that fasted overnight from which a baseline blood sample was obtained, followed by intraperitoneal injection of D-glucose (2 mg/g body weight) and collection of blood for measurement of glucose level after 30, 60, and 120 min. Insulin tolerance tests were performed in mice with free access to water and chow that received an intraperitoneal injection (0.75 U/kg body weight) of human regular insulin (Lilly, Indianapolis, IN, USA), followed by collection of blood after 30, 60, and 120 min for glucose level determinations [24,26].

#### *2.7. Area Under the Curve (AUC) Calculations for Glucose Tolerance Tests (GTTs)*

As described previously [23,24,57], the AUC for the GTT curves was calculated by the method of Sakaguchi et al. [58], where the blood glucose concentration at t = x min is designated G(x):

$$\text{AUC} = [0.25 \times \text{G(0)}] + [0.5 \times \text{G(30)}] + [0.75 \times \text{G(60)}] + [0.5 \times \text{G(120)}] \tag{1}$$

#### *2.8. Insulin Secretion In Vivo*

Mice were fasted overnight, and baseline blood samples were obtained from the saphenous vein, followed by intraperitoneal injection of D-glucose (3 mg/kg body weight), and a blood sample was obtained 30 min thereafter for the measurement of plasma insulin levels, as described in [57].

#### *2.9. Pancreatic Islet Isolation*

Islets were isolated from pancreata removed from mice by collagenase digestion after mincing, followed by Ficoll step density gradient separation, and manual selection under stereomicroscopic visualization to exclude contaminating tissues [46,59].

#### *2.10. Insulin Secretion Ex Vivo from Isolated Pancreatic Islets in Static Incubations*

Islets were rinsed with KRB medium containing 3 mM glucose and 0.1% bovine serum albumin and placed in silanized tubes (12 × 75 mm) in the same buffer, through which 95% air/5% CO2 was bubbled before the incubation. The tubes were capped and incubated (37 ◦C, 30 min) in a shaking water bath, as described in [24,46,59]. The buffer was then replaced with KRB medium containing 1, 11, or 20 mM glucose and 0.1% BSA without or with forskolin (2.5 µM), and the samples were incubated for 30 min. Insulin secreted into the medium was measured, as described in [46,57].

#### *2.11. Other Analytical Procedures*

Serum glucose was measured using reagents from Sigma, and serum insulin was measured by an enzyme-linked immunosorbent assay (Crystal Chem. Inc., Downer's Grove, IL, USA), as described in [46].

#### *2.12. Statistical Methods*

Results are presented as mean ± SEM. Data were evaluated by an unpaired, two-tailed Student's *t* test or by an analysis of variance with appropriate post-hoc tests. Significance levels are described in the figure legends.

#### **3. Results**

#### *3.1. Mouse Genotype Characterization*

As described in the experimental procedures and illustrated in Figure 1*,* mice homozygous for a floxed-iPLA2β allele were prepared and mated with mice that express Cre recombinase in a restricted set of tissues to produce offspring with conditional iPLA2β gene deletions. Such mice fail to express iPLA2β in tissues that express Cre because the floxed gene is excised by the action of the recombinase, but those mice do express iPLA2β in all other tissues. Two breeding lines of mice with a tissue-selective expression of Cre were used, one of which expresses Cre under control of the Rat Insulin Promoter (RIP) which is active in insulin-secreting pancreatic islet β-cells and in a limited number of other cells but not in the vast majority of cells [46–50]. When mated with mice homozygous for a floxed-iPLA2β allele, some progeny, which are identified by genotyping, fail to express iPLA2β in β-cells, and their genotype is designated β-cell-iPLA2 β-KO. The second breeding line expresses Cre under control of the Lysozyme-M (Lys) promoter that is active in myelomonocytic lineage cells, including monocyte/macrophages [51,52]. When mated with mice homozygous for a floxed-iPLA2β allele, some progeny, again identified by genotyping, fail to express iPLA2β in monocyte/macrophages (MØ), and their genotype is designated MØ-iPLA2β-KO. β-Cell-iPLA2β-KO mice are thus selectively deficient in iPLA2β in β-cells, and MØ-iPLA2β-KO mice are selectively deficient in iPLA2β in monocyte/macrophages. Mice homozygous for a floxed-iPLA2β allele that do not express Cre are designated "Floxed-iPLA2β" and serve as controls when examining the metabolic behavior of the conditional iPLA2β-KO mice.

#### *3.2. Glucose Tolerance Tests*

Glucose tolerance tests (GTTs) performed with female mice 6 months of age of various genotypes after consuming food from a regular diet (RD) or high-fat diet (HFD) are illustrated in Figure 2, in which the blood glucose concentration is plotted as a function of time after an intraperitoneal administration of glucose.

Figure 2A shows that for floxed-iPLA2β control mice, glucose tolerance deteriorates significantly in HFD-fed mice compared to RD-fed mice. This effect of diet was also observed in MØ-iPLA2β-KO mice, but the peak glucose concentration and the area under the curve (AUC) of the GTTs were both significantly lower for MØ-iPLA2 β-KO mice than for floxed-iPLA2β controls, suggesting that MØ-selective iPLA2β deficiency confers some protection against diet-induced glucose intolerance.

Figure 2B illustrates that GTTs performed withβ-cell-iPLA2 β-KO mice compared to floxed-iPLA2β controls. Again, there is HFD-induced deterioration in GTTs compared to RD-fed mice for the floxed-iPLA2β control mice. This dietary effect was also observed in β-cell-iPLA2β-KO mice. In contrast to MØ-iPLA2β-KO mice, the peak glucose concentration and the Area Under the Curve (AUC) of the GTT were both significantly higher in β-cell-iPLA2β-KO mice than in floxed-iPLA2β controls, suggesting that β-cell-selective iPLA2β deficiency exacerbates diet-induced glucose intolerance.

Similar effects of genotype and diet were observed in male mice 6 months of age, as illustrated in Figure 3, in which the AUC of the GTT is plotted for female (F) or male (M) mice fed a regular (R) or high-fat (HF) diet. Figure 3A shows that for a given diet, males exhibit higher GTT AUC values than females and that a HFD causes deterioration in glucose tolerance, as reflected by a higher AUC, compared to RD-fed mice. For both males and females, the diet-induced rise in GTT AUC was significantly lower for MØ-iPLA2β-KO mice than for floxed-iPLA2β controls. In contrast, the diet-induced rise in GTT was significantly higher for β-KO (RIP) mice than for floxed-iPLA2β controls for both males and females. *Biomolecules* **2020**, *10*, x FOR PEER REVIEW 8 of 24

**Figure 2.** Glucose tolerance tests for iPLA2β conditional knockout mice and floxed-iPLA2β controls. D-glucose (2 mg/g body weight) was administered by intraperitoneal injection to female (**A,B**) or male (**C**,**D**) floxed-iPLA2β control mice (circles), MØ- iPLA2β-KO mice (**A**,**C**, squares), or β-cell-iPLA2β-KO mice (**B**,**D**, squares) 6 months of age that had been fed a regular diet (open symbols) or high-fat diet (HFD, closed symbols) after the age of 8 weeks, and blood was collected at baseline and at 30, 60, and 120 min after glucose administration to measure blood glucose concentration. Values are displayed as means ± SEM (n = 6 to 24, as specified by condition in Table S1). An asterisk (\*) denotes *p* < 0.05 for comparisons between genotypes. The symbol x denotes *p* < 0.05 for the comparison between diets. **Figure 2.** Glucose tolerance tests for iPLA2β conditional knockout mice and floxed-iPLA2β controls. D-glucose (2 mg/g body weight) was administered by intraperitoneal injection to female (**A,B**) or male (**C**,**D**) floxed-iPLA2β control mice (circles), MØ- iPLA2β-KO mice (**A**,**C**, squares), or β-cell-iPLA2β-KO mice (**B**,**D**, squares) 6 months of age that had been fed a regular diet (open symbols) or high-fat diet (HFD, closed symbols) after the age of 8 weeks, and blood was collected at baseline and at 30, 60, and 120 min after glucose administration to measure blood glucose concentration. Values are displayed as means ± SEM (n = 6 to 24, as specified by condition in Table S1). An asterisk (\*) denotes *p* < 0.05 for comparisons between genotypes. The symbol x denotes *p* < 0.05 for the comparison between diets.

*Biomolecules* **2020**, *10*, x FOR PEER REVIEW 10 of 24

**Figure 3.** Areas under the curve for glucose tolerance tests for iPLA2β conditional knockout mice and floxed-iPLA2β controls. Glucose tolerance tests (GTTs) were performed as in Figure 2 for male (M) or female (F) MØ-iPLA2β-KO mice (**A**), β-cell-iPLA2β-KO mice (**B**), or floxed-iPLA2β control mice 6 months of age that had been fed a regular (R) or high-fat (HF) diet, and the Areas Under the Curves (AUCs) were calculated from the measured glucose concentration values by the trapezoidal method of Sakaguchi et al. [58]. Values are displayed as means ± SEM (n = 6 to 24, as specified by condition in Table S1). An asterisk (\*) denotes *p* < 0.05 for comparisons between genotypes. **Figure 3.** Areas under the curve for glucose tolerance tests for iPLA2β conditional knockout mice and floxed-iPLA2β controls. Glucose tolerance tests (GTTs) were performed as in Figure 2 for male (M) or female (F) MØ-iPLA2β-KO mice (**A**), β-cell-iPLA2β-KO mice (**B**), or floxed-iPLA2β control mice 6 months of age that had been fed a regular (R) or high-fat (HF) diet, and the Areas Under the Curves (AUCs) were calculated from the measured glucose concentration values by the trapezoidal method of Sakaguchi et al. [58]. Values are displayed as means ± SEM (n = 6 to 24, as specified by condition in Table S1). An asterisk (\*) denotes *p* < 0.05 for comparisons between genotypes.

*3.3. Ex Vivo Insulin Secretion from Isolated Pancreatic Islets*  The magnitude of GSIS from pancreatic islet β-cells has an important influence on glucose tolerance, and the secretory behavior of pancreatic islets isolated from RD-fed or HFD-fed mice of various genotypes was therefore examined ex vivo, as illustrated in Figure 4. Islets isolated from both male and female RD-fed floxed-iPLA2β control mice exhibited insulin secretion that increased with medium glucose concentration over a range from 1 to 20 mM, and this response was amplified in the presence of the adenylyl cyclase activator, forskolin (Figures 4A–D), as observed in previous studies Together, Figures 2 and 3 demonstrate that glucose tolerance is affected by diet, gender, and genotype, with a higher GTT AUC for males compared to females and for HFD-fed compared to RD-fed mice. Compared to flox mice, MØ-iPLA2β-KO mice exhibit a significantly lower HFD-diet induced deterioration of GTT than floxed-iPLA2β controls. In contrast, β-cell-iPLA2β-KO mice exhibit significantly poorer glucose tolerance, as reflected by a higher GTT AUC than floxed-iPLA2β controls after the introduction of an RD, and HFD-induced deterioration of glucose tolerance is greater for β-cell-iPLA2β-KO mice than for floxed-iPLA2β controls.

[22–24]. Insulin secretory responses from RD-fed MØ-iPLA2β-KO male or female mice were not statistically different from those for floxed-iPLA2β controls (Figures 4A,B). In contrast, insulin secretory responses from RD-fed β-cell-iPLA2β-KO male or female mice were significantly lower from those for floxed-iPLA2β controls (Figures 4C,D), and this was also observed in islets from HFDfed β-cell-iPLA2β-KO male and female mice (Figures 4D,E), which exhibited even lower insulin secretory responses relative to floxed-iPLA2β controls than did islets from RD-fed mice. These observations indicate that selective iPLA2β deficiency in β-cells results in impaired insulin secretion from pancreatic islets but that selective iPLA2β deficiency in MØ does not. The impaired islet insulin secretion of β-cell-iPLA2β-KO mice in Figure 4C–F is thus likely to contribute to the impaired glucose tolerance in these mice (Figures 2B,3B). Table S1 illustrates an effect of age on glucose tolerance. Metabolic abnormalities that have developed in mice aged 6 months were found to be nascent but attenuated in mice aged 3 months. For a given condition, GTT AUC is lower for mice aged 3 months than for mice aged 6 months. A HFD also induces deterioration in glucose tolerance for both female and male mice at an age of 3 months, although the effect is smaller than with mice aged 6 months. For female mice aged 3 months, the GTT AUC was significantly lower for MØ-iPLA2β-KO mice than for floxed-iPLA2β control mice after RD-consumption, and the GTT AUC was significantly higher for β-cell-iPLA2 β-KO mice than for floxed-iPLA2β mice after RD-consumption. No other comparisons were statistically significant for female or male mice aged 3 months, although there was a strong trend for higher GTT AUC for HFD-fed β-Cell-iPLA2β-KO mice than for floxed-iPLA2β controls and a trend for higher GTT AUC for RD-fed male β-cell-iPLA2β-KO mice than for floxed-iPLA2β controls. Weaker trends were observed for the

GTT AUC to be lower in HFD-fed female MØ-iPLA2β-KO mice than for floxed-iPLA2β mice and for the GTT AUC to be higher in HFD-fed male β-cell-iPLA2β-KO mice than for floxed-iPLA2β controls.

#### *3.3. Ex Vivo Insulin Secretion from Isolated Pancreatic Islets*

The magnitude of GSIS from pancreatic islet β-cells has an important influence on glucose tolerance, and the secretory behavior of pancreatic islets isolated from RD-fed or HFD-fed mice of various genotypes was therefore examined ex vivo, as illustrated in Figure 4. Islets isolated from both male and female RD-fed floxed-iPLA2β control mice exhibited insulin secretion that increased with medium glucose concentration over a range from 1 to 20 mM, and this response was amplified in the presence of the adenylyl cyclase activator, forskolin (Figure 4A–D), as observed in previous studies [22–24]. Insulin secretory responses from RD-fed MØ-iPLA2β-KO male or female mice were not statistically different from those for floxed-iPLA2β controls (Figure 4A,B). In contrast, insulin secretory responses from RD-fed β-cell-iPLA2β-KO male or female mice were significantly lower from those for floxed-iPLA2β controls (Figure 4C,D), and this was also observed in islets from HFD-fed β-cell-iPLA2β-KO male and female mice (Figure 4D,E), which exhibited even lower insulin secretory responses relative to floxed-iPLA2β controls than did islets from RD-fed mice. These observations indicate that selective iPLA2β deficiency in β-cells results in impaired insulin secretion from pancreatic islets but that selective iPLA2β deficiency in MØ does not. The impaired islet insulin secretion of β-cell-iPLA2β-KO mice in Figure 4C–F is thus likely to contribute to the impaired glucose tolerance in these mice (Figures 2B and 3B).

#### *3.4. In Vivo Insulin Secretion in Mice After Intraperitoneal Glucose Administration*

To determine whether insulin secretion in vivo would reflect the effects of genotype on insulin secretion ex vivo from isolated pancreatic islets, we determined the increment in blood insulin concentration that occurred 30 min after an intraperitoneal administration of glucose to mice of various genotypes, gender, and dietary history (Figure 5). Both RD-fed male and HFD-fed female floxed-iPLA2β control mice exhibited a significant increment in blood insulin concentrations after an intraperitoneal administration of glucose (Figure 5A,B and Figure S1). This was also true for MØ-iPLA2β-KO mice, and the magnitude of the rise in blood insulin level was similar and not significantly different when comparing MØ-iPLA2β-KO mice to floxed-iPLA2β controls. This is consistent with the observation that insulin secretion from pancreatic islets isolated from MØ-iPLA2β-KO mice is not impaired compared to floxed-iPLA2β controls (Figure 4).

In contrast, neither RD-fed female or HFD-fed male β-cell-iPLA2β-KO mice exhibited a significant increase in blood insulin levels 30 min after intraperitoneal glucose administration (Figure 5A,B and Figure S1). This is consistent with the impairment of ex vivo glucose-dependent insulin secretion that was observed with pancreatic islets isolated from β-cell-iPLA2β-KO mice relative to floxed-iPLA2β controls (Figure 4), suggesting that inadequate insulin secretion contributes to the substantially impaired glucose tolerance observed with β-cell-iPLA2β-KO mice (Figures 2 and 3).

#### *3.5. Insulin Tolerance Tests*

In addition to the magnitude of GSIS from pancreatic islets, the responsivity of peripheral tissues, including skeletal muscle, to insulin is an important determinant of glucose tolerance. To evaluate insulin responsivity of mice of various genders, genotypes, and dietary history, insulin tolerance tests (ITTs) were performed by measuring the blood glucose concentrations at various times after an intraperitoneal injection of a fixed dose of insulin, as illustrated in Figure 6.

*Biomolecules* **2020**, *10*, x FOR PEER REVIEW 11 of 24

**Figure 4.** Insulin secretion by pancreatic islets isolated from iPLA2β conditional knockout mice and floxed-iPLA2β controls. Insulin secretion was stimulated by D-glucose and forskolin from pancreatic islets isolated from male (**A**,**C**,**E**) or female (**B**,**D**,**F**) MØ-iPLA2β-KO mice (**A**,**B**, solid bars), β-celliPLA2β-KO mice (**C–F**, solid bars), or floxed-iPLA2β control mice **(A–F**, cross-hatched bars) 6 months of age that had been fed a RD (**A–D**) or a HFD (**E**,**F**). Incubations (30 islets per condition, 30 min, 37 °C) were performed in buffer containing 1, 11, or 20 mM D-glucose without or with 2.5 µM forskolin, and an aliquot of medium was then removed for measurement of insulin. Mean values ± SEM are displayed (n = 4, in triplicate). An asterisk (\*) denotes *p* < 0.05 for the comparison between genotypes. **Figure 4.** Insulin secretion by pancreatic islets isolated from iPLA2β conditional knockout mice and floxed-iPLA2β controls. Insulin secretion was stimulated by d-glucose and forskolin from pancreatic islets isolated from male (**A**,**C**,**E**) or female (**B**,**D**,**F**) MØ-iPLA2β-KO mice (**A**,**B**, solid bars), β-cell-iPLA2β-KO mice (**C–F**, solid bars), or floxed-iPLA2β control mice (**A**–**F**, cross-hatched bars) 6 months of age that had been fed a RD (**A–D**) or a HFD (**E**,**F**). Incubations (30 islets per condition, 30 min, 37 ◦C) were performed in buffer containing 1, 11, or 20 mM d-glucose without or with 2.5 µM forskolin, and an aliquot of medium was then removed for measurement of insulin. Mean values ± SEM are displayed (n = 4, in triplicate). An asterisk (\*) denotes *p* < 0.05 for the comparison between genotypes.

*3.4. In Vivo Insulin Secretion in Mice After Intraperitoneal Glucose Administration*

To determine whether insulin secretion in vivo would reflect the effects of genotype on insulin secretion ex vivo from isolated pancreatic islets, we determined the increment in blood insulin concentration that occurred 30 min after an intraperitoneal administration of glucose to mice of various genotypes, gender, and dietary history (Figure 5). Both RD-fed male and HFD-fed female floxed-iPLA2β control mice exhibited a significant increment in blood insulin concentrations after an intraperitoneal administration of glucose (Figures 5A,B and Figure S1). This was also true for MØiPLA2β-KO mice, and the magnitude of the rise in blood insulin level was similar and not significantly

observation that insulin secretion from pancreatic islets isolated from MØ-iPLA2β-KO mice is not

impaired compared to floxed-iPLA2β controls (Figure 4).

**Figure 5.** In vivo insulin secretion for iPLA2β conditional knockout mice and floxed-iPLA2β control mice fed a regular (RD) or high-fat diet (HFD). After an overnight fast, baseline blood samples were obtained from the saphenous vein of male (**A**) or female (**B**) MØ-iPLA2β-KO mice, β-cell-iPLA2β-KO mice, or floxed-iPLA2β control mice 6 months of age that had been fed a regular diet (RD, **A**) or highfat diet (HFD, **B**). D-glucose (3 mg/kg body weight) was administered by intraperitoneal injection, and a blood sample was obtained 30 min thereafter. The insulin contents of the baseline and 30 min samples were then measured by enzyme-linked immunosorbent assay, as described [23,24,57]. Displayed values represent mean ± SEM. An asterisk (\*) denotes *p* < 0.05 for the comparison between the time 0 and 30 min values (n = 5 to 28). **Figure 5.** In vivo insulin secretion for iPLA2β conditional knockout mice and floxed-iPLA2β control mice fed a regular (RD) or high-fat diet (HFD). After an overnight fast, baseline blood samples were obtained from the saphenous vein of male (**A**) or female (**B**) MØ-iPLA2β-KO mice, β-cell-iPLA2β-KO mice, or floxed-iPLA2β control mice 6 months of age that had been fed a regular diet (RD, **A**) or high-fat diet (HFD, **B**). d-glucose (3 mg/kg body weight) was administered by intraperitoneal injection, and a blood sample was obtained 30 min thereafter. The insulin contents of the baseline and 30 min samples were then measured by enzyme-linked immunosorbent assay, as described [23,24,57]. Displayed values represent mean ± SEM. An asterisk (\*) denotes *p* < 0.05 for the comparison between the time 0 and 30 min values (n = 5 to 28).

In contrast, neither RD-fed female or HFD-fed male β-cell-iPLA2β-KO mice exhibited a significant increase in blood insulin levels 30 min after intraperitoneal glucose administration (Figure 5A,B and Figure S1). This is consistent with the impairment of ex vivo glucose-dependent insulin secretion that was observed with pancreatic islets isolated from β-cell-iPLA2β-KO mice relative to floxed-iPLA2β controls (Figure 4), suggesting that inadequate insulin secretion contributes to the substantially impaired glucose tolerance observed with β-cell-iPLA2β-KO mice (Figures 2 and 3). For female mice aged 3 months either RD- or HFD-fed, the ITT curves for neither MØ-iPLA2β-KO nor β-cell-iPLA2β-KO conditional knockout mice were statistically distinguishable from those for floxed-iPLA2β control mice. This was also true for 3-month-old male MØ-iPLA2β-KO conditional knockout mice and for HFD-fed 3-month-old male β-cell-iPLA2β-KO conditional knockout mice compared to floxed-iPLA2β control mice. For 3-month-old male β-cell-iPLA2β-KO conditional knockout mice fed a RD, the ITT curves showed a slightly but significantly superior insulin sensitivity compared to floxed-iPLA2β control mice (not shown), and this difference was magnified further at age 6 months, as described below.

As illustrated in Figure 6, at 3 months of age the ITTs of male HFD-fed floxed-iPLA2β control mice and MØ-iPLA2β-KO mice (Figure 6A) did not differ significantly, and this was also true for β-cell-iPLA2β-KO mice compared to floxed-iPLA2β control mice aged 3 months (Figure 6C). The deterioration of insulin sensitivity with age occurs, and by 6 months of age, a statistically significant difference between the ITTs for HFD-fed male MØ-KO and floxed-iPLA2β control mice had developed (Figure 6B). The 6-month-old HFD-fed MØ-iPLA2β-KO mice achieved significantly lower blood glucose levels than floxed-iPLA2β control mice at 30 and 60 min after insulin administration (Figure 6B). This is consistent with the hypothesis that motivated the preparation of the MØ-iPLA2β-KO mice—that HFD feeding would induce a lower deterioration in glucose tolerance and insulin sensitivity in MØ-iPLA2β-KO mice compared to floxed-iPLA2β control mice, possibly because of impaired

migration of monocytes into peripheral tissues where their differentiation into cytokine-producing macrophages ordinarily contributes to insulin resistance. Similar to the phenomenon described above for 3-month-old male β-cell-iPLA2β-KO conditional knockout RD-fed mice, at 6 months of age HFD-fed β-cell-iPLA2β-KO mice were also significantly more responsive to insulin than floxed-iPLA2β control mice (Figure 6D), raising the unanticipated possibility that β-cell products might also contribute to the development of HFD-induced insulin resistance. *Biomolecules* **2020**, *10*, x FOR PEER REVIEW 14 of 24

**Figure 6.** Insulin tolerance tests of 3- or 6-month-old male conditional iPLA2β-knockout mice and floxed-iPLA2β controls fed a regular or high-fat diet. Male floxed-iPLA2β control mice (circles), MØiPLA2β-KO mice (triangles), or β-cell-iPLA2β-KO mice (squares) mice were fed a regular diet after weaning until they were 6 weeks of age and were then fed a HFD until aged 3 months (**A,C**) or 6 months (**B,D**). Insulin tolerance tests were then performed in mice with free access to water and chow until human regular insulin (0.75 U/kg body weight; Lilly, Indianapolis, IN) was administered by intraperitoneal injection. Blood specimens were collected at 0, 30, 60, and 120 min thereafter for glucose concentration measurements, which were expressed as a percentage of the time zero blood glucose concentration, as described in [23,24,57]. Displayed values represent mean ± SEM (n = 13 to 16 (**A**), n = 15 to 18 (**B**), n = 22 to 26 (**C**), and n = 21 to 22 (**D**). An asterisk (\*) denotes *p* < 0.05 for the comparison between genotypes. **Figure 6.** Insulin tolerance tests of 3- or 6-month-old male conditional iPLA2β-knockout mice and floxed-iPLA2β controls fed a regular or high-fat diet. Male floxed-iPLA2β control mice (circles), MØ-iPLA2β-KO mice (triangles), or β-cell-iPLA2β-KO mice (squares) mice were fed a regular diet after weaning until they were 6 weeks of age and were then fed a HFD until aged 3 months (**A,C**) or 6 months (**B,D**). Insulin tolerance tests were then performed in mice with free access to water and chow until human regular insulin (0.75 U/kg body weight; Lilly, Indianapolis, IN) was administered by intraperitoneal injection. Blood specimens were collected at 0, 30, 60, and 120 min thereafter for glucose concentration measurements, which were expressed as a percentage of the time zero blood glucose concentration, as described in [23,24,57]. Displayed values represent mean ± SEM (n = 13 to 16 (**A**), n = 15 to 18 (**B**), n = 22 to 26 (**C**), and n = 21 to 22 (**D**). An asterisk (\*) denotes *p* < 0.05 for the comparison between genotypes.

A similar phenomenon was observed in 6-month-old HFD-fed female β-cell-iPLA2β-KO mice, which exhibited superior insulin sensitivity compared to floxed-β-cell-iPLA2β-KO control mice, and this magnified a smaller but similar trend observed with 6-month-old female β-cell-iPLA2β-KO mice fed regular chow (Figure S2A,B). For 6-month-old female MØ-iPLA2β-KO mice, the ITT curves did not differ significantly from those of floxed-iPLA2β control mice fed either a RD or a high-fat diet HFD (Figure S2C,D). Curiously, 6-month-old male MØ-iPLA2β-KO RD-fed mice exhibited lower insulin sensitivity compared to floxed-iPLA2β control mice (Figure S3), and this phenomenon has been observed previously with global iPLA2β-KO mice fed RD compared to controls [23]. In contrast, 6-month-old HFD-fed MØ-iPLA2β-KO male mice exhibited superior insulin sensitivity compared to floxed-iPLA2β control mice (Figure S3), which may reflect less HFD-induced deterioration in insulin sensitivity for 6-month-old HFD-fed MØ-iPLA2β-KO male mice than that which occurred for floxed-iPLA2β control male mice. Discrepant ITT findings between male and female global iPLA2β-KO mice and their responses to dietary stress have also been observed previously [23,24] and are commonplace in animal models of perturbed glucose homeostasis.

#### **4. Discussion**

Our previous studies with global iPLA2β-knockout mice indicated that disturbances in glucose homeostasis, which included glucose intolerance and impaired insulin secretion by pancreatic islet β-cells, occurred as a consequence of iPLA2β gene disruption [23,24]. Although iPLA2β and its products might participate in multiple events in GSIS from β-cells, one of them is to amplify depolarization-induced [Ca2+] entry into the β-cell. In addition, iPLA2β appears to confer protection against lipid injury to β-cells that may reflect iPLA2β participation in the repair of oxidative damage to β-cell mitochondrial membranes that occurs in the context of lipid toxicity [24,25]. Loss of these actions of iPLA2β in β-cells provides a plausible explanation for the impaired glucose tolerance in global iPLA2β-null mice, the reduced insulin secretory response to glucose of islets isolated from global iPLA2β-null mice compared to their wild-type littermates, and the exaggerated deterioration in glucose tolerance for global iPLA2β-null mice fed a HFD compared to wild-type controls [23,24].

More puzzling is the superior sensitivity to insulin of global iPLA2β-null mice compared to wild-type controls in insulin tolerance tests [24], implying that iPLA2β gene deletion has opposing effects on insulin secretion from β-cells and the insulin responsiveness of peripheral tissues, including skeletal muscle. One potential explanation for these findings is that iPLA2β deficiency has distinct effects in different cells. A loss of iPLA2β in β-cells would reasonably be expected to impair insulin secretion and to amplify lipid-induced β-cell injury, but a loss of iPLA2β activity is some other cell type might be responsible for the amelioration of lipid-induced deterioration in insulin sensitivity of global iPLA2β-null mice compared to wild-type controls. Candidates include cells of the monocyte/macrophage lineage. It is postulated that in the setting of lipid stress, the migration of blood monocytes into extravascular sites, including adipose tissue, and their differentiation into tissue macrophages results in the elaboration of cytokines, including IL-1β, IL-6 and TNFα, which impair insulin sensitivity. Because iPLA2β-derived 2-lysophosphatidic acid (LPA) appears to be required for monocyte migration in response to the cytokine Monocyte Chemoattractant-1 (MCP-1) [29,37–39], we postulated that iPLA2β-deficiency in cells of the monocyte/macrophage lineage might confer protection against the HFD-induced deterioration of insulin sensitivity because of a failure of iPLA2β-null monocytes to migrate into peripheral tissues and differentiate into macrophages.

We therefore prepared mice that are selectively deficient in iPLA2β in β-cells or cells of the monocyte/macrophage lineage. Mice with "floxed" iPLA2β genes were prepared using embryonic stem cells from the EUCOMM consortium. These cells had incorporated a targeting construct by homologous recombination that replaced the wild-type iPLA2β gene. In this construct, LoxP sites recognized by Cre recombinase [41,42] flanked critical iPLA2β gene exons. Mice with the "floxed" iPLA2β genes were then mated with mice that express Cre recombinase under the control of promoters that are expressed in only a restricted set of cells. In cells that express Cre, the action of the recombinase removes the "floxed" segment of the iPLA2β gene-targeting construct, and transcription of this modified gene yields a truncated mRNA that is eliminated by a nonsense mediated decay and cannot lead to production of an active iPLA2β protein. In this way, mice were produced that are selectively iPLA2β-deficient in specifically targeted populations of cells that express Cre recombinase.

To direct Cre expression in β-cells, RIP-Cre mice were used that express Cre under control of the Rat Insulin 2 Promoter, which has been demonstrated to be appropriate for β-cell-specific gene deletion when used in mice on a pure C57BL/6J background [49], as is the case in this study, although anomalies may be encountered on other genetic backgrounds [53]. The use of β-cell-targeting promoters has been reviewed [60], and the use of RIP-Cre remains a popular means of directing β-cell-restricted gene deletion [46,50,61–69]. The LysM promoter is expressed in all myeloid cells, and LysM-Cre is widely used to study conditional macrophage-myeloid cell gene deletions [52,70–76].

Our expectations for the behavior of these conditional iPLA2β-KO mouse lines was that the β-cell-iPLA2β-KO mice would exhibit glucose intolerance, impaired insulin secretion, and increased deterioration in glucose tolerance compared to control mice in response to a HFD, and our observations largely conformed to these expectations. Expectations for the MØ-iPLA2β-KO mice were that insulin secretion and glucose tolerance would be preserved, that deterioration in glucose tolerance induced by a HFD would be blunted compared to floxed-iPLA2β control mice, and that insulin sensitivity as measured in insulin tolerance tests (ITTs) would be impaired less in HFD-fed MØ-iPLA2β-KO compared to floxed-iPLA2β control mice, and our observations also largely conformed to these expectations.

An unexpected finding is that HFD-fed β-cell-iPLA2β-KO mice also had superior insulin sensitivity compared to floxed-iPLA2β control mice as assessed with ITTs. The explanation for this finding has not been established, but of possible relevance is the phenomenon of selective insulin resistance first proposed by McGarry [75] and subsequently discussed by Brown and Goldstein [76] and others [77,78]. McGarry proposed that hyperinsulinemia itself elicits insulin resistance in peripheral tissues [75]. During the evolution of T2D (type 2 diabetes), resistance develops to the effect of insulin to decrease hepatic gluconeogenesis but such resistance does not develop the insulin-stimulated lipogenesis of fatty acids and triacylglycerols (TAGs), which is now known to involve the activation of the transcription factor SREBP-1c [76–78]. In fact, hepatic lipogenesis and Very Low Density Lipoprotein (VLDL) secretion are amplified by the hyperinsulinemia that results from the resistance of peripheral tissues to the action of insulin, and fatty acids derived from VLDL TAG exacerbate the insulin-resistant state in muscle and adipose tissue [75–79]. These events result in the classic T2DM triad of hyperglycemia, hyperinsulinemia, and hypertriglyceridemia [75,76]. McGarry suggested that VLDL TAG play a toxic role in the evolution of T2D by their deposition in muscle, where they enhance insulin resistance, and in liver, where they can produce non-alcoholic steatohepatitis (NASH). The term lipotoxicity is used to describe the detrimental effects of accumulation of TAG and other lipids in various tissues [80], and ceramides are thought to be among the lipid mediators that contribute to insulin resistance [79].

If hyperinsulinemia is involved in inducing peripheral tissue insulin resistance, then diabetes in a mouse model caused solely by a β-cell secretory defect might fail to generate sufficiently high levels of hyperinsulinemia to drive development of insulin resistance. Compared to a control population of mice that do develop sufficient hyperinsulinemia, mice with the pure secretory defect might thus exhibit superior insulin sensitivity in insulin tolerance tests. It is of interest in this regard that knockout mice with defective KATP channel activity also have impaired insulin secretion from β-cells but enhanced insulin sensitivity of peripheral tissues in insulin tolerance tests [81], which the authors postulated might be mediated by altered extracellular hormonal or neuronal signals perturbed by disruption of KATP channels [82]. In the context of the discussion in this and the preceding paragraph, one such altered extracellular hormonal signal might be the blood insulin concentration itself.

The possibility that the insulin secretory defect of global iPLA2β-knockout mice and of β-cell-iPLA2β-KO mice prevents the hyperinsulinemia-driven deposition of toxic lipids in tissues that otherwise occurs in mice subjected to a HFD is of interest in the context of observations that hepatic steatosis fails to develop in global iPLA2β-knockout mice subjected to a HFD, although it does occur in control mice [83]. Gene deletion of iPLA2β also greatly attenuates hepatic steatosis that otherwise occurs in the *ob*/*ob* mouse genetic model [84]. In addition, ceramides such as those that accumulate in tissues that develop insulin resistance [79,80], also accumulate in β-cells subjected to ER stress in an iPLA2β-dependent process that involves activation of SREBP-1 [85–88], which is of interest in the context of involvement of SREBP-1 activation in the pathogenesis of hyperinsulinemia-driven amplification of hepatic lipogenesis in the evolution of T2D [76–78].

In contrast to the unexpected resistance to HFD-induced deterioration in insulin-responsivity of β-cell-iPLA2β-KO mice discussed above, it was expected that HFD-fed MØ-iPLA2β-KO mice would exhibit superior insulin sensitivity compared to HFD-fed floxed-iPLA2β control mice as assessed with ITTs. This expectation was based on the postulates that HFD-induced deterioration in insulin sensitivity involves migration of blood monocytes into extravascular sites, such as adipose tissue, where they differentiate into macrophages and produce cytokines, including IL-β, IL-6, and TNFα, that impair insulin sensitivity in a process that involves the tissue elaboration of the cytokine Monocyte Chemoattractant-1 (MCP1) and its interaction with the CCR2 receptor on monocytes to induce their migration into tissues [31–36]. Genetic and pharmacologic evidence indicates that the monocyte chemotactic response to MCP-1 requires the action of iPLA2β to produce the lipid mediator 2-lysophosphatidic acid (LPA) [37–39]. Macrophages [29,30] and other cells [89] from our global iPLA2β knockout mice have been demonstrated to have impaired production of LPA in response to stimuli that induce robust LPA production in wild-type cells.

Moreover, macrophages from our global iPLA2β knockout mice have impaired migratory responses to MCP-1 in a mouse model of diet-induced glucose intolerance and atherogenesis, and these migratory responses are restored by provision of exogenous LPA [29]. The accumulation of macrophages in atherosclerotic lesions in this mouse model of diabetic stress is also associated with increased lesion content of iPLA2β immunoreactive protein and enzymatic activity [29]. Migratory responses of vascular smooth muscle cells (VSMCs) in a model of vascular injury are also greatly reduced in our global iPLA2β-null mice compared to wild-type mice [90]. The activation state of iPLA2β-null macrophages from our global iPLA2β knockout mice is also shifted toward an M2 anti-inflammatory phenotype compared to the inflammatory M1 phenotype exhibited by wild-type macrophages, and this is associated with reduced TNFα production by the iPLA2β-null macrophages [30,91–93], which suggests that in addition to participating in monocyte migration into extravascular tissues, iPLA2β may also be involved in their differentiation into pro-inflammatory macrophages that elaborate cytokines that impair insulin sensitivity. The pharmacologic inhibition of iPLA2β also ameliorates leukocyte infiltration into pancreatic islets and the onset of diabetes in NOD (non-obese diabetic) mice [92,93], which suggests that iPLA2β may participate in the evolution of T1D in addition to that of T2DM.

iPLA2β is in fact involved in regulating several fundamental aspects of monocyte/macrophage biology in addition to those discussed above, including the remodeling the fatty acid composition of macrophage phospholipids [94,95], selection of macrophage phospholipid pools from which to mobilize fatty acids upon cellular stimulation [27,96,97], governing the proliferation state of macrophage precursor cells [97], regulating macrophage apoptosis in the context of lipid stress [98], regulating the rate of transcription of the macrophage inducible nitric oxide synthase (iNOS) gene [26], and promoting macrophage adhesion and spreading in response to the engagement of the class A scavenger receptor [99]. Development of the monocyte/macrophage-selective conditional iPLA2β-knockout mice described here may prove to be useful in further characterizing these aspects of monocyte/macrophage biology in models of diabetes, atherosclerosis, and other pathophysiologic states.

In addition, preparation of the mouse line with floxed-iPLA2β genes described here may prove to be useful in the development of conditional knockout mouse lines with selective deletion of iPLA2β in other cell lines that might participate in the development of T2DM, including skeletal muscle cells, adipocytes, and hepatocytes. All of these cell types are involved in glucose homeostasis in T2D, and all express iPLA2β [83,84,100,101], which is involved in regulating fatty acid oxidation in skeletal muscle [100], differentiation of adipoctyes [101], and hepatic lipid synthesis [83,84]. Conditional

knockouts in skeletal muscle can be prepared from our iPLA2β-floxed mice with mice that express Cre under the control of the promoter for muscle creatine kinase (MCK) [102], conditional knockouts in adipocytes can be prepared with our floxed mice and mice that express Cre under control of the promoter for adipocyte-specific fatty acid binding protein (aP2) [103]. Conditional knockouts in hepatocytes can be prepared with our floxed mice and mice that express Cre under control of the promoter for rat albumin [54]. Such lines would permit further characterization of the mechanism of resistance of the global iPLA2β knockout to HFD-induced deterioration in insulin sensitivity [24].

### **5. Conclusions**

Cre-lox technology was used to produce mice with selective iPLA2β deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2β-KO), or in insulin-secreting β-cells (β-Cell-iPLA2β-KO), respectively. MØ-iPLA2β-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2β control mice after consuming a high-fat diet (HFD). MØ-iPLA2β-KO mice exhibited normal GSIS in vivo and from isolated islets ex vivo compared to floxed-iPLA2β controls. Male MØ-iPLA2β-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. β-Cell-iPLA2β-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further after introduced to a HFD. β-Cell-iPLA2β-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls, and male β-cell-iPLA2β-KO mice also exhibited enhanced insulin responsivity compared to controls after of introduction of the HFD. These findings suggest that MØ iPLA2β participates in HFD-induced deterioration in glucose tolerance, and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, β-cell iPLA2β plays a role in GSIS and also appears to confer some protection against deterioration in β-cell function induced by a HFD.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/10/10/1455/s1, Figure S1: In Vivo Insulin Secretion For iPLA2β Conditional Knockout and Floxed-iPLA2β Control Mice Fed a Regular or High-Fat Diet, Figure S2: Insulin Tolerance Tests of 6-month-old Female Conditional iPLA2β-Knockout Mice and Floxed-iPLA2β Controls Fed a Regular or High-Fat Diet (HFD), Figure S3: Insulin Tolerance Tests of 6-month-old Male Conditional iPLA2β-Knockout Mice and Floxed-iPLA2β Controls Fed a Regular or High-Fat Diet (HFD). Table S1: Glucose Tolerance Tests for Female and Male Mice Aged 3 or 6 months with Genotypes Floxed-iPLA2β, β-Cell-iPLA2β-KO, or MØ-iPLA2β-KO Fed a Regular or High-Fat Diet.

**Author Contributions:** Conceptualization, J.T., H.S. and S.R.; methodology, H.S., M.W. and X.L.; software, H.S.; validation, H.S., M.W. and X.L.; formal analysis, J.T., H.S. and S.R.; investigation, H.S. and M.W.; resources, J.T. and S.R.; data curation, M.W., H.S., C.F., and J.T.; writing—original draft preparation, J.T.; writing—review and editing, S.R.; visualization, J.T., C.F., and S.R.; supervision, J.T. and S.R.; project administration, J.T. and S.R.; funding acquisition, J.T. and S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** These studies were supported in part by United States Public Health Service Grants R37-DK34388, P60-DK20579, P30 DK020579, P30 DK056341, P41 RR000954, R24GM136766, P41GM103422, DK110292, R21 AI 146743, and a grant from the Dean's fund from the Washington University School of Medicine.

**Acknowledgments:** The intellectual input from Zhongmin Ma and Shunzhong Bao in discussions of these data is gratefully acknowledged, as is the technical assistance of Alan Bohrer and Min Tan.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

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*Article*

## **The Contribution of Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s to Adrenic Acid Mobilization in Murine Macrophages**

**Patricia Monge 1,2, Alvaro Garrido 1,2, Julio M. Rubio 1,2, Victoria Magrioti 3 , George Kokotos 3 , María A. Balboa 1,2 and Jesús Balsinde 1,2,\***

Received: 16 March 2020; Accepted: 1 April 2020; Published: 3 April 2020

**Abstract:** Adrenic acid (AA), the 2-carbon elongation product of arachidonic acid, is present at significant levels in membrane phospholipids of mouse peritoneal macrophages. Despite its abundance and structural similarity to arachidonic acid, very little is known about the molecular mechanisms governing adrenic acid mobilization in cells of the innate immune system. This contrasts with the wide availability of data on arachidonic acid mobilization. In this work, we used mass-spectrometry-based lipidomic procedures to define the profiles of macrophage phospholipids that contain adrenic acid and their behavior during receptor activation. We identified the phospholipid sources from which adrenic acid is mobilized, and compared the data with arachidonic acid mobilization. Taking advantage of the use of selective inhibitors, we also showed that cytosolic group IVA phospholipase A2 is involved in the release of both adrenic and arachidonic acids. Importantly, calcium independent group VIA phospholipase A2 spared arachidonate-containing phospholipids and hydrolyzed only those that contain adrenic acid. These results identify separate mechanisms for regulating the utilization of adrenic and arachidonic acids, and suggest that the two fatty acids may serve non-redundant functions in cells.

**Keywords:** adrenic acid; arachidonic acid; mass spectrometry; lipid signaling; inflammation; phospholipase A2; monocytes/macrophages

#### **1. Introduction**

Arachidonic acid (*cis*-5,8,11,14-eicosatetraenoic acid, AA), a fatty acid of the *n*-6 series, is the major polyunsaturated fatty acid present in cells of the innate immunity [1]. Cleavage of AA-containing membrane phospholipids by phospholipase A2 (PLA2) enzymes during activation results in substantial release of free AA. The free fatty acid is then metabolized by cyclooxygenases, lipoxygenases, or cytochrome P-450 enzymes into numerous oxygenated metabolites, collectively called the eicosanoids, which have key roles in inflammation [2–6]. The eicosanoids participate in immune regulation by influencing the activation of innate immune phagocytic cells at various levels, including differentiation and migration, phagocytic capacity, and cytokine production [7–10].

Innate immune cells also contain significant quantities of another *n*-6 fatty acid, adrenic acid (*cis*-7,10,13,16-docosatetraenoic acid, AdA), which is the 2-carbon elongation product of AA [1]

(Scheme 1). Similar to AA, AdA can also be metabolized via cyclooxygenase, lipoxygenase, and cytochrome P-450 pathways [11–14], giving rise to a number of oxygenated metabolites. Interestingly, a recent study using a mouse model of steatohepatitis reported elevated levels of free AdA in plasma and liver [15]. These data raised the possibility that alterations in the homeostatic mechanisms regulating AdA levels may be related to pathophysiological states [15]. Regarding the role of AdA in innate immune cells, it has been shown that the fatty acid is able to dampen inflammation by blocking leukotriene B4 formation by neutrophils and enhancing phagocytosis by macrophages [16]. These effects are consistent with a pro-resolving mediator function for AdA in osteoarthritis [16]. 1). Similar to AA, AdA can also be metabolized via cyclooxygenase, lipoxygenase, and cytochrome P-450 pathways [11–14], giving rise to a number of oxygenated metabolites. Interestingly, a recent study using a mouse model of steatohepatitis reported elevated levels of free AdA in plasma and liver [15]. These data raised the possibility that alterations in the homeostatic mechanisms regulating AdA levels may be related to pathophysiological states [15]. Regarding the role of AdA in innate immune cells, it has been shown that the fatty acid is able to dampen inflammation by blocking leukotriene B4 formation by neutrophils and enhancing phagocytosis by macrophages [16]. These effects are consistent with a pro-resolving mediator function for AdA in osteoarthritis [16].

*Biomolecules* **2020**, *10*, 2 of 16

7,10,13,16-docosatetraenoic acid, AdA), which is the 2-carbon elongation product of AA [1] (Scheme

**Scheme 1.** Structure of arachidonic acid (AA) and adrenic acid (AdA). The positions of the carbons at which double bonds occur are numbered. Note that all double bonds are in the *cis* configuration. **Scheme 1.** Structure of arachidonic acid (AA) and adrenic acid (AdA). The positions of the carbons at which double bonds occur are numbered. Note that all double bonds are in the *cis* configuration.

Recent work from our laboratory has taken advantage of mass-spectrometry-based lipidomic approaches to define at a molecular level the mechanisms regulating AA mobilization from phagocytic cells responding to stimuli of the innate immune response [17–27]. Our work has highlighted not only the importance of multiple PLA2 enzymes in the process, but also of the mechanisms regulating the reacylation of the liberated fatty acid back into phospholipids and the remodeling that places the AA in the appropriate cellular lipid pools. These findings have raised the intriguing possibility that not all AA pools may be reachable by the PLA2 form involved in its mobilization. For instance, release of lipoxygenase products by ionophore-activated human neutrophils [28] and zymosan-stimulated mouse peritoneal macrophages [22] appears to be associated with AA mobilization from choline-containing phospholipids (PC), not ethanolaminecontaining phospholipids (PE) or phosphatidylinositol (PI). Thus, depending on stimulation Recent work from our laboratory has taken advantage of mass-spectrometry-based lipidomic approaches to define at a molecular level the mechanisms regulating AA mobilization from phagocytic cells responding to stimuli of the innate immune response [17–27]. Our work has highlighted not only the importance of multiple PLA2 enzymes in the process, but also of the mechanisms regulating the reacylation of the liberated fatty acid back into phospholipids and the remodeling that places the AA in the appropriate cellular lipid pools. These findings have raised the intriguing possibility that not all AA pools may be reachable by the PLA2 form involved in its mobilization. For instance, release of lipoxygenase products by ionophore-activated human neutrophils [28] and zymosan-stimulated mouse peritoneal macrophages [22] appears to be associated with AA mobilization from choline-containing phospholipids (PC), not ethanolamine-containing phospholipids (PE) or phosphatidylinositol (PI). Thus, depending on stimulation conditions, the cellular distribution of AA between various phospholipid locations may also limit eicosanoid synthesis [3,26].

conditions, the cellular distribution of AA between various phospholipid locations may also limit eicosanoid synthesis [3,26]. In contrast to AA studies, very little is still known about the mechanisms governing cellular AdA availability. In this study we have analyzed the regulatory features of AdA mobilization in activated macrophages. Our work provides an in-depth examination of AdA homeostasis under pathophysiologically relevant conditions, and suggests that both cytosolic group IVA PLA2 (cPLA2α) and calcium-independent group VIA PLA2 (iPLA2-VIA) participate in the process. AdA mobilization shares regulatory features with AA mobilization with regard to cPLA2α involvement, but seems to be a more complex process, as it involves participation of a second PLA2 that is apparently not involved in AA release (i.e., iPLA2-VIA). In addition, our study also suggests that supplementation with AdA does not reduce AA utilization by the cells. Thus, the two fatty acids may play nonredundant biological roles that could be exploited to design selective strategies to control the In contrast to AA studies, very little is still known about the mechanisms governing cellular AdA availability. In this study we have analyzed the regulatory features of AdA mobilization in activated macrophages. Our work provides an in-depth examination of AdA homeostasis under pathophysiologically relevant conditions, and suggests that both cytosolic group IVA PLA2 (cPLA2α) and calcium-independent group VIA PLA2 (iPLA2-VIA) participate in the process. AdA mobilization shares regulatory features with AA mobilization with regard to cPLA2α involvement, but seems to be a more complex process, as it involves participation of a second PLA2 that is apparently not involved in AA release (i.e., iPLA2-VIA). In addition, our study also suggests that supplementation with AdA does not reduce AA utilization by the cells. Thus, the two fatty acids may play non-redundant biological roles that could be exploited to design selective strategies to control the production of AdA-derived products at the level of their precursor fatty acid.

#### production of AdA-derived products at the level of their precursor fatty acid. **2. Materials and Methods**

#### **2. Materials and Methods**  *2.1. Reagents*

*2.1. Reagents*  Cell culture medium was from Molecular Probes-Invitrogen (Carlsbad, CA, USA). Organic solvents (Optima® LC/MS grade) were from Fisher Scientific (Madrid, Spain). Lipid standards were from Avanti (Alabaster, AL, USA) or Cayman (Ann Arbor, MI, USA). Silicagel G thin-layer Cell culture medium was from Molecular Probes-Invitrogen (Carlsbad, CA, USA). Organic solvents (Optima® LC/MS grade) were from Fisher Scientific (Madrid, Spain). Lipid standards were from Avanti (Alabaster, AL, USA) or Cayman (Ann Arbor, MI, USA). Silicagel G thin-layer chromatography plates were from Macherey-Nagel (Düren, Germany). The cPLA2α inhibitor pyrrophenone [29] was synthesized and provided by Dr. Alfonso Pérez (Department of Organic Chemistry, University of

Valladolid, Valladolid, Spain). The iPLA2-VIA inhibitors FKGK18 and GK436 were synthesized in the Kokotos laboratory [30,31]. All other reagents were from Sigma-Aldrich (Madrid, Spain).

#### *2.2. Cell Culture*

Mouse peritoneal macrophages from Swiss mice (University of Valladolid Animal House, 10–12 weeks old) were obtained by peritoneal lavage using 5 mL cold phosphate-buffered saline and cultured in RPMI 1640 medium with 10% (*v*/*v*) fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, as described elsewhere [32,33]. All procedures involving animals were undertaken under the supervision of the Institutional Committee of Animal Care and Usage of the University of Valladolid (No. 7406000), in accordance with the guidelines established by the Spanish Ministry of Agriculture, Food, and Environment and the European Union.

Cells were placed in serum-free medium for 1 h before addition of stimuli or inhibitors. Afterward, they were challenged by the stimuli for the time indicated. Zymosan was prepared exactly as described [32,33]. Only zymosan batches that demonstrated no measurable endogenous PLA2 activity, as measured by in vitro assay under different conditions [34–37], were used in this study. Cell protein content was quantified according to Bradford [38] using a commercial kit (BioRad Protein Assay, Bio-Rad, Hercules, CA, USA).

#### *2.3. Gas Chromatography*/*Mass Spectrometry (GC*/*MS) Analyses*

Total lipids from approximately 107 cells were extracted according to Bligh and Dyer [39], and the following internal standards were added: 10 nmol of 1,2-diheptadecanoyl-*sn*-glycero-3-phosphocholine, 10 nmol of 1,2,3-triheptadecanoylglycerol, and 30 nmol of cholesteryl tridecanoate. Phospholipids were separated from neutral lipids by thin-layer chromatography, using *n*-hexane/diethyl ether/acetic acid (70:30:1, *v*/*v*/*v*) as the mobile phase [40]. Phospholipid classes were separated twice with chloroform/methanol/28% (*w*/*w*) ammonium hydroxide (60:37.5:4, *v*/*v*/*v*) as the mobile phase, using plates impregnated with boric acid [41]. The bands corresponding to the different lipid classes were scraped off from the plate, and fatty acid methyl esters were obtained from the various lipid fractions by transmethylation with 0.5 M KOH in methanol for 60 min at 37 ◦C [42–45]. Analyses were carried out using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in an electron impact mode (EI, 70 eV). The apparatus was also equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length × 0.25 mm internal diameter × 0.15 µM film thickness) (Agilent Technologies, Santa Clara, CA, USA). Data analysis was carried out with the Agilent G1701EA MSD Productivity Chemstation software, revision E.02.00 [42–45].

#### *2.4. Liquid Chromatography*/*Mass Spectrometry (LC*/*MS) Analyses of Phospholipids*

Lipids were extracted according to Bligh and Dyer [39], and the following internal standards were added: 20 pmol each of 1,2-dimyristoyl-*sn*-glycero-3-phosphoglycerol, 1,2-dilauroyl-*sn*-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-*sn*-glycero-3-phosphoethanolamine, 1,2-di- heptadecanoyl-*sn*-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-*sn*-glycero-3-phosphoserine, 1,2-dimyristoyl-*sn*-glycero-3-phosphate, 1,2-dipentadecanoyl-*sn*-glycero-3-phosphocholine, 1,2-diheptadecanoyl-*sn*-glycero-3-phosphocholine, and 1,2-dinonadecanoyl-*sn*-glycero-phosphocholine.

The samples were re-dissolved in hexanes/2-propanol/water (42:56:2, *v*/*v*/*v*) and injected into a Thermo Scientific Dionex Ultimate 3000 high-performance liquid chromatograph equipped with an Ultimate HPG-3400SD standard binary pump and an Ultimate ACC-3000 autosampler column compartment (Waltham, MA, USA). Separation was carried using a FORTIS HILIC column (150 × 3 mm, 3 µm particle size) (Fortis Technologies, Neston, UK). The mobile phase consisted of a gradient of solvent A (hexanes/isopropanol 30:40, *v*/*v*) and solvent B (hexanes/isopropanol/20 mM ammonium acetate in water, 30:40:7, *v*/*v*/*v*). The gradient started at 75% A from 0 to 5 min, then decreased from 75% A to 40% A at 15 min, from 40% A to 5% A at 20 min, was held at 5% until 40 min, and then increased to 75% at 41 min. Then, the column was re-equilibrated holding 75% A for an additional 14-min period before

the next sample injection [46]. The flow rate through the column was fixed at 0.4 mL/min. The liquid chromatography system was coupled online to an Sciex QTRAP 4500 Mass Spectrometer equipped with a Turbo V ion source and a TurbolonSpray probe for electrospray ionization (AB Sciex, Framingham, MA, USA). Source parameters were set as follows: ion spray voltage, −4500 V; curtain gas, 30 psi; nebulizer gas, 50 psi; desolvation gas, 60 psi; temperature, 425 ◦C. Phospholipid species were analyzed in scheduled multiple reaction monitoring mode with negative ionization, detecting in Q3 the *m*/*z* of either 303.2 or 331.2, corresponding to AA and AdA, respectively, as [M-H]−. Compound parameters were fixed as follows: declustering potential; −45 V (choline glycerophospholipids), −60 V (ethanolamine glycerophospholipids) −30 V (phosphatidylinositol), −50 V (phosphatidylserine), −60 V (phosphatidic acid), −50 V (phosphatidylglycerol); collision energy: −50 V (choline glycerophospholipids), −40 V (ethanolamine glycerophospholipids), −60 V (phosphatidylinositol), −50 V (phosphatidylserine), −45 V (phosphatidic acid), −45 V (phosphatidylglycerol); entrance potential, −10 V; and collision cell exit potential, −8 V. All glycerophospholipids were detected as [M-H]−, ions except choline glycerophospholipids, which were detected as [M + CH3COO]− ions. Quantification was carried out by integrating the chromatographic peaks of each species and comparing these with the peak area of the internal standard that corresponded to each class. Spectrometer equipped with a Turbo V ion source and a TurbolonSpray probe for electrospray ionization (AB Sciex, Framingham, MA, USA). Source parameters were set as follows: ion spray voltage, -4500 V; curtain gas, 30 psi; nebulizer gas, 50 psi; desolvation gas, 60 psi; temperature, 425 °C. Phospholipid species were analyzed in scheduled multiple reaction monitoring mode with negative ionization, detecting in Q3 the *m*/*z* of either 303.2 or 331.2, corresponding to AA and AdA, respectively, as [M-H]−. Compound parameters were fixed as follows: declustering potential; −45 V (choline glycerophospholipids), −60 V (ethanolamine glycerophospholipids) −30 V (phosphatidylinositol), −50 V (phosphatidylserine), −60 V (phosphatidic acid), −50 V (phosphatidylglycerol); collision energy: −50 V (choline glycerophospholipids), −40 V (ethanolamine glycerophospholipids), −60 V (phosphatidylinositol), −50 V (phosphatidylserine), −45 V (phosphatidic acid), −45 V (phosphatidylglycerol); entrance potential, −10 V; and collision cell exit potential, −8 V. All glycerophospholipids were detected as [M-H]−, ions except choline glycerophospholipids, which were detected as [M + CH3COO]− ions. Quantification was carried out by integrating the chromatographic peaks of each species and comparing these with the peak area of the internal standard that corresponded to each class.

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acetate in water, 30:40:7, *v*/*v*/*v*). The gradient started at 75% A from 0 to 5 min, then decreased from 75% A to 40% A at 15 min, from 40% A to 5% A at 20 min, was held at 5% until 40 min, and then

0.4 mL/min. The liquid chromatography system was coupled online to an Sciex QTRAP 4500 Mass

#### **3. Results 3. Results**

#### *3.1. Adrenic Acid and Arachidonic Acid Contents of Murine Peritoneal Macrophages 3.1. Adrenic Acid and Arachidonic Acid Contents of Murine Peritoneal Macrophages*

Lipid extracts from mouse peritoneal macrophages were analyzed for fatty acid content by GC/MS. Total AA content was 69.9 ± 4.2 nmol/mg cell protein (mean values ± standard error of the mean, *n* = 5), while AdA content was 15.1 ± 1.2 nmol/mg cell protein (mean values ± standard error of the mean, *n* = 5). Both AA and AdA were found almost exclusively in phospholipids. The distribution of AA and AdA between phospholipid classes is shown in Figure 1. Despite the difference in mass between AA and AdA, their distribution between phospholipid classes was remarkably similar, with the majority of both fatty acids being found in ethanolamine glycerophospholipids (PE), followed by choline glycerophospholipids (PC). Minor amounts of both fatty acids were found in phosphatidylinositol (PI) and phosphatidylserine (PS). Lipid extracts from mouse peritoneal macrophages were analyzed for fatty acid content by GC/MS. Total AA content was 69.9 ± 4.2 nmol/mg cell protein (mean values ± standard error of the mean, *n* = 5), while AdA content was 15.1 ± 1.2 nmol/mg cell protein (mean values ± standard error of the mean, *n* = 5). Both AA and AdA were found almost exclusively in phospholipids. The distribution of AA and AdA between phospholipid classes is shown in Figure 1. Despite the difference in mass between AA and AdA, their distribution between phospholipid classes was remarkably similar, with the majority of both fatty acids being found in ethanolamine glycerophospholipids (PE), followed by choline glycerophospholipids (PC). Minor amounts of both fatty acids were found in phosphatidylinositol (PI) and phosphatidylserine (PS).

**Figure 1.** Distribution of AA and AdA between phospholipid classes. The various phospholipid classes were separated by thin-layer chromatography. The distribution of AA (**A**) and AdA (**B**) between choline glycerophospholipids (PC), ethanolamine glycerophospholipids (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) was determined by gas chromatography/mass spectrometry (GC/MS) after converting the phospholipid-bound fatty acids into methyl esters. Results are shown as means ± standard error of the mean (*n* = 3). **Figure 1.** Distribution of AA and AdA between phospholipid classes. The various phospholipid classes were separated by thin-layer chromatography. The distribution of AA (**A**) and AdA (**B**) between choline glycerophospholipids (PC), ethanolamine glycerophospholipids (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) was determined by gas chromatography/mass spectrometry (GC/MS) after converting the phospholipid-bound fatty acids into methyl esters. Results are shown as means ± standard error of the mean (*n* = 3).

Figure 2 shows the distribution of AA- and AdA-containing phospholipid molecular species, as analyzed by liquid chromatography coupled to tandem mass spectrometry (LC/MS). In agreement with previous estimates [21,22], multiple AA-containing species were detected, with the alkenylacyl and diacyl ethanolamine phospholipid species PE(P-16:0/20:4), PE(P-18:0/20:4), and PE(18:0/20:4) predominating, followed by the diacyl choline phospholipid species PC(16:0/20:4) and PC(18:0/20:4), and the unique inositol phospholipid species PI(18:0/20:4) analyzed by liquid chromatography coupled to tandem mass spectrometry (LC/MS). In agreement with previous estimates [21,22], multiple AA-containing species were detected, with the alkenylacyl and diacyl ethanolamine phospholipid species PE(P-16:0/20:4), PE(P-18:0/20:4), and PE(18:0/20:4) predominating, followed by the diacyl choline phospholipid species PC(16:0/20:4) and PC(18:0/20:4), and the unique inositol phospholipid species PI(18:0/20:4)

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Figure 2 shows the distribution of AA- and AdA-containing phospholipid molecular species, as

**Figure 2.** AA and AdA-containing phospholipid molecular species in peritoneal macrophages The profiles of AA- (**A**) or AdA- (**B**) containing PC (red), PE (green), PI (yellow), and PS (pink) species in peritoneal macrophages were determined by liquid chromatography/mass spectrometry (LC/MS). Fatty chains within the different phospholipid species are designated by their numbers of carbons and double bonds. A designation of O- before the first fatty chain indicates that the *sn*-1 position is ether linked, whereas a P- designation indicates a plasmalogen form (*sn*-1 vinyl ether linkage) [47]. Phospholipids containing two ester bonds have no designation. Results are shown as mean values ± standard error of the mean (*n* = 3). **Figure 2.** AA and AdA-containing phospholipid molecular species in peritoneal macrophages The profiles of AA- (**A**) or AdA- (**B**) containing PC (red), PE (green), PI (yellow), and PS (pink) species inperitoneal macrophages were determined by liquid chromatography/mass spectrometry (LC/MS). Fattychains within the different phospholipid species are designated by their numbers of carbons and double bonds. A designation of O- before the first fatty chain indicates that the *sn*-1 position is ether linked, whereas a P- designation indicates a plasmalogen form (*sn*-1 vinyl ether linkage) [47]. Phospholipids containing two ester bonds have no designation. Results are shown as mean values ± standard error of the mean (*n* = 3).

Regarding AdA-containing species, the alkenyl acyl and diacyl ethanolamine phospholipid species PE(P-16:0/22:4), PE(P-18:0/22:4), and PE(18:0/22:4)], and the diacyl choline phospholipid species PC(16:0/22:4) and PC(18:0/22:4) also constituted the major cellular AdA reservoirs. Strikingly, the inositol phospholipid species PI(18:0/22:4) was not as prevalent as its AA equivalent, PI(18:0/20:4), was among AA-containing phospholipids. This may suggest that the acyl-CoA acyltransferase using lysoPI as the acceptor [48] shows selectivity for AA over AdA as a substrate. Regarding AdA-containing species, the alkenyl acyl and diacyl ethanolamine phospholipid species PE(P-16:0/22:4), PE(P-18:0/22:4), and PE(18:0/22:4)], and the diacyl choline phospholipid species PC(16:0/22:4) and PC(18:0/22:4) also constituted the major cellular AdA reservoirs. Strikingly, the inositol phospholipid species PI(18:0/22:4) was not as prevalent as its AA equivalent, PI(18:0/20:4), was among AA-containing phospholipids. This may suggest that the acyl-CoA acyltransferase using

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Macrophage stimulation with yeast-derived zymosan markedly decreased the cellular AA content in PC and PI. Despite PE being the major AA-containing class, AA losses from PE did not reach statistical significance (Figure 3A). It should be noted in this regard that during receptor stimulation, AA is known to be transferred from AA-containing PC (1-acyl species) to PE (plasmalogen species) by CoA-independent transacylase; hence, the decline in the amount of AA-containing PE during cellular stimulation may be greatly reduced [22,24,49]. Regarding AdA, decreases in its cellular content were also observed after zymosan stimulation. However the pattern clearly differed in that PC was the only phospholipid class that contributed to AdA mobilization; AdA reductions from PE and PI did not reach statistical significance (Figure 3B). lysoPI as the acceptor [48] shows selectivity for AA over AdA as a substrate. Macrophage stimulation with yeast-derived zymosan markedly decreased the cellular AA content in PC and PI. Despite PE being the major AA-containing class, AA losses from PE did not reach statistical significance (Figure 3A). It should be noted in this regard that during receptor stimulation, AA is known to be transferred from AA-containing PC (1-acyl species) to PE (plasmalogen species) by CoA-independent transacylase; hence, the decline in the amount of AAcontaining PE during cellular stimulation may be greatly reduced [22,24,49]. Regarding AdA, decreases in its cellular content were also observed after zymosan stimulation. However the pattern clearly differed in that PC was the only phospholipid class that contributed to AdA mobilization; AdA reductions from PE and PI did not reach statistical significance (Figure 3B).

**Figure 3.** AA and AdA mobilization in zymosan-stimulated macrophages. The cells were unstimulated (colored bars) or stimulated (open bars) with 1 mg/mL zymosan for 1 h. Afterward, total content of AA (**A**) or AdA (**B**) in various phospholipid classes (see x-axis) was measured by GC/MS. To allow for direct comparison between AA and AdA, the fatty acid contents in phospholipid classes in unstimulated cells are given as 100%. Actual mass values can be obtained from Figure 1. Results are shown as mean values ± standard error of the mean. (*n* = 3). Note: \* *p* < 0.05, significantly different from the corresponding phospholipid class. **Figure 3.** AA and AdA mobilization in zymosan-stimulated macrophages. The cells were unstimulated (colored bars) or stimulated (open bars) with 1 mg/mL zymosan for 1 h. Afterward, total content of AA (**A**) or AdA (**B**) in various phospholipid classes (see x-axis) was measured by GC/MS. To allow for direct comparison between AA and AdA, the fatty acid contents in phospholipid classes in unstimulated cells are given as 100%. Actual mass values can be obtained from Figure 1. Results are shown as mean values ± standard error of the mean. (*n* = 3). Note: \* *p* < 0.05, significantly different from the corresponding phospholipid class.

#### *3.2. Defining the Role of Various PLA2 Forms in Stimulus-Induced AA and AdA Mobilization 3.2. Defining the Role of Various PLA2 Forms in Stimulus-Induced AA and AdA Mobilization*

The differential involvement of phospholipid classes in AA and AdA mobilization suggests the existence of separate mechanisms for regulating the availability of each fatty acid. To characterize such mechanisms, we took advantage of the use of selective inhibitors of the two major intracellular PLA2 enzymes potentially effecting the fatty acid release in activated peritoneal macrophages, namely group IVA Ca2+-dependent cytosolic PLA2 (cPLA2α) and group VIA Ca2+-independent PLA2 (iPLA2-VIA) [22,50]. The use of selective chemical inhibitors to address the role of intracellular PLA2s during cell activation has some advantages over other widely used methods, such as small interfering RNA or cells from knockout mice. With chemical inhibitors, inhibition develops rapidly, which reduces the impact of unspecific effects that could occur over time. Also, no compensatory mechanisms take place that might obscure the interpretation of results [22]. In addition, chemical inhibitors directly target PLA2 effects that depend on enzymatic activity, without affecting noncatalytic functions of the enzyme [51]. The inhibitors used in this work are the most potent and selective inhibitors currently available to block cPLA2α and iPLA2-VIA in cells. Pyrrophenone potently and selectively inhibits cPLA2α activity using a number of in vitro assays without detectable effects on other PLA2 activities, blocks AA release in mammalian cells in the 0.01–1 µM range [29,52,53], and has been shown to be effective in experimental models of disease involving cPLA2α The differential involvement of phospholipid classes in AA and AdA mobilization suggests the existence of separate mechanisms for regulating the availability of each fatty acid. To characterize such mechanisms, we took advantage of the use of selective inhibitors of the two major intracellular PLA2 enzymes potentially effecting the fatty acid release in activated peritoneal macrophages, namely group IVA Ca2+-dependent cytosolic PLA2 (cPLA2α) and group VIA Ca2+-independent PLA2 (iPLA2-VIA) [22, 50]. The use of selective chemical inhibitors to address the role of intracellular PLA2s during cell activation has some advantages over other widely used methods, such as small interfering RNA or cells from knockout mice. With chemical inhibitors, inhibition develops rapidly, which reduces the impact of unspecific effects that could occur over time. Also, no compensatory mechanisms take place that might obscure the interpretation of results [22]. In addition, chemical inhibitors directly target PLA2 effects that depend on enzymatic activity, without affecting noncatalytic functions of the enzyme [51]. The inhibitors used in this work are the most potent and selective inhibitors currently available to block cPLA2α and iPLA2-VIA in cells. Pyrrophenone potently and selectively inhibits cPLA2α activity using a number of in vitro assays without detectable effects on other PLA2 activities, blocks AA release in mammalian cells in the 0.01–1 µM range [29,52,53], and has been shown to be effective in experimental

models of disease involving cPLA2α [54,55]. Fluoroketone FKGK18, a selective iPLA2-VIA inhibitor, is at least 200-fold more potent for inhibiting iPLA2-VIA than cPLA2α [30], and is useful for characterizing iPLA2-VIA-mediated functions in vivo [31,56,57]. β-Lactone GK436, another iPLA2-VIA inhibitor, has been found to be at least 1000-fold more potent for iPLA2-VIA than for cPLA2α [31]. For comparative purposes with data from the bibliography [22,58–64], we also used bromoenol lactone (BEL) to inhibit iPLA2-VIA. BEL inhibits calcium-independent PLA2s and exerts little or no effect on Ca2+-dependent enzymes [65,66], albeit it may exhibit off-target effects depending on cell type [67–69]. [54,55]. Fluoroketone FKGK18, a selective iPLA2-VIA inhibitor, is at least 200-fold more potent for inhibiting iPLA2-VIA than cPLA2α [30], and is useful for characterizing iPLA2-VIA-mediated functions in vivo [31,56,57]. β-Lactone GK436, another iPLA2-VIA inhibitor, has been found to be at least 1000-fold more potent for iPLA2-VIA than for cPLA2α [31]. For comparative purposes with data from the bibliography [22,58–64], we also used bromoenol lactone (BEL) to inhibit iPLA2-VIA. BEL inhibits calcium-independent PLA2s and exerts little or no effect on Ca2+-dependent enzymes [65,66], albeit it may exhibit off-target effects depending on cell type [67–69]. In agreement with previous observations [22,24], pyrrophenone was found to almost completely

In agreement with previous observations [22,24], pyrrophenone was found to almost completely inhibit zymosan-stimulated AA mobilization from phospholipids (Figure 4A). Importantly, pyrrophenone also partly inhibited AdA mobilization (Figure 4B). Conversely, none of the iPLA2-VIA inhibitors tested exerted appreciable effects on AA mobilization while significantly affecting AdA mobilization (Figure 4). The simultaneous addition of inhibitors of both cPLA2α and iPLA2-VIA resulted in complete inhibition of AdA mobilization (Figure 4B). These results suggest that unlike AA, AdA mobilization involves the action of both cPLA2α and iPLA2-VIA. inhibit zymosan-stimulated AA mobilization from phospholipids (Figure 4A). Importantly, pyrrophenone also partly inhibited AdA mobilization (Figure 4B). Conversely, none of the iPLA2- VIA inhibitors tested exerted appreciable effects on AA mobilization while significantly affecting AdA mobilization (Figure 4). The simultaneous addition of inhibitors of both cPLA2α and iPLA2-VIA resulted in complete inhibition of AdA mobilization (Figure 4B). These results suggest that unlike AA, AdA mobilization involves the action of both cPLA2α and iPLA2-VIA.

**Figure 4.** Release of AA and AdA by stimulated macrophages. The cells were either unstimulated (open bars) or stimulated by 1 mg/mL zymosan for 1 h (colored bars) in the absence (no inhibition) or presence of the following inhibitors: 2 µM pyrrophenone (Pyrr), 10 µM FKGK18, 5 µM GK436, 10 µM BEL, or 2 µM pyrrophenone plus 10 µM FKGK18 (see *x*-axis). Afterward, total content of AA (**A**) or AdA (**B**) was measured by GC/MS. The fatty acid release was calculated by subtracting the amount of phospholipid-bound AA or AdA in stimulated cells from that in unstimulated cells. Results are shown as mean values ± standard error of the mean. (n = 3). Note: \* *p* < 0.05, significantly different from zymosan-stimulated cells in the absence of inhibitors. **Figure 4.** Release of AA and AdA by stimulated macrophages. The cells were either unstimulated (open bars) or stimulated by 1 mg/mL zymosan for 1 h (colored bars) in the absence (no inhibition) or presence of the following inhibitors: 2 µM pyrrophenone (Pyrr), 10 µM FKGK18, 5 µM GK436, 10 µM BEL, or 2 µM pyrrophenone plus 10 µM FKGK18 (see *x*-axis). Afterward, total content of AA (**A**) or AdA (**B**) was measured by GC/MS. The fatty acid release was calculated by subtracting the amount of phospholipid-bound AA or AdA in stimulated cells from that in unstimulated cells. Results are shown as mean values ± standard error of the mean. (n = 3). Note: \* *p* < 0.05, significantly different from zymosan-stimulated cells in the absence of inhibitors.

#### *3.3. Studies with AdA-Enriched Cells 3.3. Studies with AdA-Enriched Cells*

Given the structural similarities between AA and AdA, it could be envisioned that in some instances, AdA competes with AA for incorporation into phospholipids, which might result in reduced amounts of "mobilizable" AA within phospholipids, and hence reduced formation of proinflammatory eicosanoids [16,70,71]. To address this possibility, we incubated the macrophages with exogenous AdA (10 µM, 20 h), which resulted in the cells avidly incorporating the fatty acid into cellular phospholipids. These conditions led to a 2–3-fold increase in the amount of AdA esterified into phospholipids compared with untreated cells (33.2 ± 4.6 nmol per mg protein; mean ± standard error of the mean, *n* = 4). Analysis by GC/MS of the fatty acid content of the AdA-enriched cells revealed that the fatty acids typically displaced within phospholipids by AdA upon AdA supplementation were oleic acid (18:1n-9) and the n-6 series members linoleic (18:2n-6) and dihomo-Given the structural similarities between AA and AdA, it could be envisioned that in some instances, AdA competes with AA for incorporation into phospholipids, which might result in reduced amounts of "mobilizable" AA within phospholipids, and hence reduced formation of pro-inflammatory eicosanoids [16,70,71]. To address this possibility, we incubated the macrophages with exogenous AdA (10 µM, 20 h), which resulted in the cells avidly incorporating the fatty acid into cellular phospholipids. These conditions led to a 2–3-fold increase in the amount of AdA esterified into phospholipids compared with untreated cells (33.2 ± 4.6 nmol per mg protein; mean ± standard error of the mean, *n* = 4). Analysis by GC/MS of the fatty acid content of the AdA-enriched cells revealed that the fatty acids typically displaced within phospholipids by AdA upon AdA supplementation were oleic acid (18:1n-9) and the n-6 series members linoleic (18:2n-6) and dihomo-γ-linolenic (20:3n-6) acids

γ-linolenic (20:3n-6) acids (Figure 5). Remarkably, little AA displacement occurred after AdA

(Figure 5). Remarkably, little AA displacement occurred after AdA supplementation. Analysis of AA and AdA mobilization in zymosan-stimulated cells indicated that the extent of the AA response did not substantially differ from that observed from cells not treated with AdA, while AdA mobilization expectedly increased (Figure 6). supplementation. Analysis of AA and AdA mobilization in zymosan-stimulated cells indicated that the extent of the AA response did not substantially differ from that observed from cells not treated with AdA, while AdA mobilization expectedly increased (Figure 6). supplementation. Analysis of AA and AdA mobilization in zymosan-stimulated cells indicated that the extent of the AA response did not substantially differ from that observed from cells not treated with AdA, while AdA mobilization expectedly increased (Figure 6).

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