Adipocyte P2Y14 receptors play a key role in regulating whole-body glucose and lipid homeostasis

Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.


Introduction
Accumulation of excess nutrient energy as triacylglycerol (TAG) in adipocytes results in the development of obesity. Obesity is a key risk factor for the pathogenesis of insulin resistance and type 2 diabetes (T2D) and can aggravate metabolic complications such as nonalcoholic fatty liver disease and cardiovascular diseases (1). Therefore, identifying drug targets for improving the metabolic function of adipocytes is of utmost importance to prevent the metabolic deficits caused by obesity.
Net lipid storage in adipocytes and adipose tissue mass depends on a fine balance between anabolic and catabolic lipid pathways. Following nutrient intake, de novo fatty acid (FA) synthesis from glucose and amino acids is induced, and synthesis of TAG promotes lipid storage in adipocytes. Increased post-feeding circulating insulin levels inhibit TAG breakdown and lipolysis in adipocytes (2). In contrast, adipocyte lipolysis provides alternative energy substrates (FAs and glycerol) to other tissues during periods of caloric deprivation (e.g., fasting). Lipolysis is a highly regulated process that requires the activation of different lipases (2). Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the 2 major lipases converting TAG to diacylglycerol (DAG) and DAG to monoacylglycerol (MAG), respectively, with the liberation of a FA at each step (2). MAG is hydrolyzed to release the final FA and glycerol. The rate of basal lipolysis is increased in obesity resulting in elevated plasma FA levels that are predicted to contribute to the development of insulin resistance (3,4). However, the activation of lipolysis by factors such as catecholamines activating β-adrenergic receptors is greatly impaired in obesity (5,6).
Adipocyte metabolism is regulated by various GPCRs that are coupled to different functional classes of heterotrimeric G proteins (G s , G q , or G i ) (7,8). Purinergic (P2Y) receptors are a class of GPCRs that are activated by nucleotides and nucleotide sugars (9). In the obese state, high plasma levels of uracil Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. G icoupled P2Y 14 receptor (P2Y 14 R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated G i -type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y 14 Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y 14 R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y 14 R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y 14 R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y 14 R antagonist. In conclusion, we demonstrate that adipose P2Y 14 Rs were critical regulators of wholebody glucose and lipid homeostasis, suggesting that P2Y 14 R antagonists might be beneficial for the therapy of obesity and T2D.
JCI Insight 2021;6(10):e146577 https://doi.org/10.1172/jci.insight.146577 nucleoside/nucleotides and nucleotide sugars have been reported to act on P2Y receptors (10,11). G i -coupled receptors such as the CB 1 cannabinoid receptor, GPR109A, and GPR81 are expressed in adipocytes and have been implicated in the regulation of adipocyte function such as lipolysis, insulin resistance, inflammation, and adipokine secretion (12)(13)(14). P2Y 14 R is a G i -coupled receptor activated by UDP-glucose (15,16). P2Y 14 R is expressed in human tissues, including adipose tissue, stomach, intestine, placenta, and certain brain regions (17). P2Y 14 R is also markedly expressed in immune cells and plays a significant role in regulating innate immunity and inflammation (18)(19)(20). However, the contribution of adipocyte P2Y 14 R in the development of obesity and associated metabolic deficits remains unexplored.
To address this issue, we generated a mutant mouse model lacking P2Y 14 R specifically in adipocytes to examine the receptor's role in adipocyte tissue physiology and its effect on whole-body glucose and energy homeostasis. Our data show that the lack of P2Y 14 R in adipocytes results in enhanced lipolysis during fasting conditions. This enhancement of lipolysis protected adipocyte-specific P2Y 14 R KO mice against diet-induced obesity (DIO), improving whole-body glucose and energy homeostasis. These findings suggest that P2Y 14 R may prove a useful target for the treatment of obesity and obesity-related metabolic disorders.

Results
P2Y 14 R expression level correlated with human obesity. P2Y 14 R is expressed in human adipose tissue (17). We first examined whether the P2Y 14 R expression level was altered in subcutaneous fat of obese human subjects suffering from insulin resistance compared with individuals (age-, race-, and sex-matched) without obesity and insulin resistance. Interestingly, P2Y 14 R expression levels were significantly increased in fat tissues of obese compared with the lean subjects ( Figure 1A). Linear regression analysis showed a correlation between P2Y 14 R expression levels and BMI of human subjects under study ( Figure 1B). P2Y 14 R expression was also correlated with fat (%) of lean and obese human subjects ( Figure 1C). These data suggest that human P2Y 14 R may have played a critical role in the adipocyte biology and development of obesity and associated pathophysiological deficits. P2Y 14 R expression was regulated by high-fat diet in mouse adipocytes. To determine the role of P2Y 14 R in adipose tissue physiology, we first quantified P2Y 14 R mRNA expression levels in different mouse adipose depots. RT-PCR analysis revealed that P2Y 14 R had similar expression levels in subcutaneous (iWAT), visceral (eWAT), and brown (BAT) adipose tissues ( Figure 1D). We next determined whether consumption of a high-fat diet (HFD) regulates P2Y 14 R expression levels in mouse adipocytes. Interestingly, P2Y 14 R mRNA levels were upregulated in isolated mature adipocytes from iWAT and eWAT but not BAT of HFD mice ( Figure 1E). The upregulation of P2Y 14 R expression in WAT of HFD mice may indicate that P2Y 14 R signaling played a role in regulating WAT function during obesity. P2Y 14 R activation inhibited lipolysis in mature adipocytes. Many GPCRs mediate lipolytic or antilipolytic effects through the regulation of adipocyte cAMP levels (21). Because P2Y 14 R couples to G i -type G proteins (15), we next studied the effect of P2Y 14 R activation on cAMP levels in mouse adipocytes. Preadipocytes from mouse iWAT were isolated and differentiated into mature adipocytes to study adipocyte lipolysis. As expected, P2Y 14 R activation by MRS2905, a selective P2Y 14 R agonist (22), inhibited the increase in cAMP accumulation caused by treatment with CL-316,243, a β 3 -adrenergic receptor agonist (% reduction by 1 nM MRS2905: 66.04 ± 3.93) ( Figure 1F). Similarly, stimulation with MRS2905 decreased CL-316,243-induced glycerol release in differentiated mouse mature adipocytes (% reduction: 21.02 ± 3.24) ( Figure 1G). Further, cells were treated with PPTN (P2Y 14 R antagonist, 200 nM, 30 min) (23), and the effect of adrenergic stimulation with or without insulin on lipolysis was studied. Blockade of P2Y 14 R with PPTN showed a trend toward enhanced CL-316,243-mediated lipolysis (% increase: 25.96 ± 6.47), possibly due to increased cAMP level in cells ( Figure 1H). Acute stimulation with insulin decreased CL-316,243-mediated lipolysis to similar levels in both control and PPTN-treated cells, indicating insulin-independent regulation of lipolysis by P2Y 14 R ( Figure  1H). Mechanistically, CL-316,243-induced phosphorylation of ATGL was decreased by P2Y 14 R activation in differentiated mature adipocytes (iWAT; % reduction: 60.67 ± 13.77; Figure 1, I and J). Together, these results suggest that the activation of P2Y 14 R inhibited lipolysis in differentiated mature adipocytes in vitro.
Acute activation of P2Y 14 R decreased lipolysis in vivo. To examine whether the acute activation of P2Y 14 R was able to regulate lipolysis, we injected fasted (overnight) HFD control mice with P2Y 14 R agonist (MRS2905, 10 mg/kg, i.p.) or saline (Figure 2A). MRS2905 treatment resulted in a significant reduction in plasma free fatty acid (FFA) levels 45 Figure 2B). Blocking P2Y 14 R with the antagonist prodrug MRS4741 (24) showed a trend toward rescuing the decrease in plasma FFA levels caused by the activation of P2Y 14 R by MRS2905 (Figure 2, C and D). In summary, acute activation of P2Y 14 R decreased lipolysis in vivo, resulting in reduced plasma FFA levels.
Lack of adipocyte P2Y 14 R protected against HFD-induced obesity and improves metabolism. Impaired regulation of lipolysis is associated with the development of obesity and insulin resistance (2). Because P2Y 14 R activation inhibits lipolysis, we next examined the physiological relevance of this phenomenon on whole-body glucose and energy homeostasis. To this end, we generated a mouse model lacking P2Y 14 R specifically in adipocytes (adipo-P2Y 14 Δ/Δ mice) ( Figure 3A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.146577DS1). Adipo-P2Y 14 Δ/Δ mice and their control littermates ) were maintained on regular mouse chow (RC) or an HFD. On RC, adipo-P2Y 14 Δ/Δ and control mice did not exhibit any significant differences in body weight or fat and lean mass, as determined by an MRI scan (Supplemental Figure 1, B and C). Similarly, adipo-P2Y 14 Δ/Δ and control mice consuming RC displayed no differences in glucose tolerance, insulin sensitivity, fed and fasted blood glucose levels, or plasma insulin levels (Supplemental Figure 1, D and E, and Supplemental Figure 2, A and B). Fasted adipo-P2Y 14 Δ/Δ mice showed a trend toward higher plasma FFA levels compared with fasted RC control mice, although this difference did not reach statistical significance (P = 0.18; Supplemental Figure 2C). No differences in plasma FFA levels were found in the fed state between the 2 mouse groups (Supplemental Figure 2C). Circulating levels of adipokines such as leptin and adiponectin were similar between the groups on RC (Supplemental Figure 2, D and E).
In striking contrast, adipo-P2Y 14 Δ/Δ mice maintained on an HFD gained less weight than control mice ( Figure 3B). MRI analysis revealed that the difference in body weight was due to a decrease in fat mass in adipo-P2Y 14 Δ/Δ mice ( Figure 3C). No difference in lean mass was observed between the 2 groups ( Figure  3C). H&E staining revealed reduced adipocyte size (iWAT and eWAT) in adipo-P2Y 14 Δ/Δ mice, compared with HFD control mice (Supplemental Figure 3). Moreover, HFD adipo-P2Y 14 Δ/Δ mice showed significantly improved glucose tolerance and insulin sensitivity, most likely due to reduced adiposity (Figure 3, D-G). HFD adipo-P2Y 14 Δ/Δ mice also displayed significantly decreased fasting blood glucose and fed plasma insulin levels, indicative of improved whole-body glucose homeostasis ( Figure 3, H and I). Plasma leptin levels were decreased in the fasted state in HFD adipo-P2Y 14 Δ/Δ mice (Supplemental Figure 4A), whereas adiponectin levels were increased in both fasted and fed states (Supplemental Figure 4B). Consistently, mRNA levels of leptin were decreased in both iWAT and eWAT of HFD adipo-P2Y 14 Δ/Δ mice (Supplemental Figure 4C). mRNA levels of adiponectin were increased in eWAT of HFD adipo-P2Y 14 Δ/Δ mice (Supplemental Figure  4D). Further, the acute activation of P2Y 14 R also decreased circulating levels of adiponectin (Supplemental Figure 4E), suggesting P2Y 14 R-mediated regulation of adiponectin secretion.  Figure 5). Adipocyte-specific P2Y 14 Figure 4A). In contrast, no difference in plasma FFA levels was observed between the 2 groups under fed conditions ( Figure 4A). Fasting also caused a significant decrease in P2Y 14 R mRNA expression in eWAT and BAT of HFD mice ( Figure 4B). These data indicate that the lack of P2Y 14 R in adipocytes acutely increased lipolysis to provide energy substrates to other organs during nutritional deficient conditions. This enhancement in lipolysis during fasting may have contributed to the reduced fat mass observed with HFD adipo-P2Y 14 Δ/Δ mice. Δ/Δ and control mice (n = 4-6/group). The expression of 18s rRNA was used to normalize qRT-PCR data. All data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 (A, C, E, and G-L: 2-tailed Student's t test; B, D, and F: 2-way ANOVA followed by Bonferroni's post hoc test). All experiments were conducted on mice maintained on an HFD for at least 8 weeks. Scale bar: 150 µm. P2Y, purinergic; DIO, diet-induced obesity; HFD, high-fat diet; GTT, glucose tolerance test; ITT, insulin tolerance test.
JCI Insight 2021;6(10):e146577 https://doi.org/10.1172/jci.insight.146577 Next, to decipher the mechanism by which P2Y 14 R regulates lipolysis, we analyzed the activity of HSL and ATGL in adipose depots of fasted HFD adipo-P2Y 14 Δ/Δ mice and control mice. The expression levels of total HSL (T-HSL) were significantly enhanced in iWAT of fasted adipo-P2Y 14 Δ/Δ mice ( Figure 4, C and E). The expression of the activated form of HSL (p-HSL; S563) tended to be increased (P = 0.07) in iWAT of fasted HFD adipo-P2Y 14 Δ/Δ mice (Figure 4, C and E). On the other hand, the expression levels of total ATGL (T-ATGL) were not significantly different between mutant and control mice (iWAT; Figure 4, C and E). However, the expression of an activated form of ATGL (p-ATGL; S406) was significantly enhanced in iWAT of fasted HFD adipo-P2Y 14 Δ/Δ mice (Figure 4, C and E). In eWAT, no difference was observed in T-HSL and T-ATGL between mutant and control mice (Figure 4, D and F). The expression levels of p-HSL were significantly enhanced in eWAT of fasted HFD adipo-P2Y 14 Δ/Δ mice (Figure 4, D and F). On the other hand, no significant difference in ATGL phosphorylation was observed in eWAT of fasted mice (Figure 4, D and F). These data indicate that P2Y 14 R KO enhances lipolysis in nutrient-deficient conditions by increasing the activity of HSL and ATGL enzymes in WAT.
Enhanced energy expenditure in HFD adipo-P2Y 14 Δ/Δ mice. Lipolysis is essential for energy homeostasis in mice (25,26). The observation that P2Y 14 R regulates cAMP levels and lipolysis in adipocytes led us to examine the role of adipose P2Y 14 R on energy homeostasis. Adipo-P2Y 14 Δ/Δ and control mice were subjected to indirect calorimetry analysis during regular diet and the first week of HFD feeding when the 2 mouse groups did not differ significantly in body weight. The effects of body weight and body composition on energy expenditure (EE) was normalized by plotting EE to BW (or fat/lean body mass) (27). ANCOVA analysis revealed no difference in EE between regular diet fed control and adipo-P2Y 14 Δ/Δ mice (Supplemental Figure 6, A-C). Oxygen consumption (VO 2 ) and EE (normalized to BW) were modestly but significantly increased in HFD adipo-P2Y 14 Δ/Δ mice (Figure 4, G and H). Normalizing EE to fat mass in HFD control and adipo-P2Y 14 Δ/Δ mice did not show significant difference in EE between the groups (Supplemental Figure 6D), whereas normalizing EE to lean mass showed a trend toward enhanced EE in adipo-P2Y 14 Δ/Δ mice (Supplemental Figure 6E). No differences in food intake and locomotor activity were observed between the 2 groups of mice (Figure 4, I and J). These data suggest that the increased availability of FFA due to enhanced lipolysis in adipo-P2Y 14 Δ/Δ mice may have caused increased EE and most likely resulted in reduced adiposity displayed by HFD adipo-P2Y 14 Δ/Δ mice. HFD adipo-P2Y 14 Δ/Δ mice were protected from hepatic steatosis and insulin resistance. Increased plasma FFA levels are associated with the development of hepatic steatosis and insulin resistance. Because fasting FFA levels were increased in adipo-P2Y 14 Δ/Δ mice, we next studied whether HFD adipo-P2Y 14 Δ/Δ mice develop liver steatosis and insulin resistance. Surprisingly, HFD adipo-P2Y 14 Δ/Δ mice displayed decreased liver weight, compared with HFD control mice ( Figure 5A). In agreement with this observation, HFD adipo-P2Y 14 Δ/Δ mice showed a significant decrease in liver triglyceride content in both fed and fasted conditions ( Figure 5B and Supplemental Figure  7A). Decreased liver steatosis was further confirmed by H&E staining of liver sections. The liver sections from fed and fasted adipo-P2Y 14 Δ/Δ mice displayed reduced lipid deposition than liver sections from control mice (Figure 5C and Supplemental Figure 7B). Oil red O staining of liver sections from fasted mice confirmed reduced lipid deposition in adipo-P2Y 14 Δ/Δ compared with the control mice (Supplemental Figure 7B). The mRNA levels of genes involved in hepatic triglyceride accumulation, including Srebp1 and Fas, were also reduced in HFD adipo-P2Y 14 Δ/Δ mice ( Figure 5D). To study insulin signaling in hepatic tissue, we injected HFD adipo-P2Y 14 Δ/Δ and control mice with saline or insulin (5 U/mouse, i.v.) and collected liver tissues 5 minutes later. Immunoblotting studies indicated enhanced phosphorylation of AKT and GSK3β in the liver of HFD adipo-P2Y 14 Δ/Δ mice (Figure 5, E and F). In summary, these data indicate that the lack of P2Y 14 R in adipocytes protected mice from the development of liver steatosis and hepatic insulin resistance. Reduced obesity and enhanced adiponectin levels may have contributed to improved liver function observed in HFD adipo-P2Y 14 Δ/Δ mice. Lack of P2Y 14 R improved adipose tissue insulin sensitivity. We next analyzed the effect of adipocyte P2Y 14 R deficiency on insulin sensitivity of different adipose tissues. To this end, we injected HFD adipo-P2Y 14 Δ/Δ and control mice with saline or insulin (5 U/mouse, i.v.) and collected eWAT, iWAT and BAT 10 minutes later. Western blotting studies showed that the lack of P2Y 14 R in eWAT enhanced insulin-induced phosphorylation of AKT and GSK3β, suggesting improved insulin sensitivity ( Figure  6, A and B). Enhanced phosphorylation of AKT was also observed in iWAT from HFD adipo-P2Y 14 Δ/Δ mice ( Figure 6, C and D). Similarly, phosphorylation of AKT and GSK3β was increased in BAT of HFD adipo-P2Y 14 Δ/Δ mice ( Figure 6, E and F). Together, these results indicate that the lack of P2Y 14 R in adipocytes improved insulin sensitivity in iWAT, eWAT, and BAT in mice consuming an HFD.

Discussion
Obesity is associated with an increase in basal lipolysis and elevated plasma FFA levels (3,4). Chronic unrestrained lipolysis mediates the development of insulin resistance, T2D, and fatty liver disease (28)(29)(30). Restrained lipolysis has been shown to protect against DIO by enhancing FA oxidation and energy expenditure (31)(32)(33). In this study, we identified a P2Y 14 R-mediated regulation of adipocyte lipolysis. We demonstrated that the inhibition of P2Y 14 R signaling in adipocytes increases lipolysis under fasting conditions and protects mice against DIO and associated metabolic deficits.
Regulation of lipolysis occurs in response to fluctuating metabolic conditions and nutrient intake and is mediated by hormonal and biochemical signals. Several GPCRs have been identified that play a role in regulating the rate of adipocyte lipolysis (21). Fasting triggers lipolysis and elevated plasma FFA and glycerol levels to provide substrates for oxidative metabolism in other metabolically important tissues. For example, the activation of adipocyte G s -coupled β-adrenergic receptors results in cAMP-mediated activation of PKA, resulting in the phosphorylation and activation of HSL and enhanced lipolysis. However, β-adrenergic receptor-stimulated lipolysis is impaired in obesity (5). Stimulation of adipocyte G i -coupled receptors has been shown to mediate antilipolytic effects via decrease in cAMP levels (21). In this study, we showed that stimulation of the G i -coupled P2Y 14 R decreased adipocyte cAMP levels. Further, P2Y 14 R activation resulted in reduced lipolysis due to decreased phosphorylation of lipolytic enzymes (ATGL and HSL). In vivo lack of P2Y 14 R specifically in adipocytes resulted in increased plasma FFA levels in the fasting state, with no effect on FFA levels observed in freely fed mice. In agreement with this observation, HFD adipo-P2Y 14 Δ/Δ mice showed enhanced phosphorylation of ATGL and HSL only under fasting conditions. Interestingly, fasting induced the downregulation of P2Y 14 R expression levels in eWAT and BAT of HFD mice. These data suggest that the downregulation of P2Y 14 R in WAT during fasting is essential for the activation of lipolytic enzymes, thus stimulating lipolysis.
DIO increased the expression of P2Y 14 R in mouse WAT, indicative of a potential role of this receptor in the development of obesity. Moreover, plasma UDP-glucose levels were increased in obese mice (34), indicating enhanced activity of P2Y 14 R in obese mice. In agreement with this concept, adipocyte specific P2Y 14 R KO mice were protected from DIO, indicating that adipocyte P2Y 14 R deficiency prevented fat accumulation by enhancing lipolysis. Decreased adiposity greatly improved glucose tolerance and insulin sensitivity in HFD adipo-P2Y 14 Δ/Δ mice. Xu et al. reported that whole-body HFD P2Y 14 R null mice showed improved insulin sensitivity in muscle, liver, and adipose tissue, but they did not observe any changes in overall fat mass (34). The authors further reported reduced infiltration of immune cells in liver of P2Y 14 R null mice (34). In this study, we observed marked reduced inflammation in eWAT of adipo-P2Y 14 Δ/Δ mice. Interestingly, HFD adipo-P2Y 14 Δ/Δ mice displayed a modest increase in oxygen consumption and total energy expenditure. This observation agrees with recent findings that elevated plasma FFA levels can promote FA oxidation, thus increasing energy expenditure and providing protection against obesity (31)(32)(33). Our data are consistent with a study showing that mice lacking G i -coupled CB 1 receptor showed reduced adiposity, improved insulin sensitivity, and enhanced energy expenditure (12). Surprisingly, enhanced lipolysis due to the lack of adipocyte P2Y 14 R did not result in ectopic lipid deposition in the liver. In contrast, liver weight and triglyceride content were reduced in adipo-P2Y 14 Δ/Δ mice, leading to improved hepatic insulin sensitivity. Taken together, these data demonstrate that the lack  of adipocyte P2Y 14 R increased lipolysis in the fasting state and protected against DIO, insulin resistance, adipose inflammation, and liver steatosis. G i -coupled receptors such as GPR84 and CB 1 have been shown to stimulate the secretion of adipokines such as adiponectin (12,35,36). In our study, we observed a marked increase in mRNA expression and circulating levels of adiponectin in HFD adipo-P2Y 14 Δ/Δ mice. Further, treatment of HFD WT mice with P2Y 14 R agonist reduced plasma adiponectin levels. Adiponectin is an antidiabetic adipokine, which has an insulin sensitizing effect on different metabolic tissues (37) and can prevent the development of hepatic steatosis (38). Together, these data suggest that adipocyte P2Y 14 R deficiency enhances circulating levels of adiponectin that contribute to improved systemic insulin sensitivity. In addition, enhanced adiponectin levels may contribute to the observed protection against hepatic lipid accumulation displayed by HFD adipo-P2Y 14 Δ/Δ mice, thereby improving metabolism in HFD adipo-P2Y 14 Δ/Δ mice. A recent study reported that the lack of G i signaling in adipocytes leads to a chronic increase (fed and fasted) in lipolysis, resulting in the development of insulin resistance and impaired glucose tolerance on an obesogenic diet (39). In this study, we identified a pathway where the lack of the G i -coupled P2Y 14 R specifically in adipocytes led to an increase in lipolysis only in the fasting state, thus protecting mice against DIO and improving glucose tolerance and insulin sensitivity. HFD adipo-P2Y 14 Δ/Δ mice further displayed reduced inflammation, hepatic steatosis, increased adiponectin levels, and enhanced energy expenditure (Figure 7). P2Y 14 R is predominantly activated by UDP-glucose and other nucleotide sugars (17,40). Recently, a study revealed that P2Y 14 R can also be potently activated by UDP (41), a nucleotide diphosphate that is a native agonist for Gq-coupled P2Y 6 R (42). Hence, because both receptors share a common agonist, it would be interesting to understand their role in regulating adipocyte function and whole-body glucose and lipid homeostasis. A recent study from our lab demonstrated that mouse adipocytes express functional P2Y 6 R (43). KO of P2Y 6 R specifically in adipocytes protected mice from DIO and decreased systemic inflammation, improving whole-body glucose metabolism. Mechanistically, the lack of P2Y 6 R in adipocytes decreased JNK activation and increased expression and activity of PPARα (43). Interestingly, the lack of P2Y 14 R specifically in adipocytes also protected mice from DIO, improving whole-body metabolism. However, P2Y 14 R is a Gi-coupled receptor and the lack of the receptor enhanced adipocyte lipolysis (fasting) and adiponectin levels in vivo. KO of P2Y 6 R or P2Y 14 R in adipocytes protected mice from the development of liver steatosis associated with obesity. Taken together, these studies suggest that the development of dual antagonist(s) for these receptors or combination therapy with antagonists of both receptors may prove useful in treatment of obesity and T2D.
P2Y 14 R antagonists have been under development for pulmonary diseases and acute kidney injury (44), as well as -potentially -pain and other inflammatory conditions (45). Known antagonists have demonstrated favorable preclinical safety. In this study, acute treatment of mice with P2Y 14 R antagonist prodrug (MRS4741) showed a trend in the decrease in plasma FFA levels caused by treatment of mice with P2Y 14 R agonist (MRS2905). These data show that P2Y 14 R antagonist mimicked the effect of genetic loss of P2Y 14 R in adipocytes and may have been used to enhance receptor-mediated lipolysis, thus providing protection against obesity. Treatment of metabolic conditions with a P2Y 14 R antagonist would have the added benefit of improving β cell function including promoting the release of insulin (46). These findings provide a rational basis for the development of P2Y 14 R antagonists for the treatment of obesity and T2D.
Mouse maintenance and DIO. Adult mice were used for all experiments reported in this study. Mice were kept on a 12-hour light and 12-hour dark cycle. Animals were maintained at room temperature (23°C) on standard chow (7022 NIH-07 diet, 15% kcal fat, energy density 3.1 kcal/g; Envigo). Mice had free access to food and water. To induce obesity, groups of mice were switched to an HFD (F3282, 60% kcal fat, energy density 5.5 kcal/g; Bio-Serv) at 8 weeks of age. Mice consumed the HFD for at least 8 weeks, unless stated otherwise. Human subcutaneous adipose tissue. The human adipose samples used in this study were described in a previous study by Pydi et al. (47). Briefly, subcutaneous adipose tissues were obtained from the abdominal area of human subjects under local anesthesia using an aspiration needle. RNA was isolated from fat tissues and gene expression analysis was performed as described above.
In vivo metabolic phenotyping. Glucose tolerance tests (GTTs) were carried out on mice after a 12-hour overnight fast. After checking fasted blood glucose levels, animals were injected i.p. with glucose (1 or 2 g/ kg as indicated), and blood was collected from the tail vein at 15, 30, 60, and 120 minutes after injection. Blood glucose concentrations were determined using a Contour portable glucometer (Bayer). Insulin tolerance tests (ITTs) were also carried out on mice fasted overnight for 12 hours. Fasted blood glucose levels were determined, and then mice were injected i.p. with human insulin (0.75 or 1 U/kg; Humulin, Eli Lilly) as indicated. Blood glucose levels were determined as described above for GTT. All metabolic tests were performed with adult mice that were at least 8 weeks old.
Body composition analysis. Mouse body mass composition (lean vs. fat mass) was measured using a 3-in-1 Echo MRI Analyzer (Echo Medical System).
RNA extraction and gene expression analysis. Dissected tissues were frozen immediately on dry ice. Total RNA from tissues was extracted using the RNeasy Mini Kit (QIAGEN). Total RNA (500 ng of RNA) was converted into cDNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Thermo Fisher Scientific). Quantitative PCR was performed using the SYBR green method (Applied Biosystems, Thermo Fisher Scientific). Gene expression was normalized to the expression of 18s rRNA using the ΔΔCt method. The primer sequences used in this study are provided in Supplemental Table 1.
Plasma metabolic profiling. Blood was collected from the tail vein of mice in chilled K 2 -EDTA-containing tubes (RAM Scientific). Blood was collected from a group of mice that had free access to food (fed state) or from overnight fasted mice. Blood was centrifuged at 4°C for 10 minutes at 10,000g to obtain plasma. ELISA kit (Crystal Chem) was used to measure plasma insulin levels, following the manufacturer's instructions. Leptin and adiponectin levels were measured using ELISA kits from R&D Systems, Bio-Techne. Plasma FFA levels were determined using a commercially available kit (MilliporeSigma).
Isolation, culture, and differentiation of white adipocytes. White preadipocytes were isolated, cultured, and differentiated as described previously (43). Excised fat depot was minced into small pieces and digested at 37°C for 45   3.3 mg/mL collagenase I (MilliporeSigma). Other chemicals were obtained as reagent grade (MilliporeSigma), unless noted. After digestion, 10 mL of KRH buffer was added, and the cell suspension was filtered through a 70 μm cell strainer, followed by centrifugation at 700g for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was resuspended in KRH buffer and recentrifuged to obtain a cell pellet of mesenchymal stem cells. The pellet was resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin (pen-strep). Approximately 80,000 cells were seeded per well of collagen I-coated 12-well plates (Corning) and cultured at 37°C, 10% CO 2 . Cells were allowed to reach 100% confluence by replenishing media every second day. Two days after reaching confluence, cells were induced for differentiation using DMEM supplemented with 10% FBS, 1% pen-strep, 0.5 μM insulin, 250 μM 3-isobutyl-1-methylxanthine (IBMX), 2 μM troglitazone, 0.5 μM dexamethasone, and 60 μM indomethacin. After 72 hours' exposure to the induction media, cells were incubated in differentiation DMEM media supplemented with 10% FBS, 1% pen-strep, and 0.5 μM insulin for the next 72 hours. Mature differentiated white adipocytes were used for further studies.
Isolation of mature adipocytes. The isolation of mature mouse adipocytes was performed as described previously (47). In brief, mouse fat pads were collected and digested with KRH buffer containing collagenase 1 (3 mg/mL). Digested tissues were filtered through a 250 μm cell strainer. After a 5-minute centrifugation step at 60g, the top layer containing mature adipocytes was collected and used for further experiments.
cAMP assay. Differentiated primary white adipocytes were washed twice with DPBS and serum starved in incomplete DMEM for 2 hours. Cells were incubated with agonist MRS2905 (2 nM, Tocris, Bio-Techne) for 30 minutes in DMEM+IBMX (100 μM) as indicated at 37°C. Subsequently, cells were treated with CL-316,243 (10 nM) for 30 minutes as indicated. Cells were lysed using 0.05N HCl, and intracellular cAMP levels were quantified using a detection kit from Cisbio Bioassays.
Lipolysis assay. Differentiated primary white adipocytes were washed twice with DPBS, followed by serum starvation in DMEM for 2 hours at 37°C. Cells were stimulated with P2Y 14 R agonist (MRS2905, 2 nM) for 30 minutes, followed by treatment with CL-316,243 (10 nM) for another 30 minutes, as indicated. In another set of experiments, cells were treated with vehicle or PPTN (200 nM; Tocris, Bio-Techne) for 30 minutes, followed by insulin (10 nM) for 15 minutes as indicated. Cells were then stimulated with CL-316,243 (10 nM) for another 30 minutes, as indicated. To terminate the reaction, the cell culture plates were incubated on ice, and cell media were collected for the measurement of glycerol levels using a glycerol assay kit (MilliporeSigma). Cells were lysed in RIPA buffer, and protein concentrations were determined for each well. Glycerol levels were normalized to protein levels.
In vivo insulin signaling. HFD adipo-P2Y 14 Δ/Δ and control mice were fasted for 4 hours. Mice were then anesthetized using isoflurane, and the abdominal cavity exposed to access the inferior vena cava. Subsequently, 5 U of human insulin (Humulin, Eli Lilly) dissolved in 100 μL of 0.9% saline was injected into the vena cava (48). Tissues including liver, adipose, and skeletal muscle were harvested 5-10 minutes after injection and snap-frozen in liquid nitrogen for Western blot studies.
Liver triglyceride content. Liver triglyceride levels were measured by homogenizing 20 mg of hepatic tissue in PBS. A chloroform/methanol (2:1) mixture was then added to the liver homogenate. The homogenate was centrifuged, and the organic phase was transferred to a new tube and dried overnight. Each sample was dissolved in ethanol containing 1% Triton X-100, and triglyceride levels were measured using a triglyceride reagent (MilliporeSigma). Triglyceride levels were normalized to protein levels in liver homogenates.
Western blot studies. Western blot studies were carried out as described previously (43). Briefly, adipocytes or adipose tissues were homogenized in adipocyte lysis buffer (50 mM Tris pH 7.4, 500 mM NaCl, 1%