The purinergic P2Y₂ receptor (P2Y₂R) is a G-protein coupled receptor that responds to extracellular nucleotides. Extracellular nucleotides, including ATP and UTP, activate P2Y₂R, regulating renal function, fibrosis, neural development, and inflammatory processes, including leukocyte recruitment (1, 2). Notably, the P2Y₂ receptor has been implicated in various inflammatory disease processes such as atherosclerosis, obesity, and diabetes (3). Under inflammatory conditions, the P2Y₂ receptor has been found to blunt glucose sensitivity, as demonstrated by studies using male P2Y₂ receptor knockout mice (P2Y₂R^-/-) fed a high-fat diet, which showed significantly lower blood glucose at steady state and during a glucose tolerance test (GTT) compared to wild-type mice (4). This implies that the absence or inhibition of the P2Y₂ receptor activity could enhance glucose tolerance, potentially benefiting individuals with impaired glucose metabolism or insulin resistance.

Glucose regulation is a critical to maintaining energy balance and overall metabolic health. The clearance of glucose from the blood is mainly accomplished through the uptake of glucose by various tissues, particularly skeletal muscle and adipocytes. Insulin, a hormone produced by the pancreas, plays a key role in glucose homeostasis by signaling the translocation of glucose transporter 4 (GLUT4) to the cell surface, promoting glucose uptake by target tissues. Skeletal muscle, being the major site of insulin-mediated glucose disposal, contributes significantly to the regulation of blood glucose levels (5). While insulin dependent trafficking of GLUT4 through the insulin receptor (INSR) is an important regulator of glucose absorption, a dysregulation of GLUT4 and INSR expression has been noted in adipose tissue from P2Y₂R null mice fed a high fat diet (4). A role for P2Y₂R receptor activation in regulation of insulin signaling was recently confirmed when activation of P2Y₂R in hepatocytes disrupted insulin dependent signaling, likely through competition for phosphatidylinositol 4,5-bisphosphate (PIP₂), an intermediate required for both signaling pathways (6).

Several studies have reported variations in insulin sensitivity, glucose metabolism, and the prevalence of metabolic disorders between males and females (7). Women have a higher insulin sensitivity (8). In mice, females have a greater glucose sensitivity during glucose tolerance testing (9, 10). Hormonal differences, genetic factors, and body composition are among the things that likely contribute to these sex-related disparities. However, the underlying mechanisms and specific receptors involved in the sexual dimorphism of glucose regulation remain incompletely understood. Moreover, there remains limited information regarding the potential sex differences in the role of the P2Y₂R in glucose homeostasis.

Sex difference in immune responses have been widely observed. For example, human biological females are more susceptible to autoimmune disease (11) and males have higher mortality with COVID-19 infection (12). Various cytokines have been shown to have sex-dependent expression. Female mice have greater circulating cytokines, such as IL-6, IL-10 and TNFα, in response to lipopolysaccharide (LPS) administration (13, 14). LPS, or endotoxin, induces acute inflammatory challenge by binding to toll like receptor 4 (TLR4), initiating the signaling cascade that activates proinflammatory cytokines, and also leads to hypoglycemia (15). Several studies have shown that LPS administration upregulates P2Y₂R (16–18). TLR4 is expressed at a higher level on macrophages from male mice (13) and is stimulated to a greater extent on macrophages of male mice in the acute stages of viral infection (19) and or after LPS challenge (13).

Despite the emerging understanding of the P2Y₂ receptor’s impact on glucose homeostasis, inflammation, and the established sex differences in both, there is a notable lack of information regarding sex differences in P2Y₂R activity in vivo. Understanding whether there are sex-specific differences in the P2Y₂R influence on glucose homeostasis could have important implications for basic research design, but also for designing treatment strategies for metabolic disorders or inflammatory diseases. Here, we provide the first sex-inclusive comparison of glucose tolerance in P2Y₂R null and wild-type mice, in the unstimulated and acute inflammatory state. We find that the receptor plays a male-specific role in influencing glucose homeostasis in the fasted state and during acute inflammation.

P2Y₂R plays a male-specific role in regulating glucose metabolism. Male P2Y₂R deficient animals have reduced tolerance to glucose administration compared to wild-type males, and all female mice ( Figures 1A, C ), suggesting that the receptor plays an important role in glucose sensitivity in males. The rates of glucose excursion, as well as any difference in tolerance are negated when GTT data is normalized to fasting ( Figure 1C ), indicating that the role P2Y₂R plays in regulation of glucose is primarily at the fasting level. Female mice had lower glucose at fasting and throughout GTT compared to male mice ( Figure 1 ). This is consistent with some past work (9, 10, 23) but not all (24). We show that LPS administration improved glucose tolerance in all mice, across sex and genotype ( Figure 2 ). Inflammation induces a decreased glucose excursion time in different inflammatory models, whether glucose was administered via intraperitoneal injection (25) or via oral gavage (26). Male mice may have increased levels of the LPS receptor TLR4 during the acute stages of inflammation (13, 19), and the cytokine response to LPS administration is sexually dimorphic (13, 14). However, these factors did not result in significant sex-dependent glucose response ( Figure 2E ) in our LPS-treated animals. One way to explain the reduced blood glucose in females is through the upregulation of GLUT4. We did not detect significant sex-dependent levels of GLUT4 mRNA, however, we did detect significantly more INSR expression in females ( Figures 3A, B ). Insulin signaling through the insulin receptor can trigger the translocation of GLUT4 protein on internal vesicles to the plasma membrane, promoting glucose uptake independent of transcription levels. Thus, it is possible that sex-dependent fasting blood glucose levels are at least partially dependent upon insulin signaling within the muscle. Importantly, our data does not rule out differential gene expression or insulin signaling in other tissues or cell types. Our gene expression analysis is only preliminary and more investigation is needed to determine if local sex-dependent expression of P2Y₂R, GLUT4 and/or INSR could be responsible for the observed changes in glucose homeostasis.

Sex differences in fasting glucose ( Figures 1B , 2B ) were eliminated in the presence of LPS, suggesting that inflammation overrides P2Y₂R-dependent control of glucose sensitivity. The LPS-dependent reduction in fasting blood glucose ( Figure 2 ) could be due to increased GLUT4 translocation to the surface during inflammation in skeletal muscle, but could also be influenced by increased glucose absorption in the liver or adipose tissue. The majority (80-90%) of glucose is absorbed by skeletal muscle (5), but alterations in glucose regulation in the liver or adipose tissue cannot be ruled out.

The P2Y₂ receptor was previously found to play a role in high fat diet-related obesity, a condition marked by activation of inflammatory pathways. P2Y₂R knock out animals have increased glucose tolerance (4) and improved insulin sensitivity (4, 27), compared to wild-type mice fed a high fat diet. The loss of the receptor globally (4) and myeloid-specific loss decreases the inflammatory response (18). Consistent with previous research, here we show that the P2Y₂R^-/- is protective against inflammation-stimulated effects. The sex-dependence and inflammation-dependence of blood glucose largely masked the effect of the P2Y₂ receptor on glucose excursion time. However, after normalizing glucose levels to baseline, it was apparent that the P2Y₂R deficient male mice are protected from inflammation-mediated effects on glucose tolerance. While the wild-type males had a reduced tolerance to glucose, the P2Y₂R^-/- males were similar to the females, suggesting that the P2Y₂R has a specific effect in male mice that increases their tolerance. Interestingly, a previous study testing only female mice noticed that those lacking P2Y₂R on myeloid cells had similar glucose levels and tolerance to wild-type mice in the presence of a HFD-induced obesity (18). This is in contrast to male whole body P2Y₂R^-/- mice on HFD that showed decreased blood glucose and increased glucose tolerance (4). While the models in the two studies are different, they support our work suggesting that P2Y₂R role in glucose homeostasis is more prominent in males. The resistance of females to alteration in glucose tolerance and reduced effects of LPS on baseline glucose levels may be due, in part, to greater insulin-sensitivity in females (28). Our own data suggests that there are greater levels of INSR in female skeletal muscle, compared to that of males, supporting the idea that females may be more insulin-sensitive.

It is clear that P2Y₂R plays a more important role in glucose regulation during LPS-stimulated inflammation in fed males than it does females. The underlying differences between the sexes might be due to differential inflammatory signaling. Sex-dependent differences in cytokine production and inflammatory factors have been widely observed. However, females mice are less susceptible to insulin resistance triggered by high fat diet (29), thus this male-specific effect is not isolated to LPS-treatment, but is more widely observed across inflammatory models. We show no significant differences in P2Y₂R expression between male and female mice ( Figure 3C ) and data from humans also suggests that P2Y₂R is expressed similarly between males and females ( Supplementary Figure 2 ), thus, differential expression of P2Y₂R cannot explain the dimorphic dependence on the receptor. The P2Y₂R gene expression profile in humans is similar to GLUT4 expression in that there is a high level of expression in the skeletal muscle, but low levels of expression in adipose, liver and sex-specific tissues such as the testes and ovaries. This suggests that the convergence of P2Y₂R signaling, and glucose sensitivity may occur in the muscle. Previous studies showed that P2Y₂R expression was increased by LPS treatment in cultured cells (16, 17) and in the blood (18), however, our study did not find significant induction of P2Y₂R in the muscle with LPS-treatment. While effect sizes suggest a possible important effect of sex and LPS treatment on P2Y₂R gene expression, more data is be needed to rule out LPS-dependent expression of P2Y₂R in muscle of mice. It is possible that P2Y₂R induction is cell-type specific, and that P2Y₂R signaling can exert its effects over long ranges. It is also possible that the dose of LPS used here was not sufficient to induce P2Y₂R expression in the muscle. However, plasma IL-6 ( Supplementary Figure 1 ) and expected LPS-dependent effects on fasting glucose levels ( Figure 2 ) confirm that the dose and timing of LPS used here was sufficient to stimulate systemic inflammation.

While sex-dependent regulation of glucose metabolism seems to be isolated to steady-state, the role of the P2Y₂ receptor is most prominent during glucose challenge, affecting both the amplitude and rate of glucose accumulation. P2Y₂ receptor signaling may interact with the insulin signaling pathway to affect the ability of insulin to signal glucose uptake in peripheral tissues, making the P2Y₂R^-/- males less insulin sensitive. A recent study showed that, in cultured hepatocytes, P2Y₂ activation reduced insulin-dependent signaling, ultimately reducing glucose uptake there (6), illustrating that P2Y₂R and insulin signaling pathways intersect to regulate glucose homeostasis locally. Of course, P2Y₂R may also reduce the levels of insulin released during glucose challenge. The role of receptor may be isolated to males due to expression of metabolism gene(s) from sex chromosomes, or perhaps by androgen-regulation or estrogen-repression of P2Y₂R activity. Indeed, sex differences in glucose regulation in HFD mouse models is minimized by gonadectomy, though androgens were primarily responsible for sex-differences (30). Testosterone also shown to decrease the levels of IL-6 in response to LPS administration in mice, providing more suggestion that the male sex hormones may be the key to understanding sexual dimorphism in glucose regulation (31).

The purinergic receptor plays important roles in inflammation and in glucose regulation, though its role appears to be more critical in males, at least among animals in the young adult age rage (8-12 weeks) tested here. Overall, this work highlights the sexual dimorphic nature of glucose regulation, but also displays a new sex-specific role for purinergic signaling. Studies that systematically address sex-dependent regulation provide crucial insights into an inclusive understanding of physiological processes, dysregulation, and treatment. Indeed, recent evidence suggests that metformin, a common pharmaceutical treatment for insulin resistance, may exert its regulation primarily through P2Y₂R (6). P2Y₂ receptor agonist have been approved to treat dry eye in some countries, and purinergic signaling is an enticing drug target for treatment of many conditions, including immune and infection-related disease (32). As these therapeutic treatments become reality, sex may be an important consideration in clinical trials and in clinical practice.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Missouri State Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.

RU and JW were the main advisors on the project, overseeing experimental design, data analysis and manuscript preparation. RU and SZ drafted the manuscript. CR, HM, DJ, NJ and ES collected GTT data and harvested tissues. CR, RU and HM analyzed GTT data. JM collected INSR gene expression data while RU collected and analyzed all other gene expression data. All authors contributed to the article and approved the submitted version.