T2D is one of the major risk factors for cardiovascular disease 
. Adenosine receptors have been studied in the context of insulin homeostasis and glucose metabolism 
, lipolysis, development of hepatic steatosis, and reverse cholesterol transport (e.g., 
). Our current study identifies a novel role for the A2bAR in the context of T2D, including insulin and glucose homeostasis, chronic inflammation and obesity under HFD.
A2bAR KO mice fed with high fat, high cholesterol diet display altered glucose clearance and insulin secretion and elevated fat to lean mass ratio, compared to WT mice, suggestive of a T2D compensatory phase. Tissue insulin resistance was evident in the A2bAR KO post HFD, noted by alterations in phosphorylation of Akt, an important downstream signaling component of the insulin pathway. In accordance with recent studies in our lab exploring the role of the A2bAR in the context of atherosclerosis and lipid metabolism on an ApoE null background 
, we determined here that the A2bAR on normal ApoE background also regulates hepatic SREBP-1 as well as plasma lipids post HFD challenge. SREBP-1 not only controls the lipid profile via its effect on enzymes involved in lipid synthesis 
, but it also regulates the expression of IRS-2 
. Additionally, IRS-2 levels are also known to be increased by cAMP 
and hepatocyte levels of cAMP are lower upon A2bAR elimination 
. Indeed, here we show that livers of A2bAR null mice under HFD have significantly reduced levels of IRS-2 compared to control mice, which is expected to result in impaired insulin signaling 
as confirmed in our system as well.
A central inquiry at hand is whether hyperglycemia in A2bAR KO mice under HFD is a result of an autonomous regulation of pancreatic ß-cells by the A2bAR and/or a result of systemic influences on the pancreas, stemming from A2bAR-induced changes in other tissues, including liver upregulation of triglycerides and cholesterol. This is a relevant inquiry, since insulin resistance can cause hyperglycemia, and hyperinsulinemia, which in turn results in resistance. We propose that both processes contribute to T2D in A2bAR KO mice. HFD leads to vast upregulation of A2bAR in the liver and elimination of A2bAR results in hyperlipidemia, while the latter has been shown to acutely trigger ß-cells to hyper-secrete insulin (e.g., 
). This raises the possibility that changes in the liver A2bAR affect pancreatic insulin secretion. On the other hand, we (Fig. S3A, S3B
), and others 
found that genetic or pharmacological ablation of the A2bAR leads to increased insulin secretion from isolated pancreatic islets. The mechanism of effect of pancreatic A2bAR on insulin secretion is yet to be explored.
The impaired insulin signaling in non-hepatic tissues of A2bAR KO mice is thought to be mediated primarily by systemic effects, suggested by the slight upregulation of A2bAR with HFD seen in the adipose and muscle tissue relative to robust upregulation noted in the liver. Hepatic A2bAR regulates the lipid profile 
, and expression of the A2bAR in sites such as the liver or macrophages 
controls the level of inflammatory cytokines. High levels of TNF-α and IL-6 have been reported in multiple diabetic and insulin resistant states 
. TNF-α disrupts insulin signaling through both inhibitory phosphorylation and reduction in total levels of IRS-1/IRS-2 and downstream signaling (
, reviewed in 
). The systemic and tissue pro-inflammatory profile in the A2bAR KO could contribute to the observed downregulated IRS-2, and impaired Akt signaling, and to the peripheral tissue insulin resistance. Future studies could explore the contribution of inflammation induced by the macrophage A2bAR, to the insulin resistance seen in the A2bAR KO. Chronic inflammation associated with obesity is thought to be an important factor in the pathogenesis of T2D 
. Recent studies have identified sensors of “danger signals”, such as the Nlrp3 inflammasome, to contribute to obesity-related inflammation and insulin resistance 
. A2bAR is known to regulate inflammatory cytokines as TNF-alpha and IL-6 
, and perhaps other danger signals, such as ceramides or free fatty acids could potentially synergize or integrate with A2bAR signals to activate the inflammasome; exploring this possibility would constitute a new line of investigation.
Our findings in subcutaneous fat from obese patients suggest that the A2bAR should be further explored as a marker of IRS-2 expression and of obesity, as well as considered as a therapeutic target. Indeed, by using a selective A2bAR agonist in vivo in mice, we showed that the A2bAR is protective against HFD-induced T2D phenotypes. A recent study 
reported that A2bAR ablation improves glucose homeostasis and clearance in mice fed a regular chow diet, while we find that under HFD, this receptor deletion has an opposite effect. Under both diets, A2bAR ablation leads to pancreatic insulin hypersecretion (Fig. S3A, S3B
. Under regular diet, this might lead to a rapid glucose clearance, as reported in 
, as there is no impairment of insulin signaling. Under HFD, A2bAR KO mice, despite elevated insulin levels, demonstrate impaired glucose clearance. This is indicative of tissue insulin resistance in A2bAR KO mice. This suggests that in this case, hyperinsulimia alone is not sufficient to cause insulin resistance. On the other hand, following HFD, liver A2bAR is vastly upregulated, resulting in maintenance of adequate levels of IRS-2 and reduced lipids, which are expected to guard against HFD-induced insulin resistance.
In summary, our study elucidates a role for the A2bAR in maintenance of glucose and insulin homeostasis, modulation of inflammatory mediated events, and lipid metabolism. We establish a role for A2bAR in the regulation of IRS-2 expression and effects on downstream insulin signaling. In absence of the A2bAR, elevated plasma and tissue inflammatory markers contribute to impaired insulin response, while pharmacological activation of the A2bAR modulates chronic inflammation and restores IRS-2 signaling. The translational value of this receptor is further enhanced with data that shows a positive association between the A2bAR and BMI, parameters of obesity (e.g. waist and hip circumference) and IRS-2 in subcutaneous fat samples obtained from obese patients. It is of interest to study if activation of the A2bAR in type 2 diabetic patients can improve insulin resistance and inflammatory parameters.