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Given its glycemic efficacy and ability to reduce the body weight, glucagon-like peptide 1 receptor (GLP-1R) agonism has emerged as a preferred treatment for diabetes associated with obesity. We here report that a small-molecule Class 1 histone deacetylase (HDAC) inhibitor Entinostat (MS-275) enhances GLP-1R agonism to potentiate glucose-stimulated insulin secretion and decrease body weight in diet-induced obese (DIO) mice. MS-275 is not an agonist or allosteric activator of GLP-1R but enhances the sustained receptor-mediated signaling through the modulation of the expression of proteins involved in the signaling pathway. MS-275 and liraglutide combined therapy improved fasting glycemia upon short-term treatment and a chronic administration causes a reduction of obesity in DIO mice. Overall, our results emphasize the therapeutic potential of MS-275 as an adjunct to GLP-1R therapy in the treatment of diabetes and obesity.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

Glucagon-like peptide-1 receptor (GLP-1R) plays a major role in promoting glucose-stimulated insulin secretion in pancreatic β-cells. In the present study, we synthesized a novel functional analog of GLP-1 conjugated to tetramethyl rhodamine to monitor the internalization of the receptor. Our data show that after being internalized the receptor is sorted to lysosomes. In endosomes, receptor-ligand complex is found to be colocalized with adenylate cyclase. Pharmacological inhibition of endocytosis attenuates GLP-1R-mediated cAMP generation and consequent downstream protein kinase A substrate phosphorylation and glucose-stimulated insulin secretion. Our study underlines a paradigm shift in GLP-1R signaling and trafficking. The receptor ligand complex triggers cAMP generation both in plasma membrane and in endosomes, which has implications for receptor-mediated regulation of insulin secretion.

Objective Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. Methods We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein–protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Results Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. Conclusions The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex., Graphical abstract Image 1, Highlights • Prolonged association of Gαs with activated GLP-1 R contributes to sustained c AMP generation in pancreatic beta cells. • Beta arrestin-1 association with the receptor does not attenuate GLP-1R mediated cyclic AMP generation. • Rab5A endosomes serve as a niche for sustained endosomal cyclic AMP generation upon GLP-1 receptor activation.

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.