Phytonutrient genistein is a survival factor for pancreatic β-cells via GPR30-mediated mechanism

We previously discovered that phytonutrient genistein rapidly activates cAMP signaling in β-cells and improves islet mass in diabetic mice. However, the mechanism underlying these actions of genistein is still unclear. Here, we show that pharmacological or molecular inhibition of Gαs blocked genistein-stimulated adenylate cyclase activity in plasma membrane and intracellular cAMP production in INS1 cells and islets. Further, genistein stimulation of cAMP generation was abolished in islets exposed to a specific GPR30 inhibitor G15 or islets from GPR30 deficient (GPR30−/−) mice. In vivo, dietary provision of genistein (0.5 g/kg diet) significantly mitigated streptozotocin-induced hyperglycemia in male WT mice, which was associated with improved blood insulin levels and pancreatic islet mass and survival, whereas these effects were absent in Gpr30−/− mice. Genistein treatment promoted survival of INS1 cells and human islets chronically exposed to palmitate and high glucose. At molecular level, genistein activated CREB phosphorylation and subsequently induced Bcl-2 expression, and knockdown of CREB diminished the protective effect of genistein on β-cells induced by lipoglucotoxicity. Finally, deletion of GPR30 in β-cells or islets ablated genistein-induced CREB phosphorylation and its cytoprotective effect. These findings demonstrate that genistein is a survival factor for β-cells via GPR30-initiated, Gαs-mediated activation of CREB.     

The relationship between GPR40 and lipotoxicity of the pancreatic β-cells as well as the effect of pioglitazone

Free fatty acids (FFAs) acutely stimulate insulin secretion from pancreatic β-cells, whereas impair β-cell function following long term exposure. GPR40, a FFAs receptor, has been demonstrated to be activated by both medium and long chain FFAs and played an important role in insulin release. This study was performed to determine the contribution of GPR40 to short- and/or long-term effects of FFAs on glucose-stimulated insulin secretion (GSIS) and the expression of PDX-1 and GLUT2 in pancreatic β-cells, as well as the intervenient effects of pioglitazone on lipotoxicity of β-cells. βTC6 cell line stably expressing GPR40shRNA were established and the intervention of FFAs and pioglitazone on GSIS and expression of PDX-1 and GLUT2 in βTC6 cells was investigated. Results showed that 1-h exposure to FFAs significantly enhanced GSIS and increased expression of PDX-1 and GLUT2 in pSilencer-control transfected cells, but not in cells transfected with GPR40shRNA. While 48-h exposure to FFAs significantly impaired GSIS in pSilencer-control transfected cells as well as cells transfected with GPR40shRNA. Furthermore, pioglitazone enhanced insulin secretion in pSilencer-control transfected cells exposed to FFAs for 48 h, but not in cells transfected with GPR40shRNA. These results indicate that GPR40 mediates the short-term effects of FFAs on GSIS, but does not mediate the chronic lipotoxicity on β-cells. The reverse role of pioglitazone on lipotoxicity of β-cells may be related to GPR40.     

Dual effects of the non-esterified fatty acid receptor ‘GPR40’ for human health

G protein-coupled receptor 40 (GPR40), a receptor for diverse non-esterified fatty acids, is expressed predominantly in the wide variety of neurons of the central nervous system and β-cells in the pancreatic islets. Since deorphanization of GPR40 in 2003, the past decade has seen major advances in our understanding of its role in the insulin secretion. However, there is still a great deal to be elucidated about the role of GPR40 in the brain, because the latter shows the most abundant GPR40 mRNA expression among the human tissues. Since a substantial expression of GPR40 is also seen in the hypothalamus, ‘brain-lipid sensing’ might be involved in the control of insulin secretion and energy balance. The preceding experiments using monkeys after transient global brain ischemia, have highlighted implication of GPR40 for amplifying adult hippocampal neurogenesis. Although GPR40-mediated intracellular signaling was recently found to result in phosphorylation of cAMP response element-binding protein (CREB) necessary for the neuronal differentiation and synaptic plasticity, the signaling cascade is still incompletely understood. Furthermore, in response to conjugated linoleic acids or trans isomers of arachidonic acid, GPR40 was recently demonstrated in rodents to mediate lipotoxicity to β-cells, neurons, or microvessels, which result in diabetes, retinopathy, stroke, etc. However, it still remains undetermined in humans whether and how oxidized, conjugated, or excessive fatty acids evoke lipotoxicity. Although literature about GPR40 is limited especially about the brain or the brain–pancreas interaction, this review aims at summarizing beneficial as well as detrimental effects of this receptor in the brain and pancreas in response to diverse fatty acids.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

Role of ERβ and GPR30 in the endocrine pancreas: A matter of estrogen dose

The endocrine pancreas has emerged as a target for estrogens. The functions of pancreatic α-, β- and δ-cells are modulated by the endogenous hormone, 17β-estradiol (E2). Low physiological concentrations (100pM-1nM) of E2 rapidly decrease the activity of the ATP-sensitive potassium channel (K(ATP)) and enhance glucose-induced insulin release in β-cells in an estrogen receptor β (ERβ)-dependent manner. In addition to the insulinotropic action of ERβ, the newly described estrogen receptor, GPR30, is involved in the insulinotropic effects of high doses of E2 (100nM-5μM). The specific GPR30 agonist G1 also increases insulin secretion in β-cells. Low glucose-induced calcium oscillations and glucagon secretion are suppressed by E2. The effects on glucagon secretion may be mediated by GPR30. Somatostatin release is also decreased by E2 and G1. In this review we summarize all the data published up to date on the rapid insulinotropic effects of estrogens in the endocrine pancreas and propose a model to integrate the estrogen actions mediated through both receptors.     

Dnmt1 activity is dispensable in δ-cells but is essential for α-cell homeostasis

In addition to β-cells, pancreatic islets contain α- and δ-cells, which respectively produce glucagon and somatostatin. The reprogramming of these two endocrine cell types into insulin producers, as observed after a massive β-cell ablation in mice, may help restoring a functional β-cell mass in type 1 diabetes. Yet, the spontaneous α-to-β and δ-to-β conversion processes are relatively inefficient in adult animals and the underlying epigenetic mechanisms remain unclear. Several studies indicate that the conserved chromatin modifiers DNA methyltransferase 1 (Dnmt1) and Enhancer of zeste homolog 2 (Ezh2) are important for pancreas development and restrict islet cell plasticity. Here, to investigate the role of these two enzymes in α- and δ-cell development and fate maintenance, we genetically inactivated them in each of these two cell types. We found that loss of Dnmt1 does not enhance the conversion of α- or δ-cells toward a β-like fate. In addition, while Dnmt1 was dispensable for the development of these two cell types, we noticed a gradual loss of α-, but not δ-cells in adult mice. Finally, we found that Ezh2 inactivation does not enhance α-cell plasticity, and, contrary to what is observed in β-cells, does not impair α-cell proliferation. Our results indicate that both Dnmt1 and Ezh2 play distinct roles in the different islet cell types.     

GLP-1 receptor antagonist as a potential probe for pancreatic β-cell imaging

We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic β-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [¹²⁵I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [¹²⁵I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120 min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [¹²⁵I]BH-exendin(9-39) injection into transgenic mice with pancreatic β-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic β-cell imaging.     

SNAP-251–180 enhances insulin secretion by blocking Kv2.1 channels in rat pancreatic islet β-cells

Voltage-gated outward K⁺ currents from pancreatic islet β-cells are known to repolarize the action potential during a glucose stimulus, and consequently to modulate Ca²⁺ entry and insulin secretion. The voltage gated K⁺ (Kv) channel, Kv2.1, which is expressed in rat islet β-cells, mediates over 60% of the Kv outward K⁺ currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-25_1–180 (S180), an N-terminal SNAP-25 domain, but not SNAP-25_1–206 (S206), inhibits Kv current and enhances glucose-dependent insulin secretion from rat pancreatic islet β-cells, and furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner.     

Can pancreatic duct-derived progenitors be a source of islet regeneration?

The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing β-cells. The functional mass of β-cells is decreased in type 1 diabetes, so replacing missing β-cells or triggering their regeneration may allow for improved type 1 diabetes treatment. Therefore, expansion of the β-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding β-cell regeneration. One potential source is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. Neogenesis, or that the new pancreatic islets can derive from progenitor cells present within the ducts has been reported, but the existence and identity of the progenitor cells have been debated.In this review, we focus on pancreatic ductal cells, which are islet progenitors capable of differentiating into islet β-cells. Islet neogenesis, seen as budding of hormone-positive cells from the ductal epithelium, is considered to be one mechanism for normal islet growth after birth and in regeneration, and has suggested the presence of pancreatic stem cells. Numerous results support the neogenesis hypothesis, the evidence for the hypothesis in the adult comes primarily from morphological studies that have in common the production of damage to all or part of the pancreas, with consequent inflammation and repair. Although numerous studies support a ductal origin for new islets after birth, lineage-tracing experiments are considered the “gold standard” of proof. Lineage-tracing experiments show that pancreatic duct cells act as progenitors, giving rise to new islets after birth and after injury. The identification of differentiated pancreatic ductal cells as an in vivo progenitor for pancreatic β-cells has implications for a potentially important, expandable source of new islets for diabetic replenishment therapy.     

DC260126: A Small-Molecule Antagonist of GPR40 that Protects against Pancreatic β-Cells Dysfunction in db/db Mice

G protein-coupled receptor 40 (GPR40) mediates both acute and chronic effects of free fatty acids (FFAs) on insulin secretion. However, it remains controversial whether inhibition of GPR40 would be beneficial in prevention of type 2 diabetes. This study is designed to evaluate the potential effects of DC260126, a small molecule antagonist of GPR40, on β-cell function following administration of 10 mg/kg dose of DC260126 to obese diabetic db/db mice. Oral glucose tolerance test, glucose stimulated insulin secretion and insulin tolerance test were used to investigate the pharmacological effects of DC260126 on db/db mice after 21-days treatment. Immunohistochemistry and serum biochemical analysis were also performed in this study. Although no significant change of blood glucose levels was found in DC260126-treated mice, DC260126 significantly inhibited glucose stimulated insulin secretion, reduced blood insulin level and improved insulin sensitivity after 3 weeks administration in db/db mice. Moreover, DC260126 reduced the proinsulin/insulin ratio and the apoptotic rate of pancreatic β-cells remarkably in DC260126-treated db/db mice compared to vehicle-treated mice (p<0.05, n = 8). The results suggest that although DC260126 could not provide benefit for improving hyperglycemia, it could protect against pancreatic β-cells dysfunction through reducing overload of β-cells, and it increases insulin sensitivity possibly via alleviation of hyperinsulinemia in db/db mice.     

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet β-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to β-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic β-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. β-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in β-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic β-cells.     

Exendin-4, a glucagon-like peptide-1 receptor agonist, suppresses pancreatic β-cell destruction induced by encephalomyocarditis virus

Viral infection is one of the important factors for the pathogenesis of type 1 diabetes. Particularly, in fulminant type 1 diabetes, rapid β-cell destruction is suggested to be triggered by viral infection. Recently, glucagon-like peptide 1 (GLP-1) receptor agonists have been reported to have direct beneficial effects on β-cells, such as anti-apoptotic effect, increasing β-cell mass, and improvement of β-cell function. However, their effects on β-cell destruction induced by viral infections have not been elucidated. In this study, we used an encephalomyocarditis virus (EMCV)-induced diabetic model mouse to show that a GLP-1 receptor agonist, exendin-4, prevents β-cell destruction. Nine-week-old male DBA/2 mice were intraperitoneally injected with EMCV (200 plaque forming units (PFU) mouse⁻¹). Low (20 nmol kg⁻¹ d⁻¹) or high (40 nmol kg⁻¹ d⁻¹) doses of exendin-4 were administered for 10 d, starting from 2 d before the infection, and the rate of diabetic onset was evaluated. In addition, the number of infiltrating macrophage per islet and the ratio of β-cell area to islet area were determined. The effects of exendin-4 on infected β-cells and macrophages were investigated by using MIN6 and RAW264 mouse macrophages. The incidence of diabetes was significantly lower in the high-dose exendin-4-treated group than in the control group. Furthermore, the β-cell area was significantly more preserved in the high-dose exendin-4-treated group than in the control. In addition, the number of macrophages infiltrating into the islets was significantly less in the high-dose exendin-4-treated group than in the control group. In vitro, exendin-4 reduced β-cell apoptosis, and tumor necrosis factor α (TNFα), interleukin β (IL-β), and inducible nitric oxide synthase (iNOS) production of infected or lipopolysaccharide (LPS)-stimulated macrophages. These results suggested that exendin-4 limits β-cell destruction by protecting β cells and reducing the inflammatory response of macrophages.     

Absence of spontaneous regeneration of endogenous pancreatic β-cells after chemical-induced diabetes and no effect of GABA on α-to-β cell transdifferentiation in rhesus monkeys

β-cell deficiency is common feature of type 1 and late-stage of type 2 diabetes mellitus. Thus, β?cell replacement therapy has been the focus of regenerative medicine past several decades. Particularly, evidences suggest that β?cell regeneration via transdifferentiation from sources including α-cells is promising. However, data using higher mammals besides rodents are scarce. Here, we examined whether endogenous pancreatic β-cells could regenerate spontaneously or under normoglycemia following porcine islet transplantation for varied periods up to 1197 days after streptozotocin-induced diabetes, and remaining α-cells transdifferentiate into β-cells by GABA treatment in vivo and in vitro. The results showed that endogenous β-cells rarely regenerate in both conditions as evidenced by stagnant serum C-peptide levels and β-cell number in the pancreas, and the remaining α-cells did not transdifferentiate into β-cells by GABA treatment. Collectively, we concluded that monkey β-cells had relatively low regenerative potential compared with rodent counterpart and GABA treatment could not induce α-to-β-cell transdifferentitation.     

Remodeling of insulin producing β-cells during Xenopus laevis metamorphosis

Insulin-producing β-cells are present as single cells or in small clusters distributed throughout the pancreas of the Xenopus laevis tadpole. During metamorphic climax when the exocrine pancreas dedifferentiates to progenitor cells, the β-cells undergo two changes. Insulin mRNA is down regulated at the beginning of metamorphic climax (NF62) and reexpressed again near the end of climax. Secondly, the β-cells aggregate to form islets. During climax the increase in insulin cluster size is not caused by cell proliferation or by acinar-to-β-cell transdifferentiation, but rather is due to the aggregation of pre-existing β-cells. The total number of β-cells does not change during the 8 days of climax. Thyroid hormone (TH) induction of premetamorphic tadpoles causes an increase in islet size while prolonged treatment of tadpoles with the goitrogen methimazole inhibits this increase. Expression of a dominant negative form of the thyroid hormone receptor (TRDN) driven by the elastase promoter not only protects the exocrine pancreas of a transgenic tadpole from TH-induced dedifferentiation but also prevents aggregation of β-cells at climax. These transgenic tadpoles do however undergo normal loss and resynthesis of insulin mRNA at the same stage as controls. In contrast transgenic tadpoles with the same TRDN transgene driven by an insulin promoter do not undergo down regulation of insulin mRNA, but do aggregate β-cells to form islets like controls. These results demonstrate that TH controls the remodeling of β-cells through cell-cell interaction with dedifferentiating acinar cells and a cell autonomous program that temporarily shuts off the insulin gene.     

Discovery and pharmacological effects of a novel GPR142 antagonist

GPR142 is a G-protein-coupled receptor (GPCR), whose most potent and efficacious ligand has been reported as being the natural amino acid l-tryptophan. GPR142 is highly expressed in pancreatic β-cells and immune cells, suggesting the receptor may play a role in the pathogenesis and development of diabetes or inflammatory diseases. In a previous report, we developed GPR142 agonists as insulin secretagogues. In this report, we show the discovery of a selective, potent small-molecule GPR142 antagonist, CLP-3094, and its pharmacological characteristics. These data support targeting this receptor for the treatment of chronic inflammatory diseases.     

The cannabinoid CB1 receptor is expressed in pancreatic delta-cells

Antagonists of cannabinoid CB1 receptor (CB1, CNR1) promote weight loss and decrease hyperglycemia in patients with type 2 diabetes. While the endocannabinoid system may modulate islet hormone secretion, the cell-type expressing CB1 receptor in islets has not been fully resolved. In this study, we verified receptor gene expression in rodent islets and cell lines and examined the distribution of CB1 receptor in mouse, rat, and human islets by confocal immunofluorescence (IF) microscopy. IF demonstrated CB1 receptor was present in β-cell lines, but co-localized solely with somatostatin in the islet δ-cells of Zucker rats, C57BL/6 mice, and humans; no CB1 receptor expression was observed in α-, β-, or pp-cells. Similarly, a rat somatostatinoma cell line, MSL-G2-Tu6, was found to express CB1 receptor. We also found monoacylglycerol lipase (MAGL) to be expressed in δ-cells and fatty acid amide hydrolase (FAAH) to be expressed in α-cells. The specific expression of CB1 in δ-cells suggests that the ECS may play a role in modulating islet hormone secretion. As there are some differences between our findings and previous reports, further studies, including detailed physiological studies of the effects of the ECS on islet function, are warranted.     

AS1907417, a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes

G-protein-coupled receptor (GPR) 119 is involved in glucose-stimulated insulin secretion (GSIS) and represents a promising target for the treatment of type 2 diabetes as it is highly expressed in pancreatic β-cells. Although a number of oral GPR119 agonists have been developed, their inability to adequately directly preserve β-cell function limits their effectiveness. Here, we evaluated the therapeutic potential of a novel small-molecule GPR119 agonist, AS1907417, which represents a modified form of a 2,4,6-tri-substituted pyrimidine core agonist, AS1269574, we previously identified. The exposure of HEK293 cells expressing human GPR119, NIT-1 cells expressing human insulin promoter, and the pancreatic β-cell line MIN-6-B1 to AS1907417, enhanced intracellular cAMP, GSIS, and human insulin promoter activity, respectively. In in vivo experiments involving fasted normal mice, a single dose of AS1907417 improved glucose tolerance, but did not affect plasma glucose or insulin levels. Twice-daily doses of AS1907417 for 4 weeks in diabetic db/db, aged db/db mice, ob/ob mice, and Zucker diabetic fatty rats reduced hemoglobin A1c levels by 1.6%, 0.8%, 1.5%, and 0.9%, respectively. In db/db mice, AS1907417 improved plasma glucose, plasma insulin, pancreatic insulin content, lipid profiles, and increased pancreatic insulin and pancreatic and duodenal homeobox 1 (PDX-1) mRNA levels. These data demonstrate that novel GPR119 agonist AS1907417 not only effectively controls glucose levels, but also preserves pancreatic β-cell function. We therefore propose that AS1907417 represents a new type of antihyperglycemic agent with promising potential for the effective treatment of type 2 diabetes.     

Hexose Metabolism in Pancreatic Islets: Glycogen Synthase and Glycogen Phosphorylase Activities

The activity of glycogen synthase and glycogen phosphorylase was measured in rat pancreatic islet homogenates. For this purpose, the sensitivity of current radioisotopic procedures for the assay of these enzymes in liver extracts was increased by about two orders of magnitude. Even so, the measurement of glycogen synthase and phosphorylase in islet homogenates was hampered by a potent amylase-like activity, resulting in the hydrolysis of preformed or newly formed ¹⁴C-labeled glycogen. Acarbose suppressed the latter phenomenon which was found attributable to both minute contamination of isolated islets by acinar cells and genuine α-amylase activity in purified islet β-cells. As measured by the more sensitive method in the presence of acarbose, the a/(a + b) ratio for glycogen synthase activity in islet homogenates was increased in islets preincubated in the presence as distinct from absence of D-glucose and decreased after preincubation with forskolin. These changes represented a mirror image of those evoked by D-glucose and forskolin in the a/(a + b) ratio for glycogen phosphorylase activity. It is concluded that glycogen synthesis and breakdown are regulated in the endocrine pancreas in a manner qualitatively comparable to that prevailing in hepatocytes, the possible participation of an amylase-like activity to glycogen metabolism in intact islet β-cells requiring further investigation.     

Pharmacophore-guided repurposing of fibrates and retinoids as GPR40 allosteric ligands with activity on insulin release

A classical drug repurposing approach was applied to find new putative GPR40 allosteric binders. A two-step computational protocol was set up, based on an initial pharmacophoric-based virtual screening of the DrugBank database of known drugs, followed by docking simulations to confirm the interactions between the prioritised compounds and GPR40. The best-ranked entries showed binding poses comparable to that of TAK-875, a known allosteric agonist of GPR40. Three of them (tazarotenic acid, bezafibrate, and efaproxiral) affect insulin secretion in pancreatic INS-1 832/13 β-cells with EC₅₀ in the nanomolar concentration (5.73, 14.2, and 13.5 nM, respectively). Given the involvement of GPR40 in type 2 diabetes, the new GPR40 modulators represent a promising tool for therapeutic intervention towards this disease. The ability to affect GPR40 was further assessed in human breast cancer MCF-7 cells in which this receptor positively regulates growth activities (EC₅₀ values were 5.6, 21, and 14 nM, respectively).