Β-cell Activity Research Articles

Association between xanthinoxidase activity andparameters of glucose homeostasis in patients with type 2 diabetes mellitus

Background. The purpose of the study is to determine the associations between clinical and anthropometric parameters, glucose homeostasis and serum xanthinoxidase (XO) activity in patients with type 2 diabetes mellitus (T2DM) taking into account gender, glycemic control and serum XO activity. Materials and methods. One hundred and twenty-five T2DM patients aged 34 to 81 years were examined, with an average age of 58.9±9.4 years, disease duration from 1 month to 29 years (average of 8.9±6.6 years). The age of patients at the time of the disease manifestation in the general sample was from 29 to 71 years, on average 50.6±9.1 years. Results. The authors have found a nonlinear dependence of serum XO activity on fasting insulin concentration in patients with T2DM in the total sample, described by the multiplicative model (r=0.45; p=0.001). Serum XO activity in patients with T2DM in the general sample increases with adaptive increase in secretory activity of β-cells on an empty stomach according to the HOMA_β%. Serum ХO activity was highest in T2DM patients with low fasting insulin sensitivity (HOMA_S% < 50%). In addition, it has been determined that the serum ХО activity in the subjects is nonlinearly associated with the QUICKI (r=–0.35; p=0.016) and Caro indices (r=–0.40; p=0.007). We have found a nonlinear dependence of serum XO activity on fasting insulin (r=0.50; p=0.08), HOMA_β% (r=–0.53; p=0.06), HOMA_S% (r=–0.48; p=0.09), HOMA-IR (r=–0.48; p=0.09) in men with optimal glycemic control (HbA1c < 7.5%) at the trend level and Caro (r=–0.64; p=0.02). In women of this group, there was a nonlinear dependence of serum XO activity on fasting insulin (r=0.56; p=0.004), HOMA_β% (r=0.56; p=0.003), HOMA_S% (r=–0.54; p=0.005), HOMA-IR (r=0.54; p=0.005), QUICKI (r=–0.50; p=0.01) and Caro (r=–0.61; p=0.003). Conclusions. In patients with T2DM, the serum uric acid is linearly associated with the level of serum XO activity, which determines 34% of its variability. In patients with T2DM, regardless of the state of glycemic control, serum XO activity is nonlinearly associated with parameters characterizing the state of glucose homeostasis (fasting insulin, HOMA_S%, HOMA_β%, QUICKI and Caro indices). Predictors of high serum XO activity in patients with T2DM are the level of postprandial blood glucose (t=–3.53; p=0.004) and serum uric acid (t=4.73; p=0.0005).

L-Asparaginase regulates mTORC1 activity via a TSC2-dependent pathway in pancreatic beta cells

Eif2ak4, a susceptibility gene for type 2 diabetes, encodes GCN2, a molecule activated by amino acid deficiency. Mutations or deletions in GCN2 in pancreatic β-cells increase mTORC1 activity by decreasing Sestrin2 expression in a TSC2-independent manner. In this study, we searched for molecules downstream of GCN2 that suppress mTORC1 activity in a TSC2-dependent manner. To do so, we used a pull-down assay to identify molecules that competitively inhibit the binding of the T1462 phosphorylation site of TSC2 to 14-3-3. l-asparaginase was identified. Although l-asparaginase is frequently used as an anticancer drug for acute lymphoblastic leukemia, little is known about endogenous l-asparaginase. l-Asparaginase, which is expressed downstream of GCN2, was found to bind 14-3-3 and thereby to inhibit its binding to the T1462 phosphorylation site of TSC2 and contribute to TSC2 activation and mTORC1 inactivation upon TSC2 dephosphorylation. Further investigation of the regulation of mTORC1 activity in pancreatic β-cells by l-asparaginase should help to elucidate the mechanism of diabetes and insulin secretion failure during anticancer drug use.

Increased pancreatic β-cell electrical activity reduces diabetes susceptibility in female mice.

In humans, the incidence of Type 2 Diabetes Mellitus (T2DM) in premenopausal women is higher compared to men, phenotype also recapitulated by many rodent models. The T2DM etiology involves impaired or total lack of insulin release from pancreatic β-cells. High voltage-gated Ca2+ channels (HVCCs) are multi-subunit protein complexes responsible for β-cell electrical activity and insulin vesicle exocytosis. Previously, we showed that genetic deletion of α2δ-1 HVCC auxiliary subunit equally reduced Ca2+ influx by 60% in β-cells of both males and females but caused diabetes only in males because female pancreatic islets released significantly more insulin (Mastrolia et al., 2017). Functional characterization shows that both male and female β-cells display a similar glucose-induced increase in the membrane potential under all stimulatory glucose levels (e.g. ≅10mM glucose RMPfemales= −64.7±5.8 mV; RMPmales= −63.1±3.6 mV) consistent with similar HVCC Ca2+ influx and KATP, Kv, BK and SK K+ effluxes. The glucose induced intracellular Ca2+ transients are also similar between sexes consistent with identical Ca2+-induced Ca2+-release and intracellular store Ca2+ content. However, females have a higher percentage of β-cells that respond to glucose stimulation with increased electrical activity (≅10 mM glucose 58% of male β-cells fire action potentials (AP) compared to 90% of female β-cells). Additionally, female β-cells displayed a higher frequency of glucose-induced AP-trains (≅10 mM, female β-cells 2.2±0.5 AP-trains/min, males 0.41±0.1, p<0.05). The increased electrical activity resulted in a significantly higher insulin release in females compared to males therefore lowering the risk of developing T2DM. Current experiments are on the way to elucidate the mechanism leading to the higher electrical activity in female β-cells. Support: FWF P31434, P36053, and DOC30-B30 to PT.

The potent contraceptive gestodene exerts insulinotropic effects through its a-ring reduced metabolites with intrinsic estrogen-like activity in pancreatic β-cells.

The contraceptive gestodene is a potent synthetic progestin used in several low-dose contraceptive formulations. Clinical studies reported a relationship between long-term use of combined oral contraceptives containing gestodene (GDN) and profound alterations in glucose metabolism in women. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether GDN may induce estrogen-like effects, even though GDN does not interact with estrogen receptors. The aim of this study was to investigate whether GDN affect pancreatic β-cell activity, directly or through its conversion to other bioactive metabolites. The effects of GDN and its two derivatives 3β,5α-tetrahydro-GDN and 3α,5α-tetrahydro-GDN on insulin 2 (Ins II) and glucokinase (Gk) expression and glucose-stimulated insulin secretion were determined in pancreatic islets from female rats. Gestodene did exert significant effects on islet β-cells activity. The most striking finding was that 3β,5α-tetrahydro-GDN and 3α,5α-tetrahydro-GDN had greater stimulatory effects on Ins II and Gk expression than that observed with GDN, consistent with their effects on glucose-stimulated insulin secretion. The effects on gene expression induced by GDN-derivatives were abolished by ICI 182,780 and MPP. In addition, the presence of inhibitors of androgen and progestin-metabolizing enzymes eliminated gene expression induced by GDN. These results indicated that GDN is metabolized to A-ring reduced metabolites with estrogen-like activities and through this mechanism, GDN may affect β-cell activity. Altogether, the data suggest that 19-nortestosterone-derived contraceptives such as GDN, possess insulinotropic effects through their conversion into metabolites with intrinsic estrogen-like activity in pancreatic β-cells.

The prerequisites for the development of type 2 diabetes or prediabetes in rats fed a high-fat diet

It is known that the pathogenesis of type 2 diabetes in humans is based on two main factors – insulin resistance and inappropriate secretory activity of β-cells of the pancreas. In animals, the role of these mechanisms has not been clearly characterized, and the differences in the manifestations of experimental diabetes under the same conditions are not sufficiently substantiated. In order to study the prerequisites and mechanisms of the development of experimental type 2 diabetes or prediabetes under lipid overload, 6-month-old male Wistar rats were fed a high-fat diet for 4 weeks; after 2 weeks of the experiment, 20 or 25 mg/kg of streptozotocin was administrated. The development of insulin resistance was assessed using the insulin tolerance test. We evaluated the dynamics of glycemia in animals, subcellular signs of liver steatosis, and determined expression of the precursor and mature protein SREBP-1 by immunoblotting. It was found that in rats fed with a high-fat diet during the 2–4th weeks of the experiment, regardless of the administration of streptozotocin, stable insulin resistance and symptoms of prediabetes were detected. The severity of carbohydrate metabolism lesion, which appeared as type 2 diabetes or prediabetes after streptozotocin administration, depended on the level of hepatosteatosis due to high-fat diet, whereas the dose of streptozotocin influenced severity of type 2 diabetes. The use of a high-fat diet led to increased processing and activation of SREBP-1, which was clearly inhibited in type 2 diabetes. Therefore, the level of lipid infiltration of the liver and deregulation of the transcription factor SREBP-1 are risk factors defining development of type 2 diabetes or prediabetes in experimental rats with lipid overloading. Changes in the maturation of SREBP-1 with the use of a high-fat diet confirm that insulin resistance in rats revealed β-cell dysfunction, which closely approximates the mechanisms of experimental type 2 diabetes to main pathways in humans. At the same time, the predisposition to β-cell dysfunction can be a prerequisite that determines compensatory reserves for maintaining carbohydrate and lipid homeostasis under the influence of lipid load in both humans and laboratory animals.

Genome-edited zebrafish model of ABCC8 loss-of-function disease

ABSTRACT ATP-sensitive potassium channel (KATP)gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. While transgenic mice expressing incomplete KATP LOF do reiterate mild hyperinsulinism, KATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical KATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human HI. Patch-clamp analysis confirmed the complete absence of channel activity in β-cells from K499X (SUR1−/−) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish nor in homozygous SUR1−/− fish, mimicking findings in SUR1 knockout mice. Mutant fish did, however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent LOF mutations, these gene-edited animals provide valid zebrafish models of KATP -dependent pancreatic diseases.

Three-in-one customized bioink for islet organoid: GelMA/ECM/PRP orchestrate pro-angiogenic and immunoregulatory function

Islet organoids open up new strategies for diabetes treatment and pancreatic tissue engineering. Digital light processing (DLP) bioprinting has been extensively applied to the construction of organoids due to its ability to provide precisely patterned scaffolds with fast printing time while specific tailored bioink is indispensable for islet organoid. Customized bioinks that meet different needs were created and frequently applied based on gelatin methacryloyl (GelMA) mixed with other ingredients. Decellularized extracellular matrix (ECM) retains many organic specific structural and functional components and is widespread utilized to reconstruct the native niche like environment. However, considerable cytokines and growth factors were inevitably lost during the decellularized process, while platelet rich plasma (PRP) contains a string of growth factors which often exerted pro-angiogenic role. Therefore, a customized specific bioink for constructing islet organoid based on GelMA, pancreatic ECM and PRP was prepared in our research. In vitro, tube formation assay, CD31 immunofluorescence and relative mRNA expression of vascular genes indicated that the bioink with distinctively promote angiogenesis potential. Macrophages polarization was also conducted, which exhibited superior expression of CD206 (M2 marker) and inferior expression of iNOS (M1 marker). 3D printed organoids maintain the activity of mouse islet β-cells (MIN6) with enhanced glucose sensitivity. In vivo, the results of CD31, CD206 and iNOS immunofluorescence were consistent with that in vitro. In summary, we prepared and characterized specific custom-made bioink with orchestrating immune-regulation response indicated by abundant M2-polarized macrophages, attenuated inflammation, and promoted angiogenesis, which provides an underlying bioink for the fabrication of 3D printed islet organoid.

Protective effect of acetylcysteine, histidine, and their combination against diabetes vascular complications in type-2 diabetic rats via reducing NF-kβ pathway signaling.

The nuclear factor-kappa B (NF-κB) signaling participates in diabetes complications. Therefore, the reduction of NF-κB signaling may be a goal to prevent or improve them. Thus, we investigated the effects of acetylcysteine (AC), histidine (His), and their combination on the NF-κB expression and its different activators in type 2 diabetic rats. The survey was performed on 50 rats that were allotted equally into five groups composed of control, diabetic, diabetic treated with (AC, 0.06%), (His, 0.1%), and (AC & His) groups. Treated groups have received the treatments daily in drinking water for two months. Metabolic profile (glucose, insulin resistance indices, lipid profile, and cardiovascular indices) and renal dysfunction parameters (creatinine and urinary protein excretion) were measured. Plus, diverse glycation (early, intermediate, and end), oxidative stress (Oxidized LDL, Reduced glutathione), and inflammatory markers (interleukine-1β, myeloperoxidase, and NF-kβ expression) were determined. Glucose, insulin resistance indices, cardiovascular indices, renal dysfunction parameters, different markers of glycation, oxidative stress, and inflammation as well as NF-κB expression, were the lowest in the (AC & His) treated diabetic rats. Besides, the cited parameter was lower in the Ac treated one than His treated (p > 0.001). The combination of AC and His had the most protective effect against diabetes complications and advantageous effect on metabolism, β-cell activity, and insulin function due to the most reductive effect on the NF-κB pathway rather than More than any of the amino acids alone.

319-OR: Na+/K+ ATPase Activation Is a Conserved Mechanism for Gi/o Protein-Coupled Receptor Control of Pancreatic ß-Cell Electrical Excitability and Insulin Secretion

Gi/o protein-coupled receptor (Gi/o-GPCR) inhibition of pancreatic β-cell Ca2+ entry limits islet insulin secretion; however, the mechanism has not been identified. As Gi/o-GPCRs activate G protein-gated inwardly rectifying K+ (GIRK) channels, we examined their contribution to β-cell Gi/o signaling. However, Gi/o-GPCR-induced β-cell GIRK currents were not observed; this was demonstrated with a pharmacological approach and by genetic knockout of the predominant β-cell GIRK channel subunit. Thus, we increased the chemical driving force for K+ movement through GIRK channels by removing extracellular K+. Interestingly, under these conditions Gi/o-GPCR-induced β-cell membrane potential (Vm) hyperpolarization was lost even though K+ channel activation could still hyperpolarize Vm, which indicated that Gi/o-GPCRs do not hyperpolarize β-cell Vm by activating K+ channels. As extracellular K+ is required for Na+/K+ ATPase (NKA) activity, we next tested and found that mouse and human β-cell NKAs were activated by Gi/o-GPCRs. Stimulation of Gi/o-GPCRs initiated cyclical β-cell NKA activity, resulting in islet Ca2+ oscillations. Moreover, paracrine activation of β-cell NKAs by intraislet δ-cell somatostatin (SST) secretion decelerated islet Ca2+ oscillations and diminished insulin secretion, which was mediated by direct activation of β-cell Gi/o-GPCRs. Gi/o signaling induced islet Ca2+ oscillations through Src tyrosine kinase-dependent NKA phosphorylation and activation; an effect that was recapitulated by stimulating insulin receptor tyrosine kinases. Whereas, islet NKA function was completely inhibited by cAMP-dependent PKA activation. Therefore, these findings reveal that NKA-mediated hyperpolarization of β-cell Vm is the primary and conserved mechanism for Gi/o-GPCR control of electrical excitability, Ca2+ handling, and insulin secretion. Disclosure M.Dickerson: None. D.Jacobson: None. P.Dadi: None. K.E.Zaborska: None. A.Y.Nakhe: None. C.Schaub: None. J.Dobson: None. N.M.Wright: None. J.Lynch: None. C.Scott: None. Funding American Diabetes Association (1-17-IBS-024) ; Vanderbilt Integrated Training in Engineering and Diabetes Grant (T32DK101003) , National Institutes of Health Grants (DK-097392 and DK-115620) , Juvenile Diabetes Research Foundation Grant (2-SRA-2019-701-S-B) , and a Pilot and Feasibility grant through the Vanderbilt Diabetes Research and Training Center Grant (P60-DK-20593) .

Accelerated Generation of Extra-Islet Insulin-Producing Cells in Diabetic Rats, Treated with Sodium Phthalhydrazide.

β-cells dysfunction plays an important role in the pathogenesis of type 2 diabetes (T2D), partially may be compensated by the generation of extra-islet insulin-producing cells (IPCs) in pancreatic acini and ducts. Pdx1 expression and inflammatory level are suggested to be involved in the generation of extra-islet IPCs, but the exact reasons and mechanisms of it are unclear. Macrophages are key inflammatory mediators in T2D. We studied changes in mass and characteristics of extra-islet IPCs in rats with a streptozotocin-nicotinamide model of T2D and after i.m. administration of 20 daily doses of 2 mg/kg b.w. sodium aminophthalhydrazide (APH). Previously, we found that APH modulates macrophage production and increases the proliferative activity of pancreatic β-cells. Expressions of insulin and Pdx1, as well as F4/80 (macrophage marker), were detected at the protein level by immunohistochemistry analysis, the concentration of pro- and anti-inflammatory cytokines in blood and pancreas—by ELISA. Diabetic rats treated with APH showed an increasing mass of extra-islet IPCs and the content of insulin in them. The presence of Pdx1+ cells in the exocrine pancreas also increased. F4/80+ cell reduction was accompanied by increasing TGF-β1 content. Interestingly, during the development of diabetes, the mass of β-cells decreased faster than the mass of extra-islet IPCs, and extra-islet IPCs reacted to experimental T2D differently depending on their acinar or ductal location.

Oscillations in K(ATP) conductance drive slow calcium oscillations in pancreatic β-cells

ATP-sensitive K+ (K(ATP)) channels were first reported in the β-cells of pancreatic islets in 1984, and it was soon established that they are the primary means by which the blood glucose level is transduced to cellular electrical activity and consequently insulin secretion. However, the role that the K(ATP) channels play in driving the bursting electrical activity of islet β-cells, which drives pulsatile insulin secretion, remains unclear. One difficulty is that bursting is abolished when several different ion channel types are blocked pharmacologically or genetically, making it challenging to distinguish causation from correlation. Here, we demonstrate a means for determining whether activity-dependent oscillations in K(ATP) conductance play the primary role in driving electrical bursting in β-cells. We use mathematical models to predict that if K(ATP) is the driver, then contrary to intuition, the mean, peak, and nadir levels of ATP/ADP should be invariant to changes in glucose within the concentration range that supports bursting. We test this in islets using Perceval-HR to image oscillations in ATP/ADP. We find that mean, peak, and nadir levels are indeed approximately invariant, supporting the hypothesis that oscillations in K(ATP) conductance are the main drivers of the slow bursting oscillations typically seen at stimulatory glucose levels in mouse islets. In conclusion, we provide, for the first time to our knowledge, causal evidence for the role of K(ATP) channels not only as the primary target for glucose regulation but also for their role in driving bursting electrical activity and pulsatile insulin secretion.

Glycerol-3-phosphate phosphatase operates a glycerol shunt in pancreatic β-cells that controls insulin secretion and metabolic stress

ObjectiveThe recently identified glycerol-3-phosphate (Gro3P) phosphatase (G3PP) in mammalian cells, encoded by the PGP gene, was shown to regulate glucose, lipid and energy metabolism by hydrolyzing Gro3P and to control glucose-stimulated insulin secretion (GSIS) in β-cells, in vitro. However, whether G3PP regulates β-cell function and insulin secretion in vivo is not known. MethodsWe now examined the role of G3PP in the control of insulin secretion in vivo, β-cell function and glucotoxicity in inducible β-cell specific G3PP-KO (BKO) mice. Inducible BKO mice were generated by crossing floxed-G3PP mice with Mip-Cre-ERT (MCre) mice. All the in vivo studies were done using BKO and control mice fed normal diet and the ex vivo studies were done using pancreatic islets from these mice. ResultsBKO mice, compared to MCre controls, showed increased body weight, adiposity, fed insulinemia, enhanced in vivo GSIS, reduced plasma triglycerides and mild glucose intolerance. Isolated BKO mouse islets incubated at high (16.7 mM), but not at low or intermediate glucose (3 and 8 mM), showed elevated GSIS, Gro3P content as well as increased levels of metabolites and signaling coupling factors known to reflect β-cell activation for insulin secretion. BKO islets also showed reduced glycerol release and increased O2 consumption and ATP production at high glucose only. BKO islets chronically exposed to elevated glucose levels showed increased apoptosis, reduced insulin content and decreased mRNA expression of β-cell differentiation markers, Pdx-1, MafA and Ins-2. ConclusionsThe results demonstrate that β-cells are endowed with a “glycerol shunt”, operated by G3PP that regulates β-cell metabolism, signaling and insulin secretion in vivo, primarily at elevated glucose concentrations. We propose that the glycerol shunt plays a role in preventing insulin hypersecretion and excess body weight gain and contributes to β-cell mass preservation in the face of hyperglycemia.

Circular RNA PIP5K1A act as microRNA-552-3p sponge to regulates inflammation, oxidative damage in glucolipotoxicity-induced pancreatic INS-1 β-cells via Janus kinase 1

ABSTRACT Elevated level of glucolipotoxicity induces the loss of pancreatic β-cells functions and plays an important role in the development of type 2 diabetes (T2DM). Previous studies have indicated the importance of developing therapies against T2DM, while circular RNA (circRNA) has gained attraction as a modulator of pancreatic β-cell function. In the present study role of circPIP5K1A in dysfunctional β cells and mouse pancreas was comprehensively analyzed. INS-1E, as it has close similarity with naïve pancreatic β-cells, and clinical samples of T2DM patients were used to investigate the effect of circPIP5K1A, miR-552-3p, and Janus kinase 1 (JAK1). While, INS-1E cells were exposed to PAHG conditions (0.5 mM palmitic acid and 28 mM glucose) as studies have suggested that increased level of fatty acid and glucose resulted in autophagy activation of pancreatic β-cells that leads to T2DM. Key player of JAK1-STAT3 pathway and the level of Reactive Oxygen Species, inflammatory factors, and insulin secretion was detected to analyze the of the active association of circPIP5K1A, miR-552-3p with JAK1pathway. Our study has revealed the elevated level ofcircPIP5K1A and JAK1, but reduced level of miR-552-3pin the serum of T2DM patients. Furthermore, we also found that reduced expression ofcircPIP5K1A leads to decreased rate of inflammation, oxidative damage and apoptosisinINS-1E cells induced by glucolipotoxicity. CircPIP5K1A was available to competitively combine with miR-552-3p, while whose direct target was JAK1. In conclusion, our study suggested a novel involvement of circPIP5K1A in a cross talk between miR5523p/JAK1/STAT3 pathways in β-cells as a new therapeutic target for T2DM.

Glucose-6-phosphatase catalytic subunit 2 negatively regulates glucose oxidation and insulin secretion in pancreatic β-cells

Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on β-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in βTC3 cells, a murine pancreatic β-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to ∼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in β-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.

Molecular mechanisms responsible for the sexual dimorphism in pancreatic β-cell insulin release

In humans, type 2 diabetes mellitus (T2DM) has a higher incidence in males compared to females, a phenotype recapitulated by many rodent models. While the sex difference in insulin sensitivity partially accounts for this phenomenon, hitherto uncharacterized differences in pancreatic β-cell insulin release strongly contribute. Here we show that stepwise increase in extracellular glucose concentration (2, 5, 7.5, 10, 15, 20 mM) induced electrical activity in β-cells of both sexes with similar glucose sensitivity (Female EC50 =9.45±0.15 mM, Male EC50 =9.42±0.16 mM).

Eugenosedin-A improves obesity-related hyperglycemia by regulating ATP-sensitive K+ channels and insulin secretion in pancreatic β cells

Eugenosedin-A (Eu-A) has been shown to protect against hyperglycemia- and hyperlipidemia-induced metabolic syndrome. We investigated the relationship of KATP channel activities and insulin secretion by Eu-A in vitro in pancreatic β-cells, and examined the effect of Eu-A on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetes mellitus (T2DM) in vivo. We isolated pancreatic islets from adult male Wistar rats (250-350g) and identified pancreatic β-cells by the cell size, capacitance and membrane potential. Perforated patch-clamp and inside-out recordings were used to monitor the membrane potential (current-clamp mode) and channel activity (voltage-clamp mode) of β-cells. The membrane potential of β-cells was raised by Eu-A and reversed by the KATP channel activator diazoxide. Eu-A inhibited the KATP channel activity measured at -60mV and increased the intracellular calcium concentration ([Ca2+]i), resulting in enhanced insulin secretion. Eu-A also reduced Kir6.2 protein on the cell membrane and scattered in the cytosol under normal glucose conditions (5.6mM). In our animal study, rats were divided into normal and STZ/NA-induced T2DM groups. Normal rats fed with regular chow were divided into control and control+Eu-A (5mg/kg/day, i.p.) groups. The STZ/NA-induced diabetic rats fed with a high-fat diet (HFD) were divided into three groups: T2DM, T2DM+Eu-A (5mg/kg/day, i.p.), and T2DM+glibenclamide (0.5mg/kg/day, i.p.; a KATP channel inhibitor). Both Eu-A and glibenclamide decreased the rats' blood glucose, prevented weight gain, and enhanced insulin secretion. We found that Eu-A blocked pancreatic β-cell KATP channels, caused membrane potential depolarization, and stimulated Ca2+ influx, thus increasing insulin secretion. Furthermore, Eu-A decreased blood glucose and increased insulin levels in T2DM rats. These results suggested that Eu-A might have clinical benefits for the control of T2DM and its complications.

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1.

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo β-Cell Function.

The pancreatic β-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in β-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, β-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying β-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how β-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate β-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate β-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in β-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect β-cell function.

Воглібоз у комбінації трьох цукрознижувальних препаратів при лікуванні цукрового діабету 2-го типу

Мета дослідження — клініко-метаболічне обґрунтування вибору третього цукрознижувального препарату для підвищення ефективності лікування хворих на цукровий діабет (ЦД) 2-го типу, оцінка впливу інтенсифікації терапії за рахунок додавання воглібозу на показники вуглеводного обміну, що не досягли цільових рівнів при комбінованій терапії метформіном з глімепіридом. Матеріали й методи. Під спостереженням перебувало 45 хворих на ЦД 2-го типу, які не досягнули цільових показників вуглеводного обміну під впливом попереднього лікування (метформін і глімепірид). Після початкового клінічного обстеження хворі були розподілені на дві групи. У першій групі (n = 30) до терапії додавали воглібоз (препарат Воксид, виробництво ТОВ «Кусум Фарм», Україна) по 0,2 мг перед сніданком, обідом і вечерею впродовж 12 тижнів. Результати. Через 12 тижнів лікування рівень глікованого гемоглобіну (НbА1с) у першій групі знизився на 1,5 [1,1; 1,9] % (р &lt; 0,05), у другій групі — на 0,1 [0,09; 0,6] % (р &gt; 0,05). У 80 % пацієнтів досягнуті цільові рівні глікемії. У першій групі пацієнтів виявлено вірогідне підвищення індексу HOMA-β на 24,9 %, а також зниження інсулінорезистентності (HOMA-IR) на 31,5 %. Через 12 тижнів терапії відзначалося статистично вірогідне зниження маси тіла на 2,1 кг у пацієнтів першої групи й відсутність динаміки в пацієнтів другої групи. Доведена безпека з боку функціонального стану печінки та нирок при цьому варіанті терапії. Висновки. Додавання воглібозу для інтенсифікації терапії у хворих на ЦД 2-го типу, які отримували до цього метформін і глімепірид, призвело до вірогідного покращання показників вуглеводного обміну (зниження HbA1c на 1,5 %). Покращання контролю вуглеводного обміну при комбінованій терапії метформіном, глімепіридом і воглібозом супроводжується підвищенням функціональної активності β-клітин підшлункової залози, зниженням інсулінорезистентності на тлі зменшення маси тіла.

Lipid Droplets Protect Human β-Cells From Lipotoxicity-Induced Stress and Cell Identity Changes.

Free fatty acids (FFAs) are often stored in lipid droplet (LD) depots for eventual metabolic and/or synthetic use in many cell types, such a muscle, liver, and fat. In pancreatic islets, overt LD accumulation was detected in humans but not mice. LD buildup in islets was principally observed after roughly 11 years of age, increasing throughout adulthood under physiologic conditions, and also enriched in type 2 diabetes. To obtain insight into the role of LDs in human islet β-cell function, the levels of a key LD scaffold protein, perilipin 2 (PLIN2), were manipulated by lentiviral-mediated knockdown (KD) or overexpression (OE) in EndoCβH2-Cre cells, a human cell line with adult islet β-like properties. Glucose-stimulated insulin secretion was blunted in PLIN2KD cells and improved in PLIN2OE cells. An unbiased transcriptomic analysis revealed that limiting LD formation induced effectors of endoplasmic reticulum (ER) stress that compromised the expression of critical β-cell function and identity genes. These changes were essentially reversed by PLIN2OE or using the ER stress inhibitor, tauroursodeoxycholic acid. These results strongly suggest that LDs are essential for adult human islet β-cell activity by preserving FFA homeostasis.