Rat Beta-cells Research Articles

Stereological study of the effects of orally administrated Otostegia persica extract on pancreatic beta cells in male diabetic rats

Otostegia persica extract was used as a traditional medicine for the management of diabetes mellitus in humans in some parts of Iran. This study investigated the effects of O. persica oral extract on pancreatic beta cells in streptozotocin-induced diabetic rats by stereological methods. Thirty-two matured normoglycemic male Sprague–Dawley rats, weighing 180–220 g, were selected and randomly divided into four groups: Control group consists of normal rats which did not receive the extract during the study. Diabetic group comprises diabetic rats but did not receive any extract. Treated control group rats were normal but received the extract at a dose of 500 mg/kg/day. Treated diabetic group (TD) was made up of diabetic rats and received the extract (500 mg/kg/day). Diabetes was induced by a single intraperitoneal injection of 50 mg/kg of streptozotocin. After 1 month, all the rats received deep anesthesia with ether, and then, the pancreas was dissected and processed. Isotropic uniform and random sections were obtained by orientator method. Volume of pancreatic beta cells and also volume density of pancreatic island were studied, using stereologic and ultrastereological methods. Blood glucose level (BGL) and blood insulin level (BIL) were measured in different phases during the investigation. Statistical analysis by one-way analysis of variance test showed the presence of hypertrophic changes in the volume of the remaining beta cells in diabetic group, not in the controls. But such changes reduced significantly (P < 0.05) in the TD group which received the extract. Also, a significant reduction in pancreatic islet volume in diabetic and TD groups was seen, in comparison with the controls (P < 0.01). BGL decreased significantly in TD group, compared to the diabetic group (P < 0.001). BIL decreased in diabetic and TD groups in comparison to the controls (P < 0.05). This study demonstrated that O. persica oral extract can play an effective role in the management of diabetes and probably by controlling the hyperglycemic condition and not stimulating the beta cell to increase insulin secretion can help prevent to a large extent the entering of the remaining beta cells to some pathologic changes, like hypertrophy.

Expression profiling of putative type 2 diabetes susceptibility genes in human islets and in rat beta cell lines

Over 50 single nucleotide polymorphisms (SNPs) have been identified by genome wide association studies (GWAS) to be associated with susceptibility to type 2 diabetes (T2D); however the causal gene in most cases is not known. In this study we sought to identify which may be the most likely causal genes at five T2D GWAS loci by measuring their expression in control and T2D islets, as well as observing their regulation by glucose. We measured the expression of ten genes at five loci (CDKN2A/2B, CDC123/CAMK-1D, HHEX/IDE, TSPAN8/LGR5, and DGKB/TMEM 195), in control and human pancreatic islets by real-time PCR. We then measured the expression of these genes in the rodent pancreatic beta cell line INS-1 exposed to 5.6 mmol/l, 11 mmol/l and 28 mmol/l glucose for 48 hours. We found differential expression of the longest isoform of CDKN2B specifically between control and T2D human islets, whereas the shortest isoform of this gene had no expression in islets. Tmem195 was the only gene to show differential expression in response to increasing glycemia in INS-1 cells under the conditions described. Our study is an example of how the differential expression of genes in loci spanning more than one gene can aid identification of the more likely causal gene.

Effect of Ginger Juice Water on Blood Glucose Levels and Histopathology of Pancreatic Beta Cells Experimental Studies on Alloxan Induced Wistar Male Rats

Background: Hyperglycemia is one of the signs of diabetes mellitus, the disease is still being suffered by the people of Indonesia. Oral antidiabetic drugs mostly give unwanted side effects, then the experts developed a system of traditional medicine for diabetes mellitus. The research aimed to determine the effect of ginger juice on blood glucose levels and histopathological of pancreatic beta cells in male wistar rats induced by alloxan.Design and methods: An experimental study with a post-test only control group design, the study subjects white male wistar rats. Rats were divided into 5 groups, each group consisted of 5 rats, the group I was the control group (no treatment), group II is diabetic group (negative control), group III is Ginger + diabetes group, diabetes group is a group IV + Ginger + glibenclamide at a dose of each half, the group is the group of diabetic + V Glibenklamid.Results: The mean blood glucose levels were highest 172.02 ± 21.40 mg / dl is the K II. Lowest mean glucose levels were 112.72 ± 14.49 mg / dl for KV. Score the highest rates of pancreatic beta cell damage that is at the K II, while the lowest in the K V. Conclusion: Ginger Juice effect on blood glucose levels alloxan-induced rats. Ginger juice histopathologic effect on pancreatic beta cells alloxan-induced rats (Sains Medika, 4(2):165-173).

The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation.

Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children

Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes. The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1-3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence. Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1β, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls. These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia

Insulin-producing beta cells are known to be highly susceptible to hypoxia, which is a major factor in their destruction after pancreatic islet transplantation. However, whether the glucagon-producing pancreatic islet alpha cells are sensitive to hypoxia is not known. Our objective was to compare the sensitivity of alpha and beta cells to hypoxia. Isolated rat pancreatic islets were exposed to hypoxia (1% oxygen, 94% N(2), 5% CO(2)) for 3 days. The viability of the alpha and beta cells, as well as the stimulus-specific secretion of glucagon and insulin, was evaluated. A quantitative analysis of the proportion of beta to alpha cells indicated that, under normoxic conditions, islet cells were composed mainly of beta cells (87 ± 3%) with only 13 ± 3% alpha cells. Instead, hypoxia treatment significantly increased the proportion of alpha cells (40 ± 13%) and decreased the proportion of beta cells to 60 ± 13%. Using the fluorescent TUNEL assay we found that only a few percent of beta cells and alpha cells were apoptotic in normoxia. In contrast, hypoxia induced an abundance of apoptotic beta cells (61 ± 22%) and had no effect on the level of apoptosis in alpha cells. In conclusion, this study demonstrates that hypoxia results in severe functional abnormality in both beta and alpha cells while alpha cells display significantly decreased rate of apoptosis compared to intensive apoptotic injury of beta cells. These findings have implications for the understanding of the possible role of hypoxia in the pathophysiology of diabetes.

Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling.

Aims/hypothesisKnowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size.MethodsElectron micrographs of rat beta cells fixed either chemically or by high-pressure freezing were compared using a high-content analysis approach. These data were used to develop three-dimensional in silico beta cell models, the slicing of which would reproduce the experimental datasets.ResultsAs previously reported, chemically fixed insulin secretory granules appeared as hollow spheres with a mean diameter of ∼350 nm. Remarkably, most granules fixed by high-pressure freezing lacked the characteristic halo between the dense core and the limiting membrane and were smaller than their chemically fixed counterparts. Based on our analyses, we conclude that the mean diameter of rat insulin secretory granules is 243 nm, corresponding to a surface area of 0.19 μm2. Rat beta cells have a mean volume of 763 μm3 and contain 5,000–6,000 granules.Conclusions/interpretationA major reason for the lower mean granule number/rat beta cell relative to previous accounts is a reduced estimation of the mean beta cell volume. These findings imply that each granule contains about twofold more insulin, while its exocytosis increases membrane capacitance about twofold less than assumed previously. Our integrated approach defines new standards for quantitative image analysis of beta cells and could be applied to other cellular systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2438-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Glucose-Induced O2 Consumption Activates Hypoxia Inducible Factors 1 and 2 in Rat Insulin-Secreting Pancreatic Beta-Cells

BackgroundGlucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner.Methodology/Principal FindingsIsolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO2 values or treated with the HIF activator CoCl2. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl2 or a four-fold reduction in pO2. Islets also showed signs of HIF activation in diabetic Leprdb/db but not non-diabetic Leprdb/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO2 or by inhibitors of Ca2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene.Conclusions/SignificanceGlucose-induced O2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes.

Multivesicular exocytosis in rat pancreatic beta cells

Aims/hypothesisTo establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells.MethodsExocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis.ResultsCompound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15–20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca2+]i from 0.2 to 2 μmol/l Ca2+. Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1–2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol.Conclusions/interpretationAlthough contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2400-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

AMP-Activated Protein Kinase (AMPK) Mediates Nutrient Regulation of Thioredoxin-Interacting Protein (TXNIP) in Pancreatic Beta-Cells

Thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK) has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids) effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP). Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes.

Interleukin‐6 treatment induces beta‐cell apoptosis via STAT‐3‐mediated nitric oxide production

Type 2 diabetes is characterized by progressive beta-cell failure and apoptosis is probably the main form of beta-cell death in this disease. It was reported that circulating levels of interleukin-6 are elevated in type 2 diabetic patients, but whether this is involved in the pathogenesis of type 2 diabetes is still debated. In this study, we examined whether interleukin-6 can induce beta-cell damage in vitro and elucidated its mechanisms. To examine the effect of interleukin-6 on beta cells, glucose-stimulated insulin secretion (GSIS) by enzyme immunoassay (EIA) method and cell apoptosis by propidium iodide and annexin-V staining were measured in a rat beta-cell line (INS-1 or INS-832/13) after treatment with interleukin-6. The expression of apoptosis-related molecules was measured using western blotting and nitric oxide (NO) production was measured using Griess assay. AG490 and N-monomethyl-L-arginine were used to inhibit Janus kinase-mediated signal transducers and activators of transcription signalling and NO production, respectively. Exposure (48 h) of INS-1 cells to 20 ng/mL interleukin-6 significantly decreased GSIS as well as cell viability. We found that sub-G1/G0 population was increased as compared with untreated cells and expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, phosphorylated p38 mitogen-activated protein kinase and phosphorylated nuclear factor-κB was increased in interleukin-6-treated INS-1 cells. Interleukin-6 increased the amount of early apoptotic cells; this increase was blocked by AG490 or N-monomethyl-L-arginine treatment. Moreover, NO production, which is known to induce apoptosis, was increased by interleukin-6 treatment but abrogated in AG490-treated cells. Our results show that exposure to interleukin-6 for 48 h can induce beta-cell death, in part via signal transducers and activators of transcription-3-mediated NO production.

Effect of aging on insulin receptor in islet beta-cells of Wistar rats

Objective To evaluate the effect of aging on the insulin receptor in islet beta-cells of Wistar rats,so as to investigate the mechanism of impaired glucose tolerance in elderly population.Methods Thirty healthy male Wistar rats were divided into young group(4-month old,n=15) and elderly group(24-monfh old,n=15).The fasting blood glucose,blood insulin,free fatty acid,and blood lipid were observed.Insulin sensitivity and the function of islet cells were assessed by the euglycaemic-hyperinsulinaemic clamp technique.The insulin receptor in islet beta-cells was observed using immunofluorescence staining,and the fluorescence intensity was analyzed using software.Results The body mass of rats in young group and elderly group was(490.60±7.72)g and(728.90±7.57)g respectively(P0.01).There was no significant difference in fasting blood glucose between the two groups(P0.05).The fasting blood insulin was(0.59±0.14)ng/ml and(2.37±0.04)ng/ml in young group and elderly group respectively(P0.01).The blood free fatty acid was significantly lower in young group than in elderly group[(0.76±0.14) vs (1.51±0.04) mmol/L,P0.01].The euglycaemic-hyperinsulinaemic clamp test displayed that the glucose infusion rate was significantly higher in young group than in elderly group[(26.02±2.83) vs(10.73±0.72)mg/(kg·mm),P0.01].Homeostasis model insulin resistance index(HOMA-IR) was(3.09±0.80) and(13.14±1.59) in young group and elderly group(P0.01).The fluorescence intensity was(63.55±5.20) and(23.26±2.50) respectively in young group and elderly group respectively(P0.01).Conclusion The elderly rats present with insulin resistance and compensatory high level insulin secretion.Meanwhile,insulin receptor in islet beta-cells is decreased.It is possible that the peripheral insulin resistance and the insulin resistance in islet beta-cells itself coexist in elderly rats.

Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity

Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans. Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy. Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules. Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.

Identification of an intracellular metabolic signature impairing beta cell function in the rat beta cell line INS-1E and human islets

Chronic hyperglycaemia promotes the progressive failure of pancreatic beta cells in patients with type 2 diabetes mellitus, a clinically highly relevant phenomenon known as glucotoxicity. The intracellular metabolic consequences of a chronically high availability of glucose in beta cells are, as yet, poorly understood in its full complexity. An unbiased metabolite profiling analysis (GC-time-of-flight-MS) was used to identify the time course of core metabolite patterns in rat beta cell line INS-1E during exposure to high glucose concentrations and its relation to insulin expression. We report here that pentose phosphate pathway (PPP) metabolites accumulate remarkably during chronic but not acute glucose treatment, indicating altered processing of glucose through the pentose phosphate pathway. Subsequent functional studies in INS-1E cells and human islets revealed that a disturbance in this pathway contributes to decreases in insulin gene expression and a lack of glucose-stimulated insulin secretion. These effects were found to depend on the activation of extracellular-regulated-kinase (ERK1/2). Long-term inhibition of 6-phosphogluconic acid dehydrogenase resulted in accumulation of PPP metabolites, induced ERK1/2 activation independently of high glucose and impaired beta cell function. In turn, inhibition of ERK1/2 overstimulation during chronic glucose exposure partly inhibited metabolite accumulation and restored beta cell function. Based on unbiased metabolite analyses, the data presented here provide novel targets, namely the inhibition of PPP metabolite accumulation towards the therapeutic goal to preserve and potentially improve beta cell function in diabetes.

Synthesis of a novel suppressor of beta-cell apoptosis via diversity-oriented synthesis.

The synthesis of a stereochemically diverse library of medium-sized rings accessible via a 'build/couple/pair' strategy is described. Key aspects of the synthesis include S(N)Ar cycloetherification of a linear amine template to afford eight stereoisomeric 8-membered lactams and subsequent solid-phase diversification of these scaffolds to yield a 6488-membered library. Screening of this compound collection in a cell-based assay for the suppression of cytokine-induced beta-cell apoptosis resulted in the identification of a small-molecule suppressor capable of restoring glucose-stimulated insulin secretion in a rat beta-cell line. The presence of all stereoisomers in the screening collection enabled preliminary determination of the structural and stereochemical requirements for cellular activity, while efficient follow-up chemistry afforded BRD-0476 (probe ML187), which had an approximately three-fold increase in activity. These results demonstrate the utility of diversity-oriented synthesis to probe discovery using cell-based screening, and the importance of including stereochemical diversity in screening collections for the development of stereo/structure-activity relationships.

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Glucokinase mediates coupling of glycolysis to mitochondrial metabolism but not to beta cell damage at high glucose exposure levels

Glucose is the main stimulus of insulin secretion in pancreatic beta cells. However, high glucose has also been considered to damage beta cells. In this study we examined, with special emphasis on the role of the glucose sensor enzyme glucokinase, whether elevated glucose metabolism evokes toxicity to beta cells. RINm5F-R-EYFP-GK cells, producing glucokinase in response to a synthetic inducer, and rat beta cells were incubated at different glucose concentrations. Glucokinase enzyme activity, insulin secretion, cell viability and mitochondrial metabolism were analysed. Glucokinase production evoked a concentration-dependent increase in glucose-induced insulin secretion from RINm5F-R-EYFP-GK cells without reducing cell viability. Pre-culture at high glucose (30 mmol/l) in the absence of high concentrations of NEFA neither reduced viability nor significantly increased apoptosis in RINm5F-R-EYFP-GK cells and rat beta cells. The integrity of the mitochondrial respiratory chain and mitochondrial dynamics, namely fusion and fission, were not impaired by high glucose pre-culture. As previously demonstrated in mouse beta cells, pre-culture at high glucose significantly decreased the mitochondrial membrane potential heterogeneity in RINm5F-R-EYFP-GK cells. Indeed, after starvation, in response to glucose, rat beta cells and RINm5F-R-EYFP-GK cells with glucokinase production pre-cultured for 48 h at high glucose showed the fastest increase in the mitochondrial membrane potential. Our experiments do not support the hypothesis that glucokinase and the glucose metabolism on its own act as a mediator of beta cell toxicity. By contrast, rather a beneficial effect on glucose-induced insulin secretion after glucokinase production was observed, based on an improved coupling of the glucose stimulus to the mitochondrial metabolism.

Pancreas-protective effects of chlorella in STZ-induced diabetic animal model: insights into the mechanism

The aim of this study is to examine the effect of intragastric administration of chlorella (1 g/kg body weight) for a period of 30 days to treat normal and diabetic male Wistar rats. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (60 mg/kg - 1 body weight). A significant (p < 0.05) reduction of blood glucose level in diabetic chlorella-treated rats was observed compared to diabetic untreated. Chlorella increased the number of glutathione-positive cell in diabetic rats compared to untreated diabetics. Chlorella administration increased the percentage of insulin secreting pancreatic beta cells both in normal and diabetic treated rats. Percentage of glucagon producing alpha cells of the pancreas were reduced both in normal and diabetic chlorella-treated rats. Chlorella-induced regenerative ability on pancreas was mediated by up-regulation of Ki67 and down-regulation of P53 and by its potent anti-oxidant ability. The present results suggest that chlorella may play an important role in improving the overall condition of diabetic patients and delay its complication by restoring the function of pancreatic insulin-secreting cells.

Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells.

Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells. By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA). At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells. In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role.

Protein Markers for Insulin-Producing Beta Cells with Higher Glucose Sensitivity

Background and MethodologyPancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins.Principal FindingsAll tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain.ConclusionsQuantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.