Aims/hypothesis
The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta ...cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1–3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration.
Methods
Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3–7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1–3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca
2+
concentration (Ca
2+
c
).
Results
Culture of ND-islets for 1–3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the Ca
2+
c
and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K
+
. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose.
Conclusions/interpretation
Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in Ca
2+
c
and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.
Graphical abstract
Insulin secretion from pancreatic β-cells is critical for maintaining glucose homeostasis and deregulation of circulating insulin levels is associated with the development of metabolic diseases. ...While many factors have been implicated in the stimulation of insulin secretion, the mechanisms that subsequently reduce insulin secretion remain largely unexplored. Here we demonstrate that mice with β-cell specific ablation of the Y1 receptor exhibit significantly upregulated serum insulin levels associated with increased body weight and adiposity. Interestingly, when challenged with a high fat diet these β-cell specific Y1-deficient mice also develop hyperglycaemia and impaired glucose tolerance. This is most likely due to enhanced hepatic lipid synthesis, resulting in an increase of lipid accumulation in the liver. Together, our study demonstrates that Y1 receptor signaling negatively regulates insulin release, and pharmacological inhibition of Y1 receptor signalling for the treatment of non-insulin dependent diabetes should be taken into careful consideration.
High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional β-cell mass in type 2 diabetes. To test that ...hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on β-cell stimulus-secretion coupling events and on β-cell apoptosis.
After 1–2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ∼1.7-fold higher in NOX2-KO vs. WT islets at 20–30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration (Ca2+i) and no differences in cytosolic GRX1-roGFP2 oxidation.
After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and β-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, Ca2+i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with β-cell apoptosis, the glucose sensitivity of the NADPH, Ca2+i and insulin secretion responses increased, the maximal Ca2+i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types.
In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of β-cell survival and function.
•NOX2 was confirmed as a negative modulator of glucose-stimulated insulin secretion.•1 day-culture under glucotoxic condition increased β-cell cytosolic thiol oxidation.•3 weeks-culture under glucotoxic condition altered β-cell function and survival.•NOX2 did not contribute to in vitro β-cell glucotoxicity.
It is well established that regular physiological stimulation by glucose plays a crucial role in the maintenance of the beta-cell differentiated phenotype. In contrast, prolonged or repeated exposure ...to elevated glucose concentrations both in vitro and in vivo exerts deleterious or toxic effects on the beta-cell phenotype, a concept termed as glucotoxicity.
The normal beta-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with beta-cells that are susceptible to failure. Here, we ...investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for beta-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several beta-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with beta-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of beta-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.
Glucose 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. Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO.sub.2 values or treated with the HIF activator CoCl.sub.2 . 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 CoCl.sub.2 or a four-fold reduction in pO.sub.2 . Islets also showed signs of HIF activation in diabetic Lepr.sup.db/db but not non-diabetic Lepr.sup.db/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO.sub.2 or by inhibitors of Ca.sup.2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1alpha and Hif2alpha, 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. Glucose-induced O.sub.2 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.
Endoplasmic reticulum (ER) stress and the consecutive activation of the Unfolded Protein Response (UPR) contribute to the pathogenesis of several diseases including diabetes, neurodegenerative ...diseases and inflammation. However, the UPR also plays a crucial adaptive role in the acquisition and maintenance of the phenotype of cells that secrete large amounts of proteins. After a brief overview of this physiological role of the UPR in immunoglobulin-secreting plasmocytes and pancreatic acinar cells, this chapter will mainly focus on insulin-secreting pancreatic b-cells that play a critical role in glucose homeostasis. Upon their stimulation with glucose and other nutrients, these cells display a rise in mitochondrial metabolism, ATP production and Ca2+ pumping in the ER, in parallel to the stimulation of protein (preferentially proinsulin) biosynthesis. These metabolic and functional features give rise to a peculiar pattern of acute regulation of the UPR by nutrients. At low non-stimulatory glucose concentrations, when intracellular ATP, Ca2+ER and protein synthesis are low, the IRE1-XBP1 branch of the UPR is at its lowest level of activation while the PERK-eIF2α-ATF4 branch of the UPR is maximally activated, with strong upregulation of Integrated Stress Response (ISR) genes. Upon glucose stimulation, the rise in ATP and Ca2+ER leads to PERK-eIF2α dephosphorylation, inhibition of the ISR and derepression of protein synthesis. Consequent activation of the IRE1-XBP1 branch of the UPR upregulates the expression of chaperones, foldases, ER to Golgi transport and ER-associated degradation machinery that help the b-cell coping with the large increase in proinsulin biosynthesis. This opposite glucose regulation of the PERK and IRE1 arms of the UPR is rapid and dynamic, suggesting its importance in the physiological adaptation of the b-cell to changes in nutrient supply.
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