Pancreatic ß-cell identity in diabetes.

Recovery of functional ß-cell mass continues to be an ongoing challenge in treating diabetes. Initial work studying ß-cells suggested apoptotic ß-cell death as a main contributor for the loss of ß-cell mass in diabetes. Restoration of ß-cells either by transplant or stimulating proliferation of remaining ß-cells or precursors would then logically be a viable therapeutic option for diabetes. However, recent work has highlighted the inherent ß-cell plasticity and the critical role of loss of ß-cell identity in diabetes, and has suggested that ß-cells fail to maintain a fully differentiated glucose-responsive and drug-responsive state, particularly in diabetic individuals with poorly controlled, long-lasting periods of hyperglycaemia. Understanding the underlying mechanisms of loss of ß-cell identity and conversion in other cell types, as well as how to regain their mature differentiated functional state, is critical to develop novel therapeutic strategies to prevent or reverse these processes. In this review, we discuss the role of plasticity and loss of ß-cell identity in diabetes, the current understanding of mechanisms involved in altering this mature functional ß-cell state and potential progresses to identify novel therapeutic targets providing better opportunities for slowing or preventing diabetes progression.

The diabetic ß-cell: hyperstimulated vs. hyperexcited.

Hyperglycaemia has multiple effects on ß-cells, some clearly prosecretory, including hyperplasia and elevated insulin content, but eventually, a 'glucotoxic' effect which leads to pancreatic ß-cell dysfunction, reduced ß-cell mass and insulin deficiency, is an important part of diabetes pathophysiology. Myriad underlying cellular and molecular processes could lead to such dysfunction. High glucose will stimulate glycolysis and oxidative phosphorylation, which will in turn increase ß-cell membrane excitability through K(ATP) channel closure. Chronic hyperexcitability will then lead to persistently elevated [Ca(2+)](i), a key trigger to insulin secretion. Thus, at least a part of the consequence of 'hyperstimulation' by glucose has been suggested to be a result of 'hyperexcitability' and chronically elevated [Ca(2+)](i). This link is lost when the [glucose], K(ATP) -channel activity link is broken, either pharmacologically or genetically. In isolated islets, such studies reveal that hyperexcitability causes a largely reversible chronic loss of insulin content, but in vivo chronic hyperexcitability per se does not lead to ß-cell death or loss of insulin content. On the other hand, chronic inexcitability in vivo leads to systemic diabetes and consequential ß-cell death, even while [Ca(2+)](i) remains low.

The mitochondria and insulin release: Nnt just a passing relationship.

Nicotinamide nucleotide transhydrogenase (Nnt) detoxifies reactive oxygen species (ROS), byproducts of mitochondrial metabolism that, when accumulated, can decrease mitochondrial ATP production. In this issue of Cell Metabolism, demonstrate that Nnt in pancreatic beta cells is important for insulin release. Their compelling data highlight the critical roles for ATP generation and subsequent closure of KATP channels for insulin secretion.

Defects in beta cell Ca²+ signalling, glucose metabolism and insulin secretion in a murine model of K(ATP) channel-induced neonatal diabetes mellitus.

AIMS/HYPOTHESIS: Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. METHODS: We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. RESULTS: In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. CONCLUSIONS/INTERPRETATION: The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.

Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases.

The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.

beta-cell hyperexcitability: from hyperinsulinism to diabetes.

Nutrient oxidation in beta cells generates a rise in [ATP]:[ADP] ratio. This reduces K(ATP) channel activity, leading to depolarization, activation of voltage-dependent Ca(2+) channels, Ca(2+) entry and insulin secretion. Consistent with this paradigm, loss-of-function mutations in the genes (KCNJ11 and ABCC8) that encode the two subunits (Kir6.2 and SUR1, respectively) of the ATP-sensitive K(+) (K(ATP)) channel underlie hyperinsulinism in humans, a genetic disorder characterized by dysregulated insulin secretion. In mice with genetic suppression of K(ATP) channel subunit expression, partial loss of K(ATP) channel conductance also causes hypersecretion, but unexpectedly, complete loss results in an undersecreting, mildly glucose-intolerant phenotype. When challenged by a high-fat diet, normal mice and mice with reduced K(ATP) channel density respond with hypersecretion, but mice with more significant or complete loss of K(ATP) channels cross over, or progress further, to an undersecreting, diabetic phenotype. It is our contention that in mice, and perhaps in humans, there is an inverse U-shaped response to hyperexcitabilty, leading first to hypersecretion but with further exacerbation to undersecretion and diabetes. The causes of the overcompensation and diabetic susceptibility are poorly understood but may have broader implications for the progression of hyperinsulinism and type 2 diabetes in humans.

Hyperinsulinism in mice with heterozygous loss of K(ATP) channels.

AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic beta cells. In humans, loss-of-function mutations of beta cell K(ATP) subunits (SUR1, encoded by the gene ABCC8, or Kir6.2, encoded by the gene KCNJ11) cause congenital hyperinsulinaemia. Mice with dominant-negative reduction of beta cell K(ATP) (Kir6.2[AAA]) exhibit hyperinsulinism, whereas mice with zero K(ATP) (Kir6.2(-/-)) show transient hyperinsulinaemia as neonates, but are glucose-intolerant as adults. Thus, we propose that partial loss of beta cell K(ATP) in vivo causes insulin hypersecretion, but complete absence may cause insulin secretory failure. MATERIALS AND METHODS: Heterozygous Kir6.2(+/-) and SUR1(+/-) animals were generated by backcrossing from knockout animals. Glucose tolerance in intact animals was determined following i.p. loading. Glucose-stimulated insulin secretion (GSIS), islet K(ATP) conductance and glucose dependence of intracellular Ca(2+) were assessed in isolated islets. RESULTS: In both of the mechanistically distinct models of reduced K(ATP) (Kir6.2(+/-) and SUR1(+/-)), K(ATP) density is reduced by approximately 60%. While both Kir6.2(-/-) and SUR1(-/-) mice are glucose-intolerant and have reduced glucose-stimulated insulin secretion, heterozygous Kir6.2(+/-) and SUR1(+/-) mice show enhanced glucose tolerance and increased GSIS, paralleled by a left-shift in glucose dependence of intracellular Ca(2+) oscillations. CONCLUSIONS/INTERPRETATION: The results confirm that incomplete loss of beta cell K(ATP) in vivo underlies a hyperinsulinaemic phenotype, whereas complete loss of K(ATP) underlies eventual secretory failure.

Hyperinsulinism induced by targeted suppression of beta cell KATP channels.

ATP-sensitive K+ (K(ATP)) channels couple cell metabolism to electrical activity. To probe the role of K(ATP) in glucose-induced insulin secretion, we have generated transgenic mice expressing a dominant-negative, GFP-tagged K(ATP) channel subunit in which residues 132-134 (Gly-Tyr-Gly) in the selectivity filter were replaced by Ala-Ala-Ala, under control of the insulin promoter. Transgene expression was confirmed by both beta cell-specific green fluorescence and complete suppression of channel activity in those cells ( approximately 70%) that did fluoresce. Transgenic mice developed normally with no increased mortality and displayed normal body weight, blood glucose levels, and islet architecture. However, hyperinsulinism was evident in adult mice as (i) a disproportionately high level of circulating serum insulin for a given glucose concentration ( approximately 2-fold increase in blood insulin), (ii) enhanced glucose-induced insulin release from isolated islets, and (iii) mild yet significant enhancement in glucose tolerance. Enhanced glucose-induced insulin secretion results from both increased glucose sensitivity and increased release at saturating glucose concentration. The results suggest that incomplete suppression of K(ATP) channel activity can give rise to a maintained hyperinsulinism.