OBJECTIVE -␤-Cell function in type 1 diabetes clinical trials is commonly measured by C-peptide response to a secretagogue in either a mixed-meal tolerance test (MMTT) or a glucagon stimulation test (GST). The Type 1 Diabetes TrialNet Research Group and the European Cpeptide Trial (ECPT) Study Group conducted parallel randomized studies to compare the sensitivity, reproducibility, and tolerability of these procedures. RESEARCH DESIGN AND METHODS -In randomized sequences, 148TrialNet subjects completed 549 tests with up to 2 MMTT and 2 GST tests on separate days, and 118 ECPT subjects completed 348 tests (up to 3 each) with either two MMTTs or two GSTs.RESULTS -Among individuals with up to 4 years' duration of type 1 diabetes, Ͼ85% had measurable stimulated C-peptide values. The MMTT stimulus produced significantly higher concentrations of C-peptide than the GST. Whereas both tests were highly reproducible, the MMTT was significantly more so (R 2 ϭ 0.96 for peak C-peptide response). Overall, the majority of subjects preferred the MMTT, and there were few adverse events. Some older subjects preferred the shorter duration of the GST. Nausea was reported in the majority of GST studies, particularly in the young age-group.CONCLUSIONS -The MMTT is preferred for the assessment of ␤-cell function in therapeutic trials in type 1 diabetes.

Activated mononuclear cells appear to be important effector cells in autoimmune beta cell destruction leading to insulin-dependent (type 1) diabetes mellitus. Conditioned medium from activated mononuclear cells (from human blood) is cytotoxic to isolated rat and human islets of Langerhans. This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1 and recombinant IL-1 derived from the predominant pI 7 form of human IL-1, consistently inhibited the insulin response. The pI 6 and pI 5 forms of natural IL-1 were ineffective. Natural and recombinant IL-1 exhibited similar dose responses in their islet-inhibitory effect and their thymocyte-stimulatory activity. Concentrations of IL-1 that inhibited islet activity were in the picomolar range. Hence, monocyte-derived pI 7 IL-1 may contribute to islet cell damage and therefore to the development of insulin-dependent diabetes mellitus.

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Pancreatic ␤-cells are sensitive to a number of proapoptotic stimuli. Thus, apoptosis is an important part of the physiological neonatal remodeling of the endocrine pancreas, and a number of pathological stimuli involved in type 1 and type 2 diabetes have been shown to elicit ␤-cell apoptosis. Factors of relevance to type 1 diabetes include proinflammatory cytokines, nitric oxide, and reactive oxygen species as well as Fas ligand. Recent findings that free fatty acids, glucose, sulfonylurea, and amylin cause ␤-cell apoptosis in vitro suggest that programmed cell death may also be involved in the pathogenesis of type 2 diabetes. Furthermore, there is evidence favoring a convergence in signaling pathways toward common effectors of ␤-cell apoptosis elicited by stimuli implicated in the pathogenesis of type 1 and type 2 diabetes. Therefore, recent studies involving the stimuli and signaling pathways of ␤-cell apoptosis-in particular, mitogen-and stressactivated protein kinases-will be reviewed. It is concluded that immunological, inflammatory, and metabolic signals cause ␤-cell apoptosis, and the possibility that these signals converge toward a common ␤-cell death signaling pathway should be investigated further. Diabetes 50 (Suppl. 1):S58-S63, 2001 G iven the vital necessity of insulin, but also the hazards of release in excess of needs, ␤-cell function and ␤-cell mass are tightly regulated. This evolutionary pressure has resulted in a highly specialized cell that is receptive to replicatory but also apoptotic stimuli to fulfill the need for dynamic regulation. Apoptosis, or programmed cell death, is a physiological mode of remodeling tissue during organogenesis but is also involved in physiology later in life, for example, in relation to thymic involution. It has become clear that the endocrine pancreas is actively remodeled postpartum and that apoptosis is an important mechanism for this (1).Apoptotic cell death is an energy-requiring process that involves de novo protein synthesis. The process is characterized by morphological changes including condensation of nuclear chromatin, cellular shrinkage, membrane blebbing, and the formation of apoptotic bodies that are membrane-surrounded cellular constituents and undergo phagocytosis before leakage of intracellular contents, thereby avoiding the stimulation of an immune response, hence an autoimmune reaction.In many chronic degenerative diseases, apoptosis leads to inappropriate deletion of cells. The apoptotic cascade may be elicited by a number of varying stimuli, including intracellular events, such as metabolic imbalance, cell cycle perturbation, or DNA damage, and extracellular factors, such as activation of death receptors (Fas and tumor necrosis factor [TNF] receptors) and withdrawal of growth factors, metabolic factors, certain hormones, and inflammatory mediators such as cytokines. Intracellular signals involve ceramide; increases in intracellular calcium; free oxygen and nitric oxide (NO) radicals; and protein kinases such as mitogen-activated protein kinas...

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Antibodies in sera from newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients are directed to a human islet cell protein of relative molecular mass (Mr) 64,000. Since Il)DM seems to develop after a prodromal period of,8-cell autoimmunity, this study has examined whether 64,000 Mr antibodies could be detected in 14 individuals who subsequently developed IDDM and five first degree relatives who have indications of altered (3-cell function. Sera were screened by immunoprecipitation on total detergent lysates of human islets and positive sera retested on membrane protein preparations. Antibodies to the 64,000 Mr membrane protein were consistently detected in 11/14 IDDM patients, and in all 5 first degree relatives. 10 IDDM patients were already positive in the first samples, obtained 4-91 mo before the clinical onset of IDIM, whereas 1 patient progressed to a high 64,000 M, immunoreactivity, at a time where a commencement of a decline in (3-cell function was detected. 64,000 M, antibodies were detected before islet cell cytoplasmic antibodies (ICCA) in two patients. In the control groups of 21 healthy individuals, 36 patients with diseases of the thyroid and 5 SLE patients, the 64,000 Mr antibodies were detected in only one individual, who was a healthy sibling to an IDDM patient. These results suggest that antibodies against the Mr 64,000 human islet protein are an early marker of (-cell autoimmunity and may be useful to predict a later development of IDDM.

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within b-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucosedependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by postER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in b-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

Aims/hypothesis Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. Methods We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. Results Glucagon output decreased three-to fivefold within minutes of shifting from low (3.5 mmol/l) to high (10 mmol/l) glucose (4.0 ± 0.5 pmol/15 min vs 1.3 ± 0.3 pmol/15 min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. Conclusions/interpretation Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucoselowering effects.