Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids.

Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to determine whether or not this phenomenon is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of [14C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14C]palmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial beta-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids.

Prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by apoptosis.

Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting beta-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by [3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 microg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 microg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10-100 microM for 2-3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces beta-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the beta-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.

Use of a soluble tetrazolium compound to assay metabolic activation of intact beta cells.

Although assessments of metabolic activation are central to studies of beta-cell function, available techniques are tedious, insensitive, and/or require cell disruption. We have investigated the use of a new water-soluble tetrazolium salt, MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4- sulfophenyl]-2H-tetrazolium, inner salt), in the presence of phenazine methosulfate (PMS), an intermediate electron acceptor that amplifies its signal (fluorescence at 490 nm). During static incubations of glucose-responsive (HIT-T15 or INS-1) dispersed beta cells with increasing glucose concentrations, there was a progressive increase in MTS reduction, with a maximum signal-to-noise (S/N) ratio of 24 with HIT-T15 cells and 10 with INS-1 cells. This was associated with, but not attributable to, parallel increases in insulin secretion. Pure mitochondrial fuels (alpha-ketoisocaproate [KIC], methyl pyruvate [MP], or L-glutamine [GLN] + L-leucine [LEU]) also increased the reduction of MTS in INS-1 cells (6.5-, 4.8-, and 14.4-fold, respectively), but generally less than glucose, suggesting a major role of glycolysis in the signal induced by glucose. Inhibitors of glucose metabolism (mannoheptulose [MH], lodoacetate [IA], or 2-deoxyglucose [2-DG]) markedly reduced the glucose-stimulated MTS signal. In comparison to another tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MTS assay provided a better S/N ratio with glucose or other nutrient secretagogues. Extant theory holds that activation of mitochondrial dehydrogenases by increments in Ca2+ influx couples glycolysis to mitochondrial oxidation of glucose-derived fuels. However, reduction of fuel-induced calcium influx (by Ca2+-free medium or diazoxide [DZX]) or direct stimulation of calcium influx (by 40 mmol/L K+) failed to significantly modulate the signal, arguing against this theory. We conclude that the MTS assay is a facile test that reflects the global metabolic function of insulin-secreting beta cells. Furthermore, since this assay does not require disruption of cells to solubilize the formazan product, and therefore also allows concomitant measurement of insulin secretion, it offers considerable advantages over earlier methods.

Use of In-111 pentetreotide scintigraphy in the diagnosis of a midgut carcinoid causing Cushing's syndrome.

A 57-year-old man presented with clinical features of hypercortisolism and was diagnosed with ACTH-dependent Cushing's syndrome. Biochemical testing showed partial suppression of urinary free cortisol with high dose dexamethasone. Initial computed tomography (CT) of the chest and abdomen, and magnetic resonance imaging of the pituitary were negative. In-111 pentetreotide scintigraphy with single photon emission computerized tomography revealed two 'hot' lesions in the abdomen which were then confirmed by subsequent directed thin-slice abdominal CT and small bowel barium study. At surgery, two segments of ileum, adjoining mesentery and lymph nodes were resected. Histopathology was consistent with a malignant carcinoid tumor of the ileum which stained intensely for ACTH. Plasma ACTH, and serum and urinary cortisol normalized postoperatively. To our knowledge, this is the first reported case of ileal carcinoid tumor causing Cushing's syndrome with premortem diagnosis. Another unique feature of this case is that In-111 pentetreotide scan provided the decisive clue to localization of the tumor.

Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.

We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).