Rat Beta-cells Research Articles

Double-stranded ribonucleic acid (RNA) induces beta-cell Fas messenger RNA expression and increases cytokine-induced beta-cell apoptosis.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells. For this purpose, fluorescence-activated cell sorting-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta and IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1beta and a combination of PIC and IFN-gamma (but not PIC or IFN-gamma alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and IFN-gamma requires nuclear factor-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. Combinations of IL-1beta plus IFN-gamma, PIC plus IFN-gamma, or PIC plus IL-1beta induced a 2- to 3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity significantly decreased PIC- plus IL-1beta-induced, but not PIC- plus IFN-gamma-induced, apoptosis. In conclusion, PIC alone or in combination with cytokines modifies the expression of several genes in pancreatic beta-cells. Two of these genes, Fas and iNOS, may contribute to beta-cell death. The transcription factor nuclear factor-kappaB is required for PIC-induced iNOS expression. PIC has an additive effect on cytokine-induced beta-cell death by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. These findings suggest that viral intermediates in synergism with local cytokine production may play an important role in beta-cell apoptosis in early T1DM.

Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1 beta-exposed human and rat islet cells.

Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes and T lymphocytes, and could thus contribute to mononuclear cell infiltration in Type I (insulin-dependent) diabetes mellitus. Cytokines induce MCP-1 mRNA expression in pancreatic rat beta cells. To investigate this issue, we analysed the signal transduction for IL-1 beta-induced MCP-1 expression in rat beta cells and in vitro MCP-1 mRNA expression and protein release by human islets as well as in vivo islet MCP-1 mRNA expression in prediabetic non-obese diabetic mice. Fluorescence-activated cell sorting-purified rat beta cells were cultured for 6 h with IL-1 beta (30 U/ml) or MAPK inhibitors or both. Human islets were cultured for 6-72 h with the cytokines IL-1 beta, IFN-gamma or the inducible nitric oxide synthase (iNOS) inhibitor NG-methyl-L-arginine or both. We measured MCP-1 mRNA by RT-PCR and protein by ELISA. The MCP-1 mRNA expression in islets from male and female non-obese diabetic mice (2-12 weeks of age) was measured by real time reverse transcription-polymerase chain reaction (RT-PCR). Interleukin-1 beta induced MCP-1 mRNA expression in rat beta cells, with a maximum induction after 6 h. A combination of p38 and ERK1/2 inhibitors decreased MCP-1 expression by 70%. IL-1 beta induced both MCP-1 mRNA expression and a threefold increase in medium MCP-1 protein accumulation in human islet cells. This effect was not prevented by iNOS blockers. In vivo there was an age-related increase in MCP-1 mRNA expression in islets from male and female non-obese diabetic mice, reaching a peak at 8 weeks. In rat and human islet cells MCP-1 mRNA is induced by IL-1 beta. Both ERK1/2 and p38 MAPK, but not nitric oxide, contribute to MCP-1 expression. In non-obese diabetic mice MCP-1 mRNA expression increases with age, peaking at the early phases of insulitis. The production of MCP-1 by pancreatic beta cells could contribute to the recruitment of mononuclear cells into pancreatic islets in early Type I diabetes.

Distinction between interleukin-1-induced necrosis and apoptosis of islet cells.

Interleukin (IL)-1beta is known to cause beta-cell death in isolated rat islets. This effect has been attributed to induction of nitric oxide (NO) synthase in beta-cells and subsequent generation of toxic NO levels; it was not observed, however, in dispersed rat beta-cells. The present study demonstrates that IL-1beta induces NO-dependent necrosis in rat beta-cells cultured for 3 days at high cell density or in cell aggregates but not as single cells. Its cytotoxic condition is not explained by higher NO production rates but might result from higher intercellular NO concentrations in statically cultured cell preparations with cell-to-cell contacts; nitrite levels in collected culture medium are not a reliable index for these intercellular concentrations. Absence of IL-1-induced necrosis in rat alpha-cells or in human beta-cells is attributed to the cytokine's failure to generate NO in these preparations, not to their reduced sensitivity to NO: the NO donor GEA 3162 (15 min, 50-100 micromol/l) exerts a comparable necrotic effect in rat and human alpha- or beta-cells. In preparations in which IL-1beta does not cause beta-cell necrosis, its combination with gamma-interferon (IFN-gamma) results in NO-independent apoptosis, starting after 3 days and increasing with the duration of exposure. Because IFN-gamma alone was apoptotic for rat alpha-cells, it is proposed that IL-1beta can make beta-cells susceptible to this effect, conceivably through altering their phenotype. It is concluded that IL-1beta can cause NO-dependent necrosis or NO-independent apoptosis of islet cells, depending on the species and on the environmental conditions. The experiments in isolated human beta-cell preparations suggest that these cells may preferentially undergo apoptosis when exposed to IL-1beta plus IFN-gamma unless neighboring non-beta-cells produce toxic NO levels.

Cytokine-mediated apoptosis in rat beta-cells is preceded by downregulation of Bcl-2 and Bax-omega expression

Proinflammatory cytokines have been shown to cause death in isolated rodent and human islet cell preparations. Depending on the species and the experimental conditions, they predominantly induce necrosis or apoptosis. In single rat β-cells, the combination of interleukin-1β and γ-interferon leads to a progressive increase in the number of apoptotic cells: after 7 days of culture, these cytokines cause apoptosis in ±30% of the cells. Their effect was blocked in the presence of N-methyl-L-arginine (NMA), a specific inhibitor of inducible NO synthase, indicating its NO dependency. It was not reproduced with one of the cytokines alone. We have investigated whether the cytokine-induced apoptosis is the result of an alteration in the cellular expression of anti- and proapoptotic proteins. After 3 days' exposure to interleukin-1β + γ-interferon, Bcl-2 mRNA and protein were clearly decreased, whereas Bax-α mRNA and protein levels remained unchanged. However, a strong downregulation was measured in Bax-Ω, a Bax-splice-variant protein, which has been described in other rat tissues. The reduction in Bcl-2 and Bax-Ω expression was not observed when NMA was added in the medium. It was absent after culture with one of the cytokines. It is concluded that cytokine-mediated apoptosis in rat β-cells is preceded by an NO-dependent downregulation of Bcl-2 and Bax-Ω, alleged antiapoptotic proteins, whereas no changes occur in Bax-α, a proapoptotic regulator. These data suggest that downregulation of Bcl-2-related antiapoptotic proteins can be responsible for apoptosis of rat β-cells.

Effect of phensuccinal on pancreatic beta-cells in rats with neonatally induced streptozotocin diabetes mellitus.

The effect of phensuccinal, a low-toxic succinic acid derivative, on the function of pancreatic beta-cells in the evolution of absolute insulin insufficiency was studied in rats with neonatally induced streptozotocin diabetes mellitus. Phensuccinal (25 mg/kg body weight) prevented disorders in the secretory response of beta-cells to glucose load at all stages of the study (2, 5, and 14 days after diabetes induction). This effect was realized via stimulation of the regenerative processes in the insulin-producing system of the pancreas and activation of the antioxidant system in diabetic animals.

Differential patterns of glucose-induced electrical activity and intracellular calcium responses in single mouse and rat pancreatic islets.

Although isolated rat islets are widely used to study in vitro insulin secretion and the underlying metabolic and ionic processes, knowledge on the properties of glucose-induced electrical activity (GIEA), a key step in glucose-response coupling, has been gathered almost exclusively from microdissected mouse islets. Using a modified intracellular recording technique, we have now compared the patterns of GIEA in collagenase-isolated rat and mouse islets. Resting membrane potentials of rat and mouse beta-cells were approximately -50 and -60 mV, respectively. Both rat and mouse beta-cells displayed prompt membrane depolarizations in response to glucose. However, whereas the latter exhibited a bursting pattern consisting of alternating hyperpolarized and depolarized active phases, rat beta-cells fired action potentials from a nonoscillating membrane potential at all glucose concentrations (8.4-22.0 mmol/l). This was mirrored by changes in the intracellular Ca2+ concentration ([Ca2+]i), which was oscillatory in mouse and nonoscillatory in rat islets. Stimulated rat beta-cells were strongly hyperpolarized by diazoxide, an activator of ATP-dependent K+ channels. Glucose evoked dose-dependent depolarizations and [Ca2+]i increases in both rat (EC50 5.9-6.9 mmol/l) and mouse islets (EC50 8.3-9.5 mmol/l), although it did not affect the burst plateau potential in the latter case. We conclude that there are important differences between beta-cells from both species with respect to early steps in the stimulus-secretion coupling cascade based on the following findings: 1) mouse beta-cells have a larger resting K+ conductance in 2 mmol/l glucose, 2) rat beta-cells lack the compensatory mechanism responsible for generating membrane potential oscillations and holding the depolarized plateau potential in mouse beta-cells, and 3) the electrical and [Ca2+]i dose-response curves in rat beta-cells are shifted toward lower glucose concentrations. Exploring the molecular basis of these differences may clarify several a priori assumptions on the electrophysiological properties of rat beta-cells, which could foster the development of new working models of pancreatic beta-cell function.

The constitutive secretory pathway is a major route for islet amyloid polypeptide secretion in neonatal but not adult rat islet cells.

Islet amyloid polypeptide (IAPP or amylin) is a normal secretory product of the pancreatic beta-cell that is cosecreted with insulin and is the major constituent of islet amyloid deposits in individuals with type 2 diabetes or insulinomas. We have previously reported that glucose stimulates IAPP, but not insulin secretion, from neonatal rat beta-cells when regulated secretion is prevented by use of calcium-free media, suggesting that IAPP secretion occurs via a constitutive secretory pathway. To directly test this hypothesis, we examined the effects of 2 substances-brefeldin A (BFA) and cycloheximide (CHX)-that are predicted to selectively block constitutive secretion on the release of IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI) from neonatal rat islet cell monolayer cultures. When regulated release was prevented by use of calcium-free media, glucose-stimulated IAPP-LI release was nearly abolished by blocking constitutive release with 10 microg/ml BFA (mean +/- SD: 8.7 +/- 7.7 vs. 29.3 +/- 14.3 pmol/l; n = 5; P < 0.05), an inhibitor of constitutive vesicle formation. Similarly, calcium-independent, glucose-stimulated IAPP-LI secretion was markedly suppressed when new protein synthesis was blocked by administration of 20 microg/ml CHX (4.6 +/- 2.1 vs. 29.5 +/- 14.0 pmol/l; n = 5; P < 0.005). Secretion of IRI was low in the absence of calcium, and neither BFA nor CHX had any further effect. When calcium was added to the incubation media to allow regulated secretion of both IRI and IAPP-LI, both BFA (47.7 micro 8.7 vs. 80.7 micro 10.3 pmol/l; P < 0.001) and CHX (37.3 +/- 5.8 vs. 73.3 +/- 6.2 pmol/l; n = 5; P < 0.0001) inhibited glucose-stimulated IAPP-LI secretion by approximately 40%, but again had no inhibitory effect on IRI secretion. These data indicate that approximately 40% of glucose-stimulated IAPP-LI release occurs via a constitutive secretory pathway in neonatal rat islet cells. By contrast, in adult rat islets, glucose-stimulated IAPP-LI release was almost abolished in the absence of calcium (86 +/- 3% inhibition; P < 0.05) and unaffected by addition of BFA (275 +/- 28 vs. 205 +/- 89 pmol/l; NS) or CHX (160 +/- 20 vs. 205 +/- 89 pmol/l; NS), suggesting that constitutive secretion of IAPP does not occur in mature beta-cells. Collectively, these data suggest that a significant proportion of glucose-stimulated IAPP secretion from neonatal, but not adult, rat islet cells occurs via a constitutive secretory pathway.

Interleukin-1β Regulates Phospholipase D-1 Expression in Rat Pancreatic β-Cells.

The cytokine interleukin (IL)-1beta induces a biphasic effect in rat pancreatic islets, with an early and transitory stimulation of insulin release followed by progressive functional suppression. To clarify the mechanisms involved in these effects, we have recently performed a differential display of messenger RNA (mRNA) by RT-PCR (DDRT-PCR) on rat beta-cells exposed for 6 or 24 h to IL-1beta. Among the different IL-1beta-induced genes, there was an early and transient increase in phospholipase D-1 (PLD1) expression. PLD1 can induce phosphatidic acid formation and subsequent activation of protein kinase C, a process which stimulates insulin release. In the present study, we characterized the regulation of PLD isoforms by IL-1beta in pancreatic beta-cells. By using different combinations of primers and RT-PCR, we observed that IL-1beta induces an early increase (2 and 6 h) in the expression of both alternatively spliced isoforms of PLD1 (PLD1alpha and 1b). Prolonged exposure to IL-1beta (12 and 24 h) caused a decrease of PLD1a mRNA expression compared with control beta-cells, and lead to a return of PLD1b mRNA to basal level. NG-methyl-L-arginine (LMA), a blocker of the inducible form of nitric oxide synthase (iNOS), prevented this late inhibitory effect of IL-1beta, suggesting that IL-1beta-induced decrease in PLD1a expression is NO-mediated. IL-1beta induced an early (2-6 h) and sustained (16-24 h) increase in PLD1a mRNA expression in insulin-producing RINm5F cells. This was paralleled by a cytokine-induced increase in PLD1 protein expression and enzyme activity. RINm5F cells, but not primary beta-cells, expressed PLD2, and the expression of this gene was not affected by IL-1beta. In conclusion, we have shown that the cytokine IL-1beta regulates PLD1 expression in primary and clonal beta-cells. The early induction of PLD1 probably contributes to the early stimulatory effects of IL-1beta on islet insulin release.

Inhibition of progressive reduction of islet β-cell mass in spontaneously diabetic Goto-Kakizaki rats by α-glucosidase inhibitor

The Goto-Kakizaki (GK) rat, an animal model of type 2 diabetes, exhibits mild hyperglycemia with a reduction of beta-cell mass. The mechanism for islet structural changes in this model and whether the changes are affected by metabolic control are not known. In the present study, we examined the process of islet changes in male GK rats aged 6, 8, 12, 24, and 36 weeks. Treatment effects with an alpha-glucosidase inhibitor (Voglibose; Takeda, Osaka, Japan) for 24 weeks (12 to 36 weeks of age) were also evaluated. The beta-cell mass increased until 8 weeks of age in both GK and control rats, but the increase was significantly (P < .01) smaller in GK rats versus at 8 weeks of age. Thereafter, the beta-cell mass decreased in GK rats, whereas it remained constant in controls. Voglibose treatment significantly (P < .01) inhibited the reduction of beta-cell mass in GK rats. Proliferative activity of beta cells as measured by bromodeoxyuridine (BrdU) uptake was significantly (P < .05) lower in GK rats versus control rats at 6 and 8 weeks, but the difference disappeared after 12 weeks of age, regardless of Voglibose treatment. The present study thus demonstrates a progressive loss of beta cells in GK rats that was mitigated by Voglibose treatment. We consider that the beta-cell loss in GK rats was due to an early impairment in proliferative activity and reduced survival. Voglibose did not appear to stimulate beta-cell proliferation, but exerted its effect via a reduction of hyperglycemia.

Exposure to glibenclamide increases rat beta cells sensitivity to glucose.

An increased sensitivity to glucose was observed in islets pre-exposed for 1 h to glibenclamide (0.1 micromol 1(-1)), but not to tolbutamide (100 micromol l(-1)), as indicated by a shift to the left of the dose-response curve (EC(50) at 5.8+/-0.3 mmol l(-1) glucose vs 10.6+/-0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l(-1) glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion-enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124+/-7 vs 51+/-9 nmol microgram prot(-1) 90 min(-1), P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l(-1) dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC(50) 10.9+/-1.3 mmol l(-1) glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial-bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.

Interleukin-1beta-induced alteration in a beta-cell phenotype can reduce cellular sensitivity to conditions that cause necrosis but not to cytokine-induced apoptosis.

Previous work has shown that interleukin-1beta (IL-1beta) alters protein expression in beta-cells. This alteration is associated with cell death in isolated rat islets but not in isolated rat beta-cells. We examined whether IL-1beta pretreatment of isolated beta-cells influences their sensitivity to toxic agents. After a 24-h culture with IL-1beta (30 U/ml), beta-cells exhibited a lower expression of the beta-cell-specific protein transcription factor pancreatic and duodenal homeobox gene (PDX)-1, glucose transporter GLUT2, and proinsulin convertase PC2, with a marked reduction (60-70%) in glucose-induced insulin production and selective sensitivity to the toxins alloxan (ALX) and streptozotocin (STZ). On the other hand, the cells presented an increased expression of Mn-superoxide dismutase, heat shock protein 70, inducible heme oxygenase, and inducible nitrite oxide synthase. This IL-1beta-induced alteration in beta-cell phenotype resulted in a reduced cellular sensitivity to the beta-cell-specific toxins ALX and STZ; the production of nontoxic conditions of nitric oxide (NO) also rendered the cells less susceptible to radical-induced damage. Exposure to IL-1beta can thus protect beta-cells against conditions that cause necrosis; however, it did not protect against apoptosis induced by the additional presence of interferon-gamma or tumor necrosis factor-alpha. Release of IL-1beta in the endocrine pancreas is thus not necessarily the cause of massive NO-dependent beta-cell destruction. On the contrary, IL-1beta may protect these cells against necrosis, though with a loss of their characteristic phenotype and homeostatic functions.

Evidence for complement activation in type 1 diabetes

Type 1 diabetes is caused by immune mediated destruction of the beta cells in the pancreatic islets of Lmgerhans. There is evidence that complement activation plays a role in this immune mediated cell death. We have demonstrated a trend towards increased terminal complement complex (TCC, SC5b-9) concentrations in cell culture supernatents of BRIN BDI 1 cells (a rat beta cell line) incubated with 10% v/v serum from patients with newly diagnosed type I diabetes for 24 hours than in supmatents from cells incubated with non-diabetic serum (mean 10,765 (sd 4640) n g / d vs 7939 (sd 1935) ng/mL). Cell viability, assessed by the internal LDH assay, was no different between cells incubated with diabetic and non-diabetic serum. This effect could be abolished by heat treating serum to inactivate complement. TCC levels measured in culture supernatent of pig kidney epithelial cell line (LLC PKI) with 10% diabetic serum were 1007 (sd 95) ng/mL after 24 hours and was 1572 (sd 197) ng/mL with 10 % normal human serum when LLC PKI cells were cultured at the same density as the BDl I cells. This indicates complement activation is cell specific. These results suggest that diabetic serum contains factors that specifically fix complement on beta cells (most likely complement fixing antiislet cell antibodies). Evidence for complement activation in type 1 diabetes 30 Immuno-modulatory mechanisms of E.coli heat labile toxin

Importance of cell-matrix interactions in rat islet beta-cell secretion in vitro: role of alpha6beta1 integrin.

It has long been recognized that islet cell function is rapidly altered in vitro, but can be maintained, at least in part, when cells are layered on defined extracellular matrices. The present work addresses the influence of short-term cell-matrix interactions on islet beta-cell function and provides first insight into the molecular basis of these interactions. When primary rat beta-cells were allowed to attach to a matrix produced by a rat carcinoma cell line (804G), there was an increased insulin secretory response to secretagogues. This change was the result of an increase in the proportion of actively secreting beta-cells and in the amount of insulin secreted per active cell, as shown using the reverse hemolytic plaque assay. In turn, the spreading or flattening of beta-cells on this matrix was enhanced by secretagogues, and flattened cells secreted more insulin than rounded cells. Using indirect immunofluorescence, it was found that 1)alpha6beta1 integrins are present at the surface of islet cells in situ, 2) alpha6beta1 expression is heterogeneous among purified beta-cells and is upregulated by insulin secretagogues, 3) alpha6beta1 expression is higher in spreading cells, and 4) anti-alpha6beta1-specific antibodies decrease spreading. These observations demonstrate that islet cell-matrix interactions can improve the sensitivity of insulin cells to glucose and are mediated, at least in part, by alpha6beta1 integrins, suggesting that outside-in signaling through alpha6beta1 integrin plays a major role in the regulation of beta-cell function.

Evidence of enhancement of malate-aspartate shuttle activity in β cells of streptozotocin-induced non—insulin-dependent diabetic rats

Glucose-induced insulin secretion is selectively impaired in beta cells from animals with non-insulin-dependent diabetes mellitus (NIDDM). This study was performed to clarify whether the malate-aspartate shuttle among the glucose metabolic pathways is intact in beta cells of NIDDM rats. The insulin secretory capacity of the islets and the K(ATP) channel activity in single beta cells were measured in control and NIDDM rats injected with streptozotocin (STZ) during the neonatal period, using a radioimmunoassay and patch-clamp technique. The increase of insulin secretion induced by 11.1 mmol/L glucose or 10 mmol/L dihydroxyacetone (DHA) was significantly reduced in NIDDM islets, suggesting an impaired glycerol-phosphate shuttle. The application of glyceraldehyde (10 mmol/L) in NIDDM or control islets elicited an increase in insulin secretion, but the difference between the 2 groups was indistinguishable. On the contrary, the increase of insulin secretion and the inhibition of K(ATP) channel activity induced by aspartate, which preferentially participates in the malate-aspartate shuttle, were significantly greater in NIDDM versus the control. However, intracellularly applied aspartate in the inside-out mode did not inhibit K(ATP) channel activity. These findings show that malate-aspartate shuttle activity is potentiated in pancreatic beta cells of NIDDM rats, suggesting the development of a compensatory mechanism for the reduced activity of the glycerol-phosphate shuttle in NIDDM.

Endocytosis of Low-Density Lipoprotein by Human Pancreatic β Cells and Uptake in Lipid-Storing Vesicles, Which Increase with Age

Studies with I(125)-labeled low-density lipoproteins (LDLs) have shown the presence of high-affinity LDL receptors on insulin-producing beta cells but not on neighboring alpha cells. By using gold-labeled lipoproteins, we demonstrate receptor-mediated endocytosis of LDLs and very low-density lipoproteins in rat and human beta cells. Specific for human beta cells is the fusion of LDL-containing endocytotic vesicles with lipid-storing vesicles (LSVs; diameter, 0.6-3.6 microm), which are absent in rodent beta cells. LSVs also occur in human pancreatic alpha and duct cells, but these sequester little gold-labeled LDL. In humans <25 years old, LSVs occupy 1% of the cytoplasmic surface area in beta, alpha, and duct cells. In humans >50 years old, LSV surface area in beta cells (11 +/- 2% of cytoplasmic surface area) is fourfold higher than in alpha and duct cells and 10-fold higher than in beta cells at younger ages (P < 0.001); the mean LSV diameter in these beta cells (1.8 +/- 0.04 microm) is larger than at younger ages (1.1 +/- 0.2 microm; P < 0.005). Oil red O staining on pancreatic sections confirms that neutral lipids accumulate in beta cells of older donors. We conclude that human beta cells can incorporate LDL and very low-density lipoprotein material in LSVs. The marked increase in the LSV area of aging human beta cells raises the question whether it is caused by prolonged exposure to high lipoprotein levels such as occurs in Western populations and whether it is causally related to the higher risk for type 2 diabetes with aging.

Decreased expression of t-SNARE, syntaxin 1, and SNAP-25 in pancreatic beta-cells is involved in impaired insulin secretion from diabetic GK rat islets: restoration of decreased t-SNARE proteins improves impaired insulin secretion.

The physiological role of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in insulin exocytosis has been reported in pancreatic beta-cells. To determine whether the beta-cells of GK rats, a nonobese rodent model of type 2 diabetes, exhibit abnormalities in their SNARE proteins, we studied the expression and function of target (t)-SNAREs, syntaxin 1A, and synaptosomal-associated protein of 25 kDa (SNAP-25) in GK rat islets. Although insulin release and insulin content of islets isolated from 12-week-old GK rats were reduced, the proinsulin biosynthetic rate was about twofold higher than that in control rat islets, and no change in the preproinsulin mRNA level was observed. Pulse-chase experiments suggested the increased degradation of insulin in GK rat islets. Immunoblot analysis revealed that protein levels of syntaxin 1A and SNAP-25 in GK rat islets decreased to approximately 60% of the levels in control rat islets. We then examined whether the restoration of the decreased expression of t-SNAREs to the normal level in GK rat islets affected insulin secretion. Restoration was achieved by the overexpression of syntaxin 1A and SNAP-25 via the recombinant adenovirus-mediated gene transduction system, which recovered levels of these proteins to almost control levels. Glucose-stimulated insulin release from AdexlCA syntaxin 1A and Adex1CA SNAP-25-infected GK rat islets increased up to approximately 135 and 200%, respectively, of those from uninfected GK rat islets, although no difference in basal (2.2 mmol/l glucose) insulin release was evident between them. We conclude that decreased expression of t-SNAREs in GK rat islets is in part the defect responsible for impaired insulin secretion.

Cellular Origin of Hexokinase in Pancreatic Islets

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K(m) hexokinases (HK) exhibit a leftward shift of the normal dose-response curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from freshly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells. We previously observed that HK activity represents 20% of total glucose phosphorylation in purified rat beta cell preparations and that HK is not homogenously distributed over these cells. The present study provides several arguments for the idea that HK detected in freshly isolated rat islets or islet cell preparations originates mainly from contaminating exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV mRNA in rat beta cells, whereas the messenger levels encoding the hexokinase II and III isoforms were undetectably low. However, immunoblots indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta cell preparations. Second, comparison of HK activity in the different pancreatic cell types resulted in 15-25-fold higher values in exocrine than in endocrine cells (acinar cells: 21 +/- 3 pmol of glucose 6-phosphate formed/h/ng of DNA; duct cells: 30 +/- 8 pmol/h/ng of DNA; islet beta cells: 1.2 +/- 0.2 pmol/h/ng DNA; alpha cells: 0.9 +/- 0.4 pmol/h/ng of DNA). Since freshly purified beta cell preparations contain 3 +/- 1% exocrine cells, at least 50% of their HK activity can be accounted for by exocrine contamination. Third, after 5 days of culture of purified islet beta cells, both HK activity and the proportion of exocrine cells decreased by more than 1 order of magnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose did not affect glucose stimulation of insulin biosynthesis and release. In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations cautions against the use of HK measurements in islet extracts in the study of these enzymes in glucose sensing by pancreatic beta cells.

Heterogeneity in glutamic acid decarboxylase expression among single rat pancreatic beta cells.

An isoform of glutamic acid decarboxylase, (GAD)65 has been identified as a pancreatic beta-cell autoantigen in Type I (insulin dependent) diabetes mellitus. We investigated the expression of GAD isoforms among single rat beta cells in culture, under different conditions and the correlation between GAD65 expression and insulin secretion-rate. Independent of culture conditions, 100 % of fresh and cultured beta cells express GAD67. In contrast, considerable heterogeneity in GAD65 expression among single beta cells was observed. After 2 days in culture in 2.6 mmol/l glucose, only 24 % of the beta cells express GAD65. This percentage increases to 39 % in 5.6 mmol/l glucose and to 54 % and 56 % in 11.6 mmol/l and 20.6 mmol/l glucose, respectively. Moreover, reducing glucose concentration from 11.6 to 2.5 mmol/l for 2 days, reduces GAD65 expression by nearly 30 %. After 11 days in culture with 11.6 mmol/l glucose, 50 % of beta cells continue expressing GAD65, this percentage is further increased to nearly 75 % by including either nerve growth factor or dibutyryl cyclic AMP or both in the culture medium. When beta cells are challenged for 1 h with 20.6 mmol/l glucose, 67 % respond forming insulin-immunoplaques. More than two-thirds of insulin-secretors are GAD65-positive, in contrast to only 11 % of the non-secreting cells. Moreover, 87 % of beta cells that have a high insulin secretory rate express GAD65. These results show that the most active beta cells, which secrete more insulin, also express GAD65 and that manipulating extracellular glucose may modify the expression of the enzyme and possibly the autoimmune attack in Type I diabetes.

Insulin resistance in HIV protease inhibitor-associated diabetes.

Fasting hyperglycemia has been associated with HIV protease inhibitor (PI) therapy. To determine whether absolute insulin deficiency or insulin resistance with relative insulin deficiency and an elevated body mass index (BMI) contribute to HIV PI-associated diabetes. Cross-sectional evaluation. 8 healthy seronegative men, 10 nondiabetic HIV-positive patients naive to PI, 15 nondiabetic HIV-positive patients receiving PI (BMI = 26 kg/m2), 6 nondiabetic HIV-positive patients receiving PI (BMI = 31 kg/m2), and 8 HIV-positive patients with diabetes receiving PI (BMI = 34 kg/m2). All patients on PI received indinavir. Fasting concentrations of glucoregulatory hormones. Direct effects of indinavir (20 microM) on rat pancreatic beta-cell function in vitro. In hyperglycemic HIV-positive subjects, circulating concentrations of insulin, C-peptide, proinsulin, glucagon, and the proinsulin/insulin ratio were increased when compared with those of the other 4 groups (p < .05). Morning fasting serum cortisol concentrations were not different among the 5 groups. Glutamic acid decarboxylase (GAD) antibody titers were uncommon in all groups. High BMI was not always associated with diabetes. In vitro, indinavir did not inhibit proinsulin to insulin conversion or impair glucose-induced secretion of insulin and C-peptide from rat beta-cells. The pathogenesis of HIV PI-associated diabetes involves peripheral insulin resistance with insulin deficiency relative to hyperglucagonemia and a high BMI. Pancreatic beta-cell function was not impaired by indinavir. HIV PI-associated diabetes mirrors that of non-insulin-dependent diabetes mellitus and impaired insulin action in the periphery.

Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes.

Reactive oxygen species are involved in a diversity of biological phenomena such as inflammation, carcinogenesis, aging, and atherosclerosis. We and other investigators have shown that the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative stress, is increased in either the urine or the mononuclear cells of the blood of type 2 diabetic patients. However, the association between type 2 diabetes and oxidative stress in the pancreatic beta-cells has not been previously described. We measured the levels of 8-OHdG and 4-hydroxy-2-nonenal (HNE)-modified proteins in the pancreatic beta-cells of GK rats, a model of nonobese type 2 diabetes. Quantitative immunohistochemical analyses with specific antibodies revealed higher levels of 8-OHdG and HNE-modified proteins in the pancreatic beta-cells of GK rats than in the control Wistar rats, with the levels increasing proportionally with age and fibrosis of the pancreatic islets. We further investigated whether the levels of 8-OHdG and HNE-modified proteins would be modified in the pancreatic beta-cells of GK rats fed with 30% sucrose solution or 50 ppm of voglibose (alpha-glucosidase inhibitor). In the GK rats, the levels of 8-OHdG and HNE-modified proteins, as well as islet fibrosis, were increased by sucrose treatment but reduced by voglibose treatment. These results indicate that the pancreatic beta-cells of GK rats are oxidatively stressed, and that chronic hyperglycemia might be responsible for the oxidative stress observed in the pancreatic beta-cells.