Meng-Chi Chen

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

Aims/hypothesis. 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-1β-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. Methods. Fluorescence-activated cell sorting-purified rat beta cells were cultured for 6 h with IL-1β (30 U/ml) or MAPK inhibitors or both. Human islets were cultured for 6–72 h with the cytokines IL-1β, IFN-γ 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). Results. Interleukin-1β 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-1β 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. Conclusion/interpretation. In rat and human islet cells MCP-1 mRNA is induced by IL-1β. 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. [Diabetologia (2001) 44: 325–332]

Inhibition of human pancreatic islet insulin release by receptor-selective somatostatin analogs directed to somatostatin receptor subtype 5.

Somatostatin (SS)-14 and SS28 are produced by pancreatic D cells and gut mucosa and inhibit pancreatic islet insulin and glucagon release. There are five distinct SS receptor (SSTR) subtypes, namely SSTR1-5, which show different affinities for SS14 and SS28. In order to identify the subtype responsible for inhibition of insulin release by human B cells, SSTR-selective SS analogs were tested in isolated human islets. Glucose-stimulated insulin secretion in human islets incubated for 1 hr at 20 mM glucose, and in islets cultured for 24 hr at a near-physiological (6.1 mM) glucose concentration, was inhibited (<50% of the control) by SSTR5-specific analogs and by SS14 and SS28. SS14, SS28, and different SSTR5 preferential analogs also inhibited islet amyloid polypeptide release during the 24-hr culture. On the other hand, a group of SSTR2-selective analogs failed to inhibit insulin release. Analysis by reverse transcription-polymerase chain reaction indicated that human islets express similar amounts of SSTR2 and SSTR5 mRNAs, while human pancreatic ductal cells express much lower levels of these mRNAs. In conclusion, our data suggest that SSTR5 is an important mediator of the insulin inhibitory action of SS in cultured human islets.

Identification of IL-1β-induced messenger RNAs in rat pancreatic beta cells by differential display of messenger RNA

Aims/hypothesis. Interleukin-1β is a putative mediator of pancreatic beta-cell dysfunction and damage in Type I (insulin-dependent) diabetes mellitus. To better understand the molecular mechanisms involved in IL-1β effects, we carried out a differential display of mRNA by RT-PCR to identify novel cytokine-regulated genes. Methods. Fluorescence activated cell sorting-purified rat pancreatic beta-cells were exposed for 6 or 24 h to IL-1β. Differentially expressed cDNA bands were cloned and then identified by comparing their sequences with data from the GenBank. Differential gene expression was confirmed by RT-PCR using specific primers. Results. Interleukin-1β increased the expression of adenine nucleotide translocator-1, phospholipase D-1 and cytokine-induced neutrophil chemoattractant-1 and decreased expression of the protein tyrosine phosphatase-like protein IA-2. Interleukin-1β-induced differential expression of these genes in beta cells was confirmed by RT-PCR. In additional studies, IL-1β was shown to induce chemokines other than cytokine-induced neutrophil chemoattractant-1, including cytokine-induced neutrophil chemoattractant-3 and monocyte chemotactic protein-1. Conclusion/interpretation. Our observations indicate that IL-1β modifies the expression of several genes in pancreatic beta cells. These genes may affect both function, viability and beta-cell recognition by the immune system. Functional characterization of the mRNAs which have been identified could facilitate a better understanding of the mechanisms leading to beta-cell destruction in Type I diabetes. [Diabetologia (1999) 42: 1199–1203]

Intercellular differences in interleukin 1beta-induced suppression of insulin synthesis and stimulation of noninsulin protein synthesis by rat pancreatic beta-cells.

The normal pancreatic beta-cell population exhibits intercellular differences in its responsiveness to glucose. This cellular heterogeneity allows glucose to regulate, in a dose-dependent manner, total rates of insulin synthesis and release. It may also predispose to intercellular differences in susceptibility to dysregulating agents. The present study examines whether this is the case for interleukin 1beta (IL-1beta), which is known to suppress glucose-induced insulin synthesis and release. The effects of the cytokine were compared on beta-cell subpopulations with, respectively, high and low sensitivity to glucose. These subpopulations were separated on the basis of differences in the cellular metabolic responsiveness to an intermediate glucose concentration (7.5 mmol/liter) and then cultured for 20 h at 5 or 20 mmol/liter with or without IL-1beta. The suppressive action of IL-1beta (0.1 ng/ml) occurred predominantly in glucose-activated beta cells, reducing their high rates of insulin synthesis and release by more than 80%. Glucose-unresponsive cells became subject to a similar inhibition after their activation during culture at 20 mmol/liter glucose. On the other hand, IL-1beta induced or enhanced the expression of several noninsulin proteins in both subpopulations. The IL-1beta-stimulated expression of inducible nitric oxide synthase (iNOS) and heat shock protein 70 was more marked in the glucose-responsive subpopulation; that of heme oxygenase and Mn superoxide dismutase was comparable in the two subpopulations. Exposure to IL-1beta resulted in 10-fold higher medium nitrite levels in both subpopulations; this effect was prevented by the iNOS blocker, N(G)-methyl-L-arginine, which also prevented the IL-1beta-induced suppression in the glucose-responsive subpopulation. This study demonstrates that the cellular heterogeneity in glucose responsiveness predisposes to intercellular differences in the IL-1-induced suppression of insulin synthesis and release. While the cytokine induces the expression of noninsulin proteins such as iNOS in both glucose responsive and unresponsive cells, the subsequent nitric oxide production appears to predominantly affect glucose-stimulated functions in the glucose-activated cells.

Interleukin-1b regulates phospholipase D-1 expression in rat pancreatic b-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.