[Protective effects of pravastatin against P38MAPK signaling pathway-mediated inflammatory toxicity in islet micro-endothelial cells].

OBJECTIVE: To study the signaling pathways associated with lipopolysaccharide (LPS)-induced inflammation in islet micro-endothelial cells (IMECs) and the mechanism of pravastatin intervention. METHODS: IMECs exposed to LPS, SB203580, pravastatin, or SB203580+pravastatin were examined for cell apoptosis with Hoechst staining and flow cytometry and for expression levels of total-p38, photophosphorylation-p38 (p-p38) and iNOS with Western blotting. RESULTS: The apoptosis rate and expression levels of total-p38, p-p38, iNOS in IMECs all increased after LPS exposure. Pravastatin, SB203580, and their combination significantly attenuated LPS-induced enhancement of cell apoptosis and total-p38, p-p38, and iNOS expressions in IMECs. CONCLUSION: LPS-induced inflammatory toxicity in IMECs is associated with the activation of P38MAPK and iNOS/NO signaling pathways. Pravastatin can inhibit these pathways and suppress the apoptosis and necrosis of IMECs to relieve the cell inflammatory injuries.

Measurement of pancreatic islet cell proliferation by heavy water labeling.

We describe a sensitive technique for measuring long-term islet cell proliferation rates in vivo in rats. Pancreatic islets were isolated and the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of DNA was measured by GC-MS. The results of heavy water labeling and BrdU staining were compared. The two methods were highly correlated (r = 0.9581, P < 0.001). Based on long-term heavy water labeling, approximately 50% of islet cells divided in rats between 8 and 15 wk of age. Of interest, long-term BrdU administration suppressed proliferation of islet cells significantly, but not of bone marrow cells. Physiological evidence further supported the validity of the method: older animals (24 wk old) had 60% lower islet cell proliferation rates than younger rats (5 wk old), and partial (50%) pancreatectomy increased proliferation by 20%. In addition, cholecystokinin-8 treatment significantly stimulated proliferation in pancreatectomized rats only. In summary, heavy water labeling is a quantitative approach for measuring islet cell proliferation and testing therapeutic agents.

Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.

In this study, we investigated the role of acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) in glucose and lipid metabolism in obese mice by reducing its expression in liver and fat with an optimized antisense oligonucleotide (ASO). High-fat diet-induced obese (DIO) C57BL/6J mice and ob/ob mice were treated with DGAT2 ASO, control ASO, or saline. DGAT2 ASO treatment reduced DGAT2 messenger RNA (mRNA) levels by more than 75% in both liver and fat but did not change DGAT1 mRNA levels in either of these tissues, which resulted in decreased DGAT activity in liver but not in fat. DGAT2 ASO treatment did not cause significant changes in body weight, adiposity, metabolic rate, insulin sensitivity, or skin microstructure. However, DGAT2 ASO treatment caused a marked reduction in hepatic triglyceride content and improved hepatic steatosis in both models, which was consistent with a dramatic decrease in triglyceride synthesis and an increase in fatty acid oxidation observed in primary mouse hepatocytes treated with DGAT2 ASO. In addition, the treatment lowered hepatic triglyceride secretion rate and plasma triglyceride levels, and improved plasma lipoprotein profile in DIO mice. The positive effects of the DGAT2 ASO were accompanied by a reduction in the mRNA levels of several hepatic lipogenic genes, including SCD1, FAS, ACC1, ACC2, ATP-citrate lyase, glycerol kinase, and HMG-CoA reductase. In conclusion, reduction of DGAT2 expression in obese animals can reduce hepatic lipogenesis and hepatic steatosis as well as attenuate hyperlipidemia, thereby leading to an improvement in metabolic syndrome.

Induction of cyclooxygenase-2 gene in pancreatic beta-cells by 12-lipoxygenase pathway product 12-hydroxyeicosatetraenoic acid.

Cyclooxygenase-2 (COX-2) gene and 12-lipoxygenase (12-LO) gene are preferentially expressed over other types of cyclooxygenase and lipoxygenase in pancreatic beta-cells. Inhibition of either COX-2 or 12-LO can prevent cytokine-induced pancreatic beta-cell dysfunction as defined by inhibition of glucose-stimulated insulin secretion. As cellular stress induces both genes and their respective end products in pancreatic beta-cells, we evaluated the role of 12-hydroxyeicosatetraenoic acid (HETE) on COX-2 gene expression, protein expression, and prostaglandin E2 (PGE2) production. We demonstrate that 12-HETE significantly increases COX-2 gene expression and consequent product formation, whereas a closely related lipid, 15-HETE, does not. In addition, IL-1beta-stimulated prostaglandin E2 production is completely inhibited by a preferential lipoxygenase inhibitor cinnaminyl-3,4-dihydroxy-alpha-cyanocinnamate. We then evaluated IL-1beta-induced PGE2 production in islets purified from control C57BL/6 mice and 12-LO knockout mice lacking cytokine-inducible 12-HETE. IL-1beta stimulated an 8-fold increase in PGE2 production in C57BL/6 islets but failed to stimulate PGE2 in 12-LO knockout islets. Addition of 12-HETE to 12-LO knockout islet cells produced a statistically significant rise in PGE2 production. Furthermore, 12-HETE, but not 15-HETE, stimulated COX-2 promoter and activator protein-1 binding activity. These data demonstrate that 12-HETE mediates cytokine-induced COX-2 gene transcription and resultant PGE2 production in pancreatic beta-cells.