The Effect And Mechanism Of NO-1886 On Glycometabolism And Lipid Metabolism

Glucose metabolism disorders can cause abnormal lipid metabolism, on the contrary, improving lipid metabolism may also make glucose metabolism improved.Diabetes is a disorder of glucose metabolism, often accompanied by abnormal lipid metabolism. And lipid metabolism disorder is a high risk factor of cardiovascular disease. Currently a few drugs that treated diabetes are directly effective to atherosclerosis.Cardia cerebral vascular disease caused by atherosclerosis(As) seriously threat human health and its incidence increasing year by year become one of the major health problems to be solved at present. As is a complex pathological process involved in a multiple factors, and macrophage lipid excessive accumulation and vascular wall inflammation is the key link. Therefore, reducing cholesterol accumulation in macrophage and inhibitting inflammation in vascular wall has important significance on prevention of As.NO-1886(World Health Organization published in 2003, commonly known as Ibrolipim, WHO Drug Information 2003, 17(1): 55) is lipoprotein lipase(LPL) activator. It can increase the plasma LPL activity, and increased high density lipoprotein cholesterol accompanying with reducing plasma triglyceride concentration. NO-1886 can also improve insulin resistance in diabetes mice induced by high-fat diet. These results suggest that NO-1886 by long-term may have a better effect on improving glucose metabolism.In order to investigate the effects and the mechanism of NO-1886 on the metabolism of glucose and lipids, we established two cell models(Hep G2 cell model of insulin resistance and THP-1 macrophage model of lipid accumulation) and a Guangxi Bama miniature pig model of glucose and lipid metabolic disorder. On this basis, the effect and mechanism of NO-1886 regulating glucose and lipid metabolism were investigated from whole, cell, molecule and gene level by morphological, biochemical and molecular biological technology,Part I: The Role and Mechanism of NO-1886 in Glycogen Synthesis of Insulin Resistant Hep G2 CellsAims: The plasma level of free fatty acid is the important risk factor for insulin resistance and type 2 diabetes. The mechanisms that FFA induced insulin resistance were unclear, but recent evidence suggested that this lipotoxic effect of FFAs decreased skeletal muscle glucose transport stimulated by insulin, liver glycogen synthesis. It was possible that this was due to some impairment in insulin signaling pathway. Dysfunction of lipoprotein lipase(LPL) was associated with dyslipidaemia, obesity and insulin resistance. Based on previous report, oral administration of the novel synthetic compound NO-1886 could decrease plasma triglycerides, glucose, and FFAs level, but increase expression of LPLm RNA and protein activity, high density lipoprotein cholesterol levels, and furthermore prevent fat accumulation in high fat-fed rats. The primary aim of this study was to ascertain the relationship between NO-1886, glycogen, and insulin resistance(IR) in hepatoma cell line(Hep G2) in vitro with insulin resistant induced by palmitate.Methods: High concentration of palmitate induced insulin resistance in Hep G2 cells. Glucose and FFAs were determined by glucose oxidase assay and colorimetric assay respectively. The content of glycogen was measured by periodic Acid Schiff staining and anthrone colorimetric assay. The m RNA expression of PI3-Kp85, PI3-Kp110, Akt1, Akt2, GSK-3α, GSK-3β and LPL were assessed by reverse transcription PCR(RT-PCR). Meanwhile, the protein content of PI3-Kp85, Akt2, GSK-3β and P-GSK-3βser9 were evaluated by western blot.Results: Palmitate induced insulin resistance in Hep G2 cells. The results showed that after pretreatment with 0.5mmol/L palmitate for 48 h, the glycogen contents in Hep G2 cells reduced significantly, but contents of FFAs were increased and glucose consumption were decreased in culture medium. The m RNA expression of LPL, PI3-Kp85 was down-regulated, but expression of GKS-3β was up-regulated. The expression of GSK-3β protein was up-regulated, but the expression of P-GKS-3βser9 was down regulated. Whereas, the glycogen content, glucose and FFAs in Hep G2 cells were significantly improved by NO-1886 after increasing the expression of PI3-Kp85 subunit m RNA and P-GSK-3βser9 protein, and inhibiting the expression of GSK-3β m RNA and protein. Furthermore, LY294002 destroyed PI3-K/Akt/GSK-3 signaling pathway, but that were recovered by NO-1886 moderately.Conclusion: NO-1886 increased glycogen content in Hep G2 cells through up-regulating the expression of PI3-Kp85 m RNA, P-GSK-3βser9 protein. Down-regulating the expression of GSK-3β protein may be a mechanism for the hepatic insulin signaling protection.Part II: Effects of NO-1886 on LPL expression and Proinflammatory Cytokine Secretion in THP-1 MacrophagesAims: To observe the effect of NO-1886 on LPL expression, lipid accumulation and proinflammatory cytokine secretion in THP-1 macrophages.Methods: THP-1 macrophage were induced to differentiation into macrophages by PMA(160nmol/L) co-incubation for 24 h, added to treatment factors in free serum culture medium. LPL m RNA and its activity in THP-1 macrophage were determined by reverse transcription-polymerase chain reaction(RT-PCR) and LPL activity detection kit respectively. Lipid accumulation in THP-1 macrophage was evaluated by Oil Red O stain. The inflammatory cytokines expression in THP-1 macrophage were determted by ELISA and quantificational rt-PCR, PPARγ m RNA and protein levels were determined by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot respectively.Results: We found that NO-1886 significantly increased LPL m RNA and activity, increased IL-1β, IL-8, TNF-α and PPARγ expression on both protein and m RNA levels,in a dose-dependent manner, promoted the Macrophage-derived foam cell formation. GW9662 the PPARγ antagonist could partly counteract the effect of NO-1886 on the increased expression of LPL m RNA and activity and the expression of IL-1β, IL-8, TNF-α and PPARγ in THP-1 macrophages.Conclusion: NO-1886 increased inflammatory cytokines levels and the lipid accumulation in THP-1 macrophages, which may be related to activation of PPARγ pathway.Part III: NO-1886 Suppressed Lipid Accumulation and increased LPL in kidneys of diet-induced Diabetic MinipigsAims: The role of renal lipoprotein lipase(LPL) per se in kidney diseases is still controversial and obscure. The purpose of this study was to observe the preventive effects of NO-1886, a LPL activator, on lipid accumulation and LPL expression in kidneys of minipigs fed a high-sucrose and high-fat diet(HFSCD).Methods: Male Chinese Bama minipigs were fed a control diet or HFSCD with or without 0.1 g/kg/day NO-1886 for 5 months. Body weight, plasma glucose, insulin, lipids, LPL activity, and urinary microalbumin were measured. Renal tissue was obtained for detecting LPL activity and contents of triglyceride and cholesterol, and for observing the renal lipid accumulation by Oil Red O staining, and for examining the m RNA and protein expression of LPL by real time PCR, Western Blot and immunohistochemistry.Results: Feeding HFSCD to minipigs caused weight gain, hyperglycemia, hyperinsulinemia, hyperlipidemia and microalbuminuria. HFSCD increased plasma LPL activity while it decreased the m RNA and protein expression and activity of LPL in the kidney. The increases in renal triglyceride and cholesterol contents were associated with the decrease in renal LPL activity of HFSCD-fed minipigs. In contrast, supplementing NO-1886 into HFSCD lowered body weight, plasma glucose, insulin,triglyceride and urinary albumin concentrations while it increased plasma total cholesterol and HDL-C. NO-1886 suppressed the renal accumulation of triglyceride and cholesterol, and stimulated the diet-induced down-regulation of LPL expression and activity in the kidney.Conclusion: NO-1886 exerts renoprotective and hypolipidemic effects via the increase in renal LPL activity and expression, and thus the increased expression and activity of renal LPL play a vital role in suppressing renal lipid accumulation and ameliorating proteinuria in diet-induced diabetic minipigs.