Rapamycin On The Study Of Type 2 Diabetes And Diabetic Nephropathy And Mechanisms

BackgroundThe prevalence of diabetes and its complications have been increaseddramaticaly over the world, especially in the developing countries because of highcaloried and high fat food intake as well as less of physical activity among thepopulation. Albuminuria, an indicator of diabetic nephropathy, can be detected inapproximately 40% of diabtic patients. Diabetic nephropathy, one of the mostcommon diabetic microvascular complications, has become the leading cause ofend-stage renal disease and accounted for 42% of all new ESRD cases annually inthe United States. In China, diabetic nephropathy is the third cause of end-stagedrenal disease and contributed 13.5% to hemodialysis and 12% to peritoneal dialysispatients. Long term and maintance hyperglycemia is the most important cause ofdiabetic nephropathy and subsequently inappropriate activation of polyol pathway,activation of the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK)pathway, increased accumulation of advanced glycation end products, andoxidative stress may contributed to the pathogenesis.Type 2 diabetes accounts for about 97% of all the diabetic patients, which ischaracterized by insulin resistance and insulin secretory insufficiency. Insulinresistance precedes the diabetes and exists during the period of type 2 diabetes.Serine residues phosphorylation of insulin receptor substrate (IRS) proteins isbelieved to be a major mechanism of suppression of IRS activities that contribute toinsulin resistance. Recently, activation of the mammlian target of rapamycin (mTOR)branch downstream of the PI3K/Akt pathway has emerged as the critical event inrendering IRS-1 and IRS-2 unresponsive to insulin/insulin like-growth factor I(IGF-I). It has been demonstrated by the recent researches that rapamycin, aspecific inhibitor of mTOR, could improve insulin resistance by inhibiting IRS serinephosphrylation as well as inhibit vascular endothelial growth factor (VEGF)expression, which is proposed to be implicated in the glomerular hyperfiltration and the occurrence of microalbuminuria. The effects of rapamycin on type 2 diabetesand type 2 diabetes-associated renal injuries have remained unclear.ObjectiveTo observe the effects of rapamycin on type 2 diabetes and type 2diabetes-associated renal injuries.To investigate the effects of rapamycin on renal VEGF expression and the roleof PI3K/mTOR pathway in modulating insulin-induced VEGF transcriptionalactivation in human mesangial cells (HMCs).Methods1. 34 KKAy mice were randomized to receive rapamycin (group R, n=12),long-effective insulin (group I, n=10), and 0.9% saline (group C, n=12). Bodyweight (BW), serum glucose, serum cholesterol and serum triglyceride wereevaluated at pretreatment, 4 weeks and 8 weeks. The insulin sensitivity wasassayed at 5th week of the study in group C and group R.Kidney weight (KW), glomerular volume (GV), urine albumin/urine creatinine(U_ALB/Cr) and the glomerular basement membrance (GBM) thickness were alsoevaluated.2. Renal VEGF and fibronection (FN) mRNA expression were dectected withreal-time PCR. VEGF and CTGF protein expression in the kidney were determinedby Westernblot and immunohistochemistry.3. VEGF transcriptional activities in HMCs were tested by luciferase gene reporterassays system. HIF-1αmRNA and protein expression were evaluated by real-timePCR and Westernblot, respectively.Results1. Obesity and higher levels of serum glucose and triglyceride could be seen in 16 week-old KKAy mice. A significant lower body weight (33.7±2.3g vs 37.4±1.1ggroup C, p＜0.05; 38.2±1.5g group I, p＜0.05) and lower serum glucose levels(17.2±7.3 vs 29.1±3.2mmol/L group C, p＜0.05) could be seen in therapamycin-treated group at 4 weeks. The lower body weight in rapamycin-treatedmice still existed as compared with the controls (32.3±3.1 vs 39.3±2.8g, p＜0.05)at 8 week, whereas the statistical difference of serum glucose levels in the twogroups (18.2±3.7mmol/L vs 24.4±5.1mmol/L, p=0.06) was not yet appeared.There were no differences in serum cholesterol and triglyceride among threegroups.2. The serum glucose levels in rapamycin-treated group decreased rapidly andsignificantly at 60 minutes after insulin injection as compared with the controls(4.88±0.73 vs 2.65±2.12mmol/L, p＜0.05) in the insulin sensitivity test.3. A significant increase in kidney weight and glomerular volume were observed in16-week-old KKAy mice when compared with aged-mached C57BL mice, p＜0.01,p＜0.01, respectively. The kidney weight was significantly lower inrapamycin-treated group as compared with the controls (445±30 vs 500±47mg,p＜0.05) at 4 weeks after the initiation of treatment. The same pattern of changeswas also seen in glomerular volume. Rapamycin administration partially preventedthe increase in glomerular volume as compared with the controlled orinsulin-treated mice at 4 weeks and 8 weeks (p＜0.01 and P＜0.05, respectively). At4 weeks, a significant decrease in glomerular volume was seen in insulin-treatedmice when compared with the controls (p＜0.05). At the end of study, a significantlyIncreased GBM thickness was also found in controled mice when compared withthat in rapamycin-treated mice (p＜0.001) or in insulin-treated mice (p＜0.01).4. The urine albumin excretion was increased significantly in KKAy mice, and theU_alb/Cr in KKAy mice and C57BL mice was significant difference (6.04±1.54 vs.2.12±0.56mg/g, p＜0.01). The increase of U_alb/Cr in KKAy mice alleviated byrapamycin and it was not found in the control group at 4th and 8th week (p＜0.05,p＜0.01, respectly), and this ameliration was stronger than insulin-treated group at8th week (p＜0.05). 5. Markedly increased VEGF, FN mRNA expression and VEGF, CTGF proteinexpression were seen in 16-week-old KKAy mice as compared with C57BL mice.The increased renal VEGF, FN mRNA expression and VEGF protein expressioncould be inhibited significantly by the asministration of rapamycin at 4th and 8thweek.6. The insulin-induced VEGF transcription activities in HMCs appeared adose-dependent manner, which were transfected with a VEGF promoter reportercontaining a 1.4kb human VEGF promoter. The peak value appeared at theconcentration of 100nM and increased 2.14±0.17 folds as compared with thecontrols. VEGF transcription activities stimulated by insulin were partially blockedby Ly294002, a specific inhibitoy of PI3K, or by rapamycin, a specific inhibitor ofmTOR. Mutation of the hypoxia-inducible factor-1 (HIF-1) binding site abolished thestimulatory effect of insulin on VEGF transcriptional activation. Significantlyincreased VEGF reporter activities were also seen in HMCs transfected withmTOR-WT or mTOR-RR plasmid, which was further enhanced by insulin. TheVEGF transcriptional activities in HMCs transfected mTOR-WT plasmid could beinhibited by rapamycin, but not in HMCs transfected with mTOR-RR.7. An increased HIF-1αprotein expression, but not HIF-1αmRNA expressionwas found in HMCs stimulated by insulin or tansfected with mTOR-WT andmTOR-RR plasmid.Conclusions1. Rapamycin could decrease the body weight, serum glucose levels and improveinsulin resistance in KKAy mice.2. Rapamycin could ameliorate the early diabetic nephropathy in KKAy mice.3. Rapamycin could delay the lately diabetic nephropathy in KKAy mice.4. Rapamycin could inhibit renal VEGF, FN expression in KKAy mice, but didn't doany effect on CTGF expression.5. VEGF transcriptional activities induced by insulin in HMCs is mediated by HIF-1 αin response to PI3K/mTOR signaling pathway.