Toshiki Inokuchi

Long-term effect of epalrestat, an aldose reductase inhibitor, on the development of incipient diabetic nephropathy in Type 2 diabetic patients.

The aim of the present study was to elucidate the long-term effect of epalrestat, an aldose reductase inhibitor (ARI), on renal function in patients with type 2 diabetes mellitus showing microalbuminuria. Patients were allocated to two groups (cases and controls) matched for age, BMI, and the extent of urinary albumin excretion (UAE). Thirty-five type 2 diabetic patients presenting microalbuminuria were included in this study: cases were treated with epalrestat (150 mg/day) for 5 years. No significant changes were found in blood pressure, HbA1c, and total cholesterol in either group during the observation period. In the control group, UAE increased significantly (P<.01) from 82+/-12 mg/g Cr at the baseline to 301+/-111 mg/g Cr at the end of the study, while UAE remained unchanged, 81+/-15 mg/g Cr at the baseline and 87+/-19 mg/g Cr at the end of the study, in the epalrestat-treated group. Reciprocal creatinine measured by an enzyme assay decreased significantly (P<.01) in both groups; however, the reduction rate in the epalrestat-treated group was significantly (P<.05) smaller than that in the control group. These results suggest the potential usefulness of ARIs in preventing the progression of incipient diabetic nephropathy in patients with type 2 diabetes mellitus.

Role of protein kinase C-angiotensin II pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels.

The intrarenal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. This study investigates the mechanisms for glucose-induced increase in angiotensin II (AII) production by human mesangial cells (MCs) in relation to protein kinase C (PKC). We also examine whether locally produced AII mediates extracellular matrix protein production in high-glucose conditions. Human MCs were cultured in 5 or 33 mmol/l glucose for 8 days, and were incubated with or without 5 mmol/l GFX, a PKC inhibitor, 0.1 micromol/l candesartan cilexetil (CC), a specific type 1 AII receptor antagonist, for another 24 h. In addition, MCs grown in 5 mmol/l glucose were incubated with 0.1 micromol/l phorbol-12,13-dibutyrate (PDBu) for 24 h. AII, TGF-beta1, fibronectin and type IV collagen in the culture media were measured by ELISA. The amount of AII secreted from MCs exposed to high-glucose levels was significantly greater (P<0.01) than that in normal glucose levels. The increase in AII production was completely prevented by GFX. The addition of PDBu mimicked the effect of glucose on AII production. The glucose-induced increases in the production of TGF-beta1, fibronectin and type IV collagen were partially, but significantly restored (P<0.01) by CC, while GFX totally abolished these effects of glucose. These results suggest that elevated glucose levels stimulate AII production via mechanisms dependent on glucose-induced PKC activation in human MCs, and that locally produced AII partly mediates the increase in mesangial matrix synthesis in high-glucose conditions.

High glucose levels enhance TGF-β1–thrombospondin-1 pathway in cultured human mesangial cells via mechanisms dependent on glucose-induced PKC activation

We have previously demonstrated that thrombospondin-1 (TSP-1) promotes activation of latent TGF-beta1 in cultured human mesangial cells (MCs) [Nephron 79 (1998) 38.]. This study was performed to clarify the relationship between protein kinase C (PKC) activity and activation of TGF-beta1 and TSP-1 production in cultured human MCs exposed to high glucose levels. MCs grown in 33 mmol/l glucose demonstrated a significant (P< .01) increase in activation of TGF-beta1 compared with those in 5 mmol/l glucose, which were evaluated by both an ELISA and a bioassay. High glucose-induced increase in active TGF-beta1 was completely inhibited by coincubation with 5 micromol/l GFX, a PKC inhibitor, and was mimicked by the addition of 0.1 micromol/l phorbol 12,13-dibutyrate (PDBu). On the other hand, increased TSP-1 production from MCs stimulated by 0.1 nmol/l recombinant TGF-beta1 and 0.1 micromol/l PDBu were significantly (P< .01) reduced by the addition of 10 nmol/l latency-associated peptide (LAP), a specific inhibitor of TGF-beta1 activity. The amount of TSP-1 secreted from MCs increased by a high ambient glucose. The glucose-induced increase in TSP-1 production was markedly attenuated by the treatment with GFX and LAP, while those agents did not affect TSP-1 production in low-glucose concentrations. Taken together, our results suggest that glucose-induced activation of TGF-beta1 is dependent on PKC activity, leading to a sequential increase in TSP-1 synthesis in cultured human MCs. Thus, we propose that high glucose conditions induce an increase in PKC-TGF-beta1 activity-TSP-1 pathway, and that glucose-induced increase in TSP-1 may synergistically facilitate TGF-beta1 activation in an autocrine manner in MCs.

Changes in carnitine metabolism with ketone body production in obese glucose-intolerant patients

To elucidate the relationship between carnitine metabolism and plasma ketone body concentrations in moderately obese patients with mild glucose intolerance, the ketone body and carnitine levels in the basal state were determined in 72 obese patients: 20 with normal glucose tolerance (NGT), 29 with impaired glucose tolerance (IGT), and 23 with non-insulin-dependent diabetes mellitus (NIDDM) having a fasting plasma glucose (FPG) level of less than 200 mg/dl. Total ketone body (TKB) levels significantly (P < 0.05) increased in the order of NGT, IGT, NIDDM, while the FPG and free fatty acid (FFA) concentrations were significantly (P < 0.05) higher in the NIDDM group than in the other two groups. In contrast, the insulin, glucagon and glycerol levels were comparable in the three groups. The plasma short-chain acylcarnitine (SCAC) concentration and the acylcarnitine/free carnitine (AC/FC) ratio were similar in the IGT and NIDDM groups, and significantly (P < 0.05) greater than those in the NGT group. The AC/FC ratio correlated significantly with the FPG and FFA, but not with the TKB. These results suggest that the combination of IGT with simple obesity may trigger the acceleration of hepatic ketogenesis in conjunction with an elevated SCAC and an increased AC/FC ratio. In addition, the data also imply that, in patients with mild NIDDM, factors other than the carnitines may play a greater role in enhancing ketonemia.

Effect of an aldose reductase inhibitor on glomerular basement membrane anionic sites in streptozotocin-induced diabetic rats

The present study was conducted in order to determine whether an aldose reductase inhibitor (ARI), epalrestat, prevents the progression of diabetic nephropathy in rats. Rats were made diabetic by intravenous injection of streptozotocin (STZ 50 mg/kg) and epalrestat (100 mg/kg) was administered orally through a gastric tube once daily for 4 weeks. Examination by electron microscope revealed that the number of anionic sites (AS) in the lamina rara externa per 1000 nm of glomerular basement membrane (GBM) was significantly decreased in diabetic rats compared to control values (17.6 + or - 0.4 vs. 21.9 + or -0.4, P < 0.01), whereas, significant recovery (20.3 + or - 0.7, P < 0.05) was observed after 4 weeks of epalrestat treatment. Urinary albumin excretion (UAE) rate was markedly increased in diabetic rats and the treatment resulted in its significant suppression from diabetic rats. In conclusion, administration of epalrestat to diabetic rats is capable of preventing a reduction in the number of AS in GBM which would ameliorate an increased permeability of the basement membrane leading to albuminuria.

Plasma fibrinopeptide A levels during insulin-induced plasma glucose falls in diabetics

Plasma fibrinopeptide A (FPA) concentration, as a measure of thrombin activity, was determined during an insulin tolerance test in 17 non-obese diabetics. FPA was measured by a modification of the Nossel method. Administration of insulin significantly lowered plasma glucose, accompanied by a significant increase in FPA from 0.9 +/- 0.1 ng/ml to 4.3 +/- 1.6 ng/ml (P less than 0.05) as well as a significant increase in circulating levels of epinephrine and growth hormone. The magnitude of the peak in epinephrine levels correlated well with both the rate of decline of plasma glucose and the magnitude of the peak of FPA. In addition, the FPA increment was suppressed by treatment with heparin. These results indicate that insulin-induced hypoglycemia or a rapid fall in plasma glucose is associated with enhanced thrombin generation and fibrin formation, which may be considered as a contributory factor to the development of diabetic microangiopathy through a hypercoagulable state.

Transforming Growth Factor-Beta-1 Latency-Associated Peptide and Soluble Betaglycan Prevent a Glucose-Induced Increase in Fibronectin Production in Cultured Human Mesangial Cells

This study was performed to investigate the effects of transforming growth factor-β1 (TGF-β1) latency-associated peptide (LAP) and betaglycan on TGF-β1 activity, and on the glucose-induced overproduction of fibronectin in cultured human mesangial cells (MCs). We found that recombinant LAP and recombinant soluble betaglycan decrease the active form of TGF-β1, measured by ELISA, in a dose-dependent manner in a cell-free system. The effective dosages of LAP and soluble betaglycan for a 50% reduction were approximately 20- and 75-fold of the TGF-β1 concentration, respectively. The active form of TGF-β1 in the media secreted from MCs was significantly (p < 0.01) reduced by the addition of 10 nmol/l LAP and 10 nmol/l soluble betaglycan with no significant change in total (active + latent) TGF-β1. Recombinant LAP and soluble betaglycan also inhibited a recombinant TGF-β1-stimulated increase in fibronectin production in MCs. Furthermore, the glucose-induced increase in fibronectin secreted from MCs was significantly (p < 0.01) suppressed by concomitant incubation with LAP or soluble betaglycan, while these agents had no effect on fibronectin production under physiological glucose concentrations. These results indicate that recombinant LAP and soluble betaglycan suppress the glucose-induced overproduction of fibronectin presumably via inhibition of TGF-β1 activity in MCs. Further in vivo studies are needed to define the possible beneficial effects of these agents in diabetic nephropathy.

The number of microvascular complications is associated with an increased risk for severity of periodontitis in type 2 diabetes patients: Results of a multicenter hospital-based cross-sectional study

To explore the relationships between periodontitis and microvascular complications as well as glycemic control in type 2 diabetes patients.