Shoji Fujitani

Leucine promotes glucose uptake in skeletal muscles of rats

Soleus muscles isolated from normal rats were incubated to evaluate whether or not leucine promotes glucose uptake under insulin-free conditions, using a labeled 2-deoxyglucose uptake assay. Glucose uptake was promoted by 2mM leucine. A metabolite of leucine, alpha-ketoisocaproic acid (alpha-KIC), also exhibited a similar stimulatory effect, although this was not as potent as leucine. Stimulation of glucose uptake by leucine was completely canceled by pre-treatment with either 10 microM LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), or 6 microM GF109203X, a specific inhibitor of protein kinase C (PKC). No significant change was observed by pre-treatment with 1 microM rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR). These results suggest that leucine stimulates glucose transport in skeletal muscle via PI3-kinase and PKC pathways independently of the mammalian target of mTOR. They also suggest that leucine stimulates glucose transport by an insulin-independent mechanism.

Hypoglycaemic and insulinotropic effects of a novel oral antidiabetic agent, (−)-N-(trans-4-isopropylcyclohexane-carbonyl)-d-phenylalanine (A-4166)

(−)‐N‐(trans‐4‐isopropylcyclohexanecarbonyl)‐d‐phenylalanine (A‐4166), a novel oral hypoglycaemic agent is a non‐sulphonylurea insulin secretagogue. We investigated the insulin‐releasing action and hypoglycaemic effect of A‐4166 compared to sulphonylureas in vitro and in vivo. A‐4166 stimulated insulin secretion from rat freshly isolated pancreatic islets at concentrations from 3×10‐6m to 3×10‐4m in the presence of 2.8 mm glucose. There was no obvious difference in glucose dependency between the insulinotropic effect of A‐4166 and that of glibenclamide, and no additive or synergistic effect was observed between these two drugs. A‐4166 displaced [3H]‐glibenclamide bound to intact HIT‐T15 cells in a concentration‐dependent manner. The Ki value was 4.34±0.04×10−7m, and the displacement potency of A‐4166 was between that of glibenclamide and tolbutamide, being similar to that of gliclazide. In fasted beagle dogs, A‐4166 showed a dose‐dependent hypoglycaemic effect after oral administration over the range 1 to 10 mg kg−1. The hypoglycaemic action of A‐4166 showed an earlier onset and a shorter duration than that of sulphonylureas. Simultaneous measurement of plasma insulin levels revealed that the hypoglycaemic effect of A‐4166 was caused by a rapid‐onset and brief burst of insulin secretion. The pharmacokinetic profile of A‐4166 was consistent with the changes of the blood glucose and plasma insulin levels. Although the in vitro insulin‐releasing effect of A‐4166 was similar to that of sulphonylureas, its hypoglycaemic effect was more rapid and shorter‐lasting, associated with rapid absorption and clearance. Thus, A‐4166 may be useful in suppressing postprandial hyperglycaemia in patients with non‐insulin‐dependent diabetes mellitus.

The ability of a new hypoglycaemic agent, A‐4166, compared to sulphonylureas, to increase cytosolic Ca2+ in pancreatic β‐cells under metabolic inhibition

N‐(trans‐4‐isopropylcyclohexanecarbonyl)‐d‐phenylalanine (A‐4166) is a new non‐sulphonylurea oral hypoglycaemic agent which stimulates insulin release by increasing cytosolic Ca2+ concentration ([Ca2+]i) in β‐cells. We studied comparative effects of A‐4166 and sulphonylureas on [Ca2+]i, measured by dual‐wavelength fura‐2 microfluorometry, in single rat pancreatic β‐cells under normal conditions and conditions where glucose metabolism was inhibited. A glucokinase inhibitor, mannoheptulose (10 mm), a mitochondrial respiratory inhibitor, KCN (100 μm), and uncouplers, dinitrophenol (DNP, 50 μm) and carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP, 0.3 μm), were used to abolish glucose‐induced increases in [Ca2+]i in a reversible manner. Under control conditions, A‐4166 was one order more potent than tolbutamide in increasing [Ca2+]i, and maximal responses were evoked by 30 μm A‐4166 and 300 μm tolbutamide. These equipotent concentrations were employed for the comparative study where glucose metabolism was inhibited. In the presence of mannoheptulose, [Ca2+]i responses to tolbutamide, but not those to A‐4166, were attenuated in a reversible manner. KCN, DNP and FCCP inhibited [Ca2+]i responses to tolbutamide to a much greater extent than those to A‐4166. Responses to tolbutamide even at 3.3 times the equipotent concentration (1000 μm) were also markedly attenuated by these inhibitors. Responses evoked by another sulphonylurea, gliclazide, were inhibited by DNP to a larger extent than A‐4166‐induced responses. The results indicate that A‐4166 acts more effectively than sulphonylureas to increase [Ca2+]i in β‐cells during metabolic inhibition.

Branched-chain amino acids inhibit the TGF-beta-induced down-regulation of taurine biosynthetic enzyme cysteine dioxygenase in HepG2 cells.

Taurine deficiency has been suggested to contribute to the pathogenesis and complications of advanced hepatic diseases. The molecular basis for a low level of taurine associated with hepatic failure is largely unknown. Using carbon tetrachloride (CCl4)-induced cirrhotic rat model, we found that the activity and expression of cysteine dioxygenase (CDO), a rate-limiting enzyme in taurine synthesis, were significantly decreased in the liver of these rats. To investigate the underlying mechanisms for the suppression, we examined the effects of pathological cytokines on CDO expression in human hepatoma HepG2 cells. Among the several cytokines, transforming growth factor-β (TGF-β), one of the key mediators of fibrogenesis, suppressed Cdo1 gene transcription through the MEK/ERK pathway. Finally, we further examined potential effects of branched-chain amino acids (BCAA) on CDO expression, as it has been reported that oral BCAA supplementation increased plasma taurine level in the patients with liver cirrhosis. BCAA, especially leucine, promoted Cdo1 gene transcription, and attenuated TGF-β-mediated suppression of Cdo1 gene expression. These results indicate that the low plasma level of taurine in advanced hepatic disease is due to decreased hepatic CDO expression, which can be partly attributed to suppressive effect of TGF-β on Cdo1 gene transcription. Furthermore, our observation that BCAA promotes Cdo1 expression suggests that BCAA may be therapeutically useful to improve hepatic taurine metabolism and further suppress dysfunctions associated with low level of taurine in hepatic diseases.