Good metabolic and safety profile of troglitazone alone and following alcohol in NIDDM subjects.

Drinking alcohol is associated with a recognised risk of hypoglycaemia. This double-blind, placebo-controlled study was designed to determine whether alcohol taken with the evening meal alters the gluco-regulatory response to troglitazone, (TR), an insulin action enhancer, in non-insulin-dependent diabetes mellitus (NIDDM) subjects to increase the risk of hypoglycaemia. In vitro studies conducted prior to the clinical study presented here showed no evidence of a pharmacokinetic interaction between the two drugs. A total of 23, diet-treated, NIDDM subjects received either TR, 200 mg once daily (n = 11) or placebo (PL) (n = 12) for 45 days. On days 42 and 45 subjects were given, on separate days, an alcohol challenge (AC), 0.6 mg/kg ethanol in orange juice and a control challenge, CC, orange juice alone, with the evening meal. Serum glucose, insulin, proinsulin-like molecules, C-peptide and lipids were measured during the study, for the 4 h after each challenge and the following morning (fasting). The over-night urine cortisol/creatinine ratio (an index of hypoglycaemia) was also determined. For the TR treated group, fasting serum glucose the next morning (adjusted geometric mean: 6.8 mmol/l for AC) and weighted mean were not statistically significantly different following AC compared to CC. Mean trough glucose for TR after the evening meal was 5.7 mmol/l following both the AC and CC. Analysis of the other parameters showed no symptomatic or pharmacodynamic evidence of an acute interaction between TR and alcohol. It can be concluded that occasional drinking of alcohol, with a meal, by TR-treated NIDDM patients is unlikely to be associated with an increased risk of hypoglycaemia.

The role of the sensory peptide calcitonin-gene-related peptide(s) in skeletal muscle carbohydrate metabolism: effects of capsaicin and resiniferatoxin.

1. The content of calcitonin-gene-related-peptide-like immunoreactivity (CGRP-LI) in various rat muscles was measured. Starvation for 24 h did not affect the content of CGRP-LI in these muscles, except for a decreased level in the starved-rat diaphragm. Higher contents of CGRP-LI were observed in well-vascularized muscles. 2. Capsaicin (at 1, 10 and 100 microM) inhibited insulin-stimulated rates of glycogen synthesis in isolated stripped incubated soleus muscle preparations by a mechanism independent of catecholamine release, since the effects of capsaicin were not altered by the beta-adrenoreceptor antagonist DL-propranolol. 3. Resiniferatoxin (10 nM), which is a potent capsaicin agonist, also significantly inhibited the insulin-stimulated rate of glycogen synthesis. Furthermore, the concentration of resiniferatoxin required to inhibit glycogen synthesis was 100 times less than the concentration of capsaicin needed for the same effect. 4. Capsaicin (10 microM) decreased the content of CGRP-LI in isolated stripped incubated soleus muscle preparations by about 40%. 5. Neonatal treatment of rats with capsaicin, which causes de-afferentation of some sensory nerves such, we hypothesize, that CGRP can no longer be released to counteract the effects of insulin in vivo, caused increased rates of glycogen synthesis and increased glycogen content in stripped soleus muscle preparations in vitro when muscles were isolated from the adult rats. 6. These findings support the hypothesis that capsaicin and resiniferatoxin elicit an excitatory response on sensory nerves in skeletal muscle in vitro to cause the efferent release of CGRP. Consequently, CGRP is delivered to skeletal muscle fibres to inhibit insulin-stimulated glycogen synthesis. The role of CGRP in recovery of blood glucose levels during hypoglycaemia is discussed.

Effects of calcium antagonists on insulin-mediated glucose metabolism in skeletal muscle.

The effect of three calcium antagonists (verapamil, diltiazem, and nifedipine) on insulin effects was investigated in isolated rat soleus muscles. Soleus muscles were incubated in the presence of insulin (100 microU/ml), a concentration that stimulates the rates of lactate formation and glycogen synthesis half-maximally and with and without a calcium antagonist. A decrease (48%; P < 0.001) was noted in the insulin-mediated rate of glycogen synthesis by verapamil at 100 microM; no effect was observed at lower concentrations of verapamil. Diltiazem decreased the insulin-mediated rates of glycogen synthesis by 36 (P < 0.001), 64 (P < 0.001), and 73% (P < 0.001) at 1, 10, and 100 microM, respectively. Nifedipine decreased the insulin-mediated rates of glycogen synthesis by 37% at 0.1 microM (P < 0.001), 36% at 1 microM (P < 0.001), 21% at 10 microM (P < 0.05), and 72% at 100 microM (P < 0.001). Verapamil at 100 microM decreased lactate formation by 48% (P < 0.001). However, diltiazem increased the rate of lactate formation by 22 (P < 0.01), 43 (P < 0.001), and 61% (P < 0.001) at 1, 10, and 100 microM, respectively. In contrast, nifedipine increased the insulin-mediated rate of lactate formation by 45% only at 100 microM (P < 0.01). The increased rate of lactate formation was probably caused by an increased rate of glycogenolysis, because high concentrations of all the calcium antagonists significantly decreased muscle glycogen content. The insulin-stimulated rate of 3-O-methyl-D-glucose transport or cAMP content was not affected by diltiazem at 1 or 10 microM. The results suggest that the calcium antagonists work by a mechanism, possibly by activating a calcium channel or an extracellular receptor, to influence markedly insulin-mediated intracellular glucose metabolism in skeletal muscle.

Biotinyl analogues of amylin as biologically active probes for amylin/CGRP receptor recognition.

Biotinyl analogues of rat amylin were synthesised with sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate (NHS-SS-Biotin). Biotinylated amylin peptides were purified by HPLC, quantitated, and the presence of the biotin group at Lys-1 confirmed by peroxidase-labelled avidin and FAB mass spectroscopy. Amylin-biotin retained a similar affinity for binding to rat liver plasma membranes compared with rat amylin and also completely inhibited insulin-stimulated glycogen synthesis in rat soleus muscle incubated in vitro. These biologically active amylin probes will enable a complete analysis of amylin/CGRP receptor expression in various cell types and facilitate the isolation and characterisation of the hormone-receptor complex.

Peroxovanadates have full insulin-like effects on glycogen synthesis in normal and insulin-resistant skeletal muscle.

1. The insulin-like effects of orthovanadate (10 mM) and peroxides of vanadate (peroxovanadates, at 1 mM) on rates of lactate formation, glucose oxidation and glycogen synthesis were measured in incubated soleus-muscle preparations isolated from non-obese Wistar rats and lean (fa/?) or insulin-resistant obese Zucker (fa/fa) rats. 2. The stimulation of the rates of lactate formation and glucose oxidation by either orthovanadate or peroxovanadates was of similar magnitude to the stimulation by a maximally effective concentration of insulin (1000 microunits/ml). 3. Peroxovanadates, but not orthovanadate, maximally stimulated the rate of glycogen synthesis in incubated soleus muscles isolated from Wistar rats. 4. When soleus-muscle preparations were incubated in the presence of both insulin (1000 microunits/ml) and peroxovanadates (1 mM), this did not result in a synergistic increase in the rate of total glucose utilization as compared with either agent alone. 5. Soleus muscles isolated from obese (fa/fa) Zucker rats exhibited a decrease in response to a physiologically relevant concentration of insulin (100 microunits/ml). Peroxovanadates, at 1 mM, maximally stimulated the rate of glycogen synthesis in soleus muscles isolated from obese (fa/fa) Zucker rats. 6. The findings indicate that peroxovanadates are useful and important agents for investigating the mechanism of action of insulin in skeletal muscle.

Physiological glucocorticoid levels regulate glutamine and insulin-mediated glucose metabolism in skeletal muscle of the rat. Studies with RU 486 (mifepristone).

This study examined the effects of antagonism of the peak level of glucocorticoids in vivo, which occurs as rats enter the feeding/activity (dark) period on glucose and glutamine metabolism in incubated isolated rat soleus muscle preparations. Thus the rats were treated with the potent glucocorticoid antagonist RU 486 2 h before and 1 and 2 h into the dark period. Both the content of glutamine in skeletal muscle in vivo and plasma glucose and glutamine concentrations were elevated midway through the dark period, compared with the beginning of the period. RU 486 prevented the increases in plasma glucose and glutamine and caused a significant decrease in both the rate of release of glutamine in soleus muscle in vitro and the content of glutamine in gastrocnemius muscle. The sensitivity of soleus muscle to insulin in vitro is markedly decreased when isolated midway through the dark period (i.e. at 03:00 h) [Leighton, Kowalchuk, Challiss & Newsholme (1988) Am. J. Physiol. 255, E41-E45]. We now show that the concentrations of insulin required to stimulate lactate formation and glycogen synthesis half-maximally were 95 and 250 muunits/ml respectively, and treatment of rats with RU 486 decreased these values to 55 and 90 muunits of insulin/ml respectively. Thus antagonism of the action of the normal circadian rise in the level of glucocorticoids in rats reverses insulin insensitivity in soleus muscles in vitro.

Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus.

The 37-amino acid peptide called amylin is a major component of the islet amyloid deposited in the pancreases of persons with type 2 diabetes mellitus. We report the isolation of a partial cDNA clone and a phage lambda genomic clone of the coding region of the amylin gene. The DNA sequence encodes a protein sequence identical to that of amylin isolated from the amyloid found in the diabetic pancreas and shows that amylin is likely to be synthesized as a precursor peptide, now named proamylin. We have demonstrated that the amylin gene is present on chromosome 12 and that it is probably transcribed in the islets of Langerhans. The sequences of the genes for amylin and the calcitonin gene-related peptides (CGRPs) show strong similarity, especially over their 5' coding regions, where both peptides have a conserved intramolecular disulfide bridge, and also over their 3' coding regions, where the presence of a glycine codon strongly suggests that the carboxyl-terminal residue of amylin, like that of CGRP, is amidated. To examine the functional relevance of these posttranslational modifications, the biological activity of amylin synthesized with or without the disulfide bridge and/or amidation was measured. It was found that both features are necessary for full biological activity, thereby confirming the functional importance of those regions of the molecule whose sequences are conserved at both protein and genetic levels.

Calcitonin gene-related peptide-1 (CGRP-1) is a potent regulator of glycogen metabolism in rat skeletal muscle.

We investigated the effects of CGRP on glucose metabolism in intact rat skeletal muscle preparations that are largely composed of either type I (soleus) or II fibres (e.g. extensor digitorum longus (EDL) or epitrochlearis muscles). CGRP-1 inhibited insulin-stimulated glycogen synthesis in both soleus and EDL muscle preparations. Rat CGRP-1 was a potent stimulator of glycogenolysis only in muscles composed of type II fibres, which depend on high rates of glycogenolysis to produce high power outputs. These results may provide the basis for understanding how CGRP regulate glycogenolysis in type II fibres in vivo.