Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2–3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48–72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.     

Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation.

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2--3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48--72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.     

Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline

Under standard conditions, liver regeneration is not impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 μg/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 μg/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Polyamine and amino acid content, and activity of polyamine-synthesizing decarboxylases, in liver of streptozotocin-induced diabetic and insulin-treated diabetic rats

1. Concentrations of polyamines, amino acids, glycogen, nucleic acids and protein, and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, were measured in livers from control, streptozotocin-diabetic and insulin-treated diabetic rats. 2. Total DNA per liver and protein per mg of DNA were unaffected by diabetes, whereas RNA per mg of DNA and glycogen per g of liver were decreased. Insulin treatment of diabetic rats induced both hypertrophy and hyperplasia, as indicated by an increase in all four of these constituents to or above control values. 3. Spermidine content was increased in the livers of diabetic rats, despite the decrease in RNA, but it was further increased by insulin treatment. Spermine content was decreased by diabetes, but was unchanged by insulin treatment. Thus the ratio spermidine/spermine in the adult diabetic rat was more typical of that seen in younger rats, whereas insulin treatment resulted in a ratio similar to that seen in rapidly growing tissues. 4. Ornithine decarboxylase activity was variable in the diabetic rat, showing a positive correlation with endogenous ornithine concentrations. This correlation was not seen in control or insulin-treated rats. Insulin caused a significant increase in ornithine decarboxylase activity relative to control or diabetic rats. 5. S-Adenosylmethionine decarboxylase activity was increased approx. 2-fold by diabetes and was not further affected by insulin. 6. Hepatic concentrations of the glucogenic amino acids, alanine, glutamine and glycine were decreased by diabetes. Their concentrations and that of glutamate were increased by injection of insulin. Concentrations of ornithine, proline, leucine, isoleucine and valine were increased in livers of diabetic rats and were decreased by insulin. Diabetes caused a decrease in hepatic concentration of serine, threonine, lysine and histidine. Insulin had no effect on serine, lysine and histidine, but caused a further fall in the concentration of threonine.     

Influence of sex,strain, and species on trace metal status of insulin-deficient diabetic rodents

Previous studies have demonstrated marked alterations in trace metal metabolism in male Sprague-Dawley rats following chemical induction of the diabetic state. To determine whether such changes represented a general response to the insulin-deficient condition the levels of zinc, copper, and maganese in liver, kidney, and intestine of normal and streptozotocin (STZ)-diabetic male rats of the Sprague-Dawley, Wistar, and Long-Evans strains, female Sprague-Dawley rats, and male mice were measured. Significantly increased concentrations of zinc, copper, and maganese in liver, and zinc and copper in kidney were found in STZ-diabetic rats, regardless of sex and strain. In contrast, the zinc and copper contents in liver and kidney of control and STZ-diabetic mice were similar, but hepatic manganese levels were significantly elevated in both organs of the diabetic mouse. The concentrations of all three metals were similar in the intestine of control and diabetic rodents. Higher amounts of zinc and copper were bound to metallothionein in the liver and kidney of the diabetic rats. Nicotinamide injection prior to STZ administration protected rats against the development of diabetes and alterations in trace metal status. These data indicate that specific alterations in the metabolism of zinc, copper and manganese during episodes of pancreatic hormonal imbalance represent a general phenomenon in the rat. A possible explanation for the differential response of the STZ-diabetic mouse is discussed.     

Effects of antioxidant stobadine on protein carbonylation, advanced oxidation protein products and reductive capacity of liver in streptozotocin-diabetic rats: role of oxidative/nitrosative stress

BACKGROUND: Increased oxidative/nitrosative stress is important in the pathogenesis of diabetic complications, and the protective effects of antioxidants are a topic of intense research. The purpose of this study was to investigate whether a pyridoindole antioxidant stobadine (STB) have a protective effect on tissue oxidative protein damage represented by the parameters such as protein carbonylation (PC), protein thiol (P-SH), total thiol (T-SH) and non-protein thiol (Np-SH), nitrotyrosine (3-NT), and advanced oxidation protein products (AOPP) in streptozotocin-diabetic rats. METHODS: Diabetes was induced in male Wistar rats by intraperitonal injection of streptozotocin (55 mg/kg). Some of the non-diabetic (control) and diabetic rats treated with STB (24.7 mg/kg/day) during 16 weeks, and the effects on blood glucose, PC, AOPP, 3-NT, P-SH, T-SH and Np-SH were studied. Biomarkers were assayed by enzyme-linked immunosorbent assay (ELISA) or by colorimetric methods. RESULTS: Administration of stobadine to diabetic animals lowered elevated blood glucose levels by approximately 16% relative to untreated diabetic rats. Although stobadine decreased blood glucose, poor glycemic control was maintained in stobadine treated diabetic rats during the treatment period. Biochemical analyses of liver proteins showed significant diminution of sulfhydryl groups, P-SH, T-SH, Np-SH, and elevation of carbonyl groups in diabetic animals in comparison to healthy controls. As a biomarker of nitrosative stress, 3-NT levels did not significantly change by diabetes induction or by stobadine treatment when compared to control animals. However, the treatment with stobadine resulted in a significant decrease in PC, AOPP levels and normalized P-SH, T-SH, Np-SH groups in liver of diabetic animals. CONCLUSIONS: The results are in accordance with the pro-oxidant role of chronic hyperglycemia, and the ability of stobadine to attenuate protein oxidation and improving tissue reductive capacity may account, at least partly for its observed beneficial effects on tissue function in diabetes.     

Effects of insulin on perfused liver from streptozotocin-diabetic and untreated rats: 13C NMR assay of pyruvate kinase flux

The effects of insulin in vitro on perfused liver from streptozotocin-diabetic rats and their untreated littermates during gluconeogenesis from either [3-13C]alanine + ethanol or [2-13C]pyruvate + NH4Cl + ethanol were studied by 13C NMR. A 13C NMR determination of the rate of pyruvate kinase flux under steady-state conditions of active gluconeogenesis was developed; this assay includes a check on the reuse of recycled pyruvate. The preparations studied provided gradations of pyruvate kinase flux within the confines of the assay's requirement of active gluconeogenesis. By this determination, the rate of pyruvate kinase flux was 0.74 +/- 0.04 of the gluconeogenic rate in liver from 24-h-fasted controls; in liver from 12-h-fasted controls, relative pyruvate kinase flux increased to 1.0 +/- 0.2. In diabetic liver, this flux was undetectable by our NMR method. Insulin's hepatic influence in vitro was greatest in the streptozotocin model of type 1 diabetes: upon treatment of diabetic liver with 7 nM insulin in vitro, a partial reversal of many of the differences noted between diabetic and control liver was demonstrated by 13C NMR. A major effect of insulin in vitro upon diabetic liver was the induction of a large increase in the rate of pyruvate kinase flux, bringing relative and absolute fluxes up to the levels measured in 24-h-fasted controls. By way of comparison, the effects of ischemia on diabetic liver were studied by 13C NMR to test whether changes in allosteric effectors under these conditions could also increase pyruvate kinase flux. A large increase in this activity was demonstrated in ischemic diabetic liver.     

Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assay detects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate (substrate). Colorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coefficients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assay can be performed in less than 7 h and has a sensitivity in the nanogram range (3–48 ng/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50–398 μg/ml) and urine RBP levels (mean 14, range 1–80 μg/ml) than normal donors (mean serum 43, range 30–60 μg/ml; mean urine RBP 0.06, range 0.04 – 0.13 μg/ml). Liver disease patients had lower than normal serum RBP values (mean 22, range 10–43 μg/ml).     

Serum esterase activities and hyperlipidaemia in the streptozotocin -diabetic rat

The levels of activity of four serum esterases were measured in control and streptozotocin-diabetic rats for a period of 6 months. Pseudocholinesterase activity was significantly elevated in the diabetic rats at all points tested, reaching 250% of control activity at 6 months. Levels of paraoxonase activity progressively decreased with time in the diabetic rats, being 36% lower than in controls at 6 months. No significant differences in either serum arylesterase or carboxylesterase activity between control and diabetic rats were observed.     

Enhanced in vivo sensitivity of vanadyl-treated diabetic rats to insulin

Vanadium has been reported to have insulin-like properties and has recently been demonstrated to be beneficial in the treatment of diabetic animals. In the present study, concentration dependence of the therapeutic effects of vanadium and the nature of interaction under in vivo conditions between vanadium and insulin were examined in streptozotocin-diabetic rats. During a 2-week period, blood glucose levels in all treated animals were decreased. At higher concentrations of vanadyl this decrease was greater and more rapid, and remained consistently lower for the entire treatment period. Daily intake of vanadyl, however, reached a similar steady state in all groups. Acute administration of submaximal doses of insulin, which had minimal effects in untreated diabetic rats, lowered blood glucose concentrations in vanadyl-treated and vanadyl-withdrawn animals to control levels. Chronic treatment of streptozotocin-diabetic rats with submaximal levels of vanadyl and insulin, ineffective alone, also produced significant decreases in blood glucose levels when used in combination. Finally, the insulin dosage required to maintain a nonglycosuric state in spontaneously diabetic (BB) rats was reduced in the presence of vanadyl. These studies indicate that chronic oral vanadyl treatment (a) produces a concentration-related lowering of blood glucose in diabetic rats, (b) potentiates the in vivo glucose lowering effects of acute and chronic administrations of insulin in streptozotocin-diabetic rats, and (c) substitutes for, or potentiates, the effects of chronic insulin therapy in spontaneously diabetic BB rats.     

Threshold level for theophylline in doping analysis

HPLC in the reversed-phase mode is used to assay methylxanthines including theobromine, paraxanthine, theophylline and caffeine in urine. The calibration graphs show good linearity in the concentration range 0–10 μg/ml. The limit for accurate quantitation of theophylline was 0.25 μg/ml. Between 6 and 20% of the parent drug is recovered in urine (0–12 h) after the oral administration of sustained release preparations containing 150 and 250 mg theophylline to four volunteers. Theophylline levels above 0.25 μg/ml were found in 1539 out of 3885 urine samples collected from athletes during unannounced doping control in Flanders. Statistical evaluation of the results gives a far outside value [75th percentile + (3× interquartile range)] of 2.25 μg/ml. The ratio theophylline paraxanthine (TP/PX) as an indicator for the non-dietary intake of theophylline seems to be more reliable. The far outside ratio was 0.20. To ensure with the greatest possible degree of certainty that no false-positive result is reported, decision limits of 5 μg/ml and 0.50, for theophylline and the ratio TP/PX respectively, are proposed.     

Streptozotocin-Induced Diabetes Reduces Brain Serotonin Synthesis in Rats

The rate of brain 5-hydroxytryptamine (serotonin) synthesis and turnover in streptozotocin-diabetic rats was assessed using three separate methods: the rate of 5-hydroxytryptophan accumulation following decarboxylase inhibition with Ro 4-4602; the decline in 5-hydroxyindoleacetic acid levels following monoamine oxidase inhibition with pargyline; and the rate of 5-hydroxyindoleacetic acid accumulation following blockade of acid transport with probenecid. Each of the three methods revealed that 5-hydroxytryptamine synthesis and turnover is decreased by 44-71% in diabetic rats with plasma glucose levels of between 500 and 600 mg%. In addition, the levels of free and bound plasma tryptophan were measured and the levels of the free amino acid were found to be the same in control and diabetic rats. Since diabetic rats exhibit a 40% decrease in brain tryptophan, the free tryptophan level in plasma does not predict brain tryptophan levels in diabetic rats. These data are discussed within the context of psychiatric disturbances experienced by diabetic patients.     

Early oxidative stress in the diabetic kidney: effect of DL-alpha-lipoic acid

Oxidative stress is implicated in the pathogenesis of diabetic nephropathy. The attempts to identify early markers of diabetes-induced renal oxidative injury resulted in contradictory findings. We characterized early oxidative stress in renal cortex of diabetic rats, and evaluated whether it can be prevented by the potent antioxidant, DL-alpha-lipoic acid. The experiments were performed on control rats and streptozotocin-diabetic rats treated with/without DL-alpha-lipoic acid (100 mg/kg i.p., for 3 weeks from induction of diabetes). Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats vs. controls (p <.01) and this increase was partially prevented by DL-alpha-lipoic acid. F(2) isoprostane concentrations (measured by GCMS) expressed per either mg protein or arachidonic acid content were not different in control and diabetic rats but were decreased several-fold with DL-alpha-lipoic acid treatment. Both GSH and ascorbate (AA) levels were decreased and GSSG/GSH and dehydroascorbate/AA ratios increased in diabetic rats vs. controls (p <.01 for all comparisons), and these changes were completely or partially (AA) prevented by DL-alpha-lipoic acid. Superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione transferase, and NADH oxidase, but not catalase, were upregulated in diabetic rats vs. controls, and these activities, except glutathione peroxidase, were decreased by DL-alpha-lipoic acid. In conclusion, enhanced oxidative stress is present in rat renal cortex in early diabetes, and is prevented by DL-alpha-lipoic acid.     

Adrenomedullin gene expression and peptide levels in the heart and blood vessels of streptozotocin-diabetic rats.

The aim of the present study was to assess the changes in gene expression and peptide adrenomedullin (AM) levels in cardiovascular and other tissues in the streptozotocin-diabetic rats. For this purpose, diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/Kg body weight). Half of the diabetic rats were subcutaneously injected with insulin in the afternoon (4 units/day) one week after STZ injection until the day before killing. Control rats received only saline injection. AM mRNA was determined in cardiovascular and other tissues of streptozotocin-diabetic rats using solution-hybridization-RNase protection assay. Circulating AM and peptide AM in cardiovascular and other tissues were estimated using a specific radioimmunoassay. There were increases in preproAM mRNA levels in the left and right ventricles and in the thoracic aorta in both the 2-week and 4-week diabetic rats, but not in the two atria, the mesenteric artery and the lung. In the 2-week diabetic rats, there were decreases in AM contents in the two atria and the lung but an increase in the thoracic aorta. In the 4-week diabetic rats, there were bigger decreases in the atria and also a decrease in the left ventricle. The plasma AM levels were not changed but there was an increase in the excretion of AM in the urine. Our results suggest a possible increase in AM release in the heart and the thoracic aorta in the 2-week and 4-week diabetic rats.     

Monoamine and diamine oxidase activities in uremic rats

The activities of monoamine and diamine oxidases in various organs and tissues and the amine levels in plasma and urine were determined in chronically uremic and pair-fed control rats. Plasma amine levels were elevated in uremic animals while the urinary excretion of amines was decreased. In uremic as compared to control animals, monomaine oxidase activity was decreased in kidney and muscle, increased in heart and plasma and not altered in liver and cerebrum. Diamine oxidase activity in uremic rats was decreased in kidney, increased in plasma and unchanged in liver and muscle. These alterations of amine oxidase activities in renal failure may affect the metabolism of many amines and thus contribute to the pathogenesis of the uremic syndrome.     

A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase

An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively.     

Copper accumulation in the soluble and particulate fractions of renal cortex in the streptozotocin-diabetic rat

The accumulation and subcellular distribution of copper in the kidney of streptozotocin-diabetic rats were investigated. Male Sprague-Dawley rats received streptozotocin (50 mg/kg body wt on two consecutive days) intraperitoneally and were fed either commercial or purified diet. The concentrations of copper, zinc, iron, and manganese present in intact kidney, renal cortex, and renal medulla were compared at various times. Chow-fed diabetic rats had a renal copper concentration 2.6 times greater than age-matched controls after 2 weeks. The concentration of zinc was only 30% higher in diabetic kidney than in control tissue, whereas the iron and manganese concentrations were similar for both groups. The additional complement of renal copper was localized entirely in the cortex and was significantly reduced by oral treatment with penicillamine, a copper chelating agent. When diabetic rats were fed purified diet (15-20 ppm Cu), the quantity of copper accumulated in the renal cortex increased from 2.3 to 8.7-fold higher than in control tissue from 1 to 4 weeks, respectively, after injection with streptozotocin. Copper levels in. both the soluble and particulate (165, 000g pellet) fractions of diabetic renal cortex were similarly increased at each time. Gel filtration Chromatographic analysis of the cytosol showed that all of the copper accumulated in the soluble fraction was associated with metallothionein. The distribution of excess copper in the particulate fraction was determined by differential centrifugation. The additional quantity of metal was localized in the crude nuclear fraction of renal cortex in the diabetic rat. Further analysis revealed that the lysosomal fraction from 3-weeek diabetic rats had a copper level 16-fold higher than in the controls. The possibility that accumulation of excessive levels of copper in the streptozotocin-diabetic kidney may contribute to the development of diabetic nephropathy is discussed.     

Effects of fructose loading in streptozotocin-diabetic and nondiabetic rats.

The present study compares the cardiovascular consequences of a 6-week fructose feeding in nondiabetic and streptozotocin-diabetic rats. Myocardial performance of these animals was determined using the isolated perfused working heart preparation. Systolic blood pressure, pulse rate, ventricular weight/body weight ratio, and plasma levels of glucose, insulin, triglycerides, and cholesterol were measured. In nondiabetic rats, fructose drinking caused significant increases in blood pressure, pulse rate, and plasma concentrations of insulin and triglycerides. Streptozotocin-diabetic animals exhibited significantly less body weight growth, slower pulse rate, higher plasma levels of cholesterol and triglycerides, ventricular enlargement, and functional impairment of the myocardium. The fructose-loaded diabetic rats had larger increases in plasma cholesterol and triglycerides than did control fructose-fed rats, but the fructose-induced increases in blood pressure and pulse rate were attenuated significantly. However, plasma levels of glucose and insulin and the degree of ventricular enlargement and myocardial dysfunction were not significantly different from those of control diabetic rats. These results show that fructose loading for 6 weeks can cause increases in blood pressure, pulse rate, and plasma lipids in both nondiabetic and diabetic rats. However, fructose ingestion does not significantly alter glycemic control or affect the development of myocardial dysfunction in streptozotocin-diabetic rats.     

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05–1.0 μg/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 μg/ml totally inhibiting cartilage formation. Concentrations of PGE₂ and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 μg/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE₂ levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE₂ and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.     

Treatment of streptozotocin-diabetic rats with metformin restores the ability of insulin to inhibit adenylate cyclase activity and demonstrates that insulin does not exert this action through the inhibitory guanine nucleotide regulatory protein Gi.

Insulin caused the inhibition of glucagon-stimulated adenylate cyclase activity in liver plasma membranes, but failed to inhibit this activity in liver membranes from rats made diabetic by treatment with either alloxan or streptozotocin. Treatment of streptozotocin-diabetic rats with insulin, to normalize their blood glucose concentrations, restored this action of insulin. Rats treated with the biguanide drug metformin exhibited a decreased content of the inhibitory guanine nucleotide regulatory protein Gi in liver plasma membranes assessed both structurally, by using a specific polyclonal antibody (AS7), and functionally. Treatment of normal rats with metformin did not alter insulin's ability to inhibit adenylate cyclase in liver plasma membranes; however, metformin treatment of streptozotocin-diabetic rats completely restored this inhibitory action of insulin. Liver plasma membranes from streptozotocin-diabetic animals which either had or had not been treated with metformin had contents of Gi which were less than 10% of those seen in control animals. We conclude that: (i) insulin does not inhibit adenylate cyclase activity through the inhibitory guanine nucleotide regulatory protein Gi; (ii) streptozotocin- and alloxan-induced diabetes elicit a selective insulin-resistant state; and (iii) metformin can exert a post-receptor effect, at the level of the liver plasma membrane, which restores the ability of insulin to inhibit adenylate cyclase.