Diet- and diabetes-induced changes of ob gene expression in rat adipose tissue.

ob gene regulation is as yet unknown. We first examined whether the ob gene is under physiological control by the nutritional state. Fasting produced a sharp (95%) decrease of ob mRNA in epididymal and inguinal fat pads from 24 h onward. Refeeding rapidly (3-6 h) re-induced ob gene expression and corrected it within 24 h. Similar changes in fatty acid synthase (FAS) and GLUT4 mRNAs were observed, whereas phosphoenolpyruvate carboxykinase (PEPCK) mRNA showed an opposite evolution. We next examined the potential role of insulin. In adipose tissue of streptozotocin-diabetic rats, ob mRNA levels were decreased by 80%. Insulin treatment (4 days) only marginally increased ob mRNA, but restored euglycemia and overcorrected FAS, GLUT4 and PEPCK expression. In conclusion, we provide evidence for a physiological regulation of ob gene by variations in the nutritional state. We also show that ob expression is impaired in streptozotocin-diabetic rats and only slightly restored by insulin treatment, which suggests that ob gene is not or only minimally regulated by the hormone.

Effects of vanadium complexes with organic ligands on glucose metabolism: a comparison study in diabetic rats.

1. Vanadium compounds can mimic actions of insulin through alternative signalling pathways. The effects of three organic vanadium compounds were studied in non-ketotic, streptozotocin-diabetic rats: vanadyl acetylacetonate (VAc), vanadyl 3-ethylacetylacetonate (VEt), and bis(maltolato)oxovanadium (VM). A simple inorganic vanadium salt, vanadyl sulphate (VS) was also studied. 2. Oral administration of the three organic vanadium compounds (125 mg vanadium element 1(-1) in drinking fluids) for up to 3 months induced a faster and larger fall in glycemia (VAc being the most potent) than VS. Glucosuria and tolerance to a glucose load were improved accordingly. 3. Activities and mRNA levels of key glycolytic enzymes (glucokinase and L-type pyruvate kinase) which are suppressed in the diabetic liver, were restored by vanadium treatment. The organic forms showed greater efficacy than VS, especially VAc. 4. VAc rats exhibited the highest levels of plasma or tissue vanadium, most likely due to a greater intestinal absorption. However, VAc retained its potency when given as a single i.p. injection to diabetic rats. Moreover, there was no relationship between plasma or tissue vanadium levels and any parameters of glucose homeostasis and hepatic glucose metabolism. Thus, these data suggest that differences in potency between compounds are due to differences in their insulin-like properties. 5. There was no marked toxicity observed on hepatic or renal function. However, diarrhoea occurred in 50% of rats chronically treated with VS, but not in those receiving the organic compounds. 6. In conclusion, organic vanadium compounds, in particular VAc, correct the hyperglycemia and impaired hepatic glycolysis of diabetic rats more safely and potently than VS. This is not simply due to improved intestinal absorption, indicating more potent insulin-like properties.

Improvement of mild hypoinsulinaemic diabetes in the rat by low non-toxic doses of vanadate.

To exert their anti-diabetic effects in animals with overt alterations of glucose homeostasis, vanadium salts must be administered in high doses, which also cause decreases in food intake and body weight gain. In this study, we evaluated the effect of low doses of vanadate in rats made mildly diabetic (fed plasma glucose levels approximately 11 mmol/l) and moderately hypoinsulinaemic by the injection of streptozotocin 4 days after birth. Vanadate was added to food and drinking water, at concentrations that led to the consumption of about 1 mg vanadium element per day (approximately 2.65 mg vanadium/kg per day in adult rats), i.e. three to fivefold less than in previous studies. The treatment was started at weaning and lasted 22 weeks (V rats), or was administered for 9 weeks only from the age of 3 months (C-V rats). Food intake and body weight gain were not affected in V rats and decreased by no more than 10% in C-V rats. In V rats, fasted and fed plasma glucose levels were decreased by about 0.5 and 2-3 mmol/l, respectively. The rises in glycaemia after three oral glucose tolerance tests were also clearly attenuated. These effects were not accompanied by any changes in plasma insulin levels. Pancreatic insulin reserves (decreased by two-thirds as compared with normal rats) were not affected by the treatment. A decrease in plasma glucose levels was also noted in C-V rats, and this improvement disappeared upon cessation of the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of vanadate on insulin counter-regulatory hormones in obese fa/fa rats.

Vanadate has been shown to improve glucose homoeostasis in mildly glucose-intolerant and severely insulin-resistant fa/fa rats. The present study examined whether changes in insulin counter-regulatory hormones contribute to this beneficial effect of vanadate. Since oral administration of Na3VO4 caused a decrease in food intake and stopped the increase in body weight, vanadate-treated fa/fa rats were compared with both controls with food available ad libitum and pair-fed rats. Slightly lower plasma glucose levels were maintained in conjunction with markedly lower plasma insulin levels in vanadate-treated rats, and this effect was not simply due to the smaller body weight of the animals. Compared with control rats, treatment with vanadate affected neither basal plasma glucagon levels nor the increase in glucagon levels observed after insulin-induced hypoglycaemia or after i.v. injection of arginine. Compared with pair-fed rats, treatment with vanadate prevented the fall in basal plasma glucagon and its exaggerated rise in response to insulin that mere food restriction produced. Plasma corticosterone levels were high in fa/fa rats. Vanadate and pair-feeding similarly decreased basal plasma levels of corticosterone as well as nocturnal corticosteronuria. Thus the attenuation of the hypercorticism of fa/fa rats results from the reduction in body weight gain rather than from a specific action of vanadate. Vanadate did not influence urinary excretion of noradrenaline, an index of neural sympathetic activity, but prevented the increase in adrenaline excretion, an index of adrenal medulla activity, that was produced by food restriction in pair-fed rats. In conclusion, vanadate administration has no or little specific effects on three major insulin counter-regulatory hormones. This reinforces the suggestion that the beneficial effects of vanadate on glucose homoeostasis in fa/fa rats are mainly due to a correction of insulin resistance in peripheral tissues.

Improvement of glucose homeostasis and hepatic insulin resistance in ob/ob mice given oral molybdate.

Molybdate (Mo) exerts insulinomimetic effects in vitro. In this study, we evaluated whether Mo can improve glucose homeostasis in genetically obese, insulin-resistant ob/ob mice. Oral administration of Mo (174 mg/kg molybdenum element) for 7 weeks did not affect body weight, but decreased the hyperglycaemia (approximately 20 mM) of obese mice to the levels of lean (L) (+/+) mice, and reduced the hyperinsulinaemia to one-sixth of pretreatment levels. Tolerance to oral glucose was improved: total glucose area was 30% lower in Mo-treated mice than in untreated ob/ob mice (O), while the total insulin area was halved. Hepatic glucokinase (GK) mRNA level and activity were unchanged in O mice compared with L mice, but the mRNA level and activity of L-type pyruvate kinase (L-PK) were increased in O mice by 3.5- and 1.7-fold respectively. Mo treatment increased GK mRNA levels and activity (by approximately 2.2-fold and 61% compared with O values), and had no, or only a mild, effect on the already increased L-PK variables. mRNA levels and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) were augmented in O liver (sixfold and by 57% respectively), and these were reduced by Mo treatment. Insulin binding to partially purified receptors from liver was reduced in O mice and restored by Mo treatment. Despite this correction, overall receptor tyrosine kinase activity was not improved in Mo mice. Moreover, the overexpression (by two- to fourfold) of the cytokine tumour necrosis factor alpha (TNF alpha) in white adipose tissue, which may have a determinant role in the insulin resistance of the O mice, was unaffected by Mo. Likewise, overexpression of the ob gene in white adipose tissue was unchanged by Mo. In conclusion, Mo markedly improved glucose homeostasis in the ob/ob mice by an insulin-like action which appeared to be exerted distal to the insulin receptor tyrosine kinase step. The blood glucose-lowering effect of Mo was unrelated to over-expression of the TNF alpha and ob genes in O mice, but resulted at least in part from attenuation of liver insulin resistance by the reversal of pre-translational regulatory defects in these mice.

Comparison of the effects of various vanadium salts on glucose homeostasis in streptozotocin-diabetic rats.

Oral administration of vanadium salts to severely diabetic rats lead to a spectacular decrease of plasma glucose levels in spite of the insulin deficiency of the animals. The insulin-like properties of vanadium have been attributed to the cationic form, vanadyl, into which the anionic form, vanadate, is reduced within cells. This has led to the suggestion that vanadyl is the form of choice for the treatment. In this study, rats made insulin-deficient and diabetic with streptozotocin were treated with three salts of vanadium: sodium orthovanadate, sodium metavanadate and vanadylsulfate. The salts were added to the drinking water, in concentrations that led to ingestion of the same amount of vanadium element by the three groups of rats (approximately 8 mg/kg per day). The initial, transient, loss of weight that affected the treated rats was slightly smaller in the vanadyl-treated group than in the vanadate-treated groups. However, during steady-state treatment, the three groups exhibited a similar food intake (lower than in controls) and growth rate (higher than in controls). The decreases in plasma glucose levels, in urinary volume and in glucosuria, and the improvement of the tolerance to an oral glucose load were similar regardless of the type of vanadium salt. Withdrawal of the treatment after 14 weeks was followed by a rapid increase in plasma glucose levels which, however, remained clearly lower than in controls for at least 4 weeks, whereas plasma insulin levels increased only transiently. A smaller glucosuria and a slightly better tolerance to oral glucose than in controls were still observed in the previously treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral vanadate decreases muscle insulin resistance in obese fa/fa rats.

Oral vanadate has been reported as improving glucose homeostasis in genetically obese and hyperinsulinaemic fa/fa rats. It has also been shown that these beneficial effects could not be ascribed to the decrease in body weight induced by the treatment, or to changes in insulin counterregulatory hormones. The present study examined therefore whether the effects of vanadate could be attributed to a direct correction of the severe insulin resistance of these animals. fa/fa Rats chronically treated with vanadate were compared to both control rats fed ad libitum and pair-fed rats. The three groups were studied in the basal state and during euglycaemic hyperinsulinaemic clamps. Slightly lower plasma glucose levels were always maintained in vanadate-treated rats in conjunction with markedly lower plasma insulin levels either during basal or clamp studies. During the clamp, the glucose infusion rate required to maintain glycaemia at basal values was consistently higher in vanadate-treated rats than in the other two groups. Experiments using [6-3H]glucose as tracer showed that this was not due to a greater inhibition of hepatic glucose production by insulin, but corresponded to a larger increment in peripheral glucose disposal. The stimulation of overall glucose metabolic clearance induced by insulin was 129% and 41% higher in vanadate-treated than in control and pair-fed rats respectively. Similar experiments with 2-deoxy-[1-3H]glucose as tracer showed that the larger increase in insulin-mediated glucose clearance occurred in various types of muscle. The action of insulin was particularly impressive on the cardiac muscle of vanadate-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific correction of lipogenic enzyme gene expression in diabetic rats given vanadate.

Vanadium is a potent insulinomimetic agent. In vivo, its blood glucose lowering action in insulin-deficient diabetic rats is associated with corrected expression of genes involved in hepatic glucose metabolism. In this study, we investigated whether vanadate treatment also reverses the impaired expression of genes coding for key enzymes of lipogenesis in diabetic liver and white adipose tissue. Oral administration of vanadate to streptozotocin-rats caused a 55% fall in plasma glucose levels after feeding without modifying low insulinaemia. It also partially corrected the low thyroid hormone concentrations. In untreated diabetic animals, hepatic mRNA levels of acetyl-CoA carboxylase and fatty acid synthase were reduced by more than 80 and 90%, respectively, in close correlation with changes in enzyme activities. Three weeks of vanadate treatment totally restored acetyl-CoA carboxylase mRNA and partially restored fatty acid synthase mRNA (71% of control levels). The activities of both lipogenic enzymes were increased 3.5 to 4-fold, to reach 45 to 65% of control values. By contrast, in white adipose tissue, vanadate modified neither expression nor activity of both lipogenic enzymes, which remained blunted (< 10% of control levels). In conclusion, vanadate treatment partially restores the activities of two key lipogenic enzymes in liver, but not in white adipose tissue, of diabetic rats. This correction results from a reversal of impaired pre-translational regulatory mechanisms possibly mediated by an improvement of thyroid function and a selective restoration of liver glycolytic flux.

Insulin and insulin-like growth factor 1 antagonize the stimulation of ob gene expression by dexamethasone in cultured rat adipose tissue.

The ob gene, specifically expressed in fat cells, encodes leptin, a hormone that induces satiety and increases energy expenditure. In this study, we investigated the interactions between glucocorticoids and insulin on ob gene expression in cultured explants of rat adipose tissue. Only low levels of ob mRNA were detected when adipose tissue from fasted rats was cultured for 12-24 h in minimal essential medium. However, the addition of dexamethasone to the medium increased ob gene expression in a concentration-dependent manner (EC50 10 nM). With 1 microM dexamethasone, ob mRNA levels were similar to those in fresh fat pads from fed rats, reaching a maximum after 12 h. The effect of dexamethasone was blocked by actinomycin D, which indicates an action on transcription. This effect was increased when a minimum amount of fuel (glucose or a mixture of lactate and pyruvate) was supplied in the medium. Unlike dexamethasone, insulin, even when combined with high glucose concentrations, did not induce ob expression, although it strongly increased the accumulation of mRNA species for fatty acid synthase (FAS), the insulin-sensitive glucose transporter GLUT4 and the gamma isoform of peroxisome proliferator-activated receptor (PPARgamma). Unexpectedly, insulin dose-dependently inhibited dexamethasone-induced ob mRNA accumulation. This effect was observed at low concentrations of insulin (IC50 1 nM) and was delayed in onset, beginning after 6-9 h of culture. It was mimicked by insulin-like growth factor 1 (IGF-1) (100 nM). The inhibition by insulin was only detectable when fuels were present and/or when a critical level of ob expression was reached. As this inhibitory effect was reversed by cycloheximide, this suggests that it required ongoing protein synthesis. In conclusion, unlike dexamethasone, insulin had no direct stimulatory effect on ob gene expression. On the other hand, insulin (and IGF-1) even inhibited the dexamethasone-induced accumulation of ob mRNA. The underlying mechanism involved ongoing synthesis of an inhibitory protein by insulin, which is in keeping with its delayed effect. Moreover, the expression of genes for FAS, GLUT4 and PPARgamma may be inversely related to that of ob.

Improvement of glucose and lipid metabolism in diabetic rats treated with molybdate.

Molybdenum mimics certain insulin actions in vitro. We have investigated the effects of oral administration of Na2MoO4 (Mo) for 8 wk on carbohydrate and lipid metabolism in streptozotocin-diabetic rats. Mo decreased hyperglycemia and glucosuria by 75% and corrected the elevation of plasma nonesterified fatty acids. Tolerance to glucose loads was improved, and glycogen stores were replenished. These effects were not due to a rise of insulinemia. In liver, Mo restored the blunted mRNA and activity of glucokinase and pyruvate kinase and decreased to normal phosphoenolpyruvate carboxykinase values. Finally, Mo totally reversed the low expression and activity of acetyl-CoA carboxylase and fatty acid synthase in liver, but not in white adipose tissue. In conclusion, Mo exerts a marked blood glucose-lowering effect in diabetic rats by an insulin-like action. This effect results in part from a restoration of hepatic glucose metabolism and is associated with a tissue-specific correction of lipogenic enzyme gene expression, both processes being essentially mediated by reversal of impaired pretranslational regulatory mechanisms. These observations raise new therapeutic perspectives in diabetes, particularly in the insulin-resistant condition.

Developmental and nutritional changes of ob and PPAR gamma 2 gene expression in rat white adipose tissue.

The ob gene encodes leptin, a hormone which induces satiety and increases energy expenditure. The peroxisome proliferator-activated receptor gamma 2 isoform (PPAR gamma 2) gene encodes a transcription factor which controls adipocyte differentiation and expression of fat-specific genes. We have studied the regulation of these two genes in white adipose tissue (WAT) during the suckling-weaning transition. Suckling rats ingest a high-fat diet (milk). Fat-pad weight barely varied during the last week of suckling. ob mRNA levels, which were very low in 15-day-old rats, rose approximately 6-fold until weaning at 21 days. When the rats were weaned on to a standard (high-carbohydrate) laboratory chow, epididymal WAT enlarged approximately 7-fold, and ob mRNA kept increasing progressively and doubled between 21 and 30 days. This evolution contrasted with that of fatty acid synthase (FAS) mRNA, which increased sharply, but only after weaning. To distinguish between the influence of developmental and nutritional factors on ob expression, a group of rats was weaned on to a high-fat diet. This prevented the rise in glycaemia and insulinaemia and the decrease in plasma non-esterified fatty acids which otherwise occurred at weaning. This also resulted in a slight (10-15%) decrease in food intake and body weight gain. Under this high-fat diet, the rise of ob mRNA in WAT was augmented (3.7-fold in 30- versus 21-day-old pups), whereas the normal rise in FAS mRNA levels was attenuated. Fat-pad weights and adipocyte cell size and number were roughly similar in high-carbohydrate- and high-fat-weaned pups. mRNA levels of PPAR gamma 2, like those of ob, were low in the WAT of 15-day-old suckling pups, doubled at 21 days, and reached a maximum as soon as 23 days. This evolution further differed from that of ob mRNA in not being influenced by diet composition. In conclusion, ob expression markedly increases during the suckling-weaning transition, and this effect is accentuated by a high-fat diet. Qualitative nutritional changes in ob mRNA were correlated with neither acute changes in adipose-tissue mass, nor cell size/number, nor variations in insulinaemia. PPAR gamma 2 also increased during suckling, but rapidly reached a plateau after weaning and no longer changed thereafter. Unlike ob, PPAR gamma 2 was not influenced by the diet composition.