Dietary modulation of circulating leptin levels: site-specific changes in fat deposition and ob mRNA expression.

In order to study the effects of diet on fat distribution, circulating leptin levels and ob mRNA expression, diets of different macronutrient composition were fed to lean mice and gold thioglucose-obese mice. A high-fat diet and 2 high-carbohydrate diets, one containing mostly high-glycaemic-index starch and the other containing low-glycaemic-index starch were fed ad libitum for 10 weeks and were compared to standard laboratory chow. Weight gain was attenuated by feeding low-glycaemic-index starch in all mice and by feeding a high-fat diet in lean mice. Reduced adiposity was seen in lean mice fed low-glycaemic-index starch, whereas increased adiposity was seen in both lean and obese mice fed on the high-fat diet. Circulating leptin levels, when corrected for adiposity, were decreased in all mice fed either the high-fat diet or the low-GI diet. In epididymal fat pads, decreased ob mRNA expression was seen after both high-fat and high-glycaemic-index starch feeding. These results show that diet macronutrient composition contributes to the variability of circulating leptin levels by the combined effects of diet on fat distribution and on site-specific changes in ob mRNA expression.

In vivo and in vitro suppression by leptin of glucose-stimulated insulin hypersecretion in high glucose-fed rats.

OBJECTIVES: Chronic feeding to rats of high glycaemic index (GI) diets results in the hypersecretion of insulin in response to an i.v. glucose load. The first aim of this study was to see if this exaggerated insulin response was accompanied by a hypersensitivity to glucose stimulation in isolated islets in vitro. The second aim was to see if the adipocyte factor, leptin, was able to alter insulin secretion in this model both in vivo and in vitro. DESIGN AND METHODS: Rats were fed for 6 weeks either a high GI diet in which the carbohydrate component was mostly glucose (GLUC diet) or a low GI diet containing mostly amylose (AMOSE diet). Rats then underwent an i.v. glucose tolerance test (ivGTT) (1g/kg) with and without a prior infusion of leptin (133 microg/kg perh). Islets were then isolated from these rats and basal and glucose-stimulated insulin secretion (GSIS) measured in both the absence and presence (100ng/ml) of leptin. RESULTS AND CONCLUSIONS: Peak insulin response during the ivGTT was 3-fold greater in GLUC rats (P<0.001). Leptin had no effect on AMOSE rat insulin response but lowered the GLUC rat response to AMOSE rat levels. In vitro, basal insulin secretion was 4-fold greater in GLUC rats (P<0.05). At 20mmol/l glucose, there was no further increase in insulin secretion in GLUC rats but a 2-fold increase in AMOSE rats. Leptin had no effect on basal insulin secretion or GSIS in AMOSE rats but reduced basal insulin secretion and GSIS in GLUC rats. These results show insulin hypersecretion in high GI-fed rats may be reduced by leptin.

Leptin has acute effects on glucose and lipid metabolism in both lean and gold thioglucose-obese mice.

Leptin is reported to have effects in peripheral tissues that are independent of its central effects on food intake and body weight. In this study, the acute effects of a single dose of recombinant mouse leptin on lipid and glucose metabolism in lean and gold thioglucose-injected obese mice were examined. Changes were measured 2 h after leptin injection. In lean mice, liver and white adipose tissue (WAT) lipogenesis was inhibited. The activity of the pyruvate dehydrogenase complex (PDHCa), the rate-determining step for glucose oxidation, was reduced in heart, liver, quadriceps muscle, and both brown and white adipose tissues. Muscle and liver glycogen and liver triglyceride (TG) content was unchanged, but muscle TG was decreased. In obese mice, liver and WAT lipogenesis was inhibited and PDHCa reduced in heart and quadriceps muscle. Muscle and liver glycogen was decreased but not TG. Serum insulin was reduced in obese but not lean mice. These results are consistent with a role for leptin in the maintenance of steady-state energy stores by decreasing lipid synthesis and increasing fat mobilization, with decreased glucose oxidation occurring as a result of increased fatty acid oxidation.

Chronic administration of BRL 26830A for 9 weeks improves insulin sensitivity but does not prevent weight gain in gold-thioglucose obese mice.

BRL 26830A, a beta adrenoceptor agonist, has been shown to have antiobesity and antidiabetic properties in rodents. The aim of this study was to study the effects of chronic BRL 26830A treatment (20 mg/kg/day for 9 weeks) on weight gain and the development of insulin resistance in gold-thioglucose-injected mice (GTG). BRL 26830A slowed the rate of weight gain in GTG such that mice weighed significantly less between 2 w and 7 w of treatment. However, at the time of sacrifice (9 w), there was no difference in body weight between treated and untreated GTG. The obesity-induced reduction in lipogenesis in brown adipose tissue (BAT) was increased 9 fold to greater than CON levels. However, weight and fatty acid (FA) content of BAT were reduced, suggesting increased lipid turnover and thermogenesis. Lipogenesis, FA content and fat pad weight were unchanged in white adipose tissue (WAT) and decreased in liver of GTG. Glucose tolerance was improved in both CON and GTG. Hyperglycemia, hyperinsulinemia and changes in cardiac and hepatic glucose oxidation as indicated by PDHC activity were normalized. Serum triglycerides and non-esterified fatty acids were reduced. Thus, chronic BRL 26830A treatment prevented the development of insulin resistance and attenuated weight gain, but did not prevent the development of obesity in this model.

Plasma insulin rise precedes rise in ob mRNA expression and plasma leptin in gold thioglucose-obese mice.

Circulating leptin levels are strongly related to the degree of adiposity, with hyperleptinemia being associated with hyperinsulinemia. In the gold thioglucose-injected mouse (GTG), hyperinsulinemia is an early abnormality in the development of insulin resistance and obesity. In this study, hyperinsulinemia occurred 1 wk post-GTG [GTG, 199 +/- 43; age-matched controls (CON), 53 +/- 5 microU/ml; P < 0.001], with leptin levels not rising until 2 wk post-GTG (CON, 3.2 +/- 0.3; GTG, 9.9 +/- 1.7 ng/ml; P < 0.001) in parallel with increases in the size of different fat pads and increased expression of ob mRNA. The ratio of serum leptin to fat pad weight was significantly higher in GTG mice 12 wk postinjection. Starvation-induced reductions in serum leptin (50%), glucose (50%), and insulin (74%) were greater than decreases in fat pad weight (18%). Adrenalectomy decreased both adiposity and serum leptin within 1 wk in both CON and GTG and altered the serum leptin level-to-fat pad weight ratio in CON. Thus hyperinsulinemia preceded increased ob expression and hyperleptinemia, which occurred in parallel with increasing adiposity, consistent with the role of leptin as an indicator of energy supplies. Changes in hormonal and nutritional status may modify this relationship.

Factors regulating the rat insulin-like growth factor-binding protein-1 response to octreotide.

Insulin-like growth factor-binding protein-1 (IGFBP-1) production is increased by somatostatin and its analogues. In order to determine the time course and identify possible mechanisms of this increase in vivo we administered octreotide to rats and determined IGFBP-1 concentrations by RIA. After 60 min of anaesthesia, the mean baseline IGFBP-1 concentrations were 166 (95% confidence interval 123 to 225) ng/ml and increased in saline-infused animals to 729 (488 to 1086) ng/ml after 180 min. IGFBP-1 was stimulated transiently in response to octreotide, with circulating IGFBP-1 concentrations peaking at 1605 (1220 to 2111) ng/ml at 105 min during a continuous infusion of octreotide (100 micrograms/kg per h). In conscious chronically cannulated rats, baseline IGFBP-1 concentrations were 22 (18 to 28) ng/ml, 8-fold less than in the anaesthetised state, and were stimulated in the short term after administration of an octreotide bolus (100 micrograms/kg s.c.) to reach 88 (62 to 126) ng/ml at 60 min. A similar response was seen after i.v. administration to conscious rats. Intravenous bolus of octreotide (100 micrograms/kg) in rats anaesthetised for 3 h resulted in an increase in IGFBP-1 to peak at 1556 (1268 to 1910) ng/ml at 60 min. The IGFBP-1 response to octreotide was diminished in high-fat fed hyperinsulinaemic rats. The pattern of disappearance of iodinated IGFBP-1 from the circulation was not influenced by octreotide. The changes in GH, insulin and glucose concentrations alone did not sufficiently account for the patterns of response observed. We conclude that, in rats, octreotide stimulates IGFBP-1 acutely and this response is potentiated by factors related to anaesthesia.

The effects of the inhibition of fatty acid oxidation on pyruvate dehydrogenase complex activity in tissues of lean and obese mice.

OBJECTIVE: To investigate the effects of an acute dose of the fatty acid oxidation inhibitor, Etomoxir, on the activity of the pyruvate dehydrogenase complex (PDHC) in different tissues in lean and obese mice. DESIGN: An acute dose of Etomoxir was given to mice in which obesity had been induced by an injection of gold thioglucose and to age-matched controls. The effects of time, dose and nutritional state were studied. MEASUREMENTS: PDHC activity in heart, quadricaps muscle, liver and white adipose tissue, glycogen content of liver and quadricaps muscle, serum glucose and insulin were measured in fed and fasted animals and in fasted animals after the ingestion of a glucose load. RESULTS: Etomoxir caused an increase in the activity of the active form of the PDHC (PDHCa) in the heart, liver and WAT of fed lean mice and in the heart and liver of fed obese mice. In fasted mice, increased PDHCa was seen in the heart of lean mice and in the liver of obese mice. Etomoxir increased the PDHC response to an oral glucose challenge in the liver and WAT of lean mice and in the liver of obese mice. Etomoxir had no effect on PDHCa in quadricaps muscle. Serum glucose levels were decreased in fasted mice with no change in the fed mice. Etomoxir decreased liver glycogen content in both fed and fasted animals and inhibited the accumulation of muscle glycogen following the glucose load. CONCLUSIONS: Acute inhibition of fatty acid oxidation results in tissue specific increases in PDHCa. Improvements in glucose oxidation in tissues other than skeletal muscle may contribute to the improved glucose tolerance seen following acute Etomoxir administration.

Hepatic gluconeogenesis and the activity of PDH in individual tissues of GTG-obese mice following adrenalectomy.

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 +/- 0.7 g; GTG: 44.1 +/- 0.4; p < 0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 +/- 1.6 mmol/L; GTG: 29.4 +/- 1.9 mmol/L; p < 0.05) and serum insulin levels (GTG ADX: 76 +/- 10 microU/mL; GTG: 470 +/- 63 microU/mL; p < 0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 +/- 10 nmol glucose/10(6) cells; GTG: 403 +/- 65 nmol glucose/10(6) cells; p < 0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 +/- 27 mumol/liver; GTG: 614 +/- 60 mumol/liver; p < 0.05) and fatty acid content (GTG ADX: 101 +/- 9 mg fatty acid/g liver; GTG: 404 +/- 40 mg fatty acid/g liver; p < 0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.

High-fat feeding alters the response of rat PDH complex to acute changes in glucose and insulin.

The activity of the pyruvate dehydrogenase complex (PDHC) was studied in tissues of controls and insulin-resistant fat-fed rats (FFR) both in the fed state and in overnight fasted animals after the induction of short-term changes in plasma insulin by an intravenous glucose load. Significant responses by the PDHC to the glucose challenge were seen in heart and white adipose tissue (WAT) in controls with smaller changes in brown adipose tissue (BAT) and quadriceps muscle (QM) and no change in liver. Reduced PDHC responses and lower fed values were seen in heart and BAT of FFR. The response in WAT of FFR was prolonged with no change in the PDHC response in QM. Plasma nonesterified fatty acids (NEFA) were decreased in response to the glucose load with no differences between controls and FFR. Tissue triglyceride levels were higher in liver and QM but not heart of FFR. These results show differential tissue PDHC responses to short-term changes in plasma insulin. The decreased PDHC activity in some tissues of the fat-fed animals despite the lack of change in plasma NEFA, together with the triglyceride accumulation seen in some tissues but not others, suggests that local intracellular fatty acid metabolism is important in the regulation of intracellular glucose oxidation.

Tissue differences in the response of the pyruvate dehydrogenase complex to a glucose load during the development of obesity in gold-thioglucose-obese mice.

The activity of pyruvate dehydrogenase (PDHC), a key enzyme complex in the oxidative disposal of glucose, was measured after an oral glucose load in the heart, liver, quadriceps muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) of gold-thioglucose (GTG)-obese mice at different stages during the development of obesity and in age-matched controls. Significant responses to the glucose load were seen 30 min post-gavage in heart, WAT and BAT of control mice but no change was observed in quadriceps muscle. The increase in activity of the active form of PDHC (PDHCa) in response to glucose in heart was reduced 2 weeks after the induction of GTG-obesity with no response in 5 or 10 week obese mice. A 2-3-fold increase in the PDHCa response in both WAT and BAT of 2 week obese mice was absent in 5 and 10 week obese animals. Basal PDHCa activity in quadriceps muscle was increased in 2 week obese mice but subsequently returned to control levels as obesity progressed. The glucose load produced no change in the activity of PDHCa in quadriceps muscle of obese mice. These results demonstrate that changes in the capacity for oxidative glucose disposal in different tissues, as indicated by changes in PDHCa activity, may contribute to glucose-intolerance and insulin-resistance in GTG-obese mice and that the response of the PDHC to insulin during the development of obesity varies in different tissues.

Complex formation by human insulin-like growth factor-binding protein-3 and human acid-labile subunit in growth hormone-deficient rats.

Insulin-like growth factor-binding protein-3 (IGFBP-3), after first associating with IGF-I or IGF-II, is able to associate with the acid-labile subunit (ALS) and form a 140-kDa complex. To investigate the factors regulating ternary complex formation in vivo, human (h) IGFBP-3, hIGF-I, and hALS were administered in various combinations to GH-deficient (dw/dw) rats. hIGFBP-3 had a complex pattern of disappearance from the rat circulation, with an initial phase lasting minutes and a prolonged phase(s) lasting hours. If coinjected with hIGF-I, significantly more hIGFBP-3 was retained over 2 h. The molecular distribution of hIGFBP-3 was determined after size-separation chromatography. After an iv bolus of hIGFBP-3, 36.1 +/- 5.0% was in the 140-kilodalton complex at 5 min; this increased to 55.1 +/- 7.1% if hIGF-I was coinjected (P < 0.05). The 140-kDa complex disappeared slowly over hours, whereas 50- and 30-kDa forms of hIGFBP-3 cleared rapidly, with half-lives of minutes. To determine the importance of ALS in regulating the molecular distribution of hIGFBP-3, hALS was coinjected. Immunoreactive hALS disappeared slowly from the circulation and was shown to retain functional activity after 2 h in vivo. Coadministration of hALS did not influence the pattern of ternary complex formation, consistent with the presence of excess endogenous rat ALS. We conclude that ALS circulates in excess even in GH deficiency, is retained in the circulation for hours, and determines the stability of the 140-kDa complex, whereas IGF-I is a limiting factor in ternary complex formation by hIGFBP-3.