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Despite numerous studies, the in vivo regulation of plasma leptin levels in response to nutritional factors continues to remain unclear. We investigated temporal and dose-response relationships of plasma leptin in response to physiological changes in insulin/glucose. After an overnight fast of 10 h, lean, healthy subjects were investigated for an additional 16 h of either extended fasting or one of three levels of glycemia/insulinemia induced by stepwise increasing iv glucose infusions. During extended fasting, plasma leptin values declined steadily and significantly. Plasma leptin levels remained constant at glucose concentrations between 5.8-6.5 mmol/liter, which maintained normoinsulinemia at 41.5-45.4 pmol/liter and FFA at 106-123 mg/liter, but leptin concentrations were increased at higher rates of glucose infusion (with plasma glucose rising to 8.7 mmol/liter). Concentrations of serum leptin were inversely related to FFA levels during extended fasting and at all levels of glycemia. Our data indicate that in lean healthy subjects, physiological changes in glycemia and insulinemia significantly alter plasma FFA and leptin concentrations. The increases in leptin concentrations demonstrate dose-dependent relationships that appear to relate to changes in FFA levels as well as to changes in glycemia/insulinemia.

Regulation of leptin production by the hormonal and metabolic milieu is poorly understood. Because abdominal obesity is commonly associated with elevated plasma free fatty acid (FFA) flux, we examined the effects of augmenting FFA on plasma leptin levels in women who were lean and of suppressing FFA in women with abdominal obesity. In study 1, nine subjects who were lean, after a 12-hour overnight fast, received either intravenous saline or Intralipid plus heparin to increase the plasma FFA concentration to approximately 1000 mumol/ L. After 3 hours of additional fasting, subjects underwent 3-hour hyperglycemic clamps. In study 2, seven subjects with abdominal obesity were evaluated by a similar protocol, but lipolysis and plasma FFA flux were instead maximally suppressed by acipimox. In the individuals who were lean, leptin levels were unchanged during clamping. Increasing plasma FFA reduced plasma leptin from 7.66 +/- 0.66 to 7.05 +/- 0.66 (p = 0.03), but 3 hours of hyperglycemia plus hyperinsulinemia had no additional effect on leptin levels (7.15 +/- 0.71). Basal leptin levels, 4-fold higher in the subjects with obesity, were reduced from 34.6 +/- 2.4 micrograms/L to 32.3 +/- 1.1 micrograms/L (p = 0.004) during the clamp period. When plasma FFA flux was suppressed, however, plasma leptin levels after clamped hyperglycemia/hyperinsulinemia were increased to 38.9 +/- 1.2 micrograms/L (p = 0.014 vs. time 0 and 0.001 vs. saline protocol). Changes in leptin concentrations are not correlated with changes in FFA. These results suggest that plasma FFA concentration does not regulate plasma leptin levels in basal, extended fasting, or hyperglycemic/hyperinsulinemic states.

The mechanism of hyperinsulinemia that accompanies insulin resistance in some abdominally obese and diabetic individuals is poorly understood. Both increased secretion of insulin and decreased clearance have been demonstrated. The present study was undertaken to examine the role of free fatty acids (FFAs) and glucose in regulating splanchnic insulin dynamics in vivo. Plasma FFA levels were raised approximately twofold via an intralipid/heparin infusion in eight lean women. Insulin dynamics were assessed using the individual's C-peptide kinetic coefficients. Studies were performed in the basal state and during two levels of glycemia, 7 and 11 mmol/l. Studies were repeated using saline, and thus each subject served as her own control. Under basal conditions, raising FFA flux resulted in a modest increase in plasma insulin concentration (PIC) secondary to an increase in insulin secretion rate (ISR); however, endogenous insulin clearance (EIC) was not influenced. During the 7 mmol/l hyperglycemic clamp, maintaining a high FFA flux resulted in a 30% increase in PIC above the effect produced by glucose alone. This represents the cumulative effects of stimulation of ISR and inhibition of EIC. Clamping plasma glucose at 11 mmol/l while maintaining a high FFA flux increased PIC twofold above that produced by glucose alone. This increase in PIC was mainly due to a significant reduction in EIC without an accompanying increase in ISR (392 +/- 159 and 787 +/- 187 ml/min with and without intralipid infusion, respectively). Analysis of variance indicated that the suppressive effect of FFA on EIC was independent of the effect of glucose. The effect of the two substrates seems to be additive.

Resistance to the capacity of insulin to suppress lipolysis may be an important link in the association between abdominal obesity and hypertension. Furthermore, a more active renin-angiotensin system in adipose tissue may contribute to insulin-resistant lipolysis in abdominally obese hypertensive subjects. We determined nonesterified fatty acid concentrations and turnover as well as lipid oxidation under basal conditions and during steady-state euglycemia with two levels of insulinemia (72 and 287 pmol/L) in lean normotensive, abdominally obese normotensive, and abdominally obese hypertensive subjects. To assess the role of the renin-angiotensin system in determining non-esterified fatty acid turnover, we repeated studies in the abdominally obese hypertensive subjects after double-blind random assignment to placebo or enalapril for 1 month each. The main findings were the following: (1) Nonesterified fatty acid flux was significantly higher in abdominally obese hypertensive subjects at both levels of insulinemia than in either abdominally obese normotensive or lean normotensive subjects and correlated significantly with both mean blood pressure and total systemic resistance during the higher level of insulinemia. (2) Enalapril significantly improved insulin-resistant lipolysis in the abdominally obese hypertensive subjects. The improvement in insulin suppressibility of nonesterified fatty acid flux at the high hormonal concentrations correlated positively with the magnitude of reduction in blood pressure. (3) Basal lipid oxidation and suppression in response to insulin were similarly impaired in both obese groups. Resistance to the antilipolytic actions of insulin is thus a characteristic feature in abdominally obese hypertensive subjects and may be linked to the elevated blood pressure in these individuals. A more active renin-angiotensin system may partly explain the insulin-resistant lipolysis in this form of hypertension.

Although resistance to insulin-mediated glucose disposal has emerged as a link between abdominal obesity and hypertension, abnormalities of nonesterified fatty acid metabolism may play a greater role. Analyses were performed on existing data from 17 abdominally obese subjects (11 hypertensive, 6 normotensive) to determine whether fatty acid concentration and turnover were related to blood pressure independently of hyperinsulinemia and resistance to insulin-mediated glucose disposal. Glucose utilization, fatty acid concentration, and fatty acid turnover were obtained fasting and during euglycemic hyperinsulinemia at 10 and 40 mU/m/min. Analyses were also performed on another group of 30 subjects with a wide range of risk factors who had blood pressure data as well as glucose and fatty acid measurements during an insulin tolerance test. Fatty acid concentration and turnover were markedly more resistant to suppression by insulin in obese hypertensive than in lean or obese normotensive individuals. In the 17 obese subjects, blood pressure measured at screening, in the laboratory, and over a period of 24 hours correlated significantly with fatty acid concentration and turnover but not with glucose disposal measured during the hyperinsulinemic clamp. These correlations remained significant after fasting insulin, the insulin area under the curve during an oral glucose tolerance test, and glucose disposal during the clamp were controlled for. In the second group of subjects, plasma fatty acids 15 minutes after intravenous insulin also correlated with blood pressure. These correlations remained significant after insulin and an index of sensitivity to insulin-mediated glucose disposal were statistically controlled for. The data indicate that blood pressure is related to the effects of insulin on fatty acid metabolism. The findings raise the possibility that resistance of hormone-sensitive lipase to insulin participates in elevating the blood pressure of abdominally obese hypertensive subjects by increasing fatty acid concentration and turnover.

In SHHF/Mcc-FAcp rats (formerly SHR/Mcc-cp), obesity and male gender synergistically modulate hyperinsulinemia, insulin resistance and predisposition to diabetes. Our previous studies showed gender and obesity modulate hepatic cell surface insulin binding and insulin clearance additively. Hepatic insulin receptors (IR) bind insulin as a first step in insulin clearance through internalization and degradation. We hypothesize that the synergistic effects of obesity and gender on hepatic insulin binding and clearance result from interaction of these two factors on hepatic IR expression. To address IR expression in SHHF/Mcc-FAcp rats, we quantitated IR protein levels in detergent-solubilized liver homogenates by Western blotting and IR mRNA levels by a solution hybridization/RNase protection assay. Obesity reduced total hepatic IR content in males and females, 50% and 68% respectively. Male gender reduced IR protein content 24% in lean, but had no effect on IR protein content in obese rats. Neither gender nor obesity affected hepatic IR mRNA content. Thus, obesity appears to affect hepatic IR protein content and cell surface binding through post-transcriptional mechanisms; similarly, male gender in lean rats reduces IR protein levels and cell surface binding through mechanisms not involving changes in mRNA levels. In obese rats, the synergistic effects of male gender appears to involve changes in IR trafficking and consequently cell surface insulin binding and processing.

Polycystic ovary (PCO) syndrome is strongly associated with insulin resistance and the accompanying adverse metabolic profile. To distinguish the mechanisms of this association, we determined the interactions of PCO with obesity and the influence of ameliorating direct androgenic actions via short-term treatment with the antiandrogen flutamide. Insulin sensitivity was determined by the hyperinsulinemic euglycemic clamp in groups of lean and obese PCO women and weight-matched controls. Compared with control values, insulin-mediated glucose utilization in PCO women was significantly lower in lean (1.96 +/- 0.17 v 1.24 +/- 0.10, P < .01) and obese (1.23 +/- 0.18 v 1.03 +/- 0.09 mmol/m2/min, P < .01) subjects. ANOVA indicated that the effects of obesity and androgenicity are independent and additive. In both lean and obese PCO women, treatment with flutamide for 1 or 3 months markedly improved the clinical and biochemical androgenic features, but did not significantly influence the overall insulin sensitivity. A large disparity between individuals in the response to treatment correlated significantly with a simultaneous reduction in plasma levels of dehydroepiandrosterone sulfate (DHEA-S). Thus in women, PCO and obesity exert synergistic effects on insulin resistance. The decreased insulin sensitivity is mediated via indirect androgenic actions or nonandrogenic mechanisms. In some individuals, a direct effect of androgens might have been masked by a decrease in DHEA-S levels.

We determined whether the palmitate effects on hepatocyte insulin receptor binding and post-receptor trafficking were mediated by accelerated mitochondrial beta-oxidation or accumulation of intracellular fatty acyl-CoA derivatives and possibly protein acylation. Preincubation of hepatocytes with moderate concentrations of palmitate (0.5 mM) resulted in a 23% decline in cell-surface binding and proportional decreases in receptor-mediated insulin internalization and degradation. Brief pretreatment of hepatocytes with the carnitine palmityltransferase-I inhibitor, methyl palmoxirate (MP), prevented 70% of the palmitate effects. At higher palmitate concentrations (2.0 mM), cell-surface binding was reduced by 34%, whereas internalization of the receptor complex was reduced by 78%. These effects were only partially prevented by MP pretreatment. Receptor-mediated insulin degradation increased by 34% and was uninfluenced by MP pretreatment. Octanoate, which is rapidly shunted into mitochondrial oxidation, produced a dose-dependent reduction in insulin binding, with proportional decreases in internalization and degradation. Similarly preincubation with 2.0 mM oleate, which, unlike palmitate, is not known to produce protein acylation, resulted in proportional decreases in insulin receptor binding and receptor-mediated internalization and degradation. High concentrations of octanoate or oleate (2.0 mM) did not reproduce the additive post-receptor effects of palmitate. We conclude that the receptor and post-receptor effects of moderate palmitate concentrations are closely linked to accelerated fatty acid oxidation. The post-receptor effects observed at higher concentrations involve other mechanisms, possibly relating to intracellular levels of palmityl-CoA derivatives.

The effects of free fatty acids (FFA) on insulin receptor binding and processing (internalization, degradation, dissociation, and release) were examined in hepatocytes isolated from 12-week-old female rats. Animals were fasted for 24 h to deplete liver glycogen and lipid content. Cells were preincubated for 30 min or 3 h at 37 degrees C in media containing 10 mM lactate, 1 mM pyruvate, and 3.5 percent albumin with increasing concentrations of palmitate (0.00, 0.05, 0.2, 0.5, 1.0 and 2.0 mM). Under these conditions palmitate is the primary substrate for cellular metabolism, and its major fate is oxidation. Equilibrium binding was determined after 18-20 h of incubation at 4 degrees C with radiolabeled insulin and increasing concentrations of unlabeled hormone. With increasing palmitate concentration, a dose-dependent decline in cell-surface insulin receptor binding was observed. Binding decreased by 35 percent and 44 percent after 30 min and 3 h of preincubation with 2 mM palmitate, respectively. This decrease was due to a reduction in insulin receptor number. Receptor-mediated insulin processing was evaluated in cells prelabeled at 4 degrees C with 125I (A14)-monoiodoinsulin at an insulin concentration of 100 pM and reincubated at 37 degrees C for up to 30 min. The amount of internalized insulin was decreased by preincubation of hepatocytes with palmitate. This decrease was proportional to the reduction in cell-surface insulin receptor density at palmitate concentrations of 0.05-0.5 mM, but was disproportionally greater at higher fatty acid concentrations. Receptor-mediated insulin degradation decreased at palmitate concentrations between 0.05 and 1.0 mM. At 2 mM, however, insulin degradation was enhanced. This enhancement was observed after 30 min or 3 h of exposure to the fatty acid. Dissociation and/or release of cell-associated internalized insulin was not influenced by the FFA exposure. The effects of FFA on hepatocyte insulin binding and processing were contingent upon cellular metabolism, since no changes were noted when cells were preincubated with palmitate at 4 degrees C under otherwise similar conditions. Thus the in vitro exposure of hepatocytes to FFA influences both receptor and postreceptor events mediating insulin metabolism. These effects may account for the altered hepatic insulin extraction and sensitivity that accompany abdominal obesity and its progression to diabetes.

The effects of obesity and sex on hepatic insulin metabolism were evaluated in the SHR/Mcc-cp rat. During in situ liver perfusion, insulin clearance rate (CLR) expressed per gram of liver tissue was reduced by 58 and 68% in obese females and males, respectively, compared with lean controls. Male sex resulted in CLR reductions of 46% in lean and 59% in obese animals. Obesity resulted in 50% reduction of insulin-receptor binding to isolated hepatocytes. In both lean and obese animals, male sex also resulted in a decrease of approximately 34% in insulin binding. Scatchard plots indicated that the reduction in insulin binding was primarily due to a decrease in number of cell surface receptors. Receptor-mediated insulin degradation was 40% less in obese than lean animals. Male sex also resulted in 27% less insulin degradation relative to females. Receptor-mediated insulin partitioning between four compartments (cell surface bound, internalized and/or cryptic, degraded, and dissociated or released intact), expressed as a percentage of the initial membrane-bound hormone, did not differ between the animal groups. Thus, male sex and obesity are independently and additively associated with a reduction in hepatic insulin clearance and a decrease in the number of cell surface insulin receptors with a proportional decrease insulin compartmentalization and degradation. This mechanism may partly account for the synergistic effects of male sex and obesity on the degree of hyperinsulinemia and insulin resistance and the predisposition to diabetes.