Thomas W. Stephens

Serum immunoreactive-leptin concentrations in normal-weight and obese humans.

BACKGROUND Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiologic actions of leptin in humans. METHODS Using a newly developed radioimmunoassay, wer measured serum concentrations of leptin in 136 normal-weight subjects and 139 obese subjects (body-mass index, > or = 27.3 for men and > or = 27.8 for women; the body-mass index was defined as the weight in kilograms divided by the square of the height in meters). The measurements were repeated in seven obese subjects after weight loss and during maintenance of the lower weight. The ob messenger RNA (mRNA) content of adipocytes was determined in 27 normal-weight and 27 obese subjects. RESULTS The mean (+/- SD) serum leptin concentrations were 31.3 +/- 24.1 ng per milliliter in the obese subjects and 7.5 +/- 9.3 ng per milliliter in the normal-weight subjects (P < 0.001). There was a strong positive correlation between serum leptin concentrations and the percentage of body fat (r = 0.85, P < 0.001). The ob mRNA content of adipocytes was about twice as high in the obese subjects as in the normal-weight subjects (P < 0.001) and was correlated with the percentage of body fat (r = 0.68, P < 0.001) in the 54 subjects in whom it was measured. In the seven obese subjects studied after weight loss, both serum leptin concentrations and ob mRNA content of adipocytes declined, but these measures increased again during the maintenance of the lower weight. CONCLUSIONS Serum leptin concentrations are correlated with the percentage of body fat, suggesting that most obese persons are insensitive to endogenous leptin production.

Nocturnal rise of leptin in lean, obese, and non-insulin-dependent diabetes mellitus subjects.

We studied 24-h profiles of circulating leptin levels using a sensitive and specific RIA in lean controls and obese subjects with or without non-insulin-dependent diabetes mellitus (NIDDM) during normal routine activity. Serum leptin levels were significantly higher in obese (41.7 +/- 9.0 ng/ml; n = 11) and obese NIDDM (30.8 +/- 6.7; n = 9) subjects compared with those in lean controls (12.0 +/- 4.4, n = 6). In all the three groups, serum leptin levels were highest between midnight and early morning hours and lowest around noon to midafternoon. The nocturnal rise in leptin levels was significant when data were analyzed by ANOVA (lean: F = 3.17, P < 0.0001, n = 4; obese: F = 2.02, P < 0.005, n = 11; and obese NIDDM: F = 4.9, P < 0.0001, n = 5). The average circadian amplitude between acrophase and nadir was 75.6% in lean, 51.7%, in obese and 60.7% in obese NIDDM groups, respectively. No significant correlations (P > 0.05) were observed between circulating levels of leptin and either insulin or glucose levels in any of the 20 subjects studied for 24-h profiles. The nocturnal rise in leptin observed in the present study resembles those reported for prolactin, thyroid-stimulating hormone, and free fatty acids. We speculate that the nocturnal rise in leptin could have an effect in suppressing appetite during the night while sleeping.

Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting.

Little is known about leptins interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

Regulation of Expression of ob mRNA and Protein by Glucocorticoids and cAMP

Regulation of obese gene (ob) expression in ob/ob and db/db mice and in cultured rat adipocytes was examined. It has been demonstrated that exogenous human OB protein (leptin) treatment reduces food intake and weight gain, as well as insulin, glucose, and corticosterone levels in ob/ob mice. In the present report we show that leptin treatment down-regulates endogenous adipose ob mRNA. However, treatment of isolated rat adipocytes with 100 ng/ml human or murine leptin had no direct effect on expression of endogenous ob mRNA, suggesting that leptin may be able to down-regulate its own expression by an indirect, non-autocrine mechanism. Glucocorticoids increased both ob mRNA levels and secreted leptin levels in vitro. Conversely, agents that increase intracellular cAMP, such as β-adrenergic agonists or BtcAMP itself, decreased ob mRNA expression and leptin secretion. Therefore, increased glucocorticoid levels and decreased sympathetic neural activity may contribute to the elevated ob mRNA expression observed in genetically obese, hyperglucocorticoid rodents. Furthermore, leptin might regulate its own expression through a feedback mechanism involving the hypothalamic pituitary axis.

The effect of exercise on leptin concentration in healthy men and in type 1 diabetic patients.

PURPOSE Leptin is a recently discovered hormone that appears as a regulator of energy balance. It is important to know whether leptin concentrations are changed under conditions of altered energy homeostasis. Consequently, we examined the effects of exercise with fasting and exercise with feeding on circulating leptin concentrations in healthy men and in type 1 diabetic patients with normal body weight and well controlled diabetes. METHODS Leptin concentrations were determined with radioimmunoassay. RESULTS During a 3-h cycle ergometer exercise with fasting, leptin decreased by 42% (P < 0.01) in nine healthy men and by 23% (P = 0.05) in eight male type 1 diabetic patients. Leptin fell equally by 12% (P < 0.03) both in nine healthy men and in eight male type 1 diabetic patients who were studied as a resting control group. The absolute fall in leptin in healthy men was similar in the exercise and resting control groups (0.8 +/- 0.1 microgram.L-1 vs 0.8 +/- 0.2 microgram.L-1). However, due to lower leptin concentration before the exercise, the relative decrease (42%) was greater than during the resting control study (12%, P < 0.005). This difference was not seen in the diabetic patients. Fasting leptin concentration correlated positively with BMI (r = 0.75, P < 0.001) and fasting insulin (r = 0.71, P < 0.01) in healthy men as well as with insulin level (r = 0.54, p < 0.05) in type 1 diabetic patients. When exercise was performed with feeding, and this was associated with a significant rise in serum cortisol level (marathon run, 14 healthy men and 7 type 1 diabetic patients), leptin concentration did not change significantly. CONCLUSIONS 1) During morning hours, leptin decreases both in healthy men and in type 1 diabetic patients, reflecting a diurnal variation of leptin concentration and the effect of fasting on leptin concentration. 2) The fall in leptin during morning hours is augmented by physical exercise in healthy men. 3) If exercise is performed with feeding and associated with a rise in serum cortisol level, leptin concentration remains unchanged. These data suggest that although exercise may reduce circulating leptin levels, the effect is small and can be counterbalanced by feeding or a rise in serum cortisol concentration.

Metabolic effects of proglycosyn

Proglycosyn, a phenylacyl imidazolium compound that lowers blood glucose levels, was demonstrated previously to promote hepatic glycogen synthesis, stabilize hepatic glycogen stores, activate glycogen synthase, inactivate glycogen phosphorylase, and inhibit glycolysis. In the present study proglycosyn was found to inhibit fatty acid synthesis, stimulate fatty acid oxidation, and lower fructose 2,6-bisphosphate levels, but to have no significant effects on cell swelling and the levels of cAMP in hepatocytes prepared from fed rats. Verapamil and atropine blocked the effects of proglycosyn on glycogen metabolism, but these compounds inhibit proglycosyn accumulation by hepatocytes. Proglycosyn stimulated phosphoprotein phosphatase activity in postmitochondrial extracts, as measured by dephosphorylation of phosphorylase a and glycogen synthase D, but this action required a very high concentration of the compound, making it unlikely to be the actual mechanism involved. It is proposed that a metabolite of proglycosyn is responsible for its metabolic effects.