Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa.

Anorexia nervosa (AN) is a complex neuropsychiatric disorder presenting with dangerously low body weight, and a deep and persistent fear of gaining weight. To date, only one genome-wide significant locus associated with AN has been identified. We performed an exome-chip based genome-wide association studies (GWAS) in 2158 cases from nine populations of European origin and 15 485 ancestrally matched controls. Unlike previous studies, this GWAS also probed association in low-frequency and rare variants. Sixteen independent variants were taken forward for in silico and de novo replication (11 common and 5 rare). No findings reached genome-wide significance. Two notable common variants were identified: rs10791286, an intronic variant in OPCML (P=9.89 × 10-6), and rs7700147, an intergenic variant (P=2.93 × 10-5). No low-frequency variant associations were identified at genome-wide significance, although the study was well-powered to detect low-frequency variants with large effect sizes, suggesting that there may be no AN loci in this genomic search space with large effect sizes.

A genome-wide association study of anorexia nervosa.

Anorexia nervosa (AN) is a complex and heritable eating disorder characterized by dangerously low body weight. Neither candidate gene studies nor an initial genome-wide association study (GWAS) have yielded significant and replicated results. We performed a GWAS in 2907 cases with AN from 14 countries (15 sites) and 14 860 ancestrally matched controls as part of the Genetic Consortium for AN (GCAN) and the Wellcome Trust Case Control Consortium 3 (WTCCC3). Individual association analyses were conducted in each stratum and meta-analyzed across all 15 discovery data sets. Seventy-six (72 independent) single nucleotide polymorphisms were taken forward for in silico (two data sets) or de novo (13 data sets) replication genotyping in 2677 independent AN cases and 8629 European ancestry controls along with 458 AN cases and 421 controls from Japan. The final global meta-analysis across discovery and replication data sets comprised 5551 AN cases and 21 080 controls. AN subtype analyses (1606 AN restricting; 1445 AN binge-purge) were performed. No findings reached genome-wide significance. Two intronic variants were suggestively associated: rs9839776 (P=3.01 × 10(-7)) in SOX2OT and rs17030795 (P=5.84 × 10(-6)) in PPP3CA. Two additional signals were specific to Europeans: rs1523921 (P=5.76 × 10(-)(6)) between CUL3 and FAM124B and rs1886797 (P=8.05 × 10(-)(6)) near SPATA13. Comparing discovery with replication results, 76% of the effects were in the same direction, an observation highly unlikely to be due to chance (P=4 × 10(-6)), strongly suggesting that true findings exist but our sample, the largest yet reported, was underpowered for their detection. The accrual of large genotyped AN case-control samples should be an immediate priority for the field.

Genetic influences on disordered eating behaviour are largely independent of body mass index.

OBJECTIVE: Prior studies suggest eating disorders and related characteristics are moderately to substantially heritable. We are interested in identifying the genes underlying disordered eating behaviour (DEB), and want to know how much of the genetic influence underlying DEB is attributable to genetic influences on body mass index (BMI). METHOD: Bivariate analyses were performed, in adolescent twins and siblings, to estimate the genetic and environmental contributions for DEB, BMI, and their overlap. RESULTS: Shared genetic risk factors explained the overlap between BMI and DEB (genetic correlation was 0.43 in women, 0.51 in men). DEB was highly heritable in women (a(2) = 0.65; a(2) independent of BMI = 0.53) and moderately heritable in men (a(2) = 0.39; a(2) independent of BMI = 0.29). BMI was highly heritable in both men (a(2) = 0.76) and women (a(2) = 0.80). CONCLUSION: The entire correlation between DEB and BMI was explained by shared genetic risk, but the majority of genetic influences on DEB were due to genetic effects independent of BMI.

Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy.

Hypophosphatasia features selective deficiency of activity of the tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (ALP) isoenzyme (TNSALP); placental and intestinal ALP isoenzyme (PALP and IALP, respectively) activity is not reduced. Three phosphocompounds (phosphoethanolamine [PEA], inorganic pyrophosphate [PPi], and pyridoxal 5'-phosphate [PLP]) accumulate endogenously and appear, therefore, to be natural substrates for TNSALP. Carriers for hypophosphatasia may have decreased serum ALP activity and elevated substrate levels. To test whether human PALP and TNSALP are physiologically active toward the same substrates, we studied PEA, PPi, and PLP levels during and after pregnancy in three women who are carriers for hypophosphatasia. Hypophosphatasemia corrected during the third trimester because of PALP in maternal blood. Blood or urine concentrations of PEA, PPi, and PLP diminished substantially during that time. After childbirth, maternal circulating levels of PALP decreased, and PEA, PPi, and PLP levels abruptly increased. In serum, unremarkable concentrations of IALP and low levels of TNSALP did not change during the study period. We conclude that PALP, like TNSALP, is physiologically active toward PEA, PPi, and PLP in humans. We speculate from molecular/crystallographic information, indicating significant similarity of structure of the substrate-binding site of ALPs throughout nature, that all ALP isoenzymes recognize these same three phosphocompound substrates.

Specificity of inhibition of calcium- and calmodulin-dependent protein kinase by alloxan.

Studies were undertaken to determine whether the effect of alloxan to inactivate a membrane-bound calcium- and calmodulin-dependent protein kinase was specific for the pancreatic islets and whether inactivation of the kinase occurred also after injection of a diabetogenic dose of alloxan into rats. The effect of alloxan was also examined on similar particulate calcium- and calmodulin-dependent kinases present in two other secretory tissues, mammary acini and forebrain. Exposure of alloxan to cell-free preparations of all secretory tissues examined inhibited the calcium- and calmodulin-dependent kinase activities, suggesting that the specificity of alloxan action was not due to the presence in islets of a kinase uniquely sensitive to alloxan. To determine whether the selective effect of alloxan action was mediated at the cellular level, experiments were performed with alloxan presented to intact cells. Whereas alloxan exposure to viable cell preparations of islets and brain decreased the subsequently measured calcium- and calmodulin-dependent protein kinase activity, the activity measured in mammary acini exposed to these alloxan concentrations was unaffected. Injection (i.v.) of a diabetogenic dose of alloxan (50 mg/kg) produced an immediate (10 min) and selective inactivation of the calcium- and calmodulin-dependent protein kinase in pancreatic islets but had no effect on the similar kinases measured in brain and mammary acini. These results indicate that the unique sensitivity of islets to alloxan may result from the ability of alloxan to rapidly gain intracellular access and then inactivate this kinase activity. The selective effect of alloxan injection on this islet protein kinase is consistent with the hypothesis that inactivation of the kinase by alloxan is related to its diabetogenic effect in vivo.

Relationship between insulin sensitivity and plasma leptin concentration in lean and obese men.

Alterations in the production of or the sensitivity to leptin, the protein encoded by the ob gene, cause obesity and diabetes in rodents. We evaluated the isolated relationship between leptin and insulin sensitivity in lean and obese humans. Three groups of subjects who were carefully matched for either insulin sensitivity (determined by the modified intravenous glucose tolerance test and minimal model analysis) or adiposity (determined by hydrodensitometry) were studied: 1) lean insulin-sensitive men (percentage body fat, 15 +/- 1%); 2) lean insulin-resistant men (percentage body fat, 16 +/- 1%), matched on percentage body fat and fat mass with the lean insulin-sensitive group; and 3) obese insulin-resistant men (percentage body fat, 31 +/- 3), matched on insulin sensitivity with the lean insulin-resistant group. Basal plasma leptin concentrations were significantly lower in the lean insulin-sensitive than in the lean insulin-resistant men (1.90 +/- 0.4 vs. 4.35 +/- 1.21 ng/ml, P < 0.05) despite identical body composition. Plasma leptin in the obese men (9.27 +/- 1.4 ng/ml) was significantly higher than values in the two lean groups (P < 0.01). Marked alterations in plasma glucose and insulin concentrations induced by glucose and tolbutamide injection did not cause any change in plasma leptin levels. These results demonstrate that insulin resistance is associated with elevated plasma leptin levels independent of body fat mass. However, plasma insulin itself does not acutely regulate leptin production.

Plasma leptin and insulin relationships in obese and nonobese humans.

Hyperinsulinemia. is associated with an overexpression of mRNA for the ob protein leptin in rodent models of genetic obesity, and insulin has been reported to directly stimulate leptin mRNA in rat adipocytes. Human obesity is also associated with increased leptin mRNA as well as plasma levels, but there have been no reports of the effect of insulin on leptin secretion. We, therefore, tested the hypothesis that insulin stimulates leptin secretion in humans. Using a newly developed leptin assay, immunoreactive leptin was measured in fasting and postprandial plasma samples from 27 healthy adults and in samples before and during euglycemic-hyperinsulinemic then stepped hypoglycemic (hourly steps at 85, 75, 65, 55, and 45 mg/dl) clamps from 10 healthy subjects and 11 patients with IDDM. Plasma leptin was correlated (r = 0.84, P = 0.0005) with BMI in obese but not nonobese subjects and with fasting (r = 0.75, P = 0.008) but not postprandial plasma insulin levels. (Leptin levels did not change postprandially.) Euglycemic hyperinsulinemia did not alter leptin levels, nor did hyperinsulinemic hypoglycemia. Thus, because circulating leptin levels are not increased during postprandial hyperinsulinemia or during euglycemic (or hypoglycemic) hyperinsulinemia, we conclude that, at least in the short term, insulin does not increase leptin secretion in humans and that hyperleptinemia in obese individuals is not likely the result of hyperinsulinemia.

Adipose tissue leptin production and plasma leptin kinetics in humans.

Abdominal adipose tissue leptin production was determined in vivo by arteriovenous balance in 14 lean and obese men (mean BMI 27.0 +/- 1.9, range 21.4-45.2). Blood samples were taken simultaneously from an abdominal vein that drains subcutaneous adipose tissue and from a radial artery. Adipose tissue blood flow was measured by xenon washout. Abdominal vein leptin concentrations (mean 8.9 +/- 2.4 ng/ml, range 2.1-36.5 ng/ml) were consistently greater than arterial values (mean 6.6 +/- 1.9 ng/ml, range 1.7-28.2 ng/ml) (P < 0.001). The net rate of abdominal adipose tissue leptin production (mean 3.2 +/- 0.5 ng x 100 g(-1) x min(-1)) correlated directly with percentage body fat (rs = 0.59, P = 0.016). Estimated whole-body leptin production rate (797 +/- 283 ng x person(-1) x min(-1)) correlated directly with percent body fat (rs = 0.93, P < 0.0001) and with regional leptin production (rs = 0.81, P < 0.001). In contrast, the rate of leptin clearance from plasma (mean 1.50 +/- 0.23 ml x kg(-1) x min(-1)) and plasma leptin half-life (mean 24.9 +/- 4.4 min) was unrelated to adiposity (rs = 0.06, P = 0.30; rs = 0.16, P = 0.30, respectively). These results provide direct evidence that leptin is produced by adipose tissue in humans and that the rate of production is directly related to adiposity. A combination of greater leptin production per unit of body fat and increased production from expanded total body fat mass, rather than alterations in leptin clearance, account for the increase in plasma leptin concentrations observed in obese humans.

Site-specific phosphorylation of synapsin I by Ca2+/calmodulin-dependent protein kinase II in pancreatic betaTC3 cells: synapsin I is not associated with insulin secretory granules.

Increasing evidence supports a physiological role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from the pancreatic beta-cell, but the precise sites of action are not known. A role of this enzyme in neuroexocytosis is implicated by its phosphorylation of a vesicle-associated protein, synapsin I. Because of emerging similarities to the neuron with respect to exocytotic mechanisms, the expression and phosphorylation of synapsin I in the beta-cell have been studied. Synapsin I expression in clonal mouse beta-cells (betaTC3) and primary rat islet beta-cells was initially confirmed by immunoblot analysis. By immunoprecipitation, in situ phosphorylation of synapsin I was induced in permeabilized betaTC3 cells within a Ca2+ concentration range shown to activate endogenous CaM kinase II under identical conditions. Proteolytic digests of these immunoprecipitates revealed that calcium primarily induced the increased phosphorylation of sites identified as CaM kinase II-specific and distinct from protein kinase A-specific sites. Immunofluorescence and immunogold electron microscopy verified synapsin I expression in betaTC3 cells and pancreatic slices but demonstrated little if any colocalization of synapsin I with insulin-containing dense core granules. Thus, although this study establishes that synapsin I is a substrate for CaM kinase II in the pancreatic beta-cell, this event appears not to be important for the mobilization of insulin granules.

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart.

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and kappa-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K0.5 = 93 +/- 5 nM), glycogen synthase (K0.5 = 127 +/- 10 nM), and kappa-casein (K0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 microM) but a low affinity for kappa-casein (21 +/- 2 microM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.

Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II.

The specificity and biochemical basis of inactivation of calmodulin-dependent protein kinase II by alloxan was studied in dispersed rat brain cells and a partially purified kinase preparation from an insulin-secreting tumor-cell line, RINm5f. When mechanically dispersed rat brain cells were incubated with [32P]-phosphate to label endogenous ATP, depolarization with 44 mM KCl produced a significant (P = 0.03) increase in phosphorylation of endogenous synapsin (132 +/- 8% of basal). Pre-treatment of the brain cells with 1.5 mM alloxan reduced depolarization-sensitive synapsin phosphorylation (109 +/- 5%). Phosphopeptide mapping of depolarization-phosphorylated synapsin showed that alloxan pre-treatment reduced phosphorylation specifically at synapsin sites phosphorylated by calmodulin-dependent protein kinase II. The results demonstrate selective inactivation of calmodulin-dependent protein kinase II activity by alloxan in an intact cell system, which may be useful in the study of the Type II kinase in cells and tissues. Using a partially purified kinase preparation from RINm5f cells, alloxan (100 microM) inactivated 76 +/- 1% calmodulin-dependent protein kinase II activity in 5 min at 37 degrees C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5'-Dithiobis (2-nitrobenzoic acid) (I50 = 2.5 microM) also inactivated the kinase. These results suggested that a sulfhydryl group was involved at the inactivation site. Iodoacetamide (1.0 mM) had no inhibitory effect; however, preincubation with iodoacetamide protected the kinase activity from subsequent inactivation by alloxan. Covalent binding of [14C]-alloxan to calmodulin-dependent protein kinase was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of calcium and calmodulin dependent protein kinase II during stimulation of insulin secretion.

Pancreatic islets contain an alloxan sensitive, calcium and calmodulin dependent protein kinase (CaM-PK) which may play an important part in the cellular control of insulin secretion. We have studied this activity in islets and the insulin secreting tumor cell line RINm5f with particular interest in the changes in kinase activity that accompany stimulation of secretion. Initial experiments showed that the CaM-PK activity enriched in microsomal preparations from RIN cells was similar to the islet cell kinase in terms of apparent endogenous substrates, Ca2+ and calmodulin dependence, and inactivation by alloxan. For studies of protein substrate specificity, tumor cell CaM-PK was isolated from other kinase activities and substantially purified by affinity chromatography with calmodulin-agarose. The major protein substrates of CaM-PK (54 kD and 57 kD) co-purified with the kinase activity, representing autophosphorylation of subunits of the enzyme. Exogenous substrates phosphorylated by these preparations included microtubule-associated protein 2, synapsin, and glycogen synthase; this pattern of substrate utilization identified the kinase as the Type II multifunctional kinase which has been extensively characterized in brain. A polyclonal antibody to rat brain CaM-PK II was employed to immunoprecipitate the kinase from RINm5f cells incubated with secretagogues to measure the effect of stimulation of secretion on autophosphorylation of CaM-PK (which reflects kinase activation). D-Glyceraldehyde (22 mM) and depolarizing concentrations of potassium increased autophosphorylation and insulin secretion in a parallel fashion. Potassium stimulated autophosphorylation was dose dependent and saturable, and was increased to near maximal levels at times as short as 1 min. These studies demonstrate that pancreatic islets and RINm5f cells contain a Type II CaM-PK which is activated during the secretion process.

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets.

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K0.5 of 10 microM) and maximum activation was observed at 50 microM arachidonate. Arachidonic acid did not affect the Ca2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 microM) inhibited (greater than 90%) calmodulin-dependent protein kinase activity (K0.5 = 50-100 microM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K0.5 = 24 microM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50-100 microM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 microM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 microM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.

Correlation of the activation of Ca2+/calmodulin-dependent protein kinase II with the initiation of insulin secretion from perifused pancreatic islets.

An experimental procedure has been designed to permit the simultaneous assessment of the activation status of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) with insulin secretion in perifused islets. By this procedure, the activation of CaM kinase II by glucose correlated closely with the initial and sustained phases of insulin secretion within a 30-min test period. By contrast, islets (160-200/tube) in static incubations neither supported second-phase insulin secretion nor CaM kinase II activation beyond 10-15 min. This was not the result of the accumulation of insulin, because the introduction of insulin (40-160 ng/ml) into the perifusion medium failed to mimic the suppression of glucose-induced insulin secretion or CaM kinase II activation. A similar addition of SRIF (0.01-1 microM) or epinephrine (1 microM) profoundly suppressed insulin secretion although failing to significantly influence CaM kinase II activation. Finally, on withdrawal of glucose from perifused islets, insulin secretion rapidly returned to basal rates, but CaM kinase II deactivation was significantly delayed. The correlation of kinase activation with the initiation of insulin secretion suggests that CaM kinase II may be important in the regulation of glucose-induced insulin secretion. The observed dissociation of these parameters in the presence of inhibitory hormones or after the withdrawal of a glucose stimulus, however, suggests that the kinase is not directly involved in the final steps of insulin exocytosis.

Serum leptin levels are reduced in response to exercise training, but not hormone replacement therapy, in older women.

The aim of this study was to evaluate the effects of exercise training and hormone replacement therapy (HRT) on serum leptin levels in older women. Previously sedentary, healthy women, aged 60-72 yr, were assigned to control (n = 16), exercise (n = 17), HRT (n = 15), or exercise + HRT (n = 13) groups. Exercise training consisted of a 2-month flexibility-exercise program followed by a 9-month exercise program that included walking, jogging, and stair climbing. HRT consisted of 11 months of continuous conjugated estrogens (0.625 mg/day) and medroxyprogesterone acetate (5 mg/day) for 13 days every third month. Body composition was assessed by dual-energy x-ray absorptiometry, and serum insulin levels were measured in the fasted state and in response to a glucose challenge. Leptin levels were reduced by 23 +/- 25% and 22 +/- 27% (both P < 0.01) in response to exercise and exercise + HRT, respectively. There was no effect of HRT on leptin. Fat mass was the strongest predictor of serum leptin concentration, both before (r = 0.81; P < 0.001) and after (r = 0.85; P < 0.001) the study period, and the change in fat mass in the exercisers was significantly correlated with the change in leptin (r = 0.55; P < 0.01). There did not seem to be an effect of exercise, independent of the reduction in fat mass, on leptin. Insulin levels were significantly correlated with leptin levels, but this was not independent of the association with adiposity. The curvilinear relationship between leptin level and fat mass and the finding that the ratio of leptin mass to fat mass decreased after weight loss suggest that fat cell size is an important determinant of circulating leptin levels.

Leptin production during moderate-intensity aerobic exercise.

Leptin, the protein product of the ob gene, may be involved in the regulation of energy balance. Although a clear relationship between energy intake and plasma leptin concentrations has been demonstrated in humans, little is known about the effect of exercise on leptin metabolism. In the present study, we evaluated abdominal adipose tissue leptin production in vivo by arteriovenous balance at rest and during 60 min of moderate-intensity cycle ergometer exercise (50% of maximal heart rate) in five sedentary male subjects (mean age 38.4 +/- 1.7 yr, body mass index (28.4 +/- 4.2 kg/m2). Blood samples were taken simultaneously from an abdominal vein, draining sc adipose tissue, and a radial artery, at rest and every 10 min during exercise. Adipose tissue blood flow was determined by the xenon washout technique. Plasma leptin concentrations did not change throughout exercise and were the same as the values obtained during resting conditions. Average net adipose tissue leptin production rates during exercise (3.07 +/- 0.89 ng/100 g-1.min-1) also were similar to resting values (3.86 +/- 0.95 ng/100 g-1.min-1). These results demonstrate that plasma leptin concentrations and leptin production do not change during an acute bout of moderate-intensity aerobic exercise.

Effect of short-term fasting on free and bound leptin concentrations in lean and obese women.

Plasma leptin exists in protein-bound and free forms, which may affect its hormonal bioactivity. Therefore, the relationship between bound and free leptin may be particularly important during physiological conditions that cause rapid alterations in total plasma leptin concentration, such as fasting. The purpose of this study was to evaluate the effect of short-term fasting on bound and free plasma leptin concentrations and leptin binding capacity (a measure of plasma leptin-binding protein content) in lean and obese women. Six lean (body mass index, 21 +/- 1 kg/m2) and 6 abdominally obese (BMI, 36 +/- 1 kg/m2) women were studied after 14 h and 22 h of fasting. Although total plasma leptin concentration was more than 6-fold greater in obese (45.4 +/- 7.6 microg/liter) compared with lean (7.4 +/- 1.0 microg/liter) women at 14 h of fasting (P < 0.05), the percentage of leptin in the bound form was greater in lean than obese subjects (29 +/- 2% vs. 12 +/- 3%; P < 0.05). Arterial total, free, and bound plasma leptin concentrations all declined between 14 h and 22 h of fasting in both lean and obese groups, but the relative decline of these fractions was greater in lean (36 +/- 4%, 60 +/- 9%, and 51 +/- 13%, respectively) than in obese (19 +/- 5%, 21 +/- 8%, and 12 +/- 7%, respectively) subjects (all P < 0.05). In contrast, leptin binding capacity was unchanged. The percentage of total plasma leptin present in bound form was constant between 14 h and 22 h of fasting in lean subjects and increased slightly but significantly in obese subjects. These data demonstrate that both free and bound fractions of leptin in plasma decrease quickly in response to energy restriction, but the decline is blunted in abdominally obese compared with lean women. In addition, the equilibrium between bound and free leptin fractions is maintained during brief fasting and is not regulated by leptin binding capacity.

Serum leptin levels in women with anorexia nervosa.

Leptin is a protein encoded by the ob gene that is expressed in adipocytes and regulates eating behavior via central neuroendocrine mechanisms. Serum leptin levels have been shown to correlate with weight and percent body fat in normal and obese individuals; however, it is not known whether the regulation of leptin is normal below a critical threshold of body fat in chronic undernutrition. We investigated serum leptin levels in 22 women, aged 23 +/- 4 yr, with anorexia nervosa. Duration of disease, weight, BMI, percent body fat, and serum leptin levels were determined for each patient. Nutritional status was assessed further by caloric intake and measurement of insulin and insulin-like growth factor I (IGF-I) levels. Twenty-three healthy women, aged 23 +/- 4 yr, taking no medications, with normal menstrual function and body mass index (BMI) between 20-26 kg/m2 (mean, 23.7 +/- 1.7 kg/m2), served as a control population for comparison of leptin levels. Subjects with anorexia nervosa were low weight (BMI, 16.3 +/- 1.6 kg/m2; normal, 20-26 kg/m2) and exhibited a striking reduction in percent body fat (7 +/- 2%; normal, 20-30%). The mean serum leptin level was significantly decreased in subjects with anorexia nervosa compared with that in age- and sex-matched controls of normal body weight (5.6 +/- 3.7 vs. 19.1 +/- 8.1 ng/mL; P < 0.0001). Serum leptin levels were correlated highly with weight, as expressed either BMI (r = 0.66; P = 0.002) or percent ideal body weight (r = 0.68; P = 0.0005), body fat (r = 0.70; P = 0.0003), and IGF-I (r = 0.64; P = 0.001), but not with caloric intake or serum levels of estradiol or insulin in subjects with anorexia nervosa. The correlation between leptin and body fat was linear, with progressively lower, but detectable, leptin levels measured even in patients with less than 5% body fat, but was not significant when the effects of weight were taken into account. In contrast, the correlation between leptin and IGF-I remained significant when the effects of weight, body fat, and caloric intake were taken into account. In normal controls, leptin correlated with BMI (r = 0.55; P = 0.007) and IGF-I (r = 0.44; P < 0.05), but not with fat mass. These data demonstrate that serum leptin levels are reduced in association with low weight and percent body fat in subjects with anorexia nervosa compared to normal controls. Leptin levels correlate highly with weight, percent body fat, and IGF-I in subjects with anorexia nervosa, suggesting that the physiological regulation of leptin is maintained in relation to nutritional status even at an extreme of low weight and body fat.

Leptin response to insulin in humans is related to the lipolytic state of abdominal subcutaneous fat.

Insulin-induced leptinemia in humans appears to be blunted by insulin resistance. We therefore examined the relationship between insulin action and plasma leptin by monitoring regional and whole body lipolysis and plasma leptin levels in 15 premenopausal women (body fat range, 14-59%) during a two-stage euglycemic clamp (insulin was infused 90 min each at 6-10 and 12-20 mU/m2 x min). Microdialysis probes were placed in abdominal and femoral sc adipose tissue. Subjects were given a primed, constant infusion of a stable isotope tracer (2H5-glycerol), and plasma glycerol isotope enrichments were analyzed by mass spectrometry to determine glycerol kinetics. Although there was no mean change in plasma leptin during the clamp (baseline, 16.6 +/- 4.5 ng/mL; final, 16.3 +/- 4.3 ng/mL), there was large interindividual variability in the changes in plasma leptin (range, -18% to +19%). Changes in plasma leptin during the clamp stages were correlated with abdominal dialysate glycerol concentrations (r = -0.44; P < 0.05), but not femoral dialysate glycerol concentrations (r = -0.15), the rate of appearance of glycerol in plasma (r = 0.005), or plasma insulin levels (r = 0.16). The results suggest that insulin-induced changes in plasma leptin are more related to the lipolytic state (i.e. low leptin response when lipolysis is high) of abdominal sc adipose tissue than that of other fat depots.

Increased plasma leptin concentration in end-stage renal disease.

Leptin is a 16-kDa protein recently identified as the obese gene product involved in body weight regulation. Administration of recombinant leptin to ob/ob mice, which have a genetic defect in leptin production, reduces food intake and increases energy expenditure. Leptin is synthesized by fat cells, and in normal humans, plasma concentrations are proportional to adiposity. The physiological actions and the degradation pathways of leptin in humans are unknown. We investigated renal elimination of leptin by comparing plasma leptin concentrations in end-stage renal disease (ESRD) patients with normal controls. Our hypothesis was that if renal filtration is a significant route of elimination, the hormone would accumulate in ESRD patients. Mean plasma levels in 141 ESRD patients (26.8 +/- 5.7 and 38.3 +/- 5.6 micrograms/L for males and females, respectively) were significantly higher (P < 0.001) than mean values obtained in normal controls (11.9 +/- 3.1 and 21.2 +/- 3.0 micrograms/L for males and females, respectively). Leptin concentrations in ESRD patients correlated directly with body mass index (BMI; r = 0.77 for men and 0.78 for women). The rate of increase in leptin concentrations with BMI was significantly greater in ESRD patients (5.5 and 6.6 micrograms/L/U BMI for men and women, respectively) than in normal controls (1.4 and 2.6 micrograms/L/U for men and women, respectively). Pre- and postdialysis leptin levels in hemodialysis patients were similar. Western blot of plasma from ESRD patients with high leptin levels showed bands corresponding to the intact protein (16 kDa) with no lesser or greater molecular mass species observed. Leptin concentrations in patients with ESRD did not correlate with measures of residual renal function (serum creatinine, beta 2-microglobulin, PTH, or GH levels). Similarly, we found no correlation between leptin levels and the number of years patients had been on dialysis or with recent weight changes. We conclude that intact leptin is increased in ESRD patients, but does not appear to cause decreased weight. As leptin levels did not correlate with residual renal function, increased production may account for the high levels observed.