Pushing the envelope on lipodystrophy.

Autosomal dominant partial lipodystrophy (PLD), in which regional adipose loss is coupled with insulin resistance, is strongly associated with missense mutations in LMNA, encoding lamin A/C-a component of the nuclear envelope. This finding indicates that other proteins and functions of the nuclear envelope may have bearing on disorders of adipose tissue and insulin action.

Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action.

The regulation of body weight and composition involves input from genes and the environment, demonstrated, for example, by the variable susceptibility of inbred strains of mice to obesity when offered a high-fat diet. The identification of the gene responsible for obesity in the ob/ob mouse provides a new approach to defining links between diet and genetics in the regulation of body weight. The ob gene protein product, leptin, is an adipocyte-derived circulating protein. Administration of recombinant leptin reduces food intake and increases energy expenditure in ob/ob mice, suggesting that it signals to the brain the magnitude of fat stores. Information on the regulation of this protein is limited. In several rodent models of obesity including db/db, fa/fa, yellow (Ay/a) VMH-lesioned, and those induced by gold thioglucose, monosodium glutamate, and transgenic ablation of brown adipose tissue, leptin mRNA expression and the level of circulating leptin are increased, suggesting resistance to one or more of its actions. We have assessed the impact of increased dietary fat on circulating leptin levels in normal FVB mice and FVB mice with transgene-induced ablation of brown adipose tissue. We find that high-fat diet evokes a sustained increase in circulating leptin in both normal and transgenic mice, with leptin levels accurately reflecting the amount of body lipid across a broad range of body fat. However, despite increased leptin levels, animals fed a high-fat diet became obese without decreasing their caloric intake, suggesting that a high content of dietary fat changes the 'set point' for body weight, at least in part by limiting the action of leptin.

Human leptin levels are pulsatile and inversely related to pituitary-adrenal function.

Leptin communicates nutritional status to regulatory centers in the brain. Because peripheral leptin influences the activity of the highly pulsatile adrenal and gonadal axes, we sought to determine whether leptin levels in the blood are pulsatile. We measured circulating leptin levels every 7 minutes for 24 hours, in six healthy men, and found that total circulating leptin levels exhibited a pattern indicative of pulsatile release, with 32.0 +/- 1.5 pulses every 24 hours and a pulse duration of 32.8 +/- 1.6 minutes. We also show an inverse relation between rapid fluctuations in plasma levels of leptin and those of adrenocorticotropic hormone (ACTH) and cortisol that could not be accounted for on the basis of glucocorticoid suppression of leptin. As leptin levels are pulsatile, we propose that a key function of the CNS is regulated by a peripheral pulsatile signal. In a separate pilot study we compared leptin pulsatility in 414 plasma samples collected every 7 minutes for 24 hours from one obese woman and one normal-weight woman. We found that high leptin levels in the obese subject were due solely to increased leptin pulse height; all concentration-independent pulsatility parameters were almost identical in the two women. Leptin pulsatility therefore can be preserved in the obese.

Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia.

Several inflammatory cytokines, most notably tumor necrosis factor (TNF) and IL-1, induce anorexia and loss of lean body mass, common manifestations of acute and chronic inflammatory conditions. In C57BL/6 female mice, the administration of TNF, IL-1, and, to a lesser extent, leukemia inhibitory factor (LIF), produced a prompt and dose-dependent increase in serum leptin levels and leptin mRNA expression in fat. IL-10, IL-4, ciliary neurotrophic factor, and IL-2, cytokines not known to induce anorexia or decrease food intake, had no effect on leptin gene expression or serum leptin levels. After administration of Escherichia coli lipopolysaccharide (LPS), leptin gene expression and leptin levels were increased. These findings suggest that leptin levels may be one mechanism by which anorexia is induced during acute inflammatory conditions.

Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes.

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.

Severely impaired adipsin expression in genetic and acquired obesity.

Adipsin, a serine protease homolog, is synthesized and secreted by adipose cells and is found in the bloodstream. The expression of adipsin messenger RNA (mRNA) and protein was analyzed in rodents during metabolic perturbations and in several experimental models of obesity. Adipsin mRNA abundance is increased in adipose tissue during fasting in normal rats and in diabetes due to streptozotocin-induced insulin deficiency. Adipsin mRNA abundance decreased during the continuous infusion of glucose, which induces a hyperglycemic, hyperinsulinemic state that is accompanied by an increased adipose mass; it is suppressed (greater than 100-fold) in two strains of genetically obese mice (db/db and ob/ob), compared to their congenic counterparts, and is also reduced when obesity is induced chemically by injection of monosodium glutamate into newborn mice. Circulating adipsin protein is decreased in these animal models of obesity, as determined by immunoblotting with antisera to adipsin. Little change in adipsin expression is observed in a model of obesity obtained by pure overfeeding of normal rats (cafeteria model). These data suggest a possible role for adipsin in the above-mentioned disordered metabolic states, and raise the possibility that adipsin expression may be used to distinguish obesities that arise from certain genetic or metabolic defects from those that result from pure overfeeding.

Antibodies that impair insulin receptor binding in an unusual diabetic syndrome with severe insulin resistance.

Six patients with a unique form of diabetes associated with extreme insulin resistance have markedly reduced insulin binding to specific receptors on their circulating monocytes. When normal insulin receptors were exposed to serum or immunoglobulin fractions from three of these patients in vitro the specific binding defect was reproduced.

Adipsin: a circulating serine protease homolog secreted by adipose tissue and sciatic nerve.

Adipsin is a serine protease homolog whose primary structure was predicted from the nucleotide sequence of a differentiation-dependent adipocyte messenger RNA. Immunoblots probed with antisera to synthetic peptides identify two forms of adipsin that are synthesized and secreted by 3T3 adipocytes. These proteins of 44 and 37 kilodaltons are converted to 25.5 kilodaltons by enzymatic deglycosylation. Although adipsin is principally synthesized in adipose tissue, it is also produced by sciatic nerve and is found in the bloodstream. Because of the apparent restriction of adipsin synthesis to tissues highly active in lipid metabolism, its presence in serum, and its modulation in altered metabolic states, this molecule may play a previously unrecognized role in systemic lipid metabolism or energy balance.

A transgenic model of visceral obesity and the metabolic syndrome.

The adverse metabolic consequences of obesity are best predicted by the quantity of visceral fat. Excess glucocorticoids produce visceral obesity and diabetes, but circulating glucocorticoid levels are normal in typical obesity. Glucocorticoids can be produced locally from inactive 11-keto forms through the enzyme 11beta hydroxysteroid dehydrogenase type 1 (11beta HSD-1). We created transgenic mice overexpressing 11beta HSD-1 selectively in adipose tissue to an extent similar to that found in adipose tissue from obese humans. These mice had increased adipose levels of corticosterone and developed visceral obesity that was exaggerated by a high-fat diet. The mice also exhibited pronounced insulin-resistant diabetes, hyperlipidemia, and, surprisingly, hyperphagia despite hyperleptinemia. Increased adipocyte 11beta HSD-1 activity may be a common molecular etiology for visceral obesity and the metabolic syndrome.

Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area.

Recent studies have reinforced the view that the lateral hypothalamic area (LHA) regulates food intake and body weight. We identified leptin-sensitive neurons in the arcuate nucleus of the hypothalamus (Arc) that innervate the LHA using retrograde tracing with leptin administration. We found that retrogradely labeled cells in the Arc contained neuropeptide Y (NPY) mRNA or proopiomelanocortin (POMC) mRNA. Following leptin administration, NPY cells in the Arc did not express Fos but expressed suppressor of cytokine signaling-3 (SOCS-3) mRNA. In contrast, leptin induced both Fos and SOCS-3 expression in POMC neurons, many of which also innervated the LHA. These findings suggest that leptin directly and differentially engages NPY and POMC neurons that project to the LHA, linking circulating leptin and neurons that regulate feeding behavior and body weight homeostasis.

Unraveling the central nervous system pathways underlying responses to leptin.

Here we summarize recent progress in the biology of leptin, concentrating on its central nervous system (CNS) actions. The product of the ob gene, leptin is a circulating hormone produced by white adipose tissue that has potent effects on feeding behavior, thermogenesis and neuroendocrine responses. Leptin regulates energy homeostasis, as its absence in rodents and humans causes severe obesity. We consider the physiological mechanisms underlying leptin action, along with several novel hypothalamic neuropeptides that affect food intake and body weight. The molecular causes of several other obesity syndromes are discussed to illuminate how the CNS regulates body weight. We describe neural circuits that are downstream of leptin receptors and propose a model linking populations of leptin-responsive neurons with effector neurons underlying leptin's endocrine, autonomic and behavioral effects.

Functionally abnormal Na+-K+ pump in erythrocytes of a morbidly obese patient.

The Na(+)-K(+) pump in the erythrocytes of a mordibly obese patient shows a unique constellation of functional abnormalities. The number of pump units, measured by [(3)H]ouabain binding to intact cells, as well as the enzymatic activity of the (Na(+)-K(+))-dependent ATPase in erythrocyte membranes were found to be markedly increased compared with control cells (18-fold and 14-fold, respectively). There was a concomitant fivefold increase in the rate of pump-mediated uptake of (86)Rubidium (a K analogue); this was balanced by an increased rate of (86)Rb efflux. In striking contrast to normal cells, however, a major portion of this efflux (80%) was inhibited by ouabain, and thus appeared to be mediated by the Na(+)-K(+) pump. Erythrocytes from this patient had elevated levels of intracellular K(+) and reduced levels of intracellular Na(+). This finding, taken together with the ouabain inhibition of K(+) efflux and the absence of associated abnormalities, argues against the possibility that the increased number of Na(+)-K(+) pump units was a compensation for a primary increase in the permeability of the erythrocyte membrane to monovalent cations, as is seen in a variety of erythrocyte disorders. Further evidence for a primary abnormality of the enzyme was our observation that the cardiac glycoside ouabain bound to these cells with reduced affinity and had a right shifted dose response for pump inhibition. The markedly increased number of Na(+)-K(+) pump units in these cells did not appear to extend to mononuclear leukocytes.In conclusion, the erythrocytes from this patient have a very large number of functionally abnormal Na(+)-K(+) ATPase units. A unique abnormality of the erythrocyte Na(+)-K(+) ATPase of these cells is the most likely explanation for these findings.

Alternatively spliced variants of the insulin receptor protein. Expression in normal and diabetic human tissues.

Two insulin receptor mRNA transcripts resulting from alternative splicing of exon 11 in the receptor gene are expressed in a highly regulated tissue-specific fashion. To date, there is no information about the relative abundance of the protein isoforms encoded by these mRNAs in tissues of normal or diabetic subjects. We employed an antibody raised against the peptide sequence encoded by exon 11 to develop a specific immunoprecipitation assay that is capable of determining the fraction of receptors that include this amino acid sequence. The assay is based on the relative ability of the exon 11 specific monoclonal antibody (alpha IR alpha) compared to a nonspecific anti-receptor antiserum (B-2) to immunoprecipitate solubilized receptors that are first labeled with 125I-insulin. The assay was validated using standard curves generated with samples composed of known ratios of the two receptor isoforms. Our results in general confirm observations regarding the relative abundance of the two mRNA species in human tissues, with marked predominance of the exon 11+ isoform in liver, and the exon 11- isoform in leukocytes. Similar amounts of both variants are present in placenta, skeletal muscle, and adipose tissue. In studies with this assay using skeletal muscle extracts from control and noninsulin-dependent diabetes mellitus (NIDDM) subjects, as well as in studies of the two mRNAs in control versus NIDDM muscle using a quantitative polymerase chain reaction assay, we could find no significant difference between control and diabetic subjects. This data contradicts a recent report claiming that normal individuals have only the exon 11- mRNA transcript in their skeletal muscle, whereas NIDDM subjects have similar expression of both mRNAs. Given the emerging evidence that functional differences exist between the two receptor isoforms, these studies are relevant to our understanding of insulin receptor function in health and disease.

Erythrocyte Na,K pump in uremia. Acute correction of a transport defect by hemodialysis.

We studied the erythrocyte Na,K-pump in chronically hemodialyzed uremic patients, immediately before and after a 4-h period of hemodialysis. Using [3H]ouabain as a probe, the number of Na,K-pump units per erythrocyte did not differ in uremic and control subjects, and hemodialysis had no acute effect on this parameter. In contrast, in these same cells the mean level of Na,K-pump-mediated 86Rb transport was 30% lower in predialysis uremic patients than in controls, and this diminution in the rate of 86Rb transport per pump unit was improved after 4 h of hemodialysis in 17 of 18 subjects. The results of in vitro incubation of normal cells with pre- and post-dialysis sera from uremic patients suggested that a serum factor is responsible for the observed inhibition of Na,K-pump activity. Changes in cell Na concentration during dialysis did not appear to be responsible for the increased rate of Na,K-pump turnover after hemodialysis. However, there was a significant correlation between the extent of rise in pump-mediated 86Rb uptake and the weight loss that occurred during dialysis. We conclude that the ion transport turnover rate of the erythrocyte Na,K-pump is impaired in uremia by a nonouabain like circulating factor. This factor, whose activity is diminished acutely by hemodialysis, may play an important role in the systemic manifestations of the uremic syndrome, and could be an important endogenous regulator of the Na,K-ATPase.

Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function.

Leptin is thought to regulate energy balance through effects on food intake and thermogenesis. In addition, leptin may serve as a mediator of the neuroendocrine response to starvation, and may modulate the stress response and the timing of puberty. A role for leptin in development is suggested by the presence of neuroendocrine and structural neuronal abnormalities in ob/ob mice with genetic leptin deficiency. Here, we sought to determine the ontogeny of leptin expression and its relationship to the developing neuroendocrine axis. Leptin increased 5-10-fold in female mice during the second postnatal week independent of fat mass, and declined after weaning. The rise in leptin preceded the establishment of adult levels of corticosterone, thyroxine, and estradiol. In contrast to adult mice, leptin was not acutely regulated by food deprivation during the early postnatal period. Circadian rhythms of leptin, corticosterone, and thyroxine were regulated by food intake in adult mice. When ad libitum feeding was restricted to the light cycle, peak corticosterone levels were shifted to the beginning of the light cycle and coincided with the nadir of leptin. The inverse relationship between leptin and corticosterone was maintained such that a rise in leptin after feeding was associated with a decline in corticosterone. To determine whether changes in corticosterone during food restriction are mediated by leptin, we compared the patterns of corticosterone levels among ob/ob, db/db, and lean mice. Despite their higher basal levels of corticosterone, leptin deficiency in ob/ ob mice did not prevent the nocturnal rise in corticosterone. In contrast, the nocturnal surge of corticosterone was blunted in db/db mice. Therefore, it is likely that factors in addition to leptin are involved in the regulation of the circadian rhythm of corticosterone. The temporal relationship between leptin and other hormones in neonatal and adult mice suggests that leptin is involved in the maturation and function of the neuroendocrine axis.

Regulation of glucose transporter-specific mRNA levels in rat adipose cells with fasting and refeeding. Implications for in vivo control of glucose transporter number.

Fasting in the rat is associated with a rapid and progressive decrease in insulin-stimulated glucose transport activity in adipose cells, which is not only restored to normal, but increased transiently to supranormal levels by refeeding. The mechanisms for these changes in glucose transport activity appear to involve alterations in both glucose transporter number and intrinsic activity (glucose turnover number). In this study, we use the human hepatoma Hep G2 glucose transporter complementary DNA clone to examine the molecular basis for these alterations. Extractable RNA per adipose cell is decreased 35% with 3 d of fasting and increased to 182% of control with 6 d of refeeding after 2 d of fasting. This parallels changes in adipose cell intracellular water, so that total RNA/water space remains relatively constant. When the changes in total RNA/cell are taken into account, Northern and slot blot analyses with quantitative densitometry reveal a 36% decrease in specific glucose transporter mRNA level in cells from the fasted rats. The mRNA level in cells from the fasted/refed rats is restored to normal. These observations correlate closely with previous measurements of glucose transporter number in adipose cell membrane fractions using cytochalasin B binding and Western blotting. The levels of specific mRNAs for tubulin and actin on a per cell basis show similar but more dramatic changes and mRNAs encoding several differentiation-dependent adipose cell proteins are also significantly affected. Thus, the levels of mRNA for multiple adipose cell genes are affected by fasting and refeeding. In particular, this demonstrates that in vivo metabolic alterations can influence the level of a glucose transporter mRNA in adipose cells. This may have implications for the regulation of glucose transporter number and glucose transport activity.

Distribution of glucose transporter messenger RNA transcripts in tissues of rat and man.

We used the complementary DNA for the human hepatoma Hep G2 glucose transporter to determine the distribution of glucose transporter messenger RNA (mRNA) in rat and human tissues. Under stringent hybridization conditions, a single 2.8-kilobase (kb) transcript is seen in all rat and human tissues examined. The mRNA is most abundant in brain, and is especially enriched in the brain microvascular fraction. The mRNA abundance in rat muscle and fat is 5% that in brain. Rat liver (both adult and fetal) and human liver have very little 2.8-kb mRNA, but it is abundant in cultured human fibroblasts and EB virus-transformed lymphoblasts. The same size mRNA is present in leg muscle of two type II diabetic patients. A very homologous glucose transporter mRNA is expressed in both insulin-sensitive and -insensitive tissues of rat and man. Hepatocytes, which have abundant glucose transport, may express a homologous but nonidentical glucose transporter.

Leptin accelerates the onset of puberty in normal female mice.

The fat-derived hormone, leptin, is proposed to serve as an adipostatic signal to the brain to reduce food intake and body weight. In addition to its effects on body weight, chronic leptin treatment restores puberty and fertility to ob/ob mice with total leptin deficiency, and acute treatment substantially corrects hypogonadism in mice starved for 2 d without affecting body weight. Leptin may therefore be a critical signal, linking adiposity and reproduction. Since body weight and adiposity appear to play a critical role in the timing of puberty in humans and rodents, and leptin levels rise with increasing adiposity, we studied the effects of once daily injections of recombinant leptin on the onset of puberty in female mice weaned on day 21 and fed ad libitum. There was a linear increase in body weight during the study period, which was not altered by the dose of leptin used. Mice injected with leptin had an earlier onset of three classic pubertal parameters (i.e., vaginal opening, estrus, and cycling) compared with saline-injected controls. Leptin is the first peripheral molecule demonstrated to accelerate the maturation of the reproductive axis in normal rodents. We propose that leptin is the signal that informs the brain that energy stores are sufficient to support the high energy demands of reproduction, and may be a major determinant of the timing of puberty.

Characterization of the insulin resistance of aging.

To clarify the nature of the insulin resistance of aging we studied the dose response for insulin-induced glucose disposal and the binding of insulin to circulating monocytes in healthy young and old men. A total of 49 two-hour euglycemic insulin clamp studies were performed in 17 young and 10 old healthy nonobese subjects. While the old group had lower estimates of lean body mass and greater estimates of total body fat than the young group, these differences did not exceed 5% and did not reach statistical significance. Insulin was infused at 20 mU/m2 per min (young = 8, old = 5); 80 mU/m2 per min (young = 13, old = 9); 200 mU/m2 per min (young = 9, old = 5). Increasing levels of hyperinsulinemia were associated with dose-dependent increases in steady-state glucose infusion rates in young and old. The maximal glucose infusion rates (milligrams per kilogram body weight per minute) were the same for young and old. However, the dose-response curve was shifted to the right in the old subjects. In the four individuals in each age group in whom studies were performed at each dose level, the Km was 54 +/- 14 microU/ml in the young and 113 +/- 11 microU/ml in the old (P less than 0.02). Correction of glucose infusion rate for lean body mass had no effect on comparisons between age groups. These data indicate an age-associated decline in sensitivity of peripheral tissues to insulin without a change in maximal tissue responsiveness. Studies of insulin binding with 14 young and 9 old subjects indicated no effect of age on the insulin binding to receptors on circulating monocytes (young = 5.25 +/- 0.35; old = 6.22 +/- 0.53% of 125I-insulin bound/10(7) cells). These studies suggest that aging may be associated with a postreceptor defect in insulin action manifested by decreased whole-body tissue sensitivity to insulin without a change in tissue responsiveness.