Leptin inhibits lysophosphatidic acid-induced intracellular calcium rise by a protein kinase C-dependent mechanism

Leptin communicates the status of body energy stores to the central nervous system, regulating appetite, metabolic rate, and neuroendocrine functions. These effects are mediated by leptin binding and activation of the cognate cell surface receptor, a member of type I cytokine receptor family, which lead to the activation of receptor-associated kinases of the Janus family. In this work, we demonstrate that leptin inhibits the l-alpha-lysophosphatidic acid (LPA)-induced intracellular calcium mobilization in a dose-dependent manner in HEK-293 cells stably expressing full-length leptin receptor (OB-Rb). This action appears to be selective, as it was not observed when other signaling families, such as VIP or EGF, were studied. Pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, reversed the effect of leptin, pointing to PI3K as an intermediate molecule involved in this process. An unspecific protein kinase C (PKC) inhibitor, staurosporine, disrupted the inhibitory action of leptin. Furthermore, intracellular levels of phosphorylated PKCepsilon and PKCdelta rose to a maximum 5 min after leptin administration, suggesting that these atypical PKC isoforms are involved in the observed cross-desensitization. To define the regions of the OB-Rb intracellular domain required for the cross-desensitization, a series of C-terminal deletion mutants were transfected into HEK-293 cells. C-terminal truncation that removed the consensus Box 3 motif of OB-Rb prevented leptin action, indicating that heterologous desensitization over LPA was exerted at the level of this intracellular motif. Our date demonstrate that leptin plays a key role in the regulation of the earliest signaling pathways activated by growth factors, such as LPA, through a signaling pathway involving PKCdelta and PKCepsilon coupled to Box 3 motif of the OB-Rb through PI3K.     

Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin.

Adipose tissue performs complex metabolic and endocrine functions. This review will focus on the recent literature on the biology and actions of three adipocyte hormones involved in the control of energy homeostasis and insulin action, leptin, acylation-stimulating protein, and adiponectin, and mechanisms regulating their production. Results from studies of individuals with absolute leptin deficiency (or receptor defects), and more recently partial leptin deficiency, reveal leptin's critical role in the normal regulation of appetite and body adiposity in humans. The primary biological role of leptin appears to be adaptation to low energy intake rather than a brake on overconsumption and obesity. Leptin production is mainly regulated by insulin-induced changes of adipocyte metabolism. Consumption of fat and fructose, which do not initiate insulin secretion, results in lower circulating leptin levels, a consequence which may lead to overeating and weight gain in individuals or populations consuming diets high in energy derived from these macronutrients. Acylation-stimulating protein acts as a paracrine signal to increase the efficiency of triacylglycerol synthesis in adipocytes, an action that results in more rapid postprandial lipid clearance. Genetic knockout of acylation-stimulating protein leads to reduced body fat, obesity resistance and improved insulin sensitivity in mice. The primary regulator of acylation-stimulating protein production appears to be circulating dietary lipid packaged as chylomicrons. Adiponectin increases insulin sensitivity, perhaps by increasing tissue fat oxidation resulting in reduced circulating fatty acid levels and reduced intramyocellular or liver triglyceride content. Adiponectin and leptin together normalize insulin action in severely insulin-resistant animals that have very low levels of adiponectin and leptin due to lipoatrophy. Leptin also improves insulin resistance and reduces hyperlipidemia in lipoatrophic humans. Adiponectin production is stimulated by agonists of peroxisome proliferator-activated receptor-gamma; an action may contribute to the insulin-sensitizing effects of this class of compounds. The production of all three hormones is influenced by nutritional status. These adipocyte hormones, the pathways controlling their production, and their receptors represent promising targets for managing obesity, hyperlipidemia, and insulin resistance.     

Cachexia in MAC16 adenocarcinoma: suppression of hunger despite normal regulation of leptin, insulin and hypothalamic neuropeptide Y

Weight loss normally stimulates hunger, through mechanisms that include falls in circulating leptin and insulin, leading to stimulation of hypothalamic neuropeptide Y (NPY). Here, we investigated the leptin, insulin and NPY to clarify why hunger is suppressed in mice with severe cachexia due to the MAC16 adenocarcinoma. MAC16-bearing mice progressively lost weight (19% below controls) and fat (- 61%) over 16 days after tumour transplantation, while total food intake fell by 10%. Pair-fed mice showed less wasting, with final weight being 9% and fat mass 25% below controls. Plasma leptin fell by 85% in MAC16 and 51% in pair-fed mice, in proportion to loss of fat. Plasma insulin was also reduced by 49% in MAC16 and 53% in pair-fed groups. Hypothalamic leptin receptor (OB-Rb) mRNA was significantly increased in both MAC16 (+ 223%) and pair-fed (+192%) mice. Hypothalamic NPY mRNA was also significantly raised in MAC16 (+152%) and pair-fed (+ 99%) groups, showing negative correlations with plasma leptin and insulin, and a positive association with OB-Rb mRNA. In MAC16-induced cachexia, leptin production and hypothalamic OB-Rb and NPY expression are regulated appropriately in response to fat depletion. Therefore, suppression of hunger is probably due to tumour products that inhibit NPY transport or release, or that interfere with neuronal targets downstream of NPY.     

Diacylglycerol kinase zeta in hypothalamus interacts with long form leptin receptor. Relation to dietary fat and body weight regulation

Leptin and its long form receptor, Ob-Rb, in hypothalamic nuclei play a key role in regulating energy balance. The mutation of Ob-Rb into one of its natural variants, Ob-Ra, results in severe obesity in rodents. We demonstrate here that diacylglycerol kinase zeta (DGKzeta) interacts, via its ankyrin repeats, with the cytoplasmic portion of Ob-Rb in yeast two-hybrid systems, in protein precipitation experiments in vitro and in vivo. It does not interact, however, with the short form, Ob-Ra, which mediates the entry of leptin into the brain. Furthermore, we show by in situ hybridization that DGKzeta is expressed in neurons of hypothalamic nuclei known to synthesize Ob-Rb and to participate in energy homeostasis. The mutant ob-/ob- and db-/db- mice exhibit increased hypothalamic DGKzeta mRNA level compared with their wild-type controls, suggesting a role for the leptin/OB-Rb system in regulating DGKzeta expression. Further experiments show that hypothalamic DGKzeta mRNA level is stimulated by the consumption of a high-fat diet. In addition, DGKzeta mRNA is statistically significantly lower in rats and inbred mice that become obese on a high-fat diet compared with their lean counterparts. In fact, it is strongly, negatively correlated with both body fat and circulating levels of leptin. Taken together, our evidence suggests that DGKzeta constitutes a downstream component of the leptin signaling pathway and that reduced hypothalamic DGKzeta mRNA, and possibly activity, is associated with obesity.     

Obesity reduced the gene expressions of leptin receptors in hypothalamus and liver.

The expression of leptin receptor (OB-R) is downregulated by leptin in some cell lines. This study investigated the expressions of leptin receptors at central nerve system and peripheral site in a dietary model of obesity. Rats in the 8 week high-diet and control group were classified based on body weight gain into obese and control groups. Serum leptin and insulin concentrations were measured and gene expressions of short form of leptin receptor (OB-Ra) and long form (OB-Rb) in hypothalamus and liver were detected by RT-PCR. The levels of serum leptin in obese rats were increased compared with control rats (p<0.05). The levels of OB-Ra and OB-Rb gene expressions in both hypothalamus and liver in obese rats were reduced significantly (p<0.01). Serum leptin concentrations of obese rats had a significant negative relationship with both of OB-Ra or OB-Rb gene expression levels in hypothalamus and liver (p<0.01). On the other hand, serum insulin levels had no relationship with OB-Ra or OB-Rb gene expression levels in neither liver nor hypothalamus. Rats with diet-induced obesity have hyperleptinemia and reduced expressions of leptin receptors in hypothalamus and liver. The results suggest that a leptin downregulated OB-R expression is one of leptin resistant mechanisms for maintaining obesity.     

Distinct impaired regulation of SOCS3 and long and short isoforms of the leptin receptor in visceral and subcutaneous fat of lean and obese women

Animal studies have illustrated the importance of the expression in adipose tissue of the leptin receptor (OB-R), and of SOCS3 an inhibitor of the leptin signaling pathway, in body weight regulation. The aim of the present study was to investigate in human adipose tissues of the same patients the OB-R isoforms and SOCS3 expression. Subcutaneous and omental adipose tissues were obtained from 6 lean and 18 morbidly obese women. The long isoform OB-Rb mRNA mediating leptin signaling, and SOCS3 mRNA are abundantly present in the subcutaneous fat of lean women, but are 90% and 70% decreased (P<0.0001) in obese women. In visceral fat from lean and obese women, both OB-Rb and SOCS3 mRNA are detected at very low levels. Subcutaneous/visceral ratios for OB-Ra the short OB-R isoform, OB-Rb, and SOCS3 mRNA abundance strongly correlate with the insulin sensitivity index, HOMA-% S, (r=0.49, P<0.0001, r=0.42, P=0.0002 and r=0.38, P=0.0002, respectively) in both lean and obese patients without type 2 diabetes. The near absence of OB-Rb mRNA and the similarly decreased SOCS3 expression in obese adipose tissue may reflect a defective leptin signaling pathway that could play a role in the impairment of insulin sensitivity associated with excess adiposity.     

p70S6 kinase phosphorylates AMPK on serine 491 to mediate leptin's effect on food intake

The PI3K-AKT, mTOR-p70S6 kinase and AMPK pathways play distinct and critical roles in metabolic regulation. Each pathway is necessary for leptin's anorexigenic effects in the hypothalamus. Here we show that these pathways converge in an integrated phosphorylation cascade to mediate leptin action in the hypothalamus. We identify serine(491) on α2AMPK as the site of convergence and show that p70S6 kinase forms a complex with α2AMPK, resulting in phosphorylation on serine(491). Blocking α2AMPK-serine(491) phosphorylation increases hypothalamic AMPK activity, food intake, and body weight. Serine(491) phosphorylation is necessary for leptin's effects on hypothalamic α2AMPK activity, neuropeptide expression, food intake, and body weight. These results identify an inhibitory AMPK kinase, p70S6 kinase, and demonstrate that AMPK is a substrate for mTOR-p70S6 kinase. This discovery has broad biologic implications since mTOR-p70S6 kinase and AMPK have multiple, fundamental and generally opposing cellular effects that regulate metabolism, cell growth, and development.     

Clusterin/ApoJ enhances central leptin signaling through Lrp2‐mediated endocytosis

Hypothalamic leptin signaling plays a central role in maintaining body weight homeostasis. Here, we show that clusterin/ApoJ, recently identified as an anorexigenic neuropeptide, is an important regulator in the hypothalamic leptin signaling pathway. Coadministration of clusterin potentiates the anorexigenic effect of leptin and boosts leptin‐induced hypothalamic Stat3 activation. In cultured neurons, clusterin enhances receptor binding and subsequent endocytosis of leptin. These effects are mainly mediated through the LDL receptor‐related protein‐2 (Lrp2). Notably, inhibition of hypothalamic clusterin, Lrp2 or endocytosis abrogates anorexia and hypothalamic Stat3 activation caused by leptin. These findings propose a novel regulatory mechanism in central leptin signaling pathways.     

Expression of leptin and its receptor genes in the ovarian follicles of cycling and early pregnant pigs

Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic–pituitary–gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs.     

Molecular mechanisms associated with leptin resistance: n-3 polyunsaturated fatty acids induce alterations in the tight junction of the brain

High-fat diets cause peripheral leptin resistance, and dietary lipid composition affects sensitivity to leptin. We examined the role of n-3 polyunsaturated fatty acid (PUFA) in peripheral leptin resistance. Dietary PUFAs (0.4% wt/wt) caused insensitivity to peripherally but not intracerebroventricularly administered leptin. n-3 PUFA increased body weight, associated with a significant reduction of leptin concentration in the cerebrospinal fluid. Dietary n-3 PUFA reduced transport of endogenous or exogenously administered leptin into the brain, associated with increased expression of hypothalamic occludin, but caused no change in expression of leptin receptors, proteins associated with leptin signaling or other tight junction proteins. Continuous intracerebroventricular infusion of an antisense morpholino oligonucleotide targeted to occludin mRNA reversed n-3 PUFA-induced insensitivity to peripherally administered leptin. We conclude that n-3 PUFA induces peripheral leptin resistance via an increase in the expression of hypothalamic occludin, reducing paracellular transport of leptin into the brain.     

Hypothalamic CaMKK2 contributes to the regulation of energy balance

Detailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPK kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPK and β. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention.     

In a randomized trial in prostate cancer patients,dietary protein restriction modifies markers of leptin and insulin signaling in plasma extracellular vesicles

Obesity, metabolic syndrome, and hyperleptinemia are associated with aging and age‐associated diseases including prostate cancer. One experimental approach to inhibit tumor growth is to reduce dietary protein intake and hence levels of circulating amino acids. Dietary protein restriction (PR) increases insulin sensitivity and suppresses prostate cancer cell tumor growth in animal models, providing a rationale for clinical trials. We sought to demonstrate that biomarkers derived from plasma extracellular vesicles (EVs) reflect systemic leptin and insulin signaling and respond to dietary interventions. We studied plasma samples from men with prostate cancer awaiting prostatectomy who participated in a randomized trial of one month of PR or control diet. We found increased levels of leptin receptor in the PR group in total plasma EVs and in a subpopulation of plasma EVs expressing the neuronal marker L1CAM. Protein restriction also shifted the phosphorylation status of the insulin receptor signal transducer protein IRS1 in L1CAM+ EVs in a manner suggestive of improved insulin sensitivity. Dietary PR modifies indicators of leptin and insulin signaling in circulating EVs. These findings are consistent with improved insulin and leptin sensitivity in response to PR and open a new window for following physiologic responses to dietary interventions in humans.     

Appetite and energy balance signals from adipocytes

Interest in the biology of white adipose tissue has risen markedly with the recent surge in obesity and its associated disorders. The tissue is no longer viewed simply as a vehicle for lipid storage; instead, it is recognized as a major endocrine and secretory organ. White adipocytes release a multiplicity of protein hormones, signals and factors, termed adipokines, with an extensive range of physiological actions. Foremost among these various adipokines is the cytokine-like hormone, leptin, which is synthesized predominantly in white fat. Leptin plays a critical role in the control of appetite and energy balance, with mutations in the genes encoding the hormone or its receptor leading to profound obesity in both rodents and man. Leptin regulates appetite primarily through an interaction with hypothalamic neuroendocrine pathways, inhibiting orexigenic peptides such as neuropeptide Y and orexin A, and stimulating anorexigenic peptides such as proopiomelanocortin. White fat also secretes several putative appetite-related adipokines, which include interleukin-6 and adiponectin, but whether these are indeed significant signals in the regulation of food intake has not been established. Through leptin and the other adipokines it is evident that adipose tissue communicates extensively with other organs and plays a pervasive role in metabolic homeostasis.     

Effects of fructose and glucose on plasma leptin, insulin, and insulin resistance in lean and VMH-lesioned obese rats

To determine the influence of dietary fructose and glucose on circulating leptin levels in lean and obese rats, plasma leptin concentrations were measured in ventromedial hypothalamic (VMH)-lesioned obese and sham-operated lean rats fed either normal chow or fructose- or glucose-enriched diets (60% by calories) for 2 wk. Insulin resistance was evaluated by the steady-state plasma glucose method and intravenous glucose tolerance test. In lean rats, glucose-enriched diet significantly increased plasma leptin with enlarged parametrial fat pad, whereas neither leptin nor fat-pad weight was altered by fructose. Two weeks after the lesions, the rats fed normal chow had marked greater body weight gain, enlarged fat pads, and higher insulin and leptin compared with sham-operated rats. Despite a marked adiposity and hyperinsulinemia, insulin resistance was not increased in VMH-lesioned rats. Fructose brought about substantial insulin resistance and hyperinsulinemia in both lean and obese rats, whereas glucose led to rather enhanced insulin sensitivity. Leptin, body weight, and fat pad were not significantly altered by either fructose or glucose in the obese rats. These results suggest that dietary glucose stimulates leptin production by increasing adipose tissue or stimulating glucose metabolism in lean rats. Hyperleptinemia in VMH-lesioned rats is associated with both increased adiposity and hyperinsulinemia but not with insulin resistance. Dietary fructose does not alter leptin levels, although this sugar brings about hyperinsulinemia and insulin resistance, suggesting that hyperinsulinemia compensated for insulin resistance does not stimulate leptin production.     

Desipramine inhibits histamine H1 receptor-induced Ca2+ signaling in rat hypothalamic cells

The hypothalamus in the brain is the main center for appetite control and integrates signals from adipose tissue and the gastrointestinal tract. Antidepressants are known to modulate the activities of hypothalamic neurons and affect food intake, but the cellular and molecular mechanisms by which antidepressants modulate hypothalamic function remain unclear. Here we have investigated how hypothalamic neurons respond to treatment with antidepressants, including desipramine and sibutramine. In primary cultured rat hypothalamic cells, desipramine markedly suppressed the elevation of intracellular Ca(2+) evoked by histamine H1 receptor activation. Desipramine also inhibited the histamine-induced Ca(2+) increase and the expression of corticotrophin-releasing hormone in hypothalamic GT1-1 cells. The effect of desipramine was not affected by pretreatment with prazosin or propranolol, excluding catecholamine reuptake activity of desipramine as an underlying mechanism. Sibutramine which is also an antidepressant but decreases food intake, had little effect on the histamine-induced Ca(2+) increase or AMP-activated protein kinase activity. Our results reveal that desipramine and sibutramine have different effects on histamine H1 receptor signaling in hypothalamic cells and suggest that distinct regulation of hypothalamic histamine signaling might underlie the differential regulation of food intake between antidepressants.