leptin and Insulinoma

Resistin promotes hypothalamic neuroinflammation and insulin resistance through Toll like receptor 4 (TLR4), this hormone is thought to be a link between obesity and insulin-resistance. Indeed, resistin plasma levels are higher in obese and insulin resistant subjects. However, the impact of maternal resistin on the predisposition of offspring to hypothalamic neuroinflammation is unknown. Here, female mice were treated with resistin during gestation/lactation periods, then hypothalamic neuroinflammation was investigated in male offspring at p28 and p90. At p28, resistin increased the expression of inflammation markers (IL6, TNFα and NFκB) and TLR4 in the hypothalamus and decreased both hypothalamic insulin and leptin receptors' expression. The hypothalamic up-regulation IL6, TNFα and TLR4 was sustained until p90 promoting most likely hypothalamic inflammation. Maternal resistin also increased IL6 and TNFα in the adipose tissue of offspring at p90 associated with a higher body weight gain. In contrast, liver and muscle were not affected. These findings reveal that the augmentation of maternal resistin during gestation and lactation promotes hypothalamic and adipose tissue inflammation of offspring as evidenced by sustained increase of inflammation markers from weaning to adulthood. Thus, maternal resistin programs offspring hypothalamic and adipose tissue inflammation predisposing then offspring to body weight gain.

Topics: Animals; Animals, Newborn; Body Weight; Female; Glucose Intolerance; Hypothalamus; Inflammation; Inflammation Mediators; Insulin Resistance; Insulinoma; Lactation; Leptin; Male; Maternal Nutritional Physiological Phenomena; Mice; Pregnancy; Resistin; Weaning; Weight Gain

Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca²(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.

Topics: Animals; Blotting, Northern; Blotting, Western; Calcium; Cell Line, Tumor; Cells, Cultured; Cytosol; Gene Expression Regulation; HeLa Cells; Hep G2 Cells; Humans; Immunohistochemistry; Insulin; Insulin Secretion; Insulin-Secreting Cells; Insulinoma; Leptin; Microscopy, Confocal; Pancreas; Protein Phosphatase 1; Rats; Reverse Transcriptase Polymerase Chain Reaction

Leptin acts not only on hypothalamic centers to control food intake but has additional functions in peripheral tissues, e.g. inhibition of insulin secretion from pancreatic islets. The leptin receptor (LEPRb) is a class I cytokine receptor that mediates activation of STAT transcription factors. In this study, we characterise the regulation of inflammation-related genes by leptin in insulinoma cells and compare the effect of transcriptional regulation by leptin with that of other cytokines.. We have used RINm5F insulinoma cells as a model system for a peripheral target cell of leptin. Six transcripts encoding inflammation-related proteins were found to be upregulated by activation of LEPRb, namely lipocalin-2, pancreatitis-associated protein, preprotachykinin-1, fibrinogen-beta, tissue-type plasminogen activator (tPA) and manganese-dependent superoxide dismutase (MnSOD). Four of these transcripts (fibrinogen-beta, lipocalin-2, tPA, MnSOD) were also induced by the proinflammatory cytokine interleukin-1beta (IL-1beta). Interferon-gamma alone had no effect on the leptin-induced transcripts but enhanced the upregulation by IL-1beta of lipocalin-2, tPA and MnSOD mRNA levels. Experiments with LEPRb point mutants revealed that the upregulation of the inflammation-related genes depended on the presence of tyrosine-1138 which mediates the activation of the transcription factors STAT1 and STAT3. Reporter gene assays showed that leptin induced the expression of preprotachykinin-1 and lipocalin-2 on the level of promoter regulation. Finally, leptin treatment increased caspase 3-like proteolytic activity in RINm5F cells.. The present data show that leptin induces a cytokine-like transcriptional response in RINm5F cells, consistent with the proposed function of leptin as a modulator of immune and inflammatory responses.

Topics: Animals; Carrier Proteins; Caspases; Cell Line, Tumor; Cricetinae; Gene Expression Regulation, Neoplastic; Genes, Reporter; Inflammation; Insulinoma; Interferon-gamma; Interleukin-1beta; Leptin; Lipocalin-2; Lipocalins; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Pancreatitis-Associated Proteins; Point Mutation; Promoter Regions, Genetic; Protein Precursors; Rats; Receptors, Cell Surface; Receptors, Leptin; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Tachykinins; Tyrosine

Cannabinoid CB(1) receptor blockade decreases weight and hyperinsulinemia in obese animals and humans in a way greatly independent from food intake.. The objective of this study was to investigate the regulation and function of the endocannabinoid system in adipocytes and pancreatic beta-cells.. Mouse 3T3-F442A adipocytes and rat insulinoma RIN-m5F beta-cells, pancreas and fat from mice with diet-induced obesity, visceral and sc fat from patients with body mass index equal to or greater than 30 kg/m(2), and serum from normoglycemic and type 2 diabetes patients were studied.. Endocannabinoid enzyme and adipocyte protein expression, and endocannabinoid and insulin levels were measured.. Endocannabinoids are present in adipocytes with levels peaking before differentiation, and in RIN-m5F beta-cells, where they are under the negative control of insulin. Chronic treatment of adipocytes with insulin is accompanied by permanently elevated endocannabinoid signaling, whereas culturing of RIN-m5F beta-cells in high glucose transforms insulin down-regulation of endocannabinoid levels into up-regulation. Epididymal fat and pancreas from mice with diet-induced obesity contain higher endocannabinoid levels than lean mice. Patients with obesity or hyperglycemia caused by type 2 diabetes exhibit higher concentrations of endocannabinoids in visceral fat or serum, respectively, than the corresponding controls. CB(1) receptor stimulation increases lipid droplets and decreases adiponectin expression in adipocytes, and it increases intracellular calcium and insulin release in RIN-m5F beta-cells kept in high glucose.. Peripheral endocannabinoid overactivity might explain why CB(1) blockers cause weight-loss independent reduction of lipogenesis, of hypoadiponectinemia, and of hyperinsulinemia in obese animals and humans.

Topics: 3T3 Cells; Adipocytes; Adiponectin; Adipose Tissue; Adult; Animals; Cannabinoid Receptor Modulators; Cell Line, Tumor; Diabetes Mellitus, Type 2; Endocannabinoids; Epididymis; Female; Gene Expression; Glucose; Homeostasis; Humans; Hyperglycemia; Insulin; Insulinoma; Intra-Abdominal Fat; Islets of Langerhans; Leptin; Male; Mice; Obesity; Pancreas; Pancreatic Neoplasms; PPAR gamma; Rats; Signal Transduction

Leptin inhibits insulin secretion and preproinsulin gene expression in pancreatic beta-cells, but signal transduction pathways and molecular mechanisms underlying this effect are poorly characterized. In this study, we analyzed leptin-mediated signal transduction and preproinsulin gene regulation at the molecular level in pancreatic beta-cells. Leptin stimulation led to janus kinase (JAK)2-dependent phosphorylation and nuclear translocation of the transcription factors signal transducer and activator of transcription (STAT)3 and STAT5b in INS-1 beta-cells. Leptin also induced mRNA expression of the JAK-STAT inhibitor suppressor of cytokine signaling (SOCS)3 in INS-1 beta-cells and human pancreatic islets in vitro and in pancreatic islets of ob/ob mice in vivo. Transcriptional activation of the rat SOCS3 promoter by leptin was observed with concomitant leptin-induced STAT3 and STAT5b DNA binding to specific promoter regions. Unexpectedly, SOCS3 inhibited both basal and STAT3/5b-dependent rat preproinsulin 1 gene promoter activity in INS-1 cells. These results suggest that SOCS3 represents a transcriptional inhibitor of preproinsulin gene expression, which is induced by leptin through JAK-STAT3/5b signaling in pancreatic beta-cells. In conclusion, although SOCS3 is believed to be a negative feedback regulator of JAK-STAT signaling, our findings suggest involvement of SOCS3 in a direct gene regulatory pathway downstream of leptin-activated JAK-STAT signaling in pancreatic beta-cells.

Topics: Animals; Base Sequence; Cell Line, Tumor; DNA Primers; Gene Expression Regulation; Genes, Reporter; Immunohistochemistry; Insulin; Insulinoma; Islets of Langerhans; Leptin; Pancreatic Neoplasms; Proinsulin; Protein Precursors; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; STAT3 Transcription Factor; STAT5 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

The effect of treatment with a 0.03% fatty acid (FA) cocktail on leptin-receptor-mediated STAT (signal transducers and activators of transcription) activation in the rat insulinoma cell line BRIN-BD11 was investigated. Leptin (10 nM) stimulated the tyrosine phosphorylation of STAT3 and STAT5b. Acute treatment with FAs prevented leptin-stimulated STAT3 tyrosine phosphorylation and significantly raised basal STAT5 phosphorylation. A chronic treatment (5 days) of BRIN-BD11 cells with FAs similarly attenuated leptin-stimulated STAT tyrosine phosphorylation. Chronic FA treatment also attenuated prolactin-stimulated STAT5b tyrosine phosphorylation but not interleukin-6-stimulated STAT3 tyrosine phosphorylation, suggesting that the effect is receptor/ligand specific. TaqMan analysis of gene expression following chronic FA treatment showed neither a decrease in the amount of leptin receptor (Ob-R) mRNA, nor an increase in the negative regulators of STAT signalling, SOCS3 (suppressors of cytokine signalling) or cytokine inducible sequence (CIS). These data demonstrate that FAs modulate leptin and prolactin signalling in beta-cells, implying that high levels of circulating FAs present in obese individuals affect the action of selective cytokines in beta-cell function.

Topics: Animals; Blotting, Western; Carrier Proteins; DNA-Binding Proteins; Fatty Acids; Immediate-Early Proteins; Insulinoma; Interleukin-6; Islets of Langerhans; Leptin; Milk Proteins; Phosphorylation; Precipitin Tests; Prolactin; Protein Isoforms; Proteins; Rats; Receptors, Cell Surface; Receptors, Leptin; Repressor Proteins; Signal Transduction; STAT3 Transcription Factor; STAT5 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Tyrosine

Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein. Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system. The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins.

Topics: Adipocytes; Amino Acid Sequence; Animals; Carrier Proteins; Cell Division; DNA, Complementary; Escherichia coli; Flow Cytometry; Gene Expression Regulation; Genes, Reporter; Humans; Inclusion Bodies; Insulinoma; Leptin; Molecular Sequence Data; Protein Folding; Protein Renaturation; Rats; Receptors, Cell Surface; Receptors, Leptin; Recombinant Fusion Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thrombin; Tumor Cells, Cultured

Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain.. This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046-38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain.. The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1.. In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance.

Topics: Animals; Carrier Proteins; Cell Line; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Gene Expression; Humans; Hyperinsulinism; Insulin; Insulinoma; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Kidney; Leptin; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Receptor, Insulin; Receptors, Cell Surface; Receptors, Leptin; Signal Transduction; Transfection; Tumor Cells, Cultured

The mechanism by which leptin increases ATP-sensitive K(+) (K(ATP)) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of K(ATP) channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) mimicked the effect of leptin by increasing K(ATP) channel activity in whole-cell and inside-out current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P(3), from increasing K(ATP) channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and K(ATP) channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P(3) as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of K(ATP) channels in this cell line. Although PtdIns(3,4,5)P(3) appears a plausible and attractive candidate for the messenger that couples K(ATP) channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P(3) demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P(3). Possible mechanisms to explain the involvement of PI 3-kinases in K(ATP) channel regulation are discussed.

Topics: Adenosine Triphosphate; Animals; Insulinoma; Leptin; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Potassium Channels; Rats; Signal Transduction; Tumor Cells, Cultured

1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 microM) prevented KATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 microg ml-1) or cytochalasin B (10 microM) to intact cells or inside-out membrane patches also increased KATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 microM) had no effect on KATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of KATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates KATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.

Topics: Actins; Adenosine Triphosphate; Animals; Cell Line; Cytochalasin B; Cytoskeleton; Deoxyribonuclease I; Insulinoma; Leptin; Microscopy, Fluorescence; Patch-Clamp Techniques; Phalloidine; Phosphatidylinositol Phosphates; Phosphoinositide-3 Kinase Inhibitors; Potassium; Potassium Channels; Rats; Signal Transduction

To further clarify the relationship between insulin and leptin, time course changes in plasma glucose, serum insulin and leptin levels were analyzed after subcutaneous administration of 100 microg octreotide acetate in two insulinoma subjects. Octreotide acetate induced a prompt decrease in serum insulin level, accompanied with an increase in plasma glucose in both patients. Following the decrease in serum insulin level, serum leptin concentrations were profoundly decreased by 66% and 44%, 8-12 hrs after octreotide injection; that is, the concentrations decreased from 41.1 to 13.8 ng/ml in patient 1, and from 17.5 to 9.8 ng/ml in patient 2. Daily profiles of plasma glucose, serum insulin and leptin without octreotide administration did not show such alterations in these indexes in patient 1. These data show that circulating leptin may be susceptible to decline dependent on the decrease in serum insulin, suggesting that insulin plays a crucial role in the maintenance of leptin secretion in humans.

Topics: Blood Glucose; Female; Hormones; Humans; Insulin; Insulinoma; Leptin; Male; Middle Aged; Octreotide

Inferential studies suggest that circulating insulin concentrations positively regulate leptin secretion by adipocytes. In humans, however, insulin requires prolonged periods of time, and relatively artificial set-ups before a relationship with leptin can be observed. In the present work, serum leptin concentrations were measured in five patients with insulinoma before and one month after surgery and in five control subjects matched by sex and body mass index (BMI). The control subjects presented a mean serum leptin concentration of 6.7+/-1.5 microg/l and a BMI of 24.9+/-1.1. The mean serum leptin concentration in patients with insulinoma was 11.8+/-3.1 microg/l (P < 0.05 vs controls), with a BMI of 26.3+/-1.9. After surgery, there was a non-significant reduction in BMI (25.8+/-1.7), and a clear reduction in serum leptin concentration (5.6+/-2.4 microg/l, P < 0.05 vs pre surgical values and no difference vs control subjects). The fasting area under the curve (AUC) of insulin concentration (in mU/l per 120 min) before surgery was 14421+/-4981 and after surgery was 1306-/+171 (P < 0.05). Before surgery, serum leptin concentrations significantly correlated with BMI (r = 0.71) and AUC of insulin (r = 0.82), a correlation that was lost after surgery. In conclusion, serum leptin concentrations are significantly elevated in patients with chronically high insulin levels due to insulinoma. After surgical treatment and normalization of insulin values, leptin levels return to normal.

Topics: Adult; Body Mass Index; Fasting; Female; Humans; Insulin; Insulinoma; Leptin; Male; Middle Aged; Osmolar Concentration; Pancreatic Neoplasms; Postoperative Period; Proteins; Reference Values

1. Using whole-cell and cell-attached recording configurations, the role of phosphorylation in leptin activation of ATP-sensitive K+ (KATP) channels was examined in the rat CRI-G1 insulinoma cell line. 2. Whole-cell current clamp recordings demonstrated that, following dialysis with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP; 3-5 mM), the leptin-induced hyperpolarization and increase in K+ conductance were completely inhibited. 3. Under current clamp conditions, application of the broad-spectrum protein kinase inhibitor H-7 (10 microM) had no effect on the resting membrane potential or slope conductance of CRI-G1 insulinoma cells and did not occlude the actions of leptin. 4. Application of the tyrosine kinase inhibitors genistein (10 microM), tyrphostin B42 (10 microM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell-attached recordings, the presence of tyrphostin B42 (10 microM) in the pipette solution activated tolbutamide-sensitive KATP channels in CRI-G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect. 5. The serine/threonine-specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 microM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 microM) prevented the actions of both leptin and tyrphostin B42. 6. In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3-kinase (PI 3-kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Androstadienes; Animals; Enzyme Inhibitors; Insulinoma; Leptin; Nitriles; Pancreatic Neoplasms; Phosphoric Monoester Hydrolases; Phosphorylation; Potassium Channels; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proteins; Rats; Tumor Cells, Cultured; Tyrosine; Tyrphostins; Wortmannin

Leptin, a hormone secreted by adipocytes, decreases food intake and increases energy expenditure. The role of insulin in the regulation of leptin secretion is poorly understood and is still a topic of debate. Insulin increases leptin mRNA synthesis in rodents, but in humans, the available data are discordant. To investigate the role of chronic hyperinsulinemia in the regulation of plasma leptin concentrations, we studied 13 patients with surgically confirmed insulinoma before and after tumor removal, along with 15 healthy control subjects matched for sex, age, and BMI. Immunoreactive plasma leptin levels were measured by radioimmunoassay; leptin mRNA levels were also determined by reverse transcription-competitive polymerase chain reaction in a subgroup of six patients with insulinoma and six control subjects. All determinations were made with subjects in the fasting state. Plasma leptin concentrations correlated positively with leptin mRNA levels (r = 0.880, P < 0.001). Leptin levels, both plasma protein and mRNA, were significantly higher in the insulinoma patients than in the control subjects (plasma protein: 17.5 +/- 3.6 vs. 2.9 +/- 0.4 ng/ml, respectively, P < 0.001; mRNA: 0.98 +/- 0.33 vs. 0.19 +/- 0.064 amol/microg RNA, respectively, P < 0.05), and they correlated positively with fasting plasma insulin levels in the patients with insulinoma (plasma protein: r = 0.686, P < 0.01; mRNA: 0.796, P < 0.05). Finally, removal of the insulin-secreting tumor was followed by the normalization of plasma leptin levels. In summary, in patients with insulinoma, 1) plasma leptin levels and leptin mRNA are elevated; 2) a direct relationship exists between leptin, both circulating protein and mRNA, and insulin concentrations; and 3) plasma leptin returns to normal levels after tumor removal. These data, therefore, support a role for insulin in the chronic regulation of leptin gene expression.

Topics: Adipose Tissue; Adult; Blood Glucose; Fasting; Female; Gene Expression; Humans; Hyperinsulinism; Insulin; Insulinoma; Leptin; Male; Middle Aged; Obesity; Pancreatic Neoplasms; Polymerase Chain Reaction; Proteins; RNA-Directed DNA Polymerase; RNA, Messenger

Leptin is a peptide secreted by white adipose tissue which has been shown to have a major influence on body weight regulation, while animal studies have revealed widespread interconnections between leptin and other endocrine systems, especially with insulin. However, its acute regulation has been little studied in the human. We have therefore investigated the effect of a 1000 kcal meal and fasting on the levels of leptin, insulin and cortisol, in both normal and obese subjects.. We have studied the effect of food and fasting on circulating leptin levels in 20 subjects of normal body mass index (BMI range 18-25) and in a group of 12 moderately-severely obese subjects (BMI range 34-61). We also studied the effect of food and fasting in a patient both before and after the successful removal of a pancreatic insulinoma as a model of excess insulin secretion.. Mean leptin levels were significantly higher in the obese than in the lean group (42.7 +/- 3.41 vs 5.35 +/- 1.55 micrograms/l, mean +/- SEM; P < 0.001), and showed a positive correlation with body mass index (r = +0.71; P < 0.001). Frequent (every 20 minutes) sampling for 3 hours after food did not show any acute changes in circulating leptin levels. On the fasting day we observed a small but significant fall in circulating leptin levels in the last 4 hours of a 20-hour fast in our subjects as a group (92 +/- 0.03% of basal, P = 0.03); however, in the lean subjects the fall was greater (86 +/- 0.04% of basal, P = 0.02) than in the obese, where it did not reach statistical significance (96 +/- 0.05% of basal). Pre-meal and peak insulin levels showed a positive correlation with circulating mean leptin levels (r = +0.65; P < 0.001 and r = +0.78; P < 0.001, respectively) in all subjects, while pre-meal and peak serum cortisol levels showed an inverse relation with leptin levels (r = -0.53; P = 0.002 and r = -0.41; P = 0.02, respectively); this effect was independent of BMI in the obese subjects. In the patient with the insulinoma the markedly elevated insulin and leptin levels measured before the operation returned to normal after removal of the tumour, in accord with reports of experimental animal data that long-term insulin excess per se is associated with increased circulating leptin concentrations.. Leptin is a robust indicator of BMI and insulin levels, both basal and stimulated, but does not change acutely following food. Fasting causes a proportionately greater decline in leptin levels in lean subjects than in obese subjects. Circulating leptin is inversely correlated with the activity of the hypothalamo-pituitary-adrenal axis: whether this is a direct influence of leptin on hypothalamo-pituitary-adrenal activity, or whether both are indirect indicators of body fat stores, requires further investigation.

Topics: Adult; Biomarkers; Eating; Female; Humans; Hydrocortisone; Insulin; Insulinoma; Leptin; Male; Obesity; Pancreatic Neoplasms; Pituitary-Adrenal System; Postprandial Period; Proteins

Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism.

Topics: Animals; Calcium; Carrier Proteins; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Leptin; Male; Mice; Mice, Mutant Strains; Mice, Obese; Obesity; Proteins; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Leptin; Second Messenger Systems; Tumor Cells, Cultured