RESUMO
Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.
Assuntos
Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Proteínas/análise , Proteínas/metabolismo , Receptores de Superfície Celular , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Leptina , Masculino , Obesidade/sangue , Obesidade/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas/fisiologia , Receptores para Leptina , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismoRESUMO
We studied 24-h profiles of circulating leptin levels using a sensitive and specific RIA in lean controls and obese subjects with or without non-insulin-dependent diabetes mellitus (NIDDM) during normal routine activity. Serum leptin levels were significantly higher in obese (41.7 +/- 9.0 ng/ml; n = 11) and obese NIDDM (30.8 +/- 6.7; n = 9) subjects compared with those in lean controls (12.0 +/- 4.4, n = 6). In all the three groups, serum leptin levels were highest between midnight and early morning hours and lowest around noon to midafternoon. The nocturnal rise in leptin levels was significant when data were analyzed by ANOVA (lean: F = 3.17, P < 0.0001, n = 4; obese: F = 2.02, P < 0.005, n = 11; and obese NIDDM: F = 4.9, P < 0.0001, n = 5). The average circadian amplitude between acrophase and nadir was 75.6% in lean, 51.7%, in obese and 60.7% in obese NIDDM groups, respectively. No significant correlations (P > 0.05) were observed between circulating levels of leptin and either insulin or glucose levels in any of the 20 subjects studied for 24-h profiles. The nocturnal rise in leptin observed in the present study resembles those reported for prolactin, thyroid-stimulating hormone, and free fatty acids. We speculate that the nocturnal rise in leptin could have an effect in suppressing appetite during the night while sleeping.
Assuntos
Ritmo Circadiano , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , Proteínas/metabolismo , Adulto , Feminino , Humanos , Leptina , Masculino , Pessoa de Meia-Idade , Proteínas/genética , RNA Mensageiro/análiseRESUMO
As one of the postulated roles of the ob gene product, leptin, is regulation of energy balance and preservation of normal body composition, we investigated the effect of acute and chronic calorie excess (weight gain) on serum leptin in humans. Two protocols were employed: 1) acute (12-h) massive (120 Cal/kg) voluntary overfeeding of eight healthy individuals; and 2) chronic overfeeding to attain 10% weight gain, with its subsequent maintenance for additional 2 weeks, involving six normal males. In the acute experiments (protocol 1), circulating leptin rose by 40% over baseline (P < 0.01) during the final hours of overfeeding; this increase persisted until the next morning. At the point of achievement and the 2-week maintenance of 10% weight gain (protocol 2), a more than 3-fold rise in the basal leptin concentration was observed (P < 0.01). A direct linear relationship was found between the magnitude of the leptin response to weight gain and the percent gain of body fat (r = 0.88; P < 0.01). In summary, 1) in contrast to normal food intake (8), short term massive overfeeding is associated with a moderate elevation of circulating leptin levels that persists until next feeding cycle is initiated; and 2) a 10% weight gain causes different changes in the body composition, and the resulting rise in circulating leptin parallels the increase in the percentage of body fat. In conclusion, these studies document acute elevation of leptin in response to positive energy balance and suggest that developing resistance to leptin is associated with bigger fat deposition during weight gain in humans.
Assuntos
Proteínas/metabolismo , Aumento de Peso/fisiologia , Tecido Adiposo/anatomia & histologia , Adulto , Glicemia/metabolismo , Regulação para Baixo , Ingestão de Energia , Metabolismo Energético , Feminino , Humanos , Insulina/sangue , Leptina , Masculino , Regulação para CimaRESUMO
Insulin resistance in adipose tissue in human obesity is associated with increased protein-tyrosine phosphatase (PTPase) activity and elevated levels of the PTPases leukocyte common antigen-related PTPase (LAR) and PTP1B. To determine whether the improved insulin sensitivity associated with weight loss in obese subjects is accompanied by reversible changes in PTPases, we obtained subcutaneous adipose tissue from seven obese subjects (mean body mass index [BMI], 40.4 kg/m2) before and after a loss of 10% of body weight and again after a 4-week maintenance period. Weight loss was accompanied by an 18.5% decrease in overall adipose tissue PTPase activity (P = .015) that was further reduced to 22.3% of the control value (P = .005) at the end of the maintenance period. By immunoblot analysis, the abundance of LAR was decreased by 21% (P = .04) and abundance of PTP1B was decreased by 40% (P < .004) after the initial weight loss, and the decreases persisted during the maintenance period. Enhanced insulin sensitivity following weight loss, evident from a 26% decrease in fasting insulin levels (P < .05), was also closely correlated with the reduction in the abundance of both LAR (R2 = .80, P < .01) and PTP1B (R2 = .64, P = .03). These results support the hypothesis that LAR and PTP1B may be reversibly involved in the pathogenesis of insulin resistance, and may be therapeutic targets in insulin-resistant states.
Assuntos
Adipócitos/enzimologia , Tecido Adiposo/enzimologia , Glicemia/metabolismo , Insulina/sangue , Obesidade/fisiopatologia , Proteínas Tirosina Fosfatases/metabolismo , Redução de Peso , Índice de Massa Corporal , Jejum , Feminino , Humanos , Insulina/metabolismo , Secreção de Insulina , Antígenos Comuns de Leucócito/metabolismo , Masculino , Obesidade/sangue , Obesidade/enzimologiaRESUMO
OBJECTIVE: This study assessed acceptability of a new 3.0-mL prefilled insulin pen device, the Humulin/Humalog Pen, as a method of delivering human insulin. Secondary objectives were to determine whether the pen device might facilitate initiation of insulin therapy in patients currently receiving oral antihyperglycemic agents and to monitor the safety of this pen device in clinical practice. BACKGROUND: For both the patient and health care provider, significant negative perceptions of the use of insulin therapy persist, including patient inconvenience, social stigma from insulin injections, and insufficient time for the provider to train the patient. METHODS: This 6-week, open-label, noncomparative study was conducted at 33 centers in the United States. Patients with type 1 or type 2 diabetes treated with insulin therapy or oral antihyperglycemic agents were enrolled in the study. Before the study, 62% (194 patients) had used a syringe and vial for insulin injection, 28% (87 patients) had used an insulin pen device, and 10% (30 patients) were insulin-naive. Prior therapy was unknown in 1% (4 patients). Patients used the Humulin/Humalog Pen for > or = 1 injection of insulin daily for 6 weeks. At the beginning and end of the study, patients completed a questionnaire designed to elicit their perceptions of the Humulin/Humalog Pen; physicians completed a questionnaire at the end of the study. Frequencies and percentages of all categoric responses were calculated and summarized. RESULTS: A total of 315 patients (136 type 1, 179 type 2 diabetes) were enrolled. Of the 299 patients who completed questionnaires at the end of the study, 76% (226 patients) were somewhat or extremely satisfied with the pen, 78% (234 patients) probably or definitely would continue to use the pen, and 80% (239 patients) probably or definitely would recommend the pen to others. Of the 33 physicians who completed questionnaires at the end of the study, 97% (32) thought that the pen was better overall compared with a vial and syringe, 88% (29) thought that it took less time to teach patients to use the pen, and 73% (24) thought that it took less time to initiate insulin therapy with the pen. CONCLUSIONS: The Humulin/Humalog Pen had an acceptable safety profile and was well accepted by patients and physicians.
Assuntos
Atitude do Pessoal de Saúde , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Injeções Intramusculares/instrumentação , Insulina/administração & dosagem , Satisfação do Paciente , Médicos , Adulto , Idoso , Feminino , Humanos , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
UNLABELLED: We investigated whether weight gain alters insulin sensitivity and leptin levels in physically active individuals. Six (5 males and 1 female; age 26.6+/-1.0 years; BMI 21.5+/-0.9, body fat 17.4+/-2.2%) healthy individuals were enrolled in an overfeeding study (caloric surplus 22.5-26.5 kcal/kg/day) to achieve up to 10% weight gain over 4-6 week period with subsequent weight maintenance over additional 2 weeks. The participants were requested to maintain their previous physical activity which in all of them included 45-60 min training sessions at the gym 2-3 times/week. RESULTS: BMI increased to 23.4+/-0.9 (4.4 kg weight gain; p<0.05) and body fat to 21.0+/-2.8% (p < 0.05) over the period of active weight gain and remained stable over the two week period of weight maintenance; fasting plasma glucose and serum insulin remained unchanged; serum leptin nearly doubled (3.8+/-1.0 vs 6.4+/-1.9 ng/ mL; p < 0.05); insulin sensitivity, when expressed per kg of the total body (11.1+/-1.6 vs 12.4+/-2.1 mg/kg/min; p = NS), and lean body mass (13.4+/-1.9 vs 15.7+/-2.6 mg/kgLBM/min; p = NS), did not decrease after weight gain. On the contrary, insulin action had improved in 5 out of 6 individuals. In conclusion, the data presented in this preliminary report indicate that a small weight gain due to overfeeding in lean, healthy, physically active individuals is associated with rise in circulating leptin levels but not with worsening of insulin action.
Assuntos
Exercício Físico , Resistência à Insulina , Aumento de Peso , Tecido Adiposo , Adulto , Glicemia/metabolismo , Composição Corporal , Índice de Massa Corporal , Ingestão de Energia , Jejum , Feminino , Técnica Clamp de Glucose , Humanos , Insulina/sangue , Leptina , Masculino , Proteínas/metabolismoRESUMO
In the present study we have examined the effect of dexamethasone on ob gene mRNA expression and leptin release from isolated human subcutaneous adipocytes. Dexamethasone stimulated leptin release from cultured adipocytes in a time- and dose-dependent manner. A two-fold increase in leptin release was detectable by 36 h of treatment with 10(-7) M dexamethasone. Leptin release was preceded by a significant 83 +/- 30% increase in ob mRNA after 24 h exposure to the compound. Co-incubation of cells with dexamethasone (10(-7) M) and insulin (10(-7) or 10(-9) M) completely blocked the dexamethasone-stimulated increase in ob mRNA and leptin release. These data demonstrate that insulin and glucocorticoids regulate leptin synthesis and release from human adipocytes in vitro.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Dexametasona/farmacologia , Insulina/farmacologia , Proteínas/metabolismo , Células Cultivadas , Humanos , Leptina , Obesidade/genética , Proteínas/genética , RNA Mensageiro/metabolismoRESUMO
In the present study mRNA from the subcutaneous adipose tissue of 68 obese (defined as a body mass index > or = 27.3 for men and > or = 27.8 for women) and 38 lean subjects was screened for mutations in the ob gene coding region. No mutations in the coding region of the human ob gene were detected using Conformation Sensitive Gel Electrophoresis in 105 subjects. A first base substitution (G to A) was detected in one individual, which changed a valine to a methionine at position 94. The mRNA of all subjects contained the codon for glutamine-49, ruling out the possibility of a splice defect occurring during the removal of intron 2. These observations suggest that defects in the ob gene sequence itself are not the primary cause of obesity in humans.
Assuntos
Tecido Adiposo/metabolismo , Processamento Alternativo , Variação Genética , Obesidade/genética , Mutação Puntual , Proteínas/genética , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Glutamina , Humanos , Íntrons , Leptina , Masculino , Metionina , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Obesidade/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Valores de Referência , Mapeamento por Restrição , Caracteres SexuaisRESUMO
BACKGROUND: A receptor for leptin has been cloned from the choroid plexus, the site of cerebrospinal-fluid (CSF) production and the location of the blood/cerebrospinal-fluid barrier. Thus, this receptor might serve as a transporter for leptin. We have studied leptin concentrations in serum and (CSF). METHODS AND FINDINGS: We demonstrated by radioimmunoassay and western blot the presence of leptin in human CSF. We then measured leptin in CSF and serum in 31 individuals with a wide range of bodyweight. Mean serum leptin was 318% higher in 8 obese (40.2 [SE 8.6] ng/mL) than in 23 lean individuals (9.6 [1.5] ng/mL, p < 0.0005). However, the CSF leptin concentration in obese individuals (0.337 [0.04] ng/mL) was only 30% higher than in lean people (0.259 [0.26] ng/mL, p < 0.1). Consequently, the leptin CSF/serum ratio in lean individuals (0.047 [0.010]) was 4.3-fold higher than that in obese individuals (0.011 [0.002], p < 0.05). The relation between CSF leptin and serum leptin was best described by a logarithmic function (r = 0 x 52, p < 0.01). INTERPRETATION: Our data suggest that leptin enters the brain by a saturable transport system. The capacity of leptin transport is lower in obese individuals, and may provide a mechanism for leptin resistance.
Assuntos
Obesidade/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular , Transporte Biológico , Barreira Hematoencefálica , Western Blotting , Índice de Massa Corporal , Proteínas de Transporte/metabolismo , Feminino , Humanos , Leptina , Masculino , Pessoa de Meia-Idade , Proteínas/fisiologia , Radioimunoensaio , Receptores de Citocinas/metabolismo , Receptores para LeptinaRESUMO
BACKGROUND: Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiologic actions of leptin in humans. METHODS: Using a newly developed radioimmunoassay, wer measured serum concentrations of leptin in 136 normal-weight subjects and 139 obese subjects (body-mass index, > or = 27.3 for men and > or = 27.8 for women; the body-mass index was defined as the weight in kilograms divided by the square of the height in meters). The measurements were repeated in seven obese subjects after weight loss and during maintenance of the lower weight. The ob messenger RNA (mRNA) content of adipocytes was determined in 27 normal-weight and 27 obese subjects. RESULTS: The mean (+/- SD) serum leptin concentrations were 31.3 +/- 24.1 ng per milliliter in the obese subjects and 7.5 +/- 9.3 ng per milliliter in the normal-weight subjects (P < 0.001). There was a strong positive correlation between serum leptin concentrations and the percentage of body fat (r = 0.85, P < 0.001). The ob mRNA content of adipocytes was about twice as high in the obese subjects as in the normal-weight subjects (P < 0.001) and was correlated with the percentage of body fat (r = 0.68, P < 0.001) in the 54 subjects in whom it was measured. In the seven obese subjects studied after weight loss, both serum leptin concentrations and ob mRNA content of adipocytes declined, but these measures increased again during the maintenance of the lower weight. CONCLUSIONS: Serum leptin concentrations are correlated with the percentage of body fat, suggesting that most obese persons are insensitive to endogenous leptin production.