RESUMEN
Low-grade inflammation associated with type 2 diabetes (T2DM) is postulated to exacerbate insulin resistance. We report that serum levels, as well as IL-13 secreted from cultured skeletal muscle, are reduced in T2DM vs. normal glucose-tolerant (NGT) subjects. IL-13 exposure increases skeletal muscle glucose uptake, oxidation, and glycogen synthesis via an Akt-dependent mechanism. Expression of microRNA let-7a and let-7d, which are direct translational repressors of the IL-13 gene, was increased in skeletal muscle from T2DM patients. Overexpression of let-7a and let-7d in cultured myotubes reduced IL-13 secretion. Furthermore, basal glycogen synthesis was reduced in cultured myotubes exposed to an IL-13-neutralizing antibody. Thus, IL-13 is synthesized and released by skeletal muscle through a mechanism involving let-7, and this effect is attenuated in skeletal muscle from insulin-resistant T2DM patients. In conclusion, IL-13 plays an autocrine role in skeletal muscle to increase glucose uptake and metabolism, suggesting a role in glucose homeostasis in metabolic disease.
Asunto(s)
Comunicación Autocrina , Diabetes Mellitus Tipo 2/patología , Glucosa/metabolismo , Interleucina-13/fisiología , MicroARNs/fisiología , Músculo Esquelético/efectos de los fármacos , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Estudios de Casos y Controles , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Interleucina-13/farmacología , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Cultivo Primario de CélulasRESUMEN
The interaction of the PPARγ Pro12Ala polymorphism with diabetes and cardiovascular risk is controversial. We studied 173 women and 309 men in the observational CARDIPP trial in which determination of left ventricular mass, carotid intima-media thickness (IMT) and pulse wave velocity (PWV) were performed. Blood pressures were measured with 24-h ambulatory technique (ABP). Heterozygotes and homozygotes of Ala were defined as Ala in the analyses. Men with Ala-isoform displayed higher waist circumference (Ala: 107 ± 14 cm, Pro: 104 ± 11 cm, p = 0.045) and body weight (Ala: 95.7 ± 18 kg, Pro: 91.6 ± 14 kg, p = 0.042) than Pro-homozygotes. Men with ALA-isoform also showed higher systolic ABP levels (Ala: 134 ± 15 mmHg, Pro: 130 ± 14 mmHg, p = 0.004), whereas left ventricular mass index, IMT and PWV were unrelated to isoforms. In contrast, carotid-radial PWV was lower in women with the Ala-isoform (Ala: 7.9 ± 1.0 m/s, Pro: 8.5 ± 1.3 m/s, p = 0.01) and levels of apolipoprotein A1 were higher (Ala: 1.43 ± 0.27 g/l, Pro: 1.35 ± 0.17 g/l, p = 0.03). In conclusion, we found that men with type 2 diabetes having the Ala-isoform of PPARγ Pro12Ala had an unfavorable cardiovascular risk profile, whereas women with this isoform had lower carotid-radial PWV and higher apolipoprotein A1 levels suggesting a beneficial prognosis. These differences according to gender of the ALA isoform in type 2 diabetes deserve further attention.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Diabetes Mellitus Tipo 2/genética , PPAR gamma/genética , Polimorfismo Genético , Anciano , Presión Sanguínea , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Caracteres SexualesRESUMEN
Type 2 diabetes (T2D) is strongly linked to obesity and an adipose tissue unresponsive to insulin. The insulin resistance is due to defective insulin signaling, but details remain largely unknown. We examined insulin signaling in adipocytes from T2D patients, and contrary to findings in animal studies, we observed attenuation of insulin activation of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). As a consequence, mTORC1 downstream effects were also affected in T2D: feedback signaling by insulin to signal-mediator insulin receptor substrate-1 (IRS1) was attenuated, mitochondria were impaired and autophagy was strongly upregulated. There was concomitant autophagic destruction of mitochondria and lipofuscin particles, and a dependence on autophagy for ATP production. Conversely, mitochondrial dysfunction attenuated insulin activation of mTORC1, enhanced autophagy and attenuated feedback to IRS1. The overactive autophagy was associated with large numbers of cytosolic lipid droplets, a subset with colocalization of perlipin and the autophagy protein LC3/atg8, which can contribute to excessive fatty acid release. Patients with diagnoses of T2D and overweight were consecutively recruited from elective surgery, whereas controls did not have T2D. Results were validated in a cohort of patients without diabetes who exhibited a wide range of insulin sensitivities. Because mitochondrial dysfunction, inflammation, endoplasmic-reticulum stress and hypoxia all inactivate mTORC1, our results may suggest a unifying mechanism for the pathogenesis of insulin resistance in T2D, although the underlying causes might differ.
Asunto(s)
Adipocitos/metabolismo , Autofagia , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Adipocitos/patología , Adulto , Anciano , Citosol/metabolismo , Diabetes Mellitus Tipo 2/patología , Glucosa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulina/metabolismo , Modelos Lineales , Metabolismo de los Lípidos , Lipofuscina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Microscopía Confocal , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Complejos Multiproteicos , Obesidad/patología , Fosforilación Oxidativa , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR , Factores de Transcripción/genéticaRESUMEN
Insulin resistance and type 2 diabetes (T2D) are closely linked to obesity. Numerous prospective studies have reported on weight gain, insulin resistance, and insulin signaling in experimental animals, but not in humans. We examined insulin signaling in adipocytes from lean volunteers, before and at the end of a 4-wk period of consuming a fast-food, high-calorie diet that led to weight gain. We also examined adipocytes from patients with T2D. During the high-calorie diet, subjects gained 10% body weight and 19% total body fat, but stayed lean (body mass index = 24.3 kg/m(2)) and developed moderate systemic insulin resistance. Similarly to the situation in T2D subjects, in subjects on the high-calorie diet, the amount of insulin receptors was reduced and phosphorylation of IRS1 at tyrosine and at serine-307 (human sequence, corresponding to murine serine-302) were impaired. The amount of insulin receptor substrate protein-1 (IRS1) and the phosphorylation of IRS1 at serine-312 (human sequence, corresponding to murine serine-307) were unaffected by the diet. Unlike the T2D subjects, in subjects on the high-calorie diet, likely owing to the ongoing weight-gain, phosphorylation of MAP-kinases ERK1/2 became hyperresponsive to insulin. To our knowledge this study is the first to investigate insulin signaling during overeating in humans, and it demonstrates that T2D effects on intracellular insulin signaling already occur after 4 wks of a high-calorie diet and that the effects in humans differ from those in laboratory animals.
Asunto(s)
Adipocitos/metabolismo , Hiperfagia/metabolismo , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Delgadez/metabolismo , Absorciometría de Fotón , Adulto , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/metabolismo , Dieta , Femenino , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Transducción de Señal , Tirosina/metabolismoRESUMEN
Leukotriene C(4) is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C(4) synthase (LTC(4)S) participate in its biosynthesis. We report evidence that LTC(4)S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC(4)S. FLAP bound to the N-terminal part/first hydrophobic region of LTC(4)S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC(4)S. Fluorescent FLAP- and LTC(4)S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC(4)S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC(4)S. Direct interaction of 5-LO and LTC(4)S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC(4)S.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Proteínas Portadoras/metabolismo , Glutatión Transferasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Animales , Células COS , Proteínas Portadoras/antagonistas & inhibidores , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia/métodos , Glutatión Transferasa/genética , Humanos , Proteínas de la Membrana/antagonistas & inhibidoresRESUMEN
MicroRNAs have emerged as important regulators of glucose and lipid metabolism in several tissues; however, their role in skeletal muscle remains poorly characterized. We determined the effects of the miR-29 family on glucose metabolism, lipid metabolism, and insulin responsiveness in skeletal muscle. We provide evidence that miR-29a and miR-29c are increased in skeletal muscle from patients with type 2 diabetes and are decreased following endurance training in healthy young men and in rats. In primary human skeletal muscle cells, inhibition and overexpression strategies demonstrate that miR-29a and miR-29c regulate glucose uptake and insulin-stimulated glucose metabolism. We identified that miR-29 overexpression attenuates insulin signaling and expression of insulin receptor substrate 1 and phosphoinositide 3-kinase. Moreover, miR-29 overexpression reduces hexokinase 2 expression and activity. Conversely, overexpression of miR-29 by electroporation of mouse tibialis anterior muscle decreased glucose uptake and glycogen content in vivo, concomitant with decreased abundance of GLUT4. We also provide evidence that fatty acid oxidation is negatively regulated by miR-29 overexpression, potentially through the regulation of peroxisome proliferator-activated receptor γ coactivator-1α expression. Collectively, we reveal that miR-29 acts as an important regulator of insulin-stimulated glucose metabolism and lipid oxidation, with relevance to human physiology and type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Glucosa/metabolismo , Metabolismo de los Lípidos/genética , MicroARNs/genética , Músculo Esquelético/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Ejercicio Físico , Ácidos Grasos/metabolismo , Femenino , Perfilación de la Expresión Génica , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Oxidación-Reducción , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Condicionamiento Físico Animal , Resistencia Física , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Fibrosis of adipose tissue (AT) increases AT rigidity, reduces its expandability, and contributes to metabolic dysfunction. Collagen type VI, α3 (COL6A3) encodes 1 subunit of a fibrotic extracellular matrix protein highly expressed in rodent AT. Knockout of collagen VI in rodent AT led to a significant improvement in metabolic health in obese, diabetic ob/ob mice. However, it is unknown whether this collagen has the same metabolic significance in human AT. We therefore aimed to undertake a comprehensive assessment of COL6A3 in relation to human AT and obesity. Characterization of COL6A3 in human AT showed 5-fold higher expression in the stromalvascular fraction compared with adipocyte expression and significantly higher expression in subcutaneous AT (SCAT) than omental AT. In both depots, COL6A3 expression appeared to be lowered in obesity, whereas diet- and surgery-induced weight loss increased COL6A3 expression in SCAT. Leptin treatment caused a dose-dependent decrease in COL6A3 expression, although no effect was seen with insulin or glucose treatment and no difference observed in subjects with diabetes. In addition, we found that the collagen expression profile in humans differs significantly from rodents, because COL6A3 does not appear to be the predominant collagen in adipose, muscle, or liver. Our findings oppose those initially seen in rodent studies and, most importantly, demonstrate a direct regulation of COL6A3 by leptin. This highlights the importance of a paracrine leptin signaling pathway in human AT and suggests an additional mechanism by which leptin can regulate extracellular matrix composition and, with it, AT expandability.
Asunto(s)
Tejido Adiposo/metabolismo , Colágeno Tipo VI/metabolismo , Leptina/metabolismo , Adulto , Restricción Calórica , Estudios de Casos y Controles , Colágeno Tipo VI/genética , Diabetes Mellitus , Femenino , Humanos , Leptina/genética , Persona de Mediana Edad , Obesidad/genética , Obesidad/metabolismoRESUMEN
The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance.
Asunto(s)
Gluconeogénesis/fisiología , Hígado/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Hígado Graso/metabolismo , Hígado Graso/patología , Interleucina-6/metabolismo , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Hígado/patología , Macrófagos/patología , Masculino , Ratones , Ratones Obesos , Obesidad/etiología , Obesidad/patología , Receptores CCR2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Interleukin 1 Receptor antagonist (IL-1Ra) is highly elevated in obesity and is widely recognized as an anti-inflammatory cytokine. While the anti-inflammatory role of IL-1Ra in the pancreas is well established, the role of IL-1Ra in other insulin target tissues and the contribution of systemic IL-1Ra levels to the development of insulin resistance remains to be defined. Using antisense knock down of IL-1Ra in vivo, we show that normalization of IL-1Ra improved insulin sensitivity due to decreased inflammation in the liver and improved hepatic insulin sensitivity and these effects were independent of changes in body weight. A similar effect was observed in IL1-R1 KO mice, suggesting that at high concentrations of IL-1Ra typically observed in obesity, IL-1Ra can contribute to the development of insulin resistance in a mechanism independent of IL-1Ra binding to IL-1R1. These results demonstrate that normalization of plasma IL-1Ra concentration improves insulin sensitivity in diet- induced obese mice.
Asunto(s)
Hepatitis/metabolismo , Resistencia a la Insulina/genética , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Hígado/metabolismo , Obesidad/metabolismo , Animales , Peso Corporal/genética , Dieta , Prueba de Tolerancia a la Glucosa , Hepatitis/genética , Inflamación/genética , Inflamación/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/genética , Ratones , Ratones Obesos , Ratones Transgénicos , Obesidad/genéticaRESUMEN
CONTEXT: Changes in energy intake have marked and rapid effects on metabolic functions, and some of these effects may be due to changes in adipocyte gene expression that precede alterations in body weight. OBJECTIVE: The aim of the study was to identify adipocyte genes regulated by changes in caloric intake independent of alterations in body weight. RESEARCH DESIGN AND METHODS: Obese subjects given a very low-caloric diet followed by gradual reintroduction of ordinary food and healthy subjects subjected to overfeeding were investigated. Adipose tissue biopsies were taken at multiple time-points, and gene expression was measured by DNA microarray. Genes regulated in the obese subjects undergoing caloric restriction followed by refeeding were identified using two-way ANOVA corrected with Bonferroni. From these, genes regulated by caloric restriction and oppositely during the weight-stable refeeding phase were identified in the obese subjects. The genes that were also regulated, in the same direction as the refeeding phase, in the healthy subjects after overfeeding were defined as being regulated by caloric intake. Results were confirmed using real-time PCR or immunoassay. RESULTS: Using a significance level of P < 0.05 for all comparisons, 52 genes were down-regulated, and 50 were up-regulated by caloric restriction and regulated in the opposite direction by refeeding and overfeeding. Among these were genes involved in lipogenesis (ACLY, ACACA, FASN, SCD), control of protein synthesis (4EBP1, 4EBP2), ß-oxidation (CPT1B), and insulin resistance (PEDF, SPARC). CONCLUSIONS: Metabolic genes involved in lipogenesis, protein synthesis, and insulin resistance are central in the transcriptional response of adipocytes to changes in caloric intake.
Asunto(s)
Adipocitos/fisiología , Peso Corporal/genética , Peso Corporal/fisiología , Ingestión de Energía/genética , Regulación de la Expresión Génica/fisiología , Adulto , Biopsia , Restricción Calórica , Ingestión de Energía/fisiología , Proteínas del Ojo/sangre , Comida Rápida , Femenino , Humanos , Inmunoensayo , Resistencia a la Insulina/genética , Lipogénesis/genética , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Factores de Crecimiento Nervioso/sangre , Obesidad/dietoterapia , Obesidad/genética , Oxidación-Reducción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serpinas/sangreRESUMEN
OBJECTIVE: Matricellular Secreted Protein, Acidic and Rich in Cysteine (SPARC), originally discovered in bone as osteonectin, is a mediator of collagen deposition and promotes fibrosis. Adipose tissue collagen has recently been found to be linked with metabolic dysregulation. Therefore, we tested the hypothesis that SPARC in human adipose tissue is influenced by glucose metabolism and adipokines. RESEARCH DESIGN AND METHODS: Serum and adipose tissue biopsies were obtained from morbidly obese nondiabetic subjects undergoing bariatric surgery and lean control subjects for analysis of metabolic markers, SPARC, and various cytokines (RT-PCR). Additionally, 24 obese subjects underwent a very-low-calorie diet of 1,883 kJ (450 kcal)/day for 16 weeks and serial subcutaneous-abdominal-adipose tissue (SCAT) biopsies (weight loss: 28 +/- 3.7 kg). Another six lean subjects underwent fast-food-based hyperalimentation for 4 weeks (weight gain: 7.2 +/- 1.6 kg). Finally, visceral adipose tissue explants were cultured with recombinant leptin, insulin, and glucose, and SPARC mRNA and protein expression determined by Western blot analyses. RESULTS: SPARC expression in human adipose tissue correlated with fat mass and was higher in SCAT. Weight loss induced by very-low-calorie diet lowered SPARC expression by 33% and increased by 30% in adipose tissue of subjects gaining weight after a fast-food diet. SPARC expression was correlated with leptin independent of fat mass and correlated with homeostasis model assessment-insulin resistance. In vitro experiments showed that leptin and insulin potently increased SPARC production dose dependently in visceral adipose tissue explants, while glucose decreased SPARC protein. CONCLUSIONS: Our data suggest that SPARC expression is predominant in subcutaneous fat and its expression and secretion in adipose tissue are influenced by fat mass, leptin, insulin, and glucose. The profibrotic effects of SPARC may contribute to metabolic dysregulation in obesity.
Asunto(s)
Tejido Adiposo/fisiología , Glucemia/metabolismo , Peso Corporal/fisiología , Insulina/sangre , Leptina/sangre , Obesidad Mórbida/sangre , Obesidad/sangre , Osteonectina/genética , Adulto , Cirugía Bariátrica , Presión Sanguínea , Índice de Masa Corporal , Dieta Reductora , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Fisiológica/genética , Obesidad/dietoterapia , Obesidad/fisiopatología , Obesidad Mórbida/fisiopatología , Obesidad Mórbida/cirugía , Osteonectina/metabolismoRESUMEN
Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.