RESUMO
Advanced glycation end products (AGEs) increase with aging and induce signaling alterations that lead to inflammation and dysfunction in several tissues. Aging reduces function and insulin signaling in lacrimal glands (LGs). To evaluate whether AGE signaling and insulin secretion in LGs are altered in aging, 24- and 2-month-old male Wistar rats were compared. Immunohistochemistry with confocal microscopy was used to evaluate AGE, AGE receptor (RAGE) and nuclear factor-kappaB (NF-kappaB) expression in LGs. Basal tear secretion volume, insulin, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) levels in tears and LGs and peroxidase activity in LG tissue were measured. Insulin secretion from isolated LGs and pancreatic beta-cells was compared in the supernatant of aging and control rats in vitro by RIA after stimulation with 2.8-16.7 mM glucose, carbachol and KCl. AGE, RAGE and NF-kappaB expression was higher in LGs of aging compared with young rats. Basal tear secretion and peroxidase activity were significantly lower in the aging group (P=0.016 for both assays). IL-1beta and TNF-alpha levels were higher in tears of aging rats compared with young rats (P=0.007 and 0.05 respectively); however, even though aging rats were insulin-resistant (as confirmed by the insulin-tolerance test), the insulin levels in the tear film of aging and control rats were similar in vivo and in vitro. The higher expression of AGEs, RAGE and NF-kappaB in LGs of aging rats is accompanied by systemic insulin resistance and may be involved in LG and tear film alterations but does not affect insulin secretion in the tear film. These observations indicate that metabolic events may be related to LG and tear film dysfunctions in aging.
Assuntos
Envelhecimento/fisiologia , Produtos Finais de Glicação Avançada/análise , Aparelho Lacrimal/metabolismo , NF-kappa B/análise , Animais , Carbacol/farmacologia , Glucose/farmacologia , Produtos Finais de Glicação Avançada/metabolismo , Imuno-Histoquímica/métodos , Insulina/metabolismo , Secreção de Insulina , Interleucina-1/análise , Masculino , Microscopia Confocal , Mióticos/farmacologia , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Pâncreas/metabolismo , Peroxidase/análise , Peroxidase/metabolismo , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/análise , Transdução de Sinais/fisiologia , Lágrimas/química , Lágrimas/metabolismo , Fator de Necrose Tumoral alfa/análiseRESUMO
Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.
Assuntos
Resistência à Insulina , Músculo Esquelético/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Glicemia/metabolismo , Modelos Animais de Doenças , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Masculino , Denervação Muscular , Músculo Esquelético/inervação , Oligonucleotídeos Antissenso/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismoRESUMO
BACKGROUND: Endurance training increases insulin-stimulated muscle glucose transport and leads to improved metabolic control in diabetic patients. OBJECTIVE: To analyze the effects of endurance training on the early steps of insulin action in muscle of rats. DESIGN: Male rats submitted to daily swimming for 6 weeks were compared with sedentary controls. At the end of the training period, anesthetized animals received an intravenous (i.v.) injection of insulin and had a fragment of their gastrocnemius muscle excised for the experiments. METHODS: Associations between insulin receptor, insulin receptor substrates (IRS)-1 and -2 and phosphatidylinositol 3-kinase (PI3-kinase) were analyzed by immunoprecipitation and immunoblotting. Akt-1 serine phosphorylation and specific protein quantification were detected by immunoblotting of total extracts, and IRS-1/IRS-2-associated PI3-kinase activity were determined by thin-layer chromatography. RESULTS: Insulin-induced phosphorylation of IRS-1 and IRS-2 increased respectively by 1.8-fold (P<0.05) and 1.5-fold (P<0.05), whereas their association with PI3-kinase increased by 2.3-fold (P<0.05) and 1.9-fold (P<0.05) in trained rats as compared with sedentary controls, respectively. The activity of PI3-kinase associated with IRS-1 and IRS-2 increased by 1.8-fold (P<0.05) and 1.7-fold (P<0.05) respectively, in trained rats as compared with their untrained counterparts. Serine phosphorylation of Akt-1/PKB increased 1.7-fold (P<0.05) in trained rats in response to insulin. These findings were accompanied by increased responsiveness to insulin as demonstrated by a reduced area under the curve for insulin during an i.v. glucose tolerance test, by increased glucose disappearance rate during an insulin tolerance test, and by increased expression of glucose transporter-4. CONCLUSIONS: The increased responsiveness to insulin induced by chronic exercise in rat skeletal muscle may result, at least in part, from the modulation of the insulin signaling pathway at different molecular levels.