RESUMO
Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKß- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKß and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA â PKCδ â NADPH oxidase and oxidative stress â IKKß/JNK â impaired hepatic insulin signaling.
Assuntos
Ácidos Graxos não Esterificados/sangue , Glucose/metabolismo , Resistência à Insulina/fisiologia , Fígado/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Proteína Quinase C/metabolismo , Animais , Feminino , Ratos , Ratos WistarRESUMO
Resistin is an adipose-derived hormone postulated to link adiposity to insulin resistance. To determine whether resistin plays a causative role in the development of diet-induced insulin resistance, we lowered circulating resistin levels in mice by use of a specific antisense oligodeoxynucleotide (ASO) directed against resistin mRNA and assessed in vivo insulin action by the insulin-clamp technique. After 3 weeks on a high-fat (HF) diet, mice displayed severe insulin resistance associated with an approximately 80% increase in plasma resistin levels. In particular, the rate of endogenous glucose production (GP) increased more than twofold compared with that in mice fed a standard chow. Treatment with the resistin ASO for 1 week normalized the plasma resistin levels and completely reversed the hepatic insulin resistance. Importantly, in this group of mice, the acute infusion of purified recombinant mouse resistin, designed to acutely elevate the levels of circulating resistin up to those observed in the HF-fed mice, was sufficient to reconstitute hepatic insulin resistance. These results provide strong support for a physiological role of resistin in the development of hepatic insulin resistance in this model.
Assuntos
Dieta , Hormônios Ectópicos/sangue , Resistência à Insulina/fisiologia , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Fígado/fisiologia , Adiponectina , Animais , Células Cultivadas , Gorduras na Dieta/metabolismo , Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Hormônios Ectópicos/genética , Humanos , Leptina/sangue , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ResistinaRESUMO
Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2'-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic beta cell function.
Assuntos
Diabetes Mellitus/metabolismo , Fígado/metabolismo , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Peptídeos/metabolismo , Receptores de Glucagon/genética , Animais , Glicemia/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Camundongos , Oligodesoxirribonucleotídeos Antissenso/genética , RatosRESUMO
Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.
Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Fígado/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Receptores de Glucocorticoides/metabolismo , Animais , Dexametasona/farmacologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperlipidemias/tratamento farmacológico , Linfopenia/induzido quimicamente , Linfopenia/fisiopatologia , Camundongos , Camundongos Obesos , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/metabolismo , RatosRESUMO
Signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is crucial for metabolic responses to insulin, and defects in PI3K signaling have been demonstrated in type 2 diabetes. PTEN (MMAC1) is a lipid/protein phosphatase that can negatively regulate the PI3K pathway by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, but it is unclear whether PTEN is physiologically relevant to insulin signaling in vivo. We employed an antisense oligonucleotide (ASO) strategy in an effort to specifically inhibit the expression of PTEN. Transfection of cells in culture with ASO targeting PTEN reduced PTEN mRNA and protein levels and increased insulin-stimulated Akt phosphorylation in alpha-mouse liver-12 (AML12) cells. Systemic administration of PTEN ASO once a week in mice suppressed PTEN mRNA and protein expression in liver and fat by up to 90 and 75%, respectively, and normalized blood glucose concentrations in db/db and ob/ob mice. Inhibition of PTEN expression also dramatically reduced insulin concentrations in ob/ob mice, improved the performance of db/db mice during insulin tolerance tests, and increased Akt phosphorylation in liver in response to insulin. These results suggest that PTEN plays a significant role in regulating glucose metabolism in vivo by negatively regulating insulin signaling.
Assuntos
Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases , Proteínas Supressoras de Tumor/genética , Células 3T3 , Adipócitos/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Genes Supressores de Tumor , Glucose/metabolismo , Hepatócitos , Insulina/metabolismo , Cinética , Metabolismo dos Lipídeos , Camundongos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , Proteínas Recombinantes/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Transfecção , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
Protein kinase C (PKC) promotes cell survival in response to ionizing radiation in a variety of experimental models including human carcinoma, human glioblastoma, and transformed mouse embryo fibroblast cell lines. We have introduced specific antisense oligonucleotides into human mammary tumor cell lines in vitro to analyze the role of individual PKC isoforms in radiation-induced cell death in breast cancer. MDA-MB-231 and MCF-7 cells treated with oligonucleotide directed against the PKC delta isoform exhibited impaired survival in response to 5.6 Gy gamma-radiation as measured by mitochondrial metabolism of tetrazolium dye. The role of PKC delta in the breast tumor cell lines was of particular interest, because contradictory reports exist in the literature regarding the role of PKC delta in cell survival and apoptosis. A comparison of the effects of the PKC delta antisense oligonucleotide and a nucleotide scrambled version of this nucleotide revealed only the antisense oligonucleotide decreased cell survival. The PKC delta antisense oligonucleotide decreased cell survival after exposure to low (1.5 Gy) radiation doses and in the absence of radiation insult. We found 3 micro M rottlerin, a selective PKC delta inhibitor, to reduce MCF-7 and MDA-MB-231 cell survival. Furthermore, MCF-7 cells transformed to express a dominant-negative mutant of PKC delta exhibited reduced survival. Comet analysis showed that PKC delta oligonucleotide treatment caused an accumulation of cells containing damaged DNA similar to that seen in 1.5 Gy radiation-treated cells. We conclude that PKC delta acts as a prosurvival factor in human breast tumor cells in vitro.
Assuntos
Neoplasias da Mama/metabolismo , Proteína Quinase C/fisiologia , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Western Blotting , Neoplasias da Mama/patologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Sobrevivência Celular/fisiologia , Ensaio Cometa , DNA de Neoplasias/genética , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Raios gama , Genes Dominantes , Humanos , Isoenzimas , Oligorribonucleotídeos Antissenso/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-delta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Human ovarian cancer cell lines derived from A2780 by stepwise exposure to increasing cisplatin concentrations show progressive resistance to cisplatin. Previous studies have shown increased cellular glutathione and elevated steady-state expression of gamma-glutamylcysteine synthetase (gamma-GCS) and of the transcription factor c-Jun, all in proportion to the level of resistance in the resistant cells. We hypothesized that c-Jun was an important locus of control of the detoxicating enzymes mediating resistance, and that resistance reversal would be achieved by specific inhibition of this mechanism. A2780 (sensitive) and C30 (resistant) cells were treated with a 20-mer c-jun phosphorothioate antisense oligodeoxynucleotide (ISIS 10582, 1 microM), and a decrease in steady-state c-jun mRNA was demonstrated in the resistant cells. The expression of gamma-GCS mRNA was down-regulated and the cellular level of glutathione was decreased in C30 cells. No change in gamma-GCS expression occurred in A2780 cells. Using the microtetrazolium (MTT) cytotoxicity assay, we determined that the c-jun antisense decreased the IC50 value for cisplatin in C30 cells from 18.2 to 3.7 microM, and had a substantially smaller effect in A2780 cells. To determine if c-jun overexpression alone could confer resistance to the sensitive cell line, we transiently transfected A2780 cells with a c-jun expression vector. The transfected cells exhibited a 10.7-fold elevation of glutathione (GSH) content, a 9.2-fold increase in c-Jun protein content, and a 2-fold increase in the IC50 for cisplatin. These data suggest that altered regulation of transcription factor expression contributes to the acquired resistance phenotype in these ovarian cancer cells, and provide a novel potential target for therapeutic intervention.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Feminino , Humanos , Neoplasias Ovarianas/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-jun/genética , Células Tumorais CultivadasRESUMO
Protein tyrosine phosphatase (PTP)-1B antagonizes insulin signaling and is a potential therapeutic target for insulin resistance associated with obesity and type 2 diabetes. To date, studies of PTP-1B have been limited by the availability of specific antagonists; however, treatment of rodents with antisense oligonucleotides (ASOs) directed against PTP-1B improves insulin sensitivity, inhibits lipogenic gene expression, and reduces triglyceride accumulation in liver and adipose tissue. Here we investigated ASO-mediated PTP-1B inhibition in primates. First, PTP-1B ASO (ISIS 113715) dose-dependently inhibited PTP-1B mRNA and protein expression in cultured monkey hepatocytes. Subcutaneous administration of ISIS 113715 reduced PTP-1B mRNA expression in liver and adipose tissue of normal-weight monkeys by 40-50% and improved insulin sensitivity during an iv glucose tolerance test (IVGTT). In obese, insulin-resistant rhesus monkeys, treatment with 20 mg/kg ISIS 113715 for 4 wk reduced fasting concentrations of insulin and glucose and reduced insulin responses during an IVGTT. In these animals, adiponectin concentrations were also increased by 70%, most of which was an increase of high-molecular-weight oligomers. These effects were not observed in monkeys on a lower, dose-escalation regimen (1-10 mg/kg over 9 wk). Overall, the increase of adiponectin concentrations during ISIS 113715 treatment was correlated with the lowering of insulin responses during IVGTT (r = -0.47, P = 0.042). These results indicate that inhibition of PTP-1B with ASOs such as ISIS 113715 may be a viable approach for the treatment and prevention of obesity-associated insulin resistance and type 2 diabetes because they potently increase adiponectin concentrations in addition to improving insulin sensitivity.
Assuntos
Adiponectina/metabolismo , Resistência à Insulina , Oligonucleotídeos Antissenso/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Glicemia/efeitos dos fármacos , Western Blotting , Peso Corporal/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Macaca fascicularis , Obesidade/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.
Assuntos
Antiporters/genética , Antiporters/metabolismo , Diabetes Mellitus Tipo 2/terapia , Doença de Depósito de Glicogênio/prevenção & controle , Fígado/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Acidose Láctica/metabolismo , Acidose Láctica/prevenção & controle , Animais , Glicemia/biossíntese , Glicemia/metabolismo , Complicações do Diabetes/metabolismo , Complicações do Diabetes/prevenção & controle , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/metabolismo , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Doença de Depósito de Glicogênio/metabolismo , Hiperlipidemias/metabolismo , Hiperlipidemias/prevenção & controle , Hiperuricemia/metabolismo , Hiperuricemia/prevenção & controle , Hipoglicemia/metabolismo , Hipoglicemia/prevenção & controle , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , RNA Mensageiro/metabolismoRESUMO
To investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in glucose metabolism and insulin action, a specific antisense oligonucleotide (ASO) was used to reduce its expression both in vitro and in vivo. Reduction of LMW-PTP expression with the ASO in cultured mouse hepatocytes and in liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice led to increased phosphorylation and activity of key insulin signaling intermediates, including insulin receptor-beta subunit, phosphatidylinositol 3-kinase, and Akt in response to insulin stimulation. The ASO-treated DIO and ob/ob animals showed improved insulin sensitivity, which was reflected by a lowering of both plasma insulin and glucose levels and improved glucose and insulin tolerance in DIO mice. The treatment did not decrease body weight or increase metabolic rate. These data demonstrate that LMW-PTP is a key negative regulator of insulin action and a potential novel target for the treatment of insulin resistance and type 2 diabetes.
Assuntos
Hiperglicemia/metabolismo , Resistência à Insulina , Insulina/metabolismo , Isoenzimas/metabolismo , Obesidade/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Peso Corporal , Teste de Tolerância a Glucose , Hepatócitos/metabolismo , Imunoprecipitação , Proteínas Substratos do Receptor de Insulina , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.