O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.

Transporte de glucosa en músculo esquelético: insulina, ejercicio físico y diabetes mellitus tipo 2 / Skeletal muscle glucose transport: insulin, physical exercise and diabetes mellitus type 2

La glucosa es un sustrato energético fundamental y su transporte y metabolismo debe mantenerse bajo un delicado control. Alteraciones por déficit o por exceso generan importes complicaciones para la salud del ser humano y el conocimiento de los mecanismos que regulan su homeostasis permite una mejor aproximación al tratamiento de disfunciones como el síndrome de insulino resistencia o la diabetes mellitus. Esta revisión muestra los aspectos moleculares fundamentales, relacionados con el control de la glicemia, haciendo énfasis en el transporte de la glucosa a través de la membrana plasmatica, así como en las rutas de señalización molecular utilizadas por los dos estímulos más importantes en su control, insulina y ejercicio físico, tanto en condiciones de salud, como en presencia de diabetes mellitus

An overview of pathophysiology and treatment of insulin resistance.

Insulin resistance has emerged out as a concept linking diabetes mellitus and hypertension. Clinically it is characterized by hyperinsulinemia, hypertension, central obesity, abnormal lipid profile and cardiovascular complications. Insulin resistance is often associated with presence of anti-insulin antibodies and absent or dysfunctional insulin receptors. At molecular level insulin resistance appears to occur at the level of G-protein, kinase activation, glucose carriers (GLUT) and gene expression. Although with advent or research, the molecular mechanisms of insulin resistance are becoming more clear and there is development of new therapeutic agents like insulin sensitizers (thizolidinediones), in clinical practice, as of today, a patient with insulin resistance is looked upon as hypertensive or having diabetes mellitus. Accordingly he is taking either antihypertensives or antidiabetic drugs or both. It is thus essential to look into effects of these agents on insulin sensitivity. In recent years some scattered studies have been conducted to evaluate the effect of various antihypertensives and antidiabetics on insulin sensitivity. An antihypertensive or antidiabetic drug should directly benefit the cardiovascular risk profile of these patients. Although various newer approaches are explored to have a therapeutic benefit in insulin resistance, it is still a long way in the research, when a suitable pharmacological agent with least untoward effects will be available for the treatment of insulin residence.

GLUT-4, tumour necrosis factor, essential fatty acids and daf-genes and their role in glucose homeostasis, insulin resistance, non-insulin dependent diabetes mellitus, and longevity.

GLUT-4 receptor, tumor necrosis factor-alpha (TNF-alpha), essential fatty acids (EFAs) and their metabolites and daf-genes seem to play an important and essential role in the maintenance of glucose homeostasis, and in the pathobiology of obesity and non-insulin dependent diabetes mellitus (NIDDM). Daf-genes encode for proteins which are 35% identical to the human insulin receptor, a transforming growth factor-beta (TGF-beta) type signal and can also enhance the expression of superoxide dismutase (SOD). On the other hand, EFAs and their metabolites can increase the cell membrane fluidity and thus, enhance the expression of GLUT-4 and insulin receptors. In addition, EFAs can suppress TNF-alpha production and secretion and thus, are capable of reversing insulin resistance. Melatonin has anti-oxidant actions similar to daf-16, TGF-beta and SOD. Hence, it is likely that there is a close interaction between GLUT-4, TNF-alpha, EFAs, daf-genes, melatonin and leptin that may have relevance to the development of insulin resistance, obesity, NIDDM, complications due to NIDDM, longevity and ageing.

A Study of the Regulation of the Glucose Transporter in the Plasma Membranes of Hepatoma Cells Induced by 3'-Me DAB

5'-nucelotidase and glucose-6-phosphatase are liver plasma and microsomal membranes markers and their respective activities were determined. In the liver homogenate, the activities of 5'-nucleotidase were 0.58 +/- 0.08 and 0.29 +/- 0.07 micromols/mg protein/10min in the control and 3'-methyl-4-dimethyl aminoazobenzene (3'-Me DAB) groups respectively. The enzyme activities m the partially purified plasma membranes were 2.15 +/- 0.25 and 1.31 +/- 0.23 micromols/mg protein/10min in the control and 3'-Me DAB groups respectively. The glucose-6-phosphatase activities in the homogenates of the control and 3'-Me DAB groups were 0.23 +/- 0.10, and 0.45 +/- 0.25 micromols/mg protein/10min, and in the microsomal fraction, 1.14 +/- 0.32, and 0.63 +/- 0.11 micromols/mg protein/10min, respectively, The concentrations of glucose carrier in the plasma membranes from the control and 3'-Me DAB group were 25, and 35 pmols/mg membrane protein, respectively, and the Ka values for cytochalsin B in each group were 5.20 X 109. and 5.14 X 109ml/mol, respectively. However in the microsomal fraction, no significant differences of glucose carrier were found between the control and 3'-Me DAB groups from the DEAE Sephadex A-50 ion exchange chromatography, fractions I and ll were obtained. Electrophoretic analysis of fraction I revealed a major protein band with a molecular weight of 45,000 and minor bands with MWs of 50,000, 55,000 and 15,000. Following AcA 34 gel filtration, a major protein band with a MW of 45,000 was obtained. From these results, it can be concluded that the glucose carrier protein was increased on plasma membrane of hepatoma induced by 3'-Me DAB, and the carrier protein showed similar molecular weight to other glucose carrier found in the RBC, muscle cells and adipocyte.