phosphorus-radioisotopes and Diabetes-Mellitus--Type-2

The study aims to characterize age-associated changes in skeletal muscle bioenergetics by evaluating the response to ischemia-reperfusion in the skeletal muscle of the Goto-Kakizaki (GK) rats, a rat model of non-obese type 2 diabetes (T2D). (31)P magnetic resonance spectroscopy (MRS) and blood oxygen level-dependent (BOLD) MRI was performed on the hindlimb of young (12 weeks) and adult (20 weeks) GK and Wistar (control) rats. (31)P-MRS and BOLD-MRI data were acquired continuously during an ischemia and reperfusion protocol to quantify changes in phosphate metabolites and muscle oxygenation. The time constant of phosphocreatine recovery, an index of mitochondrial oxidative capacity, was not statistically different between GK rats (60.8 ± 13.9 sec in young group, 83.7 ± 13.0 sec in adult group) and their age-matched controls (62.4 ± 11.6 sec in young group, 77.5 ± 7.1 sec in adult group). During ischemia, baseline-normalized BOLD-MRI signal was significantly lower in GK rats than in their age-matched controls. These results suggest that insulin resistance leads to alterations in tissue metabolism without impaired mitochondrial oxidative capacity in GK rats.

Topics: Animals; Brain Ischemia; Diabetes Mellitus, Type 2; Disease Models, Animal; Hydrogen-Ion Concentration; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Male; Mitochondria; Muscle, Skeletal; Phosphocreatine; Phosphorus Radioisotopes; Rats; Reperfusion Injury

We recently described novel regulatory roles for protein histidine phosphorylation of key islet proteins (e.g., nucleoside diphosphate kinase and succinyl thiokinase) in insulin secretion from the islet beta-cell (Kowluru A. Diabetologia 44: 89-94, 2001; Kowluru A, Tannous M, and Chen HQ. Arch Biochem Biophys 398: 160-169, 2002). In this context, we also characterized a novel, ATP- and GTP-sensitive protein histidine kinase in isolated beta-cells that catalyzed the histidine phosphorylation of islet (endogenous) proteins as well as exogenously added histone 4, and we implicated this kinase in the activation of islet endogenous G proteins (Kowluru A. Biochem Pharmacol 63: 2091-2100, 2002). In the present study, we describe abnormalities in ATP- or GTP-mediated histidine phosphorylation of nucleoside diphosphate kinase in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes. Furthermore, we provide evidence for a marked reduction in the activities of ATP- or GTP-sensitive histidine kinases in GK rat islets. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.

Topics: Adenosine Triphosphate; Animals; Diabetes Mellitus, Type 2; Female; Guanosine Triphosphate; Histidine; Islets of Langerhans; Male; Nucleoside-Diphosphate Kinase; Phosphorus Radioisotopes; Phosphorylation; Radionuclide Imaging; Rats; Rats, Mutant Strains; Rats, Wistar

Protein tyrosine phosphatases (PTPs) are required for the dephosphorylation of the insulin receptor (IR) and its initial cellular substrates, and it has recently been reported that PTP-1B may play a role in the pathogenesis of insulin resistance in obesity and type 2 diabetes mellitus (DM). We therefore determined the amount and activity of PTP-1B in abdominal adipose tissue obtained from lean nondiabetic subjects (lean control (LC)), obese nondiabetic subjects (obese control (OC)), and subjects with both type 2 DM (DM2) and obesity (obese diabetic (OD)). PTP-1B protein levels were 3-fold higher in OC than in LC (1444 +/- 195 U vs 500 +/- 146 U (mean +/- SEM), P < .015), while OD exhibited a 5.5-fold increase (2728 +/- 286 U, P < .01). PTP activity was assayed by measuring the dephosphorylating activity toward a phosphorus 32-labeled synthetic dodecapeptide. In contrast to the increased PTP-1B protein levels, PTP-1B activity per unit of PTP-1B protein was markedly reduced, by 71% and 88% in OC and OD, respectively. Non-PTP-1B tyrosine phosphatase activity was comparable in all three groups. Similar results were obtained when PTP-1B activity was measured against intact human IR. A significant correlation was found between body mass index (BMI) and PTP-1B level (r = 0.672, P < .02), whereas BMI and PTP-1B activity per unit of PTP-1B showed a strong inverse correlation (r = -0.801, P < .002). These data suggest that the insulin resistance of obesity and DM2 is characterized by the increased expression of a catalytically impaired PTP-1B in adipose tissue and that impaired PTP-1B activity may be pathogenic for insulin resistance in these conditions.

Topics: Adipose Tissue; Adult; Aged; Animals; Blotting, Western; Cell Line; Diabetes Mellitus, Type 2; Enzyme Activation; Female; Fibroblasts; Humans; Hydrolysis; Male; Middle Aged; Nerve Tissue Proteins; Obesity; Phosphorus Radioisotopes; Phosphorylation; Precipitin Tests; Protein Tyrosine Phosphatases; Rats; Receptor-Like Protein Tyrosine Phosphatases, Class 5

A sensitive microtiter well-based assay for the measurement of insulin activation of insulin receptor kinase in intact human circulating mononuclear cells has been developed and characterized. Mononuclear cells from 100-150 ml blood were incubated with various insulin concentrations to activate the receptor kinase. The cells were then solubilized in the presence of phosphatase and kinase inhibitors and the receptors immobilized to microwells coated with anti-insulin receptor antibody (efficiency of receptor immobilization > 85%). Receptor kinase activity and binding activity were then consecutively measured in the same wells. Insulin incubation of the cells increased the kinase activity three- to fourfold with a half-maximal effect at 5 nM and a maximal effect at 87 nM. In mononuclear cells from 16 subjects with NIDDM, the insulin effect on receptor kinase activation was significantly reduced compared with 16 nondiabetic control subjects (0.135 +/- 0.016 vs. 0.195 +/- 0.024 fmol P.fmol binding activity-1 x min-1, respectively; P < 0.05). We conclude that; 1) it is possible to determine insulin activation of receptor kinase in intact cells in this easily accessible human tissue; 2) insulin activation of insulin receptor kinase is impaired in intact mononuclear cells from patients with NIDDM; and 3) the finding that kinase activation in NIDDM is reduced in a tissue that, according to the literature, contains only the A isoform of the insulin receptor, suggests that mechanisms other than a different abundance of the A and B insulin receptor isoforms must exist that contribute to the decreased kinase activity in NIDDM.

Topics: Aged; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Enzyme Activation; Female; Humans; In Vitro Techniques; Insulin; Leukocytes, Mononuclear; Male; Middle Aged; Phosphorus Radioisotopes; Protein-Tyrosine Kinases; Receptor, Insulin

To study whether insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus is due to a defect in the expression of the insulin-responsive glucose transporter gene (GLUT-4) in human skeletal muscle, we measured the level of GLUT-4 mRNA and (in some of the subjects) its protein in muscle biopsies taken from 14 insulin-resistant patients with Type 2 diabetes, 10 first-degree relatives of the diabetic patients and 12 insulin-sensitive control subjects. Insulin sensitivity was measured with a + 45 mU.m2(-1).min-1 euglycaemic insulin clamp in combination with indirect calorimetry and infusion of [3-3H]glucose. GLUT-4 mRNA was measured using a human GLUT-4 cDNA probe and GLUT-4 protein with a polyclonal antibody specific for the 15 amino acid carboxy-terminal peptide. Both Type 2 diabetic patients and their relatives showed impaired stimulation of total-body glucose disposal by insulin compared with control subjects (29.5 +/- 2.1 and 34.0 +/- 4.8 vs 57.9 +/- 3.1 mumol.kg lean body mass-1.min-1; p less than 0.01). This impairment in glucose disposal was primarily accounted for by a reduction in insulin-stimulated storage of glucose as glycogen (13.0 +/- 2.4 and 15.6 +/- 3.9 vs 36.9 +/- 2.2 mumol.kg lean body mass-1.min-1; p less than 0.01). The levels of GLUT-4 mRNA expressed both per microgram of total RNA and per microgram DNA, were higher in the diabetic patients compared with the control subjects (116 +/- 25 vs 53 +/- 10 pg/microgram RNA and 177 +/- 35 vs 112 +/- 29 pg/microgram DNA; p less than 0.05, p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Autoradiography; Blood Glucose; Blotting, Western; Diabetes Mellitus, Type 2; Glycated Hemoglobin; Humans; Insulin; Insulin Resistance; Middle Aged; Monosaccharide Transport Proteins; Muscle Proteins; Muscles; Phosphorus Radioisotopes; Reference Values; RNA, Messenger

Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration [( Ca2+]i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal [Ca2+]i value was similar in the three groups, the rise in [Ca2+]i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Blood Platelets; Blood Proteins; Calcium; Diabetes Mellitus, Type 2; Female; Humans; Male; Middle Aged; Myosin-Light-Chain Kinase; Myosins; Phosphatidic Acids; Phosphatidylinositols; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate