cytochalasin-b and Diabetes-Mellitus--Type-2

Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition. A library of C-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC

Topics: Chalcones; Diabetes Mellitus, Type 2; Glucosides; HEK293 Cells; Humans; Membranes, Artificial; Molecular Docking Simulation; Phosphatidylcholines; Sodium-Glucose Transporter 1; Sodium-Glucose Transporter 2; Sodium-Glucose Transporter 2 Inhibitors

This paper investigates the effect of glycation on glucose transport in erythrocytes. Glucose transporter function, numbers and erythrocyte phosphorylation rates are simultaneously studied using 30 Caucasian patients with diabetes and 30 Caucasian control volunteers (mean +/- SD where P < or = 0.05; age 48 +/- 8 vs. 45 +/- 8 years [ns]; body mass index [BMI] 31 +/- 7 vs. 27 +/- 5 [P=0.035]; blood glucose 12 +/- 7 vs. 5 +/- 0.6 mmol/L [P=0.001]; HbA1c 8 +/- 2 vs. 5 +/- 0.3% [P=0.0001]; fructosamine 336 +/- 64 vs. 237 +/- 16 micromol/L [P=0.0001]; disease duration 13 +/- 11 years, respectively). Significant differences were found for glucose transporter function, with 3-O-methylglucose uptake rates (108 +/- 49 vs. 146 +/- 55 micromol/L/sec/10(12) cells [P=0.010]); D-glucose influx (64 +/- 30 vs. 117 +/- 45 micromol/L/sec/10(12) cells [P=0.0001]); and D-glucose net transport (31 +/- 22 vs. 74 +/- 55 micromol/L/sec/ 10(12) cells [P = 0.0001]). No differences were found for phosphorylation rates using 2-deoxyglucose (33 +/- 17 vs. 38 +/- 12 micromol/L/sec/10(12) cells [P=0.194]). The number of functional transporters using cytochalasin B studies measured via B(max), was not found to be significantly different between the groups (195 +/- 139 vs. 264 +/- 174 [P=0.206]). However, K(d) was lower for those with diabetes, suggesting higher binding affinity (12 +/- 11 vs. 32 +/- 25 nmol/L [P=0.006]). A negative correlation between HbAlc and D-glucose influx involving both groups was found (r=-0.670, P=0.0001). Glucose transport is shown to be decreased in people who have diabetes compared to normoglycaemic volunteers, whereas the number of glucose transporters is apparently unchanged; however, affinity for binding is increased.

Topics: Adult; Cell Membrane; Cytochalasin B; Diabetes Mellitus, Type 2; Erythrocytes; Glucose; Glucose Transporter Type 1; Glycosylation; Humans; In Vitro Techniques; Middle Aged

A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. The probable mechanism involves augmented reactive oxygen species (ROS) synthesis. The aim of this study was to evaluate the synthesis of ROS in human umbilical vein endothelial cells (HUVECs) obtained from healthy newborns with (experimental) and without (control) a strong family history of type 2 DM, exposed to different glucose concentrations.. HUVECs were exposed to various glucose concentrations for 24 and 48 h periods, before cell proliferation, mitochondrial activity, and mitochondrial membrane potential were determined. Intracellular ROS synthesis in the presence or absence of the mitochondrial uncoupler CCCP, cytochalasin B, or diphenyleneiodonium (DPI) was also evaluated.. As opposed to control HUVECs, we found that experimental HUVECs exposed to 30 mmol/L glucose showed a 50% decrease in cell proliferation, a 90% reduction in mitochondrial activity, and a statistically significant inhibition of ROS synthesis in the presence of CCCP or cytochalasin B; DPI had no effect.. Our results suggest that mitochondria and NAD(P)H-oxidase from HUVECs obtained from healthy newborns with a family history of DM have an innate deficient response to high glucose concentrations.

Topics: Adolescent; Adult; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Proliferation; Cells, Cultured; Cytochalasin B; Diabetes Mellitus, Type 2; Endothelium, Vascular; Family Health; Female; Glucose; Humans; Infant, Newborn; Membrane Potential, Mitochondrial; Mitochondria; Onium Compounds; Pregnancy; Reactive Oxygen Species; Umbilical Veins

We studied the distribution of nephrin in renal biopsies from 17 patients with diabetes and nephrotic syndrome (7 type 1 and 10 type 2 diabetes), 6 patients with diabetes and microalbuminuria (1 type 1 and 5 type 2 diabetes), and 10 normal subjects. Nephrin expression was semiquantitatively evaluated by measuring immunofluorescence intensity by digital image analysis. We found an extensive reduction of nephrin staining in both type 1 (67 +/- 9%; P < 0.001) and type 2 (65 +/- 10%; P < 0.001) diabetic patients with diabetes and nephrotic syndrome when compared with control subjects. The pattern of staining shifted from punctate/linear distribution to granular. In patients with microalbuminuria, the staining pattern of nephrin also showed granular distribution and reduction intensity of 69% in the patient with type 1 diabetes and of 62 +/- 4% (P < 0.001) in the patients with type 2 diabetes. In vitro studies on human cultured podocytes demonstrated that glycated albumin and angiotensin II reduced nephrin expression. Glycated albumin inhibited nephrin synthesis through the engagement of receptor for advanced glycation end products, whereas angiotensin II acted on cytoskeleton redistribution, inducing the shedding of nephrin. This study indicates that the alteration in nephrin expression is an early event in proteinuric patients with diabetes and suggests that glycated albumin and angiotensin II contribute to nephrin downregulation.

Topics: Adult; Aged; Albuminuria; Angiotensin II; Biopsy; Blotting, Western; Cells, Cultured; Cytochalasin B; Cytoskeleton; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Female; Fluorescent Antibody Technique, Indirect; Gene Expression; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Kidney; Male; Membrane Proteins; Microscopy, Fluorescence; Middle Aged; Nephrotic Syndrome; Proteins; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Albumin; Tissue Distribution

Type 2 diabetes mellitus is characterized by impaired glucose uptake. With a photometric method of recording the erythrocyte suspension absorption during the course of glucose transport across the membranes, we observed that the initial rate of glucose zero-trans entry was decreased significantly in 30 Chinese type 2 diabetic patients as compared to 25 healthy controls. The rate of glucose infinite-cis efflux exhibited no difference between the patients and controls. The measurement of temperature dependence of glucose transport showed that the activation energy for glucose entry was increased in diabetic patients. The inhibitory constant of glucose entry by cytochalasin B (CB) in patients was similar to that of the controls. However, we found that the inhibitory constant was increased significantly in the patient erythrocytes after phloretin treatment. After the erythrocytes were made into stripped white ghosts, the fluorescence quenching experiment was performed. Glucose, CB and phloretin can quench the fluorescence of tryptophan residues in the glucose transporter 1, GLUT1. The abnormality of fluorescence quenching in the erythrocyte membranes of patients was observed. The transfer tendency of tryptophan residues from the hydrophilic environment to the hydrophobic environment was decreased in patient ghosts as binding with glucose, and the opposite tendency appeared as CB and phloretin instead of glucose. We conclude that the decreased in glucose entry in the erythrocyte membranes of diabetic patients was due to the GLUT1 change in structure - mostly the outer domain of the glucose transporter.

Topics: Asian People; China; Cytochalasin B; Diabetes Mellitus, Type 2; Energy Metabolism; Erythrocyte Membrane; Glucose; Glucose Transporter Type 1; Humans; Monosaccharide Transport Proteins; Phloretin

Pancreatic islet amyloid, formed from islet amyloid polypeptide, is found in 96% of Type II (non-insulin-dependent) diabetic patients. Islet amyloidosis is progressive and apparently irreversible. Fibrils immunoreactive for islet amyloid polypeptide are found in macrophages associated with amyloid, suggesting that deposits can be phagocytosed. To determine the mechanism for the recognition and internalisation of fibrils, mouse peritoneal macrophages were cultured with fibrillar synthetic human islet amyloid polypeptide. Fibrils did not exert a cytotoxic effect over 72 h of culture. The uptake and degradation of fibrils was analysed by quantitative light-and electron-microscopic immunocytochemistry and immunoreactivity was detectable in 86+/-3% cells within 6 h of culture. Neither polyinosinic acid (200 microg/ml) nor nocodazole (10 microg/ml) inhibited fibril uptake, suggesting that internalisation is not blocked by poly-ions and is independent of microtubule assembly. Inhibition of pseudopodia formation by cytochalasin B blocked fibriI uptake. Fibril aggregates became condensed in lysosomes to form protofilaments and were resistant to intracellular proteolysis. Fibrils can be phagocytosed by macrophages in vitro but amyloid-associated factors may block the recognition of fibrils in vivo preventing the removal of islet amyloid in diabetes.

Topics: Amyloid; Animals; Cells, Cultured; Cytochalasin B; Diabetes Mellitus, Type 2; Endocytosis; Endopeptidases; Humans; Islet Amyloid Polypeptide; Lysosomes; Macrophages, Peritoneal; Male; Mice; Mice, Inbred Strains; Microtubules; Nocodazole; Pancreas; Poly I

To determine the rheological properties of polymorphonuclear leukocytes (PMN) from non-insulin-dependent diabetes mellitus (NIDDM) patients.. The deformability of PMN from 33 NIDDM subjects, 13 with impaired glucose tolerance (IGT), and 22 with normal glucose tolerance (NGT) was studied. A Cell Transit Analyzer that measures the transit time of PMN through 8-microns pores was used. Studies were performed under three different conditions: 1) basal state; 2) after incubation with cytochalasin B (20 microM) to dissociate f-actin from the cytoskeleton; and 3) following activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 nM).. PMN from diabetic patients were more rigid (i.e., had longer transit time) than those from subjects with NGT or IGT under basal conditions and after cytochalasin B, but not after stimulation with fMLP. The deformability of PMN from subjects with IGT was similar to those of the NGT group. In the pooled data, basal transit time correlated with age; systolic and diastolic blood pressure; HbA1c; and serum creatinine, cholesterol, and triglyceride concentrations (r = 0.29, 0.34, 0.37, 0.48, 0.25, 0.36, 0.29, respectively, P < 0.05 for each). Hypertensive diabetic patients had less deformable PMN than normotensive ones. No relation was found between PMN deformability and the duration of diabetes, type of treatment, or the presence of retinopathy.. These data indicate increased rigidity of PMN in NIDDM that may contribute to development of microcirculatory disturbances and microangiopathy.

Topics: Adult; Age Factors; Aged; Blood Pressure; Cytochalasin B; Diabetes Mellitus, Type 2; Female; Glucose Intolerance; Glucose Tolerance Test; Hispanic or Latino; Humans; In Vitro Techniques; Leukocyte Count; Male; Mexico; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Reference Values

We investigated the influence of altered glucose levels on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from NIDDM patients (n = 13) and non-diabetic subjects (n = 23). Whole body insulin sensitivity was 71% lower in the NIDDM patients compared to the non-diabetic subjects (p < 0.05), whereas, insulin-mediated peripheral glucose utilization in the NIDDM patients under hyperglycaemic conditions was comparable to that of the non-diabetic subjects at euglycaemia. Following a 30-min in vitro exposure to 4 mmol/l glucose, insulin-stimulated 3-0-methylglucose transport (600 pmol/l insulin) was 40% lower in isolated skeletal muscle strips from the NIDDM patients when compared to muscle strips from the non-diabetic subjects. The impaired capacity for insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle was normalized following prolonged (2h) exposure to 4 mmol/l, but not to 8 mmol/l glucose. Insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle exposed to 8 mmol/l glucose was similar to that of the non-diabetic muscle exposed to 5 mmol/l glucose, but was decreased by 43% (p < 0.01) when compared to non-diabetic muscle exposed to 8 mmol/l glucose. Despite the impaired insulin-stimulated 3-0-methylglucose transport capacity demonstrated by skeletal muscle from the NIDDM patients, skeletal muscle glycogen content was similar to that of the non-diabetic subjects. Kinetic studies revel a Km for 3-0-methylglucose transport of 9.7 and 8.8 mmol/l glucose for basal and insulin-stimulated conditions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-O-Methylglucose; Biological Transport; Blood Glucose; Cytochalasin B; Diabetes Mellitus, Type 2; Female; Glucose; Humans; In Vitro Techniques; Insulin; Insulin Resistance; Kinetics; Male; Methylglucosides; Middle Aged; Muscles

Insulin action was investigated in cultured skin fibroblasts from two consanguineous patients with a heterozygous point mutation in the insulin receptor kinase (Arg1152-Gln). In spite of normal binding, Gln1152 insulin receptor exhibited 20% increased basal kinase activity, but significantly reduced insulin-dependent autophosphorylation and kinase activity compared to controls from either weight-matched noninsulin-dependent diabetic patients (n = 4) or normal subjects (n = 5). In fibroblasts from the mutant patients, basal alpha-aminoisobutyric acid and 2-deoxyglucose (2-DG) uptake, cytochalasin-B (CB) plasma membrane binding, and glycogen synthase activity were increased to levels similar to those in maximally insulin-stimulated control cells. No insulin stimulation of these metabolic effects was detected in the mutant cells. In spite of the high basal 2-DG uptake and CB binding and the lack of further insulin response, fibroblasts from the mutant patients responded to 12-O-tetradecanoylphorbol-13-acetate with a further 50% increase in 2-DG uptake and CB binding. The magnitude of the effects of insulin and 12-O-tetradecanoylphorbol-13-acetate in control cells were nearly identical. We conclude that the Gln1152 insulin receptor impairs insulin regulation of metabolic responses in patient cells. Its presence in fibroblasts from the mutant patients appears to be accompanied by an increased pool of glucose transporters.

Topics: Adult; Aminobutyrates; Cells, Cultured; Cytochalasin B; Deoxyglucose; Diabetes Mellitus, Type 2; Female; Fibroblasts; Glucose; Glutamine; Glycogen Synthase; Humans; Insulin; Male; Middle Aged; Phosphorylation; Phosphotransferases; Point Mutation; Receptor, Insulin; Tetradecanoylphorbol Acetate

The purpose of this study was to investigate cellular changes in the glucose transport system in skeletal muscle of lean non-insulin-dependent diabetes mellitus (NIDDM) compared to lean nondiabetic control patients. NIDDM patients had significantly elevated fasting levels (means +/- SE) of serum glucose (10.1 +/- 1.3 vs. 5.4 +/- 0.4 mM, P less than 0.001) and serum insulin (110.8 +/- 31.1 vs. 35.9 +/- 3.6 pM, P less than 0.0025). Basal glucose transport (35.1 +/- 5.5 vs. 30.8 +/- 8.0 pM/mg protein) and cytochalasin-beta binding (3.5 +/- 1.2 vs 3.8 +/- 1.0 pM/mg protein) in isolated sarcolemmal vesicles were not significantly different between NIDDM and control groups. Insulin binding was reduced in NIDDM (0.82 +/- 0.03 vs. 1.63 +/- 0.18 pM/mg protein) as was the Kd (0.93 +/- 0.03 vs. 1.38 + 0.12 nM). Tyrosine kinase activity, as assessed from incorporation of [32P]ATP into Glu 4:Tyr 1, was significantly (P less than 0.005) reduced in NIDDM at insulin concentrations from 1-100 nM. Maximum kinase activity was depressed (1.88 +/- 0.04 vs. 2.97 +/- 0.07 fM 32P/fM insulin binding at 100 nM insulin). The number of glucose transporters in the low-density microsomes was not significantly different between NIDDM and control groups (7.01 +/- 1.40 vs. 7.65 +/- 0.90 pM cytochalasin-beta bound/mg protein). These results suggest that decreased insulin binding and diminished receptor tyrosine kinase activity play a substantial role in the development of skeletal muscle insulin resistance associated with NIDDM.

Topics: Adenosine Triphosphate; Adult; Aged; Blood Glucose; Cytochalasin B; Diabetes Mellitus, Type 2; Fasting; Glucose; Humans; Insulin; Kinetics; Middle Aged; Muscles; Phosphorylation; Protein-Tyrosine Kinases; Receptor, Insulin; Reference Values

The cellular mechanism of action of sulfonylureas may vary depending upon the exact nature of the drug, the tissue or cell type in question, and the status of the subject from which it was obtained. In adipocytes from patients with type II non-insulin-dependent diabetes, there is increasing evidence indicating that sulfonylureas ameliorate a post-receptor defect in insulin action by potentiating the insulin-stimulated glucose transport normally seen in these cells. Studies undertaken to elucidate the molecular mechanism of this potentiation investigated the effects of a 48-hour incubation with glyburide (2 micrograms/ml) on the recruitment of glucose carriers from microsomal storage pools to the plasma membrane. With the use of cytochalasin B, a potent competitive inhibitor of glucose transport, glucose-sensitive cytochalasin B binding was studied in basal and insulin-stimulated adipocytes from control and sulfonylurea-treated tissue. The data indicated that sulfonylurea treatment did not affect the total glucose-sensitive cytochalasin B binding capacity of adipocyte membranes. It did, however, increase the insulin-induced recruitment of the glucose carrier from the microsome to the plasma membrane by 27 to 31 percent. This suggests that the molecular mechanism of sulfonylurea-enhanced insulin-stimulated glucose transport is the recruitment of glucose transporters from an intracellular microsomal storage pool to the plasma membrane.

Topics: Adipose Tissue; Biological Transport; Blood Glucose; Cell Membrane; Cytochalasin B; Diabetes Mellitus, Type 2; Glyburide; Humans; Insulin; Kinetics; Microsomes