bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-6 gene polymorphism and insulin sensitivity.
Authors: Fernández-Real J M , Broch M , Vendrell J , Gutiérrez C , Casamitjana R , Pugeat M , Richart C , Ricart W
Published: March 2007

Type 2 diabetes and the insulin resistance syndrome have been hypothesized to constitute manifestations of an ongoing acute-phase response. We aimed to study an interleukin-6 (IL-6) gene polymorphism in relation to insulin sensitivity (IL-6 is the main cytokine involved in an acute-phase response). Subjects homozygous for the C allele at position -174 of the IL-6 gene (SfaNI genotype), associated to lower plasma IL-6 levels, showed significantly lower integrated area under the curve of serum glucose concentrations (AUCglucose) after an oral glucose tolerance test, lower blood glycosylated hemoglobin, lower fasting insulin levels, lower total and differential white blood cell count (a putative marker of peripheral IL-6 action), and an increased insulin sensitivity index than carriers of the G allele, despite similar age and body composition. A gene dosage effect was especially remarkable for AUCglucose (6.4 vs. 9.3 vs. 9.7 mmol/l in C/C, C/G, and G/G individuals, respectively). The serum concentration of fully glycosylated cortisol binding globulin (another marker of IL-6 action), suggested by concanavalin A adsorption, was lower in C/C subjects than in G/G individuals (32.6+/-2.9 vs. 37.6+/-4.6 mg/l, P = 0.03). In summary, a polymorphism of the IL-6 gene influences the relationship among insulin sensitivity, postload glucose levels, and peripheral white blood cell count.
A PC-1 amino acid variant (K121Q) is associated with faster progression of renal disease in patients with type 1 diabetes and albuminuria.
Authors: De Cosmo S , Argiolas A , Miscio G , Thomas S , Piras G P , Trevisan R , Perin P C , Bacci S , Zucaro L , Margaglione M , Frittitta L , Pizzuti A , Tassi V , Viberti G C , Trischitta V
Published: March 2007

Insulin resistance characterizes type 1 diabetes in patients with albuminuria. A PC-1 glycoprotein amino acid variant, K121Q, is associated with insulin resistance. We examined the impact of the PC-1 K121Q variant on the rate of decline of the glomerular filtration rate (GFR) by creatinine clearance derived from the Cockroft-Gault formula in 77 type 1 diabetic patients with albuminuria who were followed for an average of 6.5 years (range 2.5-15). Patients carrying the Q allele (n = 22; 20 with KQ and 2 with QQ genotypes) had a faster GFR decline than those patients with the KK genotype (n = 55) (median 7.2 vs. 3.7 ml x min(-1) x year(-1); range 0.16 to 16.6 vs. -3.8 to 16.0 ml x min(-1) x year(-1); P < 0.001). Significantly more patients carrying the Q allele belonged to the highest tertile of GFR decline (odds ratio = 5.7, 95% CI 4.1-7.2, P = 0.02). Levels of blood pressure, HbA1c, and albuminuria were comparable in the two genotype groups. Albuminuria (P = 0.001), mean blood pressure (P = 0.046), and PC-1 genotype (P = 0.036) independently correlated with GFR decline. Because all patients were receiving antihypertensive treatment, the faster GFR decline in the patients carrying the Q allele could be the result of reduced sensitivity to the renoprotective effect of antihypertensive therapy. PC-1 genotyping identifies type 1 diabetic patients with a faster progression of diabetic nephropathy.
v- and t-SNARE protein expression in models of insulin resistance: normalization of glycemia by rosiglitazone treatment corrects overexpression of cellubrevin, vesicle-associated membrane protein-2, and syntaxin 4 in skeletal muscle of Zucker diabetic fatty rats.
Authors: Maier V H , Melvin D R , Lister C A , Chapman H , Gould G W , Murphy G J
Published: March 2007

Insulin stimulation of adipose and muscle cells results in the translocation of GLUT4 from an intracellular location to the plasma membrane; this translocation is defective in insulin resistance. Studies have suggested an important role for synaptobrevin and syntaxin homologues in this event, particularly the v-soluble N-ethylmaleimide attachment protein receptors (SNAREs) cellubrevin and vesicle-associated membrane protein-2 (VAMP-2) and the t-SNARE syntaxin 4, but the expression of these proteins has not been studied in insulin-resistant tissues. Therefore, we examined SNARE protein content in skeletal muscle from Zucker diabetic fatty (ZDF) rats compared with lean controls and determined the effect of the thiazolidinedione insulin sensitizer rosiglitazone on these proteins. GLUT4 levels in skeletal muscle from ZDF rats were similar to those in lean control animals. In contrast, cellubrevin, VAMP-2, and syntaxin 4 protein levels were elevated (2.8-fold, P = 0.02; 3.7-fold, P = 0.01; and 2.2-fold, P < 0.05, respectively) in skeletal muscle from ZDF rats compared with lean controls. Restoration of normoglycemia and normoinsulinemia in ZDF rats with rosiglitazone (30 micromol/kg) normalized cellubrevin, VAMP-2, and syntaxin 4 protein to levels approaching those observed in lean control animals. These data show that elevated v- and t-SNARE protein levels are associated with insulin resistance in skeletal muscle and that these increases may be reversed by rosiglitazone treatment concomitant with a restoration of glycemic control. Such increases in SNARE protein levels were not observed in streptozotocin-induced diabetic rats, which suggests that hyperinsulinemia rather than hyperglycemia may be more important in modulating SNARE protein expression in rodent models of insulin resistance. Consistent with this hypothesis, elevated levels of SNARE proteins were also observed in 3T3-L1 adipocytes chronically treated with insulin (500 nmol/l for 24 h). These data argue that SNARE protein levels may be altered in insulin-resistant states and that the levels of these proteins are modulated by agents that increase insulin sensitivity. Moreover, these data demonstrate for the first time altered expression of proteins known to regulate GLUT4 translocation in a model of diabetes.
Can clinical factors estimate insulin resistance in type 1 diabetes?
Authors: Williams K V , Erbey J R , Becker D , Arslanian S , Orchard T J
Published: March 2007

An insulin resistance syndrome (IRS) score was developed based on clinical risk factors in adults with childhood-onset type 1 diabetes in the Epidemiology of Diabetes Complications (EDC) Study and was validated using euglycemic-hyperinsulinemic clamp studies. Hypertension, waist-to-hip ratio (WHR), triglyceride and HDL cholesterol levels, family history of type 2 diabetes, and glycemic control were risk factors used to define the score. A score of 1 (lowest likelihood IRS) to 3 (highest likelihood IRS) was assigned for each risk factor. Eligible subjects (n = 24) were recruited from the EDC cohort based on tertile of IRS score. Subjects received an overnight insulin infusion to normalize glucose levels, then underwent a 3-h euglycemic-hyperinsulinemic (60 mU x m(-2) x min(-1)) clamp. Glucose disposal rate (GDR) was determined during the last 30 min of the clamp. The GDR differed significantly by IRS group (9.65 +/- 2.99, 8.02 +/- 1.39, and 5.68 +/- 2.16 mg x kg(-1) x min(-1), P < 0.01). The GDR was inversely correlated with the IRS score (r = -0.64, P < 0.01). Using linear regression, the combination of risk factors that yielded the highest adjusted r2 value (0.57, P < 0.001) were WHR, hypertension, and HbA1. This study found that clinical risk factors can be used to identify subjects with type 1 diabetes who are insulin resistant, and it provides validation of a score based on clinical factors to determine the extent of insulin resistance in type 1 diabetes. This score will be applied to the entire EDC population in future studies to determine the effect of insulin resistance on complications.
Increase in fat oxidation on a high-fat diet is accompanied by an increase in triglyceride-derived fatty acid oxidation.
Authors: Schrauwen P , Wagenmakers A J , van Marken Lichtenbelt W D , Saris W H , Westerterp K R
Published: March 2007

The aim of this study is to investigate the mechanism behind the slow increase in fat oxidation on a high-fat diet. Therefore, we determined 24-h substrate oxidation using respiration chambers and the rate of appearance and oxidation of plasma-derived fatty acids in seven healthy nonobese men (age 23 +/- 2 years, height 1.85 +/- 0.03 m, weight 70.4 +/- 2.3 kg, % body fat 13 +/- 1). Before testing, they consumed a low-fat diet (30% fat, 55% carbohydrate) at home for 3 days. Measurements were performed after 1 day consumption of either a low-fat diet (LF), a high-fat diet (HF1, 60% fat, 25% carbohydrate), or a high-fat diet preceded by a glycogen-lowering exercise test (HF1+EX), and after 7 days on a high-fat diet (HF7). After an overnight fast, an infusion of [U-13C]palmitate (0.00806 micromol x min(-1) x kg(-1)) was started and continued for 2 h at rest followed by 1 h of exercise at 50% of maximal power output (Wmax). Whole-body fat oxidation was measured using indirect calorimetry, and plasma-derived fatty acid oxidation was evaluated by measuring breath 13CO2 enrichment and corrected with the acetate recovery factor. Twenty-four-hour fat oxidation gradually increased on the high-fat diet. Both at rest and during exercise, there was no change in rate of appearance of fatty acids and plasma-derived fatty acid oxidation. Triglyceride-derived fatty acid oxidation tended to be higher after 7 days of high-fat diet at rest (P < 0.07). This difference was significant during exercise (P < 0.05). In conclusion, the results from this study suggest that triglyceride-derived fatty acid oxidation (VLDL or intramuscular triglycerides) plays a role in the increase in fat oxidation on a high-fat diet, but plasma-derived fatty acids remain the major source for fat oxidation.
Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance.
Authors: Shankar R R , Wu Y , Shen H Q , Zhu J S , Baron A D
Published: March 2007

Studies from our laboratory using acute pharmacologic blockade of nitric oxide synthase (NOS) activity have suggested that nitric oxide (NO) has an important role in regulating carbohydrate metabolism. We now report on insulin sensitivity in mice with targeted disruptions in endothelial NOS (eNOS) and neuronal NOS (nNOS) genes compared with their wild-type (WT) counterparts. Mice underwent hyperinsulinemic-euglycemic clamp studies after a 24-h fast, during an insulin infusion of 20 mU x kg(-1) x min(-1). Glucose levels were measured at baseline and every 10 min during the clamp. Insulin levels were measured at baseline and at the end of the clamp study. Glucose infusion rates (GIRs) during the last 30 min of the clamp study were in a steady state. Tritiated glucose infusion was used to measure rates of endogenous glucose output (EGO) both at baseline and during steady-state euglycemia. Glucose disposal rates (GDRs) were computed from the GIR and EGO. Fasting and steady-state glucose and insulin levels were comparable in the 3 groups of mice. No differences in fasting EGO were noted between the groups. GIR was significantly reduced (37%, P = 0.001) in the eNOS knockout (KO) mice compared with the WT mice, with values for the nNOS mice being intermediate. EGO was completely suppressed in the nNOS and WT mice during insulin infusion, but not in the eNOS mice. Even so, the eNOS mice displayed significantly reduced whole-body GDRs compared with those of the WT mice (82.67+/-10.77 vs. 103.67+/-3.47 mg x kg(-1) x min(-1), P = 0.03). eNOS KO mice are insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS KO mice are insulin resistant only in the latter. These data indicate that NO plays a role in modulating insulin sensitivity and carbohydrate metabolism and that the eNOS isoform may play a dominant role relative to nNOS.
Secretion of tumor necrosis factor-alpha shows a strong relationship to insulin-stimulated glucose transport in human adipose tissue.
Authors: Löfgren P , van Harmelen V , Reynisdottir S , Näslund E , Rydén M , Rössner S , Arner P
Published: March 2007

Some animal models suggest that tumor necrosis factor (TNF)-alpha is a key component in obesity-linked insulin resistance because it inhibits insulin receptor signaling and glucose transport in insulin-sensitive tissues. However, in vivo data in humans have given conflicting results regarding the relationship between circulating TNF-alpha levels and insulin sensitivity. In the present study, the potential local role of TNF-alpha on insulin action in human subcutaneous adipose tissue was studied in 42 obese women (BMI 39+/-10 kg/m2). We found a strong inverse correlation between adipose TNF-alpha secretion and maximum insulin-stimulated glucose transport in adipocytes that was independent of fat cell volume, age, and BMI (P < 0.001, r = 0.58). As much as one-third of the variation in insulin-stimulated glucose transport could be accounted for by variations in TNF-alpha secretion. There was no significant correlation (r = 0.11) between secretion of adipose plasminogen activator inhibitor 1 and glucose transport. Furthermore, subcutaneous adipose tissue of 4 obese women (BMI 40+/-4) incubated with TNF-A for 24 h showed a one-third concentration-dependent inhibition of insulin-stimulated glucose transport (P < 0.01). In conclusion, adipose TNF-alpha may be an important specific and local factor in adipose tissue that influences the ability of insulin to stimulate glucose transport in human fat cells, at least in obese women.
Localization of glucokinase gene expression in the rat brain.
Authors: Lynch R M , Tompkins L S , Brooks H L , Dunn-Meynell A A , Levin B E
Published: March 2007

The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic beta-cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK+ neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K+ channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and beta-cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK+ neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism.
Cx36 preferentially connects beta-cells within pancreatic islets.
Authors: Serre-Beinier V , Le Gurun S , Belluardo N , Trovato-Salinaro A , Charollais A , Haefliger J A , Condorelli D F , Meda P
Published: March 2007

Previous studies have provided evidence for the transcripts of Cx43 and Cx45 within pancreatic islets. As of yet, however, it has proven difficult to unambiguously demonstrate the expression of these proteins by islet cells. We have investigated whether Cx36, a new connexin species recently identified in mammalian brain and retina, may also be expressed in pancreatic islets. Using probes that permitted the original identification of Cx36 in the central nervous system, we show that a transcript for Cx36 is clearly detectable in rat pancreatic islets. Using novel and affinity-purified polyclonal antibodies, we have found that Cx36 is actually expressed in pancreatic islets. Both in situ hybridization and immunolabeling indicated that this connexin is abundant in the centrally located insulin-producing beta-cells and is expressed much less, if at all, by the other endocrine cell types. This differential expression was further confirmed on fluorescence-activated cell sorter-purified preparations enriched in either beta- or non-beta-cells. The finding of a differential distribution of Cx36 within distinct regions of pancreatic islets creates the possibility that this connexin may provide the establishment of selective pathways of communication between the different types of endocrine cells comprising the pancreatic islet.
Hepatic fat content and insulin action on free fatty acids and glucose metabolism rather than insulin absorption are associated with insulin requirements during insulin therapy in type 2 diabetic patients.
Authors: Ryysy L , Häkkinen A M , Goto T , Vehkavaara S , Westerbacka J , Halavaara J , Yki-Järvinen H
Published: March 2007

To determine causes of interindividual variation in insulin requirements, we recruited 20 type 2 diabetic patients with stable glucose control and insulin doses for >1 year on combination therapy with bedtime NPH insulin and metformin. Insulin absorption (increase in free and total insulin over 8 h after a subcutaneous dose of regular insulin) and actions of intravenous (6-h 0.3 mU x kg(-1) x min(-1) euglycemic insulin clamp combined with [3-3H]glucose) and subcutaneous (glucose infusion rate required to maintain isoglycemia and suppression of free fatty acids [FFAs]) insulin, liver fat content (proton spectroscopy), visceral fat (magnetic resonance imaging), weight, and body composition were determined. We found the following variation in parameters: insulin dose range 10-176 U (mean 42 U, fold variation 17.6x) or 0.13-1.39 U/kg (0.44 U/kg, 10.7x), absorbed insulin 10.6x, action of subcutaneous insulin to suppress FFAs 7.5 x and to stimulate glucose metabolism (M value) 11.5x, body weight 67-127 kg (91 kg, 1.9x), liver fat 2-28% (12%, 14x), and visceral fat 179-2,053 ml (1,114 ml, 11.5x). The amount of insulin absorbed, measured as either free or total insulin, was significantly correlated with its ability to suppress FFAs and stimulate glucose metabolism but not with the insulin dose per se. The actions of absorbed insulin were, on the other hand, significantly correlated with the daily insulin dose (r = 0.70 for action on FFAs, P < 0.001, and r = -0.61 for M value, P < 0.005). Actions of subcutaneous and intravenous insulin to suppress FFAs were significantly correlated (r = 0.82, P < 0.001, R2 = 67%). Of the measures of adiposity, the percent hepatic fat was the parameter best correlated with the daily insulin dose (r = 0.76, P < 0.001). The percent hepatic fat was also significantly correlated with the ability of intravenous insulin to suppress endogenous glucose production (r = 0.72, P < 0.005). We conclude that the major reason for interindividual variation in insulin requirements in type 2 diabetes is the variation in insulin action. Variation in hepatic fat content may influence insulin requirements via an effect on the sensitivity of endogenous glucose production to insulin.
A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice.
Authors: Fukui Y , Masui S , Osada S , Umesono K , Motojima K
Published: March 2007

Thiazolidinediones (TZDs) reduce insulin resistance in type 2 diabetes by increasing peripheral uptake of glucose, and they bind to and activate the transcriptional factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Studies have suggested that TZD-induced activation of PPAR-gamma correlates with antidiabetic action, but the mechanism by which the activated PPAR-gamma is involved in reducing insulin resistance is not known. To examine whether activation of PPAR-gamma directly correlates with antidiabetic activities, we compared the effects of 4 TZDs (troglitazone, pioglitazone, BRL-49653, and a new derivative, NC-2100) on the activation of PPAR-gamma in a reporter assay, transcription of the target genes, adipogenesis, plasma glucose and triglyceride levels, and body weight using obese KKAy mice. There were 10- to 30-fold higher concentrations of NC-2100 required for maximal activation of PPAR-gamma in a reporter assay system, and only high concentrations of NC-2100 weakly induced transcription of the PPAR-gamma but not PPAR-alpha target genes in a whole mouse and adipogenesis of cultured 3T3L1 cells, which indicates that NC-2100 is a weak PPAR-gamma activator. However, low concentrations of NC-2100 efficiently lowered plasma glucose levels in KKAy obese mice. These results strongly suggest that TZD-induced activation of PPAR-gamma does not directly correlate with antidiabetic (glucose-lowering) action. Furthermore, NC-2100 caused the smallest body weight increase of the 4 TZDs, which may be partly explained by the finding that NC-2100 efficiently induces uncoupling protein (UCP)-2 mRNA and significantly induces UCP1 mRNA in white adipose tissue (WAT). NC-2100 induced UCP1 efficiently in mesenteric WAT and less efficiently in subcutaneous WAT, although pioglitazone and troglitazone also slightly induced UCP1 only in mesenteric WAT. These characteristics of NC-2100 should be beneficial for humans with limited amounts of brown adipose tissue.
Interaction between insulin sensitivity and muscle perfusion on glucose uptake in human skeletal muscle: evidence for capillary recruitment.
Authors: Baron A D , Tarshoby M , Hook G , Lazaridis E N , Cronin J , Johnson A , Steinberg H O
Published: March 2007

Insulin and glucose delivery (muscle perfusion) can modulate insulin-mediated glucose uptake. This study was undertaken to determine 1) to what extent insulin sensitivity modulates the effect of perfusion on glucose uptake and 2) whether this effect is achieved via capillary recruitment. We measured glucose disposal rates (GDRs) and leg muscle glucose uptake (LGU) in subjects exhibiting a wide range of insulin sensitivity, after 4 h of steady-state (SS) euglycemic hyperinsulinemia (>6,000 pmol/l) and subsequently after raising the rate of leg blood flow (LBF) 2-fold with a superimposed intrafemoral artery infusion of methacholine chloride (Mch), an endothelium-dependent vasodilator. LBF was determined by thermodilution: LGU = arteriovenous glucose difference (AVGdelta) x LBF. As a result of the 114+/-12% increase in LBF induced by Mch, the AVGdelta decreased 32+/-4%, and overall rates of LGU increased 40+/-5% (P < 0.05). We found a positive relationship between the Mch-modulated increase in LGU and insulin sensitivity (GDR) (r = 0.60, P < 0.02), suggesting that the most insulin-sensitive subjects had the greatest enhancement of LGU in response to augmentation of muscle perfusion. In separate groups of subjects, we also examined the relationship between muscle perfusion rate and glucose extraction (AVGdelta). Perfusion was either pharmacologically enhanced with Mch or reduced by intra-arterial infusion of the nitric oxide inhibitor N(G)-monomethyl-L-arginine during SS euglycemic hyperinsulinemia. Over the range of LBF, changes in AVGdelta were smaller than expected based on the noncapillary recruitment model of Renkin. Together, the data indicate that 1) muscle perfusion becomes more rate limiting to glucose uptake as insulin sensitivity increases and 2) insulin-mediated increments in muscle perfusion are accompanied by capillary recruitment. Thus, insulin-stimulated glucose uptake displays both permeability- and perfusion-limited glucose exchange properties.
Targeted upregulation of pyruvate dehydrogenase kinase (PDK)-4 in slow-twitch skeletal muscle underlies the stable modification of the regulatory characteristics of PDK induced by high-fat feeding.
Authors: Holness M J , Kraus A , Harris R A , Sugden M C
Published: March 2007

In using Western blot analysis with antibodies raised against recombinant pyruvate dehydrogenase kinase (PDK) isoforms PDK2 and PDK4, this study demonstrates selective PDK isoform switching in specific skeletal muscle types in response to high-fat feeding that is associated with altered regulation of PDK activity by pyruvate. The administration of a diet high in saturated fats led to stable (approximately 2-fold) increases in PDK activities in both a typical slow-twitch (soleus [SOL]) muscle and a typical fast-twitch (anterior tibialis [AT]) muscle. Western blot analysis revealed that high-fat feeding significantly increased (approximately 2-fold; P < 0.001) PDK4 protein expression in SOL, with a modest (1.3-fold) increase in PDK2 protein expression. The relative increase in PDK4 protein expression in SOL was associated with a 7.6-fold increase in the pyruvate concentration that was required to elicit a 50% active pyruvate dehydrogenase complex, which indicates a marked decrease in the sensitivity of PDK to inhibition by pyruvate. In AT muscle, high-fat feeding elicited comparable (1.5- to 1.7-fold) increases (P < 0.05) in PDK4 and PDK2 protein expression. Loss of sensitivity of PDK to inhibition by pyruvate was less marked. The data suggest that a positive correlation exists between increases in PDK4 expression and the propensity with which muscles use lipid-derived fuels as respiratory substrates rather than with the degree of insulin resistance induced in skeletal muscles by high-fat feeding. In conclusion, high-fat feeding leads to selective upregulation of PDK4 expression in slow-twitch muscle in response to high-fat feeding in vivo, which is associated with a pronounced loss of sensitivity of PDK activity to acute inhibition by pyruvate. Thus, increased PDK4 expression may underlie the stable modification of the regulatory characteristics of PDK observed in slow-twitch muscle in response to high-fat feeding.
Tumor necrosis factor-alpha induces hepatic insulin resistance in obese Zucker (fa/fa) rats via interaction of leukocyte antigen-related tyrosine phosphatase with focal adhesion kinase.
Authors: Cheung A T , Wang J , Ree D , Kolls J K , Bryer-Ash M
Published: March 2007

The molecular mechanism whereby tumor necrosis factor-alpha (TNF-alpha) induces insulin resistance in obesity is not well understood. Previously, we have shown that inhibition of TNF-alpha improved hepatic insulin sensitivity in obese Zucker rats without altering the tyrosine phosphorylation of liver insulin receptors (IRs), which indicates that the TNF-alpha and insulin-signaling cascades interact distally to the IR. To assess the effects of TNF-alpha on signaling molecules downstream from the IR, we analyzed the tyrosine phosphorylation patterns of liver homogenate proteins from TNF-alpha-neutralized fa/fa rats and showed that focal adhesion kinase (FAK) was consistently hyperphosphorylated (4.5-fold). Moreover, intravenous insulin increased hepatic FAK phosphorylation in a time-dependent manner in Sprague-Dawley rats, which suggests that TNF-alpha may induce hepatic insulin resistance by preventing FAK phosphorylation in response to insulin treatment. To explore the cellular mechanism whereby TNF-alpha regulates phosphorylation of FAK in the liver, we measured c-Src kinase activity and the abundance of 3 major protein tyrosine phosphatases (PTPs) (PTP-1B, leukocyte antigen-related tyrosine phosphatase [LAR], and src homology 2 domain-containing protein-tyrosine phosphatase [SHPTP-2]) in liver homogenates from obese Zucker rats after TNF-alpha blockade. Hepatic c-Src kinase activity was unaltered, but LAR protein was reduced by 75%. In addition, TNF-alpha blockade reduced hepatic PTP activity toward tyrosine phosphorylated FAK by 70%, and this was accounted for by immunodepletion of LAR. Incubation of HepG2 cells with TNF-alpha increased LAR protein levels in a dose-dependent manner. Additionally, pretreatment with TNF-alpha abolished insulin-stimulated tyrosine phosphorylation of FAK in HepG2 cells but had no effect on IR tyrosine phosphorylation or expression. These data suggest that TNF-alpha promotes LAR expression and thus prevents insulin-mediated tyrosine phosphorylation of FAK. This probably represents the interface between TNF-alpha and insulin signaling in the liver.
Presumed apoptosis and reduced arcuate nucleus neuropeptide Y and pro-opiomelanocortin mRNA in non-coma hypoglycemia.
Authors: Tkacs N C , Dunn-Meynell A A , Levin B E
Published: March 2007

Hypoglycemia reduces sympathoadrenal responses to subsequent hypoglycemic bouts by an unknown mechanism. To assess whether such hypoglycemia-associated autonomic failure is due to actual brain damage, male Sprague-Dawley rats underwent 1-h bouts of insulin-induced (5 U/kg i.v.) hypoglycemia (1.6-2.8 mmol/l) 1 or 3 times on alternate days. Rats remained alert and were rescued with intravenous glucose at 60-80 min. Plasma epinephrine and corticosterone responses were significantly reduced during the second and third bouts. Brains from these rats were processed by the terminal transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) procedure as an index of apoptotic cell death at 24, 48, or 96 h after their first bout. At 48 h, but not 24 h, TUNEL+ cells were consistently seen only in the arcuate nucleus (arcuate hypothalamic nucleus [ARC]). Hypoglycemic rats had 188% more apoptotic ARC cells (1 bout 39+/-5; 3 bouts 37+/-4) than euglycemic controls (13+/-3;P = 0.001). In situ hybridization for neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA was performed in sections of ARC containing maximal numbers of apoptotic cells as well as in other fresh frozen brains. After 1 bout, NPY (0.041+/-0.003) and POMC (0.119+/-0.022) mRNA were decreased, respectively, by 52 and 55% vs. controls (NPY 0.076+/-0.007; POMC 0.222+/-0.020; P = 0.01). NPY (0.029+/-0.002) but not POMC (0.093+/-0.013) fell 29% further after a third bout. NPY (r = -0.721; P = 0.001) and POMC (r = -0.756; P = 0.001) mRNA levels correlated negatively with the number of apoptotic ARC cells in the same sections. Thus, non-coma hypoglycemia produces apparent apoptotic cell death with reduced NPY and POMC expression selectively in the ARC. This may contribute to the reduced counterregulatory response following repeated bouts of hypoglycemia.
Nitric oxide synthesis and isoprostane production in subjects with type 1 diabetes and normal urinary albumin excretion.
Authors: O'Byrne S , Forte P , Roberts L J , Morrow J D , Johnston A , Anggård E , Leslie R D , Benjamin N
Published: March 2007

The role of nitric oxide (NO) and free radicals in the development of microvascular disease in type 1 diabetes remains unclear. We have measured NO and isoprostane (a stable marker of in vivo lipid peroxidation) production in 13 type 1 diabetic subjects with normal urinary albumin excretion and 13 healthy volunteers. Whole-body NO synthesis was quantified by measuring the urinary excretion of 15N-nitrate after the intravenous administration of L-[15N]2-arginine. The urinary excretion of the major urinary metabolite of 15-F2t-isoprostane (8-iso-prostaglandin-F2alpha), 2,3-dinor-5,6-dihydro-F2t-IsoP, was quantified as a marker of in vivo lipid peroxidation. Whole-body NO synthesis was significantly higher in diabetic subjects compared with control subjects (342 vs. 216 nmol 15N-nitrate/mmol creatinine [95% CI of the difference 45-207], P = 0.005). This increase was not explained by a difference in renal function between the 2 groups. There was no difference in 2,3-dinor-5,6-dihydro-F2t-IsoP excretion between diabetic subjects and control subjects (44.8+/-7.8 vs. 41.4+/-10.0 ng/mmol creatinine, mean +/- 95% CI). However, there was an inverse correlation between NO synthesis and free radical activity in subjects with diabetes (r = -0.62, P = 0.012) that was not observed in control subjects (r = 0.37, P = 0.107). We conclude that whole-body NO synthesis is higher in type 1 diabetic subjects with normal urinary albumin excretion than in control subjects. The inverse correlation between isoprostane production and NO synthesis in diabetic subjects is consistent with the hypothesis that NO is being inactivated by reactive oxygen species.
Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats.
Authors: Isshiki K , Haneda M , Koya D , Maeda S , Sugimoto T , Kikkawa R
Published: March 2007

Thiazolidinedione (TZD) compounds are widely used as oral hypoglycemic agents. Herein, we provide evidence showing that troglitazone, one of the TZD compounds, is able to prevent glomerular dysfunction in diabetic rats through a novel mechanism independent of its insulin-sensitizing action. We examined the effect of troglitazone on functional and biochemical parameters of glomeruli in streptozotocin-induced diabetic rats. Troglitazone was able to prevent not only diabetic glomerular hyperfiltration and albuminuria, but an increase in mRNA expression of extracellular matrix proteins and transforming growth factor-beta1 in glomeruli of diabetic rats, without changing blood glucose levels. Biochemically, an increase in diacylglycerol (DAG) contents and the activation of the protein kinase C (PKC)-extracellular signal-regulated kinase (ERK) pathway in glomeruli of diabetic rats were abrogated by troglitazone. The activation of DAG-PKC-ERK pathways in vitro in mesangial cells cultured under high glucose conditions was also inhibited by troglitazone. Troglitazone enhanced the activities of DAG kinase, which could metabolize DAG to phosphatidic acid, in both glomeruli of diabetic rats and mesangial cells cultured under high glucose conditions. Surprisingly, pioglitazone, another TZD compound without alpha-tocopherol moiety in its structure, also prevented the activation of the DAG-PKC pathway and activated DAG kinase in mesangial cells cultured under high glucose conditions. These results may identify the TZDs as possible new therapeutic agents for diabetic nephropathy that prevent glomerular dysfunction through the inhibition of the DAG-PKC-ERK pathway.
Anti-modified LDL antibodies, LDL-containing immune complexes, and susceptibility of LDL to in vitro oxidation in patients with type 2 diabetes.
Authors: Mironova M A , Klein R L , Virella G T , Lopes-Virella M F
Published: March 2007

We investigated the hypothesis that modified lipoproteins trigger an immune response leading to the production of autoantibodies and subsequently to the formation of atherogenic immune complexes (IC). We recruited 20 type 2 diabetic patients with macrovascular disease, 14 nondiabetic patients with coronary artery disease (CAD), and 34 healthy control subjects matched for age, sex, and race. Serum antibodies to oxidized and glycated LDL did not differ significantly among the 3 groups. Serum IC contained variable, but not statistically different, amounts of IgG, IgM, and IgA. In contrast, the content of cholesterol in IC isolated from diabetic patients was significantly higher than that in IC isolated from control subjects, and the content of apolipoprotein (apo)-B was significantly higher than that in IC isolated from control subjects and patients with CAD. Cholesteryl ester accumulation in human monocyte-derived macrophages incubated with IC, a measure of the atherogenic potential of IC, was significantly higher in macrophages incubated with red blood cell-adsorbed IC isolated from diabetic patients compared with IC isolated from control subjects (P < 0.03) or from patients with CAD (P < 0.04) and was strongly correlated with the content of apoB (r = 0.68, P < 0.001) and cholesterol (r = 0.61, P < 0.001) in IC. LDL from diabetic patients was more susceptible to oxidation in vitro, was significantly smaller, and contained significantly less alpha-tocopherol than LDL isolated from subjects in the other groups. In addition, the n-3 polyunsaturated fatty acid content of phospholipids and cholesteryl esters in LDL isolated from diabetic patients was significantly increased (P < 0.05) compared with that from patients with CAD or from control subjects. We postulate that LDL size, susceptibility to oxidation, and lipid fatty acid composition may play a critical role in the production of antibodies to oxidized LDL and consequently in the formation of LDL-containing IC in patients with type 2 diabetes.
Segregation analysis of urinary albumin excretion in families with type 2 diabetes.
Authors: Fogarty D G , Hanna L S , Wantman M , Warram J H , Krolewski A S , Rich S S
Published: March 2007

Elevated urinary albumin excretion (UAE) is a predictor of the development of nephropathy and cardiovascular mortality. To study whether genetic factors may determine UAE, we examined familial aggregation of UAE in 96 large multigenerational pedigrees ascertained for type 2 diabetes. A total of 1,269 subjects had UAE measured as the urinary albumin-to-creatinine ratio (ACR). This included 630 subjects with type 2 diabetes and 639 subjects without diabetes. A significant correlation (Spearman's correlation 0.34, P < 0.001) was found between the median ACR values determined separately in nondiabetic and diabetic members of the same family. To determine whether this familial aggregation of ACR could be explained by the transmission of 1 or more major genes and thus be suitable for gene mapping studies, segregation analyses were performed. In these analyses, ACR was modeled as a continuous trait with the inclusion of age, sex, and duration of diabetes as covariates. Likelihood ratio tests were performed to test competing hypotheses, and Akaike's information criterion was used to determine the most parsimonious models. The Mendelian model with multifactorial inheritance was supported more strongly than Mendelian inheritance alone. These analyses suggested that the best model for ACR levels was multifactorial with evidence for a common major gene. When the analyses were repeated for diabetic subjects only, the evidence for Mendelian inheritance was improved, although a single major locus with additional multifactorial effects was more strongly supported. The results from the current study suggest that levels of UAE are determined by a mixture of genes with large and small effects as well as other measured covariates, such as diabetes.
Effect of hyperglycemia and hyperlipidemia on atherosclerosis in LDL receptor-deficient mice: establishment of a combined model and association with heat shock protein 65 immunity.
Authors: Keren P , George J , Shaish A , Levkovitz H , Janakovic Z , Afek A , Goldberg I , Kopolovic J , Keren G , Harats D
Published: March 2007

Diabetes and atherosclerosis have been proposed to be influenced by immune and autoimmune mechanisms. A common incriminated antigen in both disorders is the heat shock protein (HSP)-60/65. In the current study, we established a model combining hyperglycemia with hyperlipidemia in LDL receptor-deficient (LDL-RD) mice and assessed its possible influences on lipid profile, HSP60/65, and atherogenesis. LDL-RD mice were injected either with streptozotocin to induce hyperglycemia or with citrate buffer (control). When hyperglycemia was induced, both study groups were challenged with a high-fat (Western) diet for 6 weeks. Plasma fasting glucose, lipid profile, and antibody levels to HSP65 and oxidized LDL were assessed. At death, the spleens from both groups were evaluated for their proliferative response to HSP65 and the consequent cytokine production. The extent of atherosclerosis was assessed at the aortic sinus. Plasma glucose, cholesterol, and triglyceride levels were elevated in mice injected with streptozotocin compared with control mice. Atherosclerotic lesions were significantly larger in the streptozotocin-injected hyperglycemic LDL-RD mice (132 +/- 23 x 10(5) microm2) in comparison to their normoglycemic litter-mates (20 +/- 6.6 x 10(5) microm2; P < 0.0001). Both humoral and cellular immune response to HSP65 was more pronounced in streptozotocin-injected mice. When challenged with HSP65 in vitro, splenocytes from streptozotocin-injected mice favored the production of the T-helper (TH)-1 cytokine gamma-interferon. In conclusion, we have established a mouse model that combines hyperglycemia with diet-induced hyperlipidemia in LDL-RD mice and studied its effect on atherosclerosis progression. The accelerated atherosclerotic process is associated with heightened immune response to HSP65 and a shift to a TH1 cytokine profile.
5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase.
Authors: Lochhead P A , Salt I P , Walker K S , Hardie D G , Sutherland C
Published: March 2007

Insulin regulates the rate of expression of many hepatic genes, including PEPCK, glucose-6-phosphatase (G6Pase), and glucose-6-phosphate dehydrogenase (G6PDHase). The expression of these genes is also abnormally regulated in type 2 diabetes. We demonstrate here that treatment of hepatoma cells with 5-aminoimidazole-4-carboxamide riboside (AICAR), an agent that activates AMP-activated protein kinase (AMPK), mimics the ability of insulin to repress PEPCK gene transcription. It also partially represses G6Pase gene transcription and yet has no effect on the expression of G6PDHase or the constitutively expressed genes cyclophilin or beta-actin. Several lines of evidence suggest that the insulin-mimetic effects of AICAR are mediated by activation of AMPK. Also, insulin does not activate AMPK in H4IIE cells, suggesting that this protein kinase does not link the insulin receptor to the PEPCK and G6Pase gene promoters. Instead, AMPK and insulin may lie on distinct pathways that converge at a point upstream of these 2 gene promoters. Investigation of the pathway by which AMPK acts may therefore give insight into the mechanism of action of insulin. Our results also suggest that activation of AMPK would inhibit hepatic gluconeogenesis in an insulin-independent manner and thus help to reverse the hyperglycemia associated with type 2 diabetes.
Exposure to exogenous insulin promotes IgG1 and the T-helper 2-associated IgG4 responses to insulin but not to other islet autoantigens.
Authors: Füchtenbusch M , Kredel K , Bonifacio E , Schnell O , Ziegler A G
Published: March 2007

Insulin immunization in animal models induces T-helper (Th) 2-like antibody subclass responses to insulin and other beta-cell antigens. The aim of this study was to determine whether exposure to insulin in humans resulted in a similar subclass bias of the humoral immune response. Levels of IgG subclass antibodies to insulin (IAs), GAD, and IA-2 were measured before and after treatment with insulin in the following groups of patients: 29 patients with newly diagnosed type 1 diabetes treated with intravenous and/or subcutaneous insulin; 10 newly diagnosed patients randomized to cyclosporin A (CsA) or placebo plus subcutaneous insulin for 12 months; and 14 islet cell antibody-positive relatives receiving either intravenous and subcutaneous insulin prophylaxis or no treatment. At the onset of diabetes, the major subclass distributions of insulin autoantibodies (IAAs) were IgG1 and, to a lesser extent, IgG4. After insulin treatment in the 29 new-onset patients, IAs were initially of the IgG1 subclass. IgG4-IAs appeared later, but at 12 months, they were at higher levels than IgG1-IAs in 11 patients. Responses were higher in children compared with adults and were higher in subjects with IAAs (P < 0.001). Insulin prophylaxis in relatives showed a similar profile, with a decline in levels of IgG1-IAs after cessation of daily subcutaneous insulin. Patients treated with CsA took longer to develop IAs and showed suppressed levels of IgG4-IAs; however, their levels of high-titer IgG1-IAs persistently rebounded after completion of CsA therapy. Despite the presence of IgG4-IAs in most insulin-treated patients and relatives, a shift to IgG4-anti-GAD or IgG4-IA-2 was not found for up to 3 years after the initiation of insulin therapy. While our findings need to be correlated with T-cell cytokine responses, we suggest that the strong IgG4-IA response in insulin-treated patients is consistent with an enhancement of Th2 immunity, but there is no evidence of subsequent spreading of potentially Th2-associated IgG4 responses to other autoantigens.
Melanin-concentrating hormone regulates leptin synthesis and secretion in rat adipocytes.
Authors: Bradley R L , Kokkotou E G , Maratos-Flier E , Cheatham B
Published: March 2007

Obesity is a common problem in Western society and is associated with significant morbidity and mortality. Energy homeostasis is regulated by a complex system involving both peripheral signals such as leptin and a number of orexigenic and anorectic neuropeptides. Obesity can result from dysregulation of the peripheral and/or central signals. Melanin-concentrating hormone (MCH) is a hypothalamic peptide that is important in the regulation of feeding behavior, primarily via uncharacterized signaling pathways in the central nervous system. Leptin, expressed in adipose tissue, mediates some of its actions through several hypothalamic neuropeptides, notably agouti-related peptide, proopiomelanocortin, and neuropeptide Y. Expression of leptin is regulated by dietary status, insulin, and glucocorticoids. Furthermore, certain neuropeptides may act on adipocytes. However, the potential effect of MCH has not been investigated. We report that MCH stimulates leptin mRNA expression and leptin secretion. MCH stimulated a 2-fold increase in leptin secretion by isolated rat adipocytes after 4 h of treatment. This increase in secreted leptin was preceded by a rapid and transient increase in ob mRNA levels; MCH stimulated a 2.5-fold increase in ob mRNA within 1 h of treatment, followed by a decline to basal levels within 4 h. In addition, we demonstrate that the MCH receptor SLC-1 is expressed in adipocytes, suggesting that fat cells may be targets of MCH or an MCH-like peptide under physiological conditions. Finally, using a radioimmunoassay, MCH/MCH-like peptide was detected in rat plasma. This study establishes a novel in vitro mammalian system for examining MCH signaling pathways.