A custom-built insulin resistance gene chip

Objectives/Aim—Microarray (gene chip) technology offers a powerful new tool for analyzing the expression of large numbers of genes in many experimental samples. The aim of this study was to design, construct, and use a gene chip to measure the expression levels of key genes in metabolic pathways related to insulin resistance.
Methods—We selected genes that were implicated in the development of insulin resistance, including genes involved in insulin signaling; glucose uptake, oxidation, and storage; fat uptake, oxidation, and storage; cytoskeletal components; and transcription factors. The key regulatory genes in the pathways were identified, along with other recently identified candidate genes such as calpain-10. A total of 242 selected genes (including 32 internal control elements) were sequence-verified, purified, and arrayed on aldehyde-coated slides.
Results—Where more than 1 clone containing the gene of interest was available, we chose those containing the genes in the 5' orientation and an insert size of around 1.5 kb. Of the 262 clones purchased, 56 (21%) were found to contain sequences other than those expected. In addition, 2 (1%) did not grow under standard conditions and were assumed to be nonviable. In these cases, alternate clones containing the gene of interest were chosen as described above. The current version of the Insulin Resistance Gene Chip contains 210 genes of interest, plus 48 control elements. A full list of the genes is available at http://www.hbs.deakin.edu.au/mru/research/gene_chip_tech/genechip_three.htm/.
Conclusions—The human Insulin Resistance Gene Chip that we have constructed will be a very useful tool for investigating variation in the expression of genes relevant to insulin resistance under various experimental conditions. Initially, the gene chip will be used in studies such as exercise interventions, fasting, euglycemic-hyperinsulinemic clamps, and administration of antidiabetic agents

Effect of epinephrine on glucose disposal during exercise in humans: role of muscle glycogen

This study examined the effect of epinephrine on glucose disposal during moderate exercise when glycogenolytic flux was limited by low preexercise skeletal muscle glycogen availability. Six male subjects cycled for 40 min at 59 ± 1% peak pulmonary O₂ uptake on two occasions, either without (CON) or with (EPI) epinephrine infusion starting after 20 min of exercise. On the day before each experimental trial, subjects completed fatiguing exercise and then maintained a low carbohydrate diet to lower muscle glycogen. Muscle samples were obtained after 20 and 40 min of exercise, and glucose kinetics were measured using [6,6-²H]glucose. Exercise increased plasma epinephrine above resting concentrations in both trials, and plasma epinephrine was higher (P < 0.05) during the final 20 min in EPI compared with CON. Muscle glycogen levels were low after 20 min of exercise (CON, 117 ± 25; EPI, 122 ± 20 mmol/kg dry matter), and net muscle glycogen breakdown and muscle glucose 6-phosphate levels during the subsequent 20 min of exercise were unaffected by epinephrine infusion. Plasma glucose increased with epinephrine infusion (i.e., 20-40 min), and this was due to a decrease in glucose disposal (R_d) (40 min: CON, 33.8 ± 3; EPI, 20.9 ± 4.9 µmol · kg^-1 · min^-1, P < 0.05), because the exercise-induced rise in glucose rate of appearance was similar in the trials. These results show that glucose R_d during exercise is reduced by elevated plasma epinephrine, even when muscle glycogen availability and utilization are low. This suggests that the effect of epinephrine does not appear to be mediated by increased glucose 6-phosphate, secondary to enhanced muscle glycogenolysis, but may be linked to a direct effect of epinephrine on sarcolemmal glucose transport.

Regulation of glucose kinetics during intense exercise in humans: effects of α- and β-adrenergic blockade

This study examined the effect of combined α- and β-adrenergic blockade on glucose kinetics during intense exercise. Six endurance-trained men exercised for 20 minutes at approximately 78% of their peak oxygen consumption (V_O 2) following ingestion of a placebo (CON) or combined α- (prazosin hydrochloride) and β- (timolol maleate) adrenoceptor antagonists (BLK). Plasma glucose increased during exercise in CON (0 minutes: 5.5 ± 0.1; 20 minutes: 6.5 ± 0.3 mmol · L⁻¹, P < .05). In BLK, the exercise-induced increase in plasma glucose was abolished (0 minutes: 5.7 ± 0.3; 20 minutes: 5.7 ± 0.1 mmol · L⁻¹). Glucose kinetics were measured using a primed, continuous infusion of [6,6-²H] glucose. Glucose production was not different between trials; on average these values were 25.3 ± 3.9 and 30.9 ± 4.4 μmol · kg⁻¹ · min⁻¹ in CON and BLK, respectively. Glucose uptake during exercise was greater (P < .05) in BLK (30.6 ± 4.6 μmol · kg⁻¹ · min⁻¹) compared with CON (18.4 ± 2.5 μmol · kg⁻¹ · min⁻¹). In BLK, plasma insulin and catecholamines were higher (P < .05), while plasma glucagon was unchanged from CON. Free fatty acids (FFA) and glycerol were lower (P < .05) in BLK. These findings demonstrate that adrenergic blockade during intense exercise results in a blunted plasma glucose response that is due to enhanced glucose uptake, with no significant change in glucose production.

Effect of creatine ingestion on glucose tolerance and insulin sensitivity in men

Purpose: This study investigated whether acute (5 d) and/or short-term (28 d) creatine (Cr) ingestion altered glucose tolerance or insulin action in healthy, untrained men (aged 26.9 ± 5.7 yr; SD). Methods : Subjects were randomly allocated to either a Cr (N = 8) or placebo group (N = 9) and were tested in the control condition (presupplementation), and after 5 and a further 28 d of supplementation. The Cr group ingested 20 g and 3 g·d-1 of Cr for the first 5 and following 28 d, respectively. The placebo group ingested similar amounts of glucose over the same time period. During each testing period, subjects underwent an oral glucose tolerance test (OGTT) to determine insulin sensitivity, and six subjects from each group underwent a muscle biopsy before each OGTT. Results : Cr supplementation resulted in an increased (P < 0.05) muscle TCr content after both the acute and short-term loading phase compared with placebo. Neither acute nor short-term Cr supplementation influenced skeletal muscle glycogen content, glucose tolerance, or measures of insulin sensitivity. Conclusions: These findings demonstrated that acute Cr supplementation (20 g·d-1 for 5 d) followed by short-term Cr supplementation (3 g·d-1 for 28 d) did not alter insulin action in healthy, active untrained men.

Australian sweet lupin flour addition reduces the glycaemic index of a white bread breakfast without affecting palatability in healthy human volunteers

The addition of some legume ingredients to bread has been associated with effects on glycaemic, insulinaemic and satiety responses that may be beneficial in controlling type 2 diabetes, cardiovascular disease and obesity. However, the effect of Australian sweet lupin (Lupinus angustifolius) flour (ASLF) is unknown. This investigation examined the effect of adding ASLF to standard white bread on post-meal glycaemic, insulinaemic and satiety responses and palatability in healthy subjects. Using a randomised, single-blind, cross-over design, 11 subjects consumed one breakfast of ASLF bread and two of standard white bread ≥ 7 days apart after fasting overnight. Each breakfast also included margarine, jam, and tea with milk and contained 50g available carbohydrate. On each test day, blood samples were taken after fasting, then several times over 2 hours post-prandially, and analysed for plasma glucose and serum insulin. Subjects rated breakfast palatability and perception of satiety, in the fasting state and over 3 hours post-prandially, after which food intake from an ad libitum buffet and for the rest of the day was recorded. Incremental areas under the curves for glucose, insulin and satiety, glycaemic index, insulinaemic index and satiety index were calculated. ASLF addition to the breakfast reduced its glycaemic index (mean ± SEM; ASLF bread breakfast = 74.0 ± 9.6. Standard white bread breakfast = 100, P=0.022), raised its insulinaemic index (ASLF bread breakfast = 127.7 ± 12.0. Standard white bread breakfast = 100, P=0.046), but did not affect palatability, satiety or food intake. ASLF addition resulted in a palatable breakfast; however, the potential benefits of the lowered glycaemic index may be eclipsed by the increased insulinaemic index.

Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes.

Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 ± 0.6 yr; body mass index: 23.8 ± 1.0 kg/m²; maximal O2 consumption: 3.85 ± 0.21 l/min). Plasma FFA concentration increased during exercise (P < 0.01) in the fasted state but remained unchanged after glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P < 0.05). Except for a significant decrease in the expression of pyruvate dehydrogenase kinase-4 (P < 0.05), glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5'-AMP-activated protein kinase-α2; P < 0.05). In conclusion, glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.

Randomised controlled trial evaluating lifestyle interventions in people with impaired glucose tolerance

Aim

To evaluate the effectiveness of lifestyle interventions in people with impaired glucose tolerance (IGT).
Methods

Participants with IGT (n = 78), diagnosed on two consecutive oral glucose tolerance tests (OGTTs), were randomly assigned to a 2-year lifestyle intervention or to a control group. Main outcome measures were changes from baseline in: nutrient intake; physical activity; anthropometry, glucose tolerance and insulin sensitivity. Measurements were repeated at 6, 12 and 24 months follow-up.
Results

After 24 months follow-up, there was a significant fall in total fat consumption (difference in change between groups (Δ intervention − Δ control) = −17.9, 95% confidence interval (CI) −33.6 to −2.1 g/day) as a result of the intervention. Body mass was significantly lower in the intervention group compared with controls after 6 months (−1.6, 95% CI −2.9 to −0.4 kg) and 24 months (−3.3, 95% CI −5.7 to −0.89 kg). Whole body insulin sensitivity, assessed by the short insulin tolerance test (ITT), improved after 12 months in the intervention group (0.52, 95% CI 0.15–0.89%/min).
Conclusions

These findings complement the findings of the Finnish Diabetes Prevention Study and the American Diabetes Prevention Study, both of which tested intensive interventions, by showing that pragmatic lifestyle interventions result in improvements in obesity and whole body insulin sensitivity in individuals with IGT, without change in other cardiovascular risk factors.

Prevention of type 2 diabetes in a primary health care setting interim results from the Greater Green Triangle (GGT) Diabetes Prevention Project

Although clinical trials have shown that lifestyle modifications reduce the risk of type 2 diabetes, translating lessons from trials to primary care remains a challenge. The aim of the study was to evaluate efficacy and feasibility of primary care-based diabetes prevention model with modest resource requirements in rural Australia. Three hundred and eleven subjects with at least a moderate risk of type 2 diabetes participated in a combined dietary and physical activity intervention. Clinical measurements and fasting blood samples were taken at the baseline and after intervention. After 3 months intervention, total (change −3.5%, p < 0.001) and LDL cholesterol (−4.8%, p < 0.001) plasma levels as well as body mass index (−2.5%, p < 0.001), weight (−2.5%, p < 0.001), and waist (−1.6%, p < 0.001) and hip (−2.7%, p < 0.001) circumferences reduced significantly. A borderline reduction was found in triglyceride levels (−4.8%, p = 0.058) while no changes were observed in HDL cholesterol (+0.6%, p = 0.525), glucose (+0.06%, p = 0.386), or systolic (−0.98%, p = 0.095) or diastolic (−1.06%, p = 0.134) blood pressure levels. In conclusion, a lifestyle intervention improved health outcomes – especially obesity and blood lipids – in a population at high risk of developing type 2 diabetes. Our results suggest that the present model is effective and feasible to carry out in primary care settings.

Secretion of the glucose-regulated selenoprotein SEPS1 from hepatoma cells

SEPS1 (also called selenoprotein S, SelS, Tanis or VIMP) is a selenoprotein, localized predominantly in the ER membrane and also on the cell surface. In this report, we demonstrate that SEPS1 protein is also secreted from hepatoma cells but not from five other types of cells examined. The secretion can be abolished by the ER-Golgi transport inhibitor Brefeldin A and by the protein synthesis inhibitor cycloheximide. Using a sandwich ELISA, SEPS1 was detected in the sera of 65 out of 209 human subjects (31.1%, average = 15.7 ± 1.1 ng/mL). Fractionation of human serum indicated that SEPS1 was associated with LDL and possibly with VLDL. The function of plasma SEPS1 is unclear but may be related to lipoprotein metabolism.

Effect of carbohydrate ingestion on glucose kinetics during exercise in the heat

Six endurance-trained men [peak oxygen uptake (VO₂) = 4.58 ± 0.50 (SE) l/min] completed 60 min of exercise at a workload requiring 68 ± 2% peak VO₂ in an environmental chamber maintained at 35°C (<50% relative humidity) on two occasions, separated by at least 1 wk. Subjects ingested either a 6% glucose solution containing 1 µCi [3-³H]glucose/g glucose (CHO trial) or a sweet placebo (Con trial) during the trials. Rates of hepatic glucose production [HGP = glucose rate of appearance (R_a) in Con trial] and glucose disappearance (R_d), were measured using a primed, continuous infusion of [6,6-^₂H]glucose, corrected for gut-derived glucose (gut R_a) in the CHO trial. No differences in heart rate, VO₂, respiratory exchange ratio, or rectal temperature were observed between trials. Plasma glucose concentrations were similar at rest but increased (P < 0.05) to a greater extent in the CHO trial compared with the Con trial. This was due to the absorption of ingested glucose in the CHO trial, because gut R_a after 30 and 50 min (16 ± 5 µmol · kg^-1 · min^-1) was higher (P < 0.05) compared with rest, whereas HGP during exercise was not different between trials. Glucose R_d was higher (P < 0.05) in the CHO trial after 30 and 50 min (48.0 ± 6.3 vs 34.6 ± 3.8 µmol · kg^-1 · min^-1, CHO vs. Con, respectively). These results indicate that ingestion of carbohydrate, at a rate of ~1.0 g/min, increases glucose Rd but does not blunt the rise in HGP during exercise in the heat.

Plasma glucose kinetics during prolonged exercise in trained humans when fed carbohydrate

Nine endurance-trained men exercised on a cycle ergometer at ~68% peak O₂ uptake to the point of volitional fatigue [232 ± 14 (SE) min] while ingesting an 8% carbohydrate solution to determine how high glucose disposal could increase under physiological conditions. Plasma glucose kinetics were measured using a primed, continuous infusion of [6,6-²H]glucose and the appearance of ingested glucose, assessed from [3-³H]glucose that had been added to the carbohydrate drink. Plasma glucose was increased (P < 0.05) after 30 min of exercise but thereafter remained at the preexercise level. Glucose appearance rate (Ra) increased throughout exercise, reaching its peak value of 118 ± 7 µmol · kg^-1 · min^-1 at fatigue, whereas gut R_a increased continuously during exercise, peaking at 105 ± 10 µmol · kg^-1 · min^-1 at the point of fatigue. In contrast, liver glucose output never rose above resting levels at any time during exercise. Glucose disposal (R_d) increased throughout exercise, reaching a peak value of 118 ± 7 µmol · kg^-1 · min^-1 at fatigue. If we assume 95% oxidation of glucose R_d, estimated exogenous glucose oxidation at fatigue was 1.36 ± 0.08 g/min. The results of this study demonstrate that glucose uptake increases continuously during prolonged, strenuous exercise when carbohydrate is ingested and does not appear to limit exercise performance.

Long-term effects of a reduced fat diet intervention on cardiovascular disease risk factors in individuals with glucose intolerance

The long-term effects on cardiovascular disease risk factors of a reduced fat (RF), ad libitum diet were compared with usual diet (control, CD) in glucose intolerance individuals.

Participants were 136 adults aged ≥40 years with ‘glucose intolerance’ (2 h blood glucose 7–11.0 mmol/l) detected at a Diabetes Survey who completed at 1 year intervention study of reduced fat, ad libitum diet versus usual diet. They were re-assessed at 2, 3 and 5 years. Main outcome measures were blood pressure, serum concentrations of total cholesterol, HDL and LDL cholesterol, total cholesterol:HDL ratio, triglycerides and body weight.

The reduced fat diet lowered total cholesterol (P<0.01), LDL cholesterol (P≤0.05), total cholesterol:HDL ratio (P≤0.05), body weight (P<0.01) and systolic blood pressure (P≤0.05) initially and diastolic blood pressure (P<0.01) long-term. No significant changes occurred in HDL cholesterol or triglycerides. In the more compliant 50% of the intervention group, systolic and diastolic blood pressure levels and body weight were lower at 1, 2 and 3 years (P<0.05).

It was concluded that a reduced fat ad libitum diet has short-term benefits for cholesterol, body weight and systolic blood pressure and long-term benefits for diastolic blood pressure without significantly effecting HDL cholesterol and triglycerides despite participants regaining their lost weight.

Creatine supplementation increases glycogen storage but not GLUT-4 expression in human skeletal muscle

It has been speculated that creatine supplementation affects muscle glucose metabolism in humans by increasing muscle glycogen storage and up-regulating GLUT-4 protein expression. In the present study, we assessed the effects of creatine loading and prolonged supplementation on muscle glycogen storage and GLUT-4 mRNA and protein content in humans. A total of 20 subjects participated in a 6-week supplementation period during which creatine or a placebo was ingested. Muscle biopsies were taken before and after 5 days of creatine loading (20 g.day(-1)) and after 6 weeks of continued supplementation (2 g.day(-1)). Fasting plasma insulin concentrations, muscle creatine, glycogen and GLUT-4 protein content as well as GLUT-4, glycogen synthase-1 (GS-1) and glycogenin-1 (Gln-1) mRNA expression were determined. Creatine loading significantly increased total creatine, free creatine and creatine phosphate content with a concomitant 18 +/- 5% increase in muscle glycogen content (P<0.05). The subsequent use of a 2 g.day(-1) maintenance dose for 37 days did not maintain total creatine, creatine phosphate and glycogen content at the elevated levels. The initial increase in muscle glycogen accumulation could not be explained by an increase in fasting plasma insulin concentration, muscle GLUT-4 mRNA and/or protein content. In addition, neither muscle GS-1 nor Gln-1 mRNA expression was affected. We conclude that creatine ingestion itself stimulates muscle glycogen storage, but does not affect muscle GLUT-4 expression.

Sustained weight loss in obese subjects has benefits that are independent of attained weight

Objective: To explore the hypothesis that sustained weight loss in severely obese patients may have benefits that are independent of their attained BMI. Research Methods and Procedures: We conducted a comparison of two weight-stable groups with BMI in the 30 to 35 kg/m2 range. Subjects (n = 79) were selected obese patients 3 years after laparoscopic adjustable gastric band surgery, and controls (n = 79) were obese patients seeking weight loss therapy. Subjects were selected in a de-identified manner from our database to best match the control group. A range of clinical, biochemical, and questionnaire measures were obtained to assess obesity-related health status Results: Subjects maintained a mean weight loss of 32.8 plusminus 18 kg after surgery. The weight loss subjects had significantly lower fasting plasma glucose, insulin, and triglyceride concentrations, along with higher high-density lipoprotein-cholesterol levels and better indirect measures of insulin sensitivity when compared with controls (p < 0.05 for all). In addition, aminotransferase levels, neutrophil counts, and globulin levels were also significantly lower in weight loss subjects. All differences in laboratory variables remained significant after controlling for BMI. The subjects also reported better health-related quality of life, fewer symptoms of depression, and greater satisfaction with their appearance than controls. Discussion: These findings suggest that the post-weight loss state conveys benefits that are greater than predicted by the attained BMI. These findings may have important implications regarding the expectations of weight loss therapy, and mechanisms for this effect should be carefully sought.