Being born small-for-gestational-age is associated with an unfavourable dietary intake in Danish adolescent girls: findings from the Danish National Birth Cohort.

Individuals born small have an increased risk for developing type 2 diabetes. Altered food preferences in these subjects seem to play a role; however, limited evidence is available on the association between being born small-for-gestational-age (SGA) at term and food intake in adolescence. Alterations in leptin, ghrelin and dopamine levels are suggested mechanisms linking SGA with later food intake. From a large prospective Danish National Birth Cohort, we compared dietary intake of adolescents being born SGA with normal-for-gestational-age (NGA) adolescents. Intake of foods and nutrients was assessed by a validated food frequency questionnaire in a subsample of 15,607 14-year-old individuals born at term. SGA was defined by birth weight (BW) <10th percentile (n = 1470) and NGA as BW between 10 and 90th percentile (n = 14,137) according to sex and gestational age-specific BW standard curves. Girls born SGA had a 7% (95% CI: 3-12%, P = 0.002) higher intake of added sugar and a 2-8% lower intake of dietary fibre, vegetables, polyunsaturated fatty acids, and total n-6, compared with NGA girls (P < 0.05). Adjusting for parental socio-occupational status, maternal smoking and diet in pregnancy did not substantially change the differences in dietary intake, except from dietary fibre, which were no longer statistically significant. No significant differences in dietary intake between SGA and NGA boys were found. In summary, girls born SGA had an unfavourable dietary intake compared with NGA girls. These differences persisted after controlling for potential confounders, thus supporting a fetal programming effect on dietary intake in girls born SGA at term. However, residual confounding by other factors operating early in childhood cannot be excluded.

A common variation of the PTEN gene is associated with peripheral insulin resistance.

AIM: Phosphatase and tensin homologue (PTEN) reduces insulin sensitivity by inhibiting the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue (Akt) pathway. This study investigated how a common single nucleotide polymorphism near PTEN, previously associated with fasting levels of plasma insulin and glucose, influences in vivo glucose metabolism and insulin signalling. The primary outcome measure was the gene variant's association with peripheral glucose disposal rate and, secondarily, whether this association was explained by altered activities of PTEN targets PI3K and Akt. METHODS: A total of 183 normoglycaemic Danes, including 158 twins and 25 singletons, were genotyped for PTEN rs11202614, which is in complete linkage disequilibrium with rs2142136 and rs10788575, which have also been reported in association with glycaemic traits and type 2 diabetes (T2D). Hepatic and peripheral insulin sensitivity was measured using tracer and euglycaemic-hyperinsulinaemic clamp techniques; insulin secretion was assessed by intravenous glucose tolerance test; and muscle biopsies were taken during insulin infusion from 150 twins for measurement of PI3K and Akt activities. RESULTS: The minor G allele of PTEN rs11202614 was associated with elevated fasting plasma insulin levels and a decreased peripheral glucose disposal rate, but not with the hepatic insulin resistance index or insulin secretion measured as the first-phase insulin response and disposition index. The single nucleotide polymorphism was not associated with either PI3K or Akt activities. CONCLUSION: A common PTEN variation is associated with peripheral insulin resistance and subsequent risk of developing T2D. However, the association with insulin resistance is not explained by decreased proximal insulin signalling in skeletal muscle.

Effects of 12 weeks of treatment with fermented milk on blood pressure, glucose metabolism and markers of cardiovascular risk in patients with type 2 diabetes: a randomised double-blind placebo-controlled study.

OBJECTIVE: Studies have indicated a blood pressure (BP)-lowering effect of milk-derived peptides in non-diabetic individuals, but the cardiometabolic effects of such peptides in patients with type 2 diabetes (T2D) are not known. We investigated the effect of milk fermented with Lactobacillus helveticus on BP, glycaemic control and cardiovascular risk factors in T2D. DESIGN: A randomised, double-blinded, prospective, placebo-controlled study. METHODS: In one arm of a factorial study design, 41 patients with T2D were randomised to receive 300 ml milk fermented with L. helveticus (Cardi04 yogurt) (n=23) or 300 ml artificially acidified milk (placebo yogurt) (n=18) for 12 weeks. BPs were measured over 24-h, and blood samples were collected in the fasting state and during a meal test before and after the intervention. RESULTS: Cardi04 yogurt did not reduce 24-h, daytime or nighttime systolic or diastolic BPs compared with placebo (P>0.05). Daytime and 24-h heart rate (HR) were significantly reduced in the group treated by Cardi04 yogurt compared with the placebo group (P<0.05 for both). There were no differences in HbA1c, plasma lipids, C-reactive protein, plasminogen activator inhibitor-1, tumour necrosis factor alpha, tissue-type plasminogen activator: Ag, and von Willebrand factor: Ag between the groups. The change in fasting blood glucose concentration differed significantly between the two groups with a larger increase in the placebo group (P<0.05). CONCLUSIONS: Ingestion of milk fermented with L. helveticus compared with placebo for 12 weeks did not significantly reduce BP in patients with T2D. Our finding of lower HRs and fasting plasma glucose levels in T2D patients during ingestion of fermented milk needs further validation.

Effects of short-term high-fat overfeeding on genome-wide DNA methylation in the skeletal muscle of healthy young men.

AIMS/HYPOTHESIS: Energy-dense diets that are high in fat are associated with a risk of metabolic diseases. The underlying molecular mechanisms could involve epigenetics, as recent data show altered DNA methylation of putative type 2 diabetes candidate genes in response to high-fat diets. We examined the effect of a short-term high-fat overfeeding (HFO) diet on genome-wide DNA methylation patterns in human skeletal muscle. METHODS: Skeletal muscle biopsies were obtained from 21 healthy young men after ingestion of a short-term HFO diet and a control diet, in a randomised crossover setting. DNA methylation was measured in 27,578 CpG sites/14,475 genes using Illumina's Infinium Bead Array. Candidate gene expression was determined by quantitative real-time PCR. RESULTS: HFO introduced widespread DNA methylation changes affecting 6,508 genes (45%), with a maximum methylation change of 13.0 percentage points. The HFO-induced methylation changes were only partly and non-significantly reversed after 6-8 weeks. Alterations in DNA methylation levels primarily affected genes involved in inflammation, the reproductive system and cancer. Few gene expression changes were observed and these had poor correlation to DNA methylation. CONCLUSIONS/INTERPRETATION: The genome-wide DNA methylation changes induced by the short-term HFO diet could have implications for our understanding of transient epigenetic regulation in humans and its contribution to the development of metabolic diseases. The slow reversibility suggests a methylation build-up with HFO, which over time may influence gene expression levels.

Differential aetiology and impact of phosphoinositide 3-kinase (PI3K) and Akt signalling in skeletal muscle on in vivo insulin action.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is a key factor in the development of type 2 diabetes and although some studies indicate that this could be partly attributed to reduced content and activity of various proximal and distal insulin signalling molecules, consensus is lacking. We therefore aimed to investigate the regulation of proximal insulin signalling in skeletal muscle and its effect on glucose metabolism in a large non-diabetic population. METHODS: We examined 184 non-diabetic twins with gold-standard techniques including the euglycaemic-hyperinsulinaemic clamp. Insulin signalling was evaluated at three key levels, i.e. the insulin receptor, IRS-1 and V-akt murine thymoma viral oncogene (Akt) levels, employing kinase assays and phospho-specific western blotting. RESULTS: Proximal insulin signalling was not associated with obesity, age or sex. However, birthweight was positively associated with IRS-1-associated phosphoinositide 3-kinase (PI3K; IRS-1-PI3K) activity (p = 0.04); maximal aerobic capacity (VO2(max)), paradoxically, was negatively associated with IRS-1-PI3K (p = 0.02) and Akt2 activity (p = 0.01). Additionally, we found low heritability estimates for most measures of insulin signalling activity. Glucose disposal was positively associated with Akt-308 phosphorylation (p < 0.001) and Akt2 activity (p = 0.05), but not with insulin receptor tyrosine kinase or IRS-1-PI3K activity. CONCLUSIONS/INTERPRETATION: With the exception of birthweight, 'classical' modifiers of insulin action, including genetics, age, sex, obesity and VO2(max) do not seem to mediate their most central effects on whole-body insulin sensitivity through modulation of proximal insulin signalling in skeletal muscle. We also demonstrated an association between Akt activity and in vivo insulin sensitivity, suggesting a role of Akt in control of in vivo insulin resistance and potentially in type 2 diabetes.

Diminished insulin-mediated forearm blood flow and muscle glucose uptake in young men with low birth weight.

BACKGROUND: Low birth weight (LBW) is associated with increased risk of type 2 diabetes and cardiovascular disease. We studied endothelial function and insulin sensitivity in young men with LBW (n = 22) and controls (n = 22). METHODS: Insulin sensitivity and endothelial function was studied with venous occlusion plethysmography and intra-arterial infusions of adenosine and acetylcholine, before and during a hyperinsulinemic isoglycemic clamp. RESULTS: Forearm blood flow response to systemic hyperinsulinemia was diminished in LBW compared to controls (p < 0.05). Fractional arteriovenous glucose extraction was similar, and consequently insulin-stimulated forearm glucose clearance was diminished in LBW compared with controls (0.8 +/- 0.09 vs. 1.4 +/- 0.36 ml x 100 ml(-1) x min(-1), respectively, p < 0.05). Forearm blood flow response to adenosine and acetylcholine with or without insulin stimulation did not differ between groups. Whole-body glucose uptake was lower in LBW than controls (8.7 +/- 0.5 and 9.1 +/- 0.6 mg x min(-1) x kg(-1) lean body mass); however, this was not significant. CONCLUSIONS: Forearm blood flow response to insulin is impaired in LBW, whereas the response to adenosine and acetylcholine is preserved. The impaired insulin-mediated increase in bulk flow in LBW may be due to an impairment of insulin-mediated capillary recruitment independent of - or preceding - whole-body insulin resistance in LBW subjects.

Pathophysiology and aetiology of impaired fasting glycaemia and impaired glucose tolerance: does it matter for prevention and treatment of type 2 diabetes?

Prior to the development of type 2 diabetes, glucose levels increase into the prediabetic states of isolated impaired fasting glycaemia (i-IFG), isolated impaired glucose tolerance (i-IGT), or combined IFG/IGT. A better understanding of the aetiology and pathophysiology of the prediabetic states might give a basis for the development of individualised prevention and treatment strategies for type 2 diabetes. Several studies have examined mechanisms and potential aetiological factors leading to the development of the different prediabetic states. The pathophysiology of i-IFG seems to include the following key defects: reduced hepatic insulin sensitivity, stationary beta cell dysfunction and/or chronic low beta cell mass, altered glucagon-like peptide-1 secretion and inappropriately elevated glucagon secretion. Conversely, the prediabetic state i-IGT is characterised by reduced peripheral insulin sensitivity, near-normal hepatic insulin sensitivity, progressive loss of beta cell function, reduced secretion of glucose-dependent insulinotropic polypeptide and inappropriately elevated glucagon secretion. Individuals developing combined IFG/IGT exhibit severe defects in both peripheral and hepatic insulin sensitivity as well as a progressive loss of beta cell function. The aetiologies of i-IFG and i-IGT also seem to differ, with i-IFG being predominantly related to genetic factors, smoking and male sex, while i-IGT is predominantly related to physical inactivity, unhealthy diet and short stature. Since the transition from the prediabetic states to overt type 2 diabetes is characterised by a non-reversible vicious cycle that includes severe deleterious effects on glucose metabolism, there are good reasons to use the well-established aetiological and pathophysiological differences in i-IFG, i-IGT and IFG/IGT to design individualised preventive strategies.

Predictors of future fasting and 2-h post-OGTT plasma glucose levels in middle-aged men and women-the Inter99 study.

AIMS: Screening and prevention strategies for Type 2 diabetes require insight into the aetiological and potentially different risk factors leading to early impairments of fasting plasma glucose (FPG) and 2-h post-load plasma glucose (2hPG) levels. We studied whether risk factors predicting subtle elevations of FPG levels were different from those predicting elevations of 2hPG levels in men and women. METHODS: We used baseline and 5-year follow-up data from middle-aged men and women with normal glucose tolerance (NGT) at baseline in the Danish population-based Inter99 study (n = 3164). Anthropometric and non-anthropometric baseline predictors of the 5-year FPG and 2hPG levels were estimated in linear regression models stratified by gender. RESULTS: In men, but not in women, smoking and family history of diabetes predicted increased FPG levels, whereas high physical activity predicted a decline in 2hPG levels. Among the anthropometric variables, large waist circumference was the strongest predictor of increased FPG levels in men, whereas high body mass index (BMI) was the strongest predictor of increased FPG levels in women. In both men and women, BMI and waist circumference were equally strong in predicting 2hPG levels. Furthermore, short height predicted increased 2hPG levels in men, and short height and low hip circumference predicted increased 2hPG levels in women. CONCLUSIONS: Risk factors that predict future FPG levels are different from those predicting future 2hPG levels. Furthermore, different risk factors predict glycaemic levels in men compared with women. These findings indicate that different aetiological pathways may lead to Type 2 diabetes in men and women.

The T allele of rs7903146 TCF7L2 is associated with impaired insulinotropic action of incretin hormones, reduced 24 h profiles of plasma insulin and glucagon, and increased hepatic glucose production in young healthy men.

AIMS/HYPOTHESIS: We studied the physiological, metabolic and hormonal mechanisms underlying the elevated risk of type 2 diabetes in carriers of TCF7L2 gene. METHODS: We undertook genotyping of 81 healthy young Danish men for rs7903146 of TCF7L2 and carried out various beta cell tests including: 24 h glucose, insulin and glucagon profiles; OGTT; mixed meal test; IVGTT; hyperglycaemic clamp with co-infusion of glucagon-like peptide (GLP)-1 or glucose-dependent insulinotropic polypeptide (GIP); and a euglycaemic-hyperinsulinaemic clamp combined with glucose tracer infusion to study hepatic and peripheral insulin action. RESULTS: Carriers of the T allele were characterised by reduced 24 h insulin concentrations (p < 0.05) and reduced insulin secretion relative to glucose during a mixed meal test (beta index: p < 0.003), but not during an IVGTT. This was further supported by reduced late-phase insulinotropic action of GLP-1 (p = 0.03) and GIP (p = 0.07) during a 7 mmol/l hyperglycaemic clamp. Secretion of GLP-1 and GIP during the mixed meal test was normal. Despite elevated hepatic glucose production, carriers of the T allele had significantly reduced 24 h glucagon concentrations (p < 0.02) suggesting altered alpha cell function. CONCLUSIONS/INTERPRETATION: Elevated hepatic glucose production and reduced insulinotropic effect of incretin hormones contribute to an increased risk of type 2 diabetes in carriers of the rs7903146 risk T allele of TCF7L2.

Age influences DNA methylation and gene expression of COX7A1 in human skeletal muscle.

AIMS/HYPOTHESIS: Reduced oxidative capacity of the mitochondria in skeletal muscle has been suggested to contribute to insulin resistance and type 2 diabetes. Moreover, a set of genes influencing oxidative phosphorylation (OXPHOS) is downregulated in diabetic muscle. Here we studied whether genetic, epigenetic and non-genetic factors influence a component of the respiratory chain, COX7A1, previously shown to be downregulated in skeletal muscle from patients with type 2 diabetes. The specific aims were to: (1) evaluate the impact of genetic (single nucleotide polymorphisms [SNPs]), epigenetic (DNA methylation) and non-genetic (age) factors on the expression of COX7A1 in human skeletal muscle; and (2) investigate whether common variants in the COX7A1 gene are associated with increased risk of type 2 diabetes. METHODS: COX7A1 mRNA expression was analysed in muscle biopsies from young (n = 110) and elderly (n = 86) non-diabetic twins and related to measures of in vivo metabolism. Genetic variants (three SNPs) from the COX7A1 locus were genotyped in the twins and in two independent type 2 diabetes case-control cohorts (n = 1466 and 6380, respectively). DNA methylation of the COX7A1 promoter was analysed in a subset of twins (ten young, ten elderly) using bisulphite sequencing. RESULTS: While DNA methylation of the COX7A1 promoter was increased in muscle from elderly compared with young twins (19.9 +/- 8.3% vs 1.8 +/- 2.7%; p = 0.035), the opposite was found for COX7A1 mRNA expression (elderly 1.00 +/- 0.05 vs young 1.68 +/- 0.06; p = 0.0005). The heritability of COX7A1 expression was estimated to be 50% in young and 72% in elderly twins. One of the polymorphisms investigated, rs753420, influenced basal COX7A1 expression in muscle of young (p = 0.0001) but not of elderly twins. The transcript level of COX7A1 was associated with increased in vivo glucose uptake and VO(2max) (p = 0.009 and p = 0.001, respectively). We did not observe any genetic association between COX7A1 polymorphisms and type 2 diabetes after correcting for multiple testing. CONCLUSIONS/INTERPRETATION: Our results provide further evidence for age as a factor influencing DNA methylation and expression of OXPHOS genes, and thereby in vivo metabolism.

Impaired fasting glycaemia vs impaired glucose tolerance: similar impairment of pancreatic alpha and beta cell function but differential roles of incretin hormones and insulin action.

AIMS/HYPOTHESIS: The impact of strategies for prevention of type 2 diabetes in isolated impaired fasting glycaemia (i-IFG) vs isolated impaired glucose tolerance (i-IGT) may differ depending on the underlying pathophysiology. We examined insulin secretion during OGTTs and IVGTTs, hepatic and peripheral insulin action, and glucagon and incretin hormone secretion in individuals with i-IFG (n = 18), i-IGT (n = 28) and normal glucose tolerance (NGT, n = 20). METHODS: Glucose tolerance status was confirmed by a repeated OGTT, during which circulating insulin, glucagon, glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) levels were measured. A euglycaemic-hyperinsulinaemic clamp with [3-3H]glucose preceded by an IVGTT was performed. RESULTS: Absolute first-phase insulin secretion during IVGTT was decreased in i-IFG (p = 0.026), but not in i-IGT (p = 0.892) compared with NGT. Hepatic insulin sensitivity was normal in i-IFG and i-IGT individuals (p > or = 0.179). Individuals with i-IGT had peripheral insulin resistance (p = 0.003 vs NGT), and consequently the disposition index (DI; insulin secretion x insulin sensitivity) during IVGTT (DI(IVGTT))) was reduced in both i-IFG and i-IGT (p < 0.005 vs NGT). In contrast, the DI during OGTT (DI(OGTT)) was decreased only in i-IGT (p < 0.001), but not in i-IFG (p = 0.143) compared with NGT. Decreased levels of GIP in i-IGT (p = 0.045 vs NGT) vs increased levels of GLP-1 in i-IFG (p = 0.013 vs NGT) during the OGTT may partially explain these discrepancies. Basal and post-load glucagon levels were significantly increased in both i-IFG and i-IGT individuals (p < or = 0.001 vs NGT). CONCLUSIONS/INTERPRETATION: We propose that differentiated preventive initiatives in prediabetic individuals should be tested, targeting the specific underlying metabolic defects.

Decreased protein levels of key insulin signalling molecules in adipose tissue from young men with a low birthweight: potential link to increased risk of diabetes?

AIMS/HYPOTHESIS: Individuals with low birthweight are at increased risk of type 2 diabetes mellitus. However, the underlying molecular mechanisms are unknown. Previously we have shown that low birthweight is associated with changes in muscle insulin signalling proteins. Here we determined whether low birthweight is associated with changes in insulin signalling proteins in adipose tissue. METHODS: Men (age 23 years) with either a low (bottom 10th percentile) (n = 17) or a normal (50th-90th percentile) (n = 17) birthweight were recruited from the Danish Medical Birth Registry and subcutaneous adipose biopsies were taken. RESULTS: Between the two groups there was no difference in protein level of the insulin receptor, protein kinase C zeta, glycogen synthase kinase-3 (GSK3) alpha, GSK3 beta, protein kinase B alpha and beta, peroxisome proliferative activated receptor gamma coactivator 1 or Src-homology-2-containing protein. However, the levels of GLUT4 (also known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) (52 +/- 10.9% reduction, p < 0.01), p85alpha subunit of phosphoinositide 3-kinase (PI3K) (45 +/- 9% reduction, p < 0.01), p110ss subunit of PI3K (48 +/- 17% reduction, p = 0.06) and IRS1 (59 +/- 24% reduction, p < 0.05) were reduced in men of low birthweight. CONCLUSIONS/INTERPRETATION: These findings show that low birthweight is associated with reduced levels of adipose insulin signalling proteins, thus providing a potential molecular framework to explain why people with low birthweight are at increased risk of developing type 2 diabetes. These differences precede the development of diabetes and thus may help predict disease risk.

Plasma cytokine levels in young and elderly twins: genes versus environment and relation to in vivo insulin action.

AIMS/HYPOTHESIS: We studied the impact of genetic versus pre- and postnatal environmental factors on the plasma levels of IL6, TNF and the soluble TNF receptor superfamily, member 1A (TNFRSF1A, previously known as TNF receptor type1 [TNFR1]). MATERIALS AND METHODS: Using the hyperinsulinaemic-euglycaemic clamp, we assessed the association between cytokine levels and both peripheral insulin sensitivity and hepatic glucose production. Fasting plasma IL6, TNF and soluble TNFRSF1A levels were measured using ELISA in 55 young and 43 elderly twin pairs. RESULTS: Plasma IL6 and TNF were influenced by genetic factors in young and elderly twins respectively, while no significant impact of genetics was found for soluble TNFRSF1A. Significant intra-twin pair correlations between birthweight and both IL6 and soluble TNFRSF1A levels were found. In addition to the effect of birthweight on IL6, age and fat % also significantly influenced IL6 levels, whereas soluble TNFRSF1A levels were significantly influenced by zygosity, age, fat % and sex. Plasma soluble TNFRSF1A was associated with higher plasma NEFA and to some extent with reduced insulin sensitivity. Plasma IL6 was associated positively with basal NEFA. TNF level was not associated with in vivo glucose or fat metabolism after correction for known confounders. CONCLUSIONS/INTERPRETATION: Plasma IL6 and soluble TNFRSF1A are influenced by the intrauterine environment. We found some evidence of a genetic component for TNF and IL6 in young and elderly twins respectively. Plasma IL6 may influence basal lipolysis, but neither plasma TNF nor IL6 levels are independently associated with hepatic or peripheral insulin action. Nevertheless, plasma soluble TNFRSF1A may play some role in the control of insulin action.

No effect of taurine on platelet aggregation in men with a predisposition to type 2 diabetes mellitus.

Any diet therapy that potentially could affect platelet function would also influence the initiation of atherosclerotic plaque formation which is an important complication of diabetes mellitus eventually resulting in myocardial infarction and stroke. Blood platelets are rich in taurine, and it has been shown that taurine inhibits platelet aggregation in healthy subjects. The purpose was to examine the effect of taurine supplementation on platelet aggregation in high-risk subjects with a positive family history of T2DM. Twenty healthy men were included in a double-blinded, randomized, crossover study, receiving daily supplementation of 1.5 g taurine or placebo for two 8-week periods. Subjects were overweight and first-degree relatives of T2DM patients. At the end of each treatment, fasting blood samples for assessment of platelet aggregation was drawn. Platelet aggregation was induced by ADP. Plasma taurine concentration was significantly greater after taurine intervention compared to placebo (131.4+/-61.7 vs. 38.9+/-6.7 micromol/l, P<0.0001). There was no difference in the threshold level for complete platelet aggregation induced by ADP in vivo between placebo and taurine intervention (placebo 3.86+/-2.21 vs. taurine 3.86+/-3.25 micromol/l). Supplementation with 1.5 g of taurine for 8 weeks had no effect on platelet aggregation in overweight prediabetic men.

Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected].

To determine whether defects in the insulin signal transduction cascade are present in skeletal muscle from prediabetic individuals, we excised biopsies from eight glucose-intolerant male first-degree relatives of patients with type 2 diabetes (IGT relatives) and nine matched control subjects before and during a euglycemic-hyperinsulinemic clamp. IGT relatives were insulin-resistant in oxidative and nonoxidative pathways for glucose metabolism. In vivo insulin infusion increased skeletal muscle insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (P = 0.01) and phosphatidylinositide 3-kinase (PI 3-kinase) activity (phosphotyrosine and IRS-1 associated) in control subjects (P < 0.02) but not in IGT relatives (NS). The incremental increase in insulin action on IRS-1 tyrosine phosphorylation was lower in IGT relatives versus control subjects (P < 0.05). The incremental defects in signal transduction noted for IRS-1 and PI 3-kinase may be attributed to elevated basal phosphorylation/activity of these parameters, because absolute phosphorylation/activity under insulin-stimulated conditions was similar between IGT relatives and control subjects. Insulin increased Akt serine phosphorylation in control subjects and IGT relatives, with a tendency for reduced phosphorylation in IGT relatives (P = 0.12). In conclusion, aberrant phosphorylation/activity of IRS-1, PI 3-kinase, and Akt is observed in skeletal muscle from relatives of patients with type 2 diabetes with IGT. However, the elevated basal activity of these signaling intermediates and the lack of a strong correlation between these parameters to glucose metabolism suggests that other defects of insulin signal transduction and/or downstream components of glucose metabolism may play a greater role in the development of insulin resistance in skeletal muscle from relatives of patients with type 2 diabetes.

Irreversibility of the defect in glycogen synthase activity in skeletal muscle from obese patients with NIDDM treated with diet and metformin.

OBJECTIVE: To assess the reversibility of the defect in glycogen synthase (GS) activity in skeletal muscle from obese patients with NIDDM treated with a hypocaloric diet and metformin. RESEARCH DESIGN AND METHODS: Eighteen obese patients newly diagnosed with NIDDM were included in a randomized placebo-controlled double-blind parallel group trial and followed for 3 months. Euglycemic-hyperinsulinemic clamp including indirect calorimetry and biopsy of m. vastus lateralis was performed before and after treatment with a hypocaloric diet plus metformin or placebo. The patients were studied at basal, low, and high insulin concentrations. RESULTS: The impaired GS activity in muscle biopsies was not reversed either by acute normalization of glycemia (for 8 h) or by chronic reduction of hyperglycemia by diet plus metformin. In both treatment groups, comparable effects on glycemic control and weight loss were found together with marked insulin suppression of nonesterified fatty acids and increased glucose oxidation. Total glucose disposal at euglycemic-hyperinsulinemic clamp increased significantly in the metformin group by 25% at high insulin level (259 +/- 31 vs. 207 +/- 21 mg x m(-2) x min(-1), P < 0.05). An insignificant increase by 13% was found in the placebo group. There were no significant changes in nonoxidative glucose metabolism. GS activity and glucose utilization showed no significant differences between the two treatment groups when regression coefficients, expressed as incremental changes by increments of insulin, were compared. CONCLUSIONS: Defective GS activity in obese NIDDM patients is not secondary to hyperglycemia. Metformin and diet had no significant influence on GS activity. The added effect of metformin to that of a hypocaloric diet in improving insulin-stimulated glucose utilization is marginal when blood glucose reduction is obtained by weight loss.

Gut incretin hormones in identical twins discordant for non-insulin-dependent diabetes mellitus (NIDDM)--evidence for decreased glucagon-like peptide 1 secretion during oral glucose ingestion in NIDDM twins.

The incremental glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) responses (areas under curves; AUCs) were determined during a standard 180-min 75-g oral glucose tolerance test in a group of 12 identical twin pairs discordant for non-insulin-dependent diabetes mellitus (NIDDM) and 13 matched controls without family history of diabetes using highly sensitive and specific radioimmunoassay hormone assays. Data were analysed using multifactor analysis of variance (ANOVA) to identify and correct for possible covariates and to correct for multiple comparisons. Fasting plasma GLP-1 and GIP concentrations were similar in all groups. The twins with frank NIDDM had a decreased incremental GLP-1 response during oral glucose ingestion compared with controls without family history of diabetes (AUC +/- SEM: 0.55 +/- 0.14 vs 1.17 +/- 0.25 (mmol/l) x min, p < 0.05). The incremental GLP-1 secretion in the non-diabetic twins was not significantly different from neither their NIDDM co-twins nor the controls without family history of diabetes. The incremental GIP responses were similar in all study groups. Gender was identified as the major independent covariate for incremental glucose, insulin, GIP and GLP-1 responses, with higher values of all parameters in females. Waist-to-hip ratio and body mass index (BMI) were identified as independent but oppositely directed covariates for the incremental GLP-1 responses (waist-to-hip ratio: r = 0.43, p < 0.02; BMI: r = -0.34, p = 0.06). Incremental GLP-1 responses correlated with incremental insulin responses in the combined study population (N = 37; R = 0.42, p = 0.01). In conclusion, a decreased intestinal GLP-1 secretion may contribute to the abnormal insulin secretion during oral glucose ingestion in NIDDM twins. However, decreased secretion of gut incretin hormones (GLP-1 or GIP) does not explain all of the defects of pancreatic insulin secretion in NIDDM patients/twins or in non-diabetic individuals (identical twins) with a genetic predisposition to NIDDM.

Intracellular glucose and fat metabolism in identical twins discordant for non-insulin-dependent diabetes mellitus (NIDDM): acquired versus genetic metabolic defects?

UNLABELLED: Intracellular glucose and lipid metabolism was studied in 12 identical twin pairs discordant for non-insulin-dependent (Type 2) diabetes mellitus (NIDDM) and 13 control subjects without family history of diabetes during low (baseline) and high plasma insulin concentrations, using the hyperinsulinaemic clamp technique combined with indirect calorimetry, tritiated water glycolytic flux rates and biopsy skeletal muscle glycogen synthase activity determinations. Baseline and insulin stimulated rates of lipid oxidation were elevated--and glucose oxidation decreased--in the NIDDM twins compared with the non-diabetic co-twins and controls (all p < 0.05). Baseline and insulin stimulated rates of glucose and lipid oxidation were similar in non-diabetic twins and controls. Exogenous glycolytic flux was decreased in NIDDM twins compared with both their non-diabetic co-twins and controls during clamp insulin measurements (p < 0.02), but similar in all study groups during baseline measurements. Insulin stimulated glucose disposal, exogenous glucose storage (glucose disposal-exogenous glycolytic flux) and skeletal muscle glycogen synthase activity were all significantly decreased in NIDDM twins compared with both their non-diabetic co-twins and controls. Furthermore, glucose disposal and glucose storage were decreased in the non-diabetic twins (n = 12) compared with controls (p < 0.05 both). However, insulin stimulated fractional skeletal muscle glycogen synthase activity was not significantly decreased in non-diabetic twins compared with controls. IN CONCLUSION: (1) the glucose fatty acid cycle plays a major role in the secondary--but not the primary--abnormalities of glucose metabolism in NIDDM; (2) insulin resistance in non-diabetic identical co-twins of NIDDM patients is restricted exclusively to the pathway of exogenous glucose storage; (3) however, the decreased glucose storage is not explained solely by an impairment of insulin stimulated skeletal muscle glycogen synthase activity; and finally (4) the impairment of skeletal muscle glycogen synthase activity in NIDDM has an apparent non-genetic component and can be escaped (or postponed) in individuals (twins) with a 100% genetic predisposition to NIDDM.

Glucose processing during the intravenous glucose tolerance test.

The impact of the dynamic changes in plasma glucose and insulin levels observed during a frequently sampled intravenous (IV) glucose tolerance test (FSIGT) on whole-body glucose processing and muscle glycogen metabolism is not known. Paired randomized FSIGTs were performed in eight healthy subjects (age, 31 years; range, 28 to 35; BMI, 25.4 kg/m2; range, 22.3 to 32.1), one with muscle biopsy samples and one without. The mean time average (0- to 40- and 0- to 120-minute) insulin levels during the test were 26.6 and 11.4 mU/1, respectively. Glucose oxidation increased following the IV glucose bolus (basal 1.34 +/- 0.21 v mean value at 0 to 120 minutes 2.09 +/- 0.22 mg/kg fat-free mass [FFM]/min, P < .02). In contrast, fractional glucose-6-phosphate [G-6-P]) (0.1/10 mmol/L) skeletal muscle glycogen synthase activity in muscle biopsies obtained before and following the IV glucose bolus (-30, 30,60, and 120 minutes, respectively) were unchanged (38.1% +/- 2.3%, 38.3% +/- 2.9%, 38.1% +/- 2.3%, 35.4% +/- 2.3%, NS). Skeletal muscle glycogen concentration decreased slightly (449 +/- 54, 439 +/- 55, and 383 +/- 29, and 438 +/- 48 mmol/kg dry weight, P =.05), indicating no net storage of glucose into glycogen during the FSIGT. G-6-P decreased (0.77 +/- 0.08, 0.64 +/- 0.07, 0.66 +/- 0.07, and 0.54 +/- 0.04 mmol/kg dry weight, P < .05). Levels of the insulin-regulatable glucose transporter, GLUT-4, were unchanged. Insulin sensitivity (Si), glucose effectiveness, and insulin secretion parameters (01 and 02) were not affected by the muscle biopsy procedure. In conclusion, the FSIGT is associated predominantly with increased whole-body glucose oxidation with no apparent activation of muscle glucose storage as glycogen. Thus, the Si measured by the FSIGT, although similar in magnitude to the clamp-derived parameter, represents primarily glucose oxidation, in contrast to the euglycemic clamp, which involves glucose oxidation and storage.

Multiple defects of both hepatic and peripheral intracellular glucose processing contribute to the hyperglycaemia of NIDDM.

Non-insulin-dependent diabetic (NIDDM) patients were studied during a modified euglycaemic state when fasting hyperglycaemia was normalized by a prior (-210 to -150 min)--and later withdrawn (-150-0 min)--intravenous insulin infusion. Glucose metabolism was assessed in NIDDM patients (n = 10) and matched control subjects (n = 10) using tritiated glucose turnover rates, indirect calorimetry and skeletal muscle glycogen synthase activity determinations. Total and non-oxidative exogenous glycolytic flux rates were measured using appearance rates of tritiated water. A + 180 min euglycaemic hyperinsulinaemic (40 mU.m-2.min-1) clamp was performed to determine the insulin responsiveness of the various metabolic pathways. Plasma glucose concentration increased spontaneously during baseline measurements in the NIDDM patients (-120 to 0 min: 4.8 +/- 0.3 to 7.0 +/- 0.3 mmol/l; p < 0.01), and was primarily due to an elevated rate of hepatic glucose production (3.16 +/- 0.13 vs 2.51 +/- 0.16 mg.kg FFM-1.min-1; p < 0.01). In the NIDDM subjects baseline glucose oxidation was decreased (0.92 +/- 0.17 vs 1.33 +/- 0.14 mg.kg FFM-1.min-1; p < 0.01) in the presence of a normal rate of total exogenous glycolytic flux and skeletal muscle glycogen synthase activity. The simultaneous finding of an increased lipid oxidation rate (1.95 +/- 0.13 vs 1.61 +/- 0.07 mg.kg FFM-1.min-1; p = 0.05) and increased plasma lactate concentrations (0.86 +/- 0.05 vs 0.66 +/- 0.03 mmol/l; p = 0.01) are consistent with a role for both the glucose-fatty acid cycle and the Cori cycle in the maintenance and development of fasting hyperglycaemia in NIDDM during decompensation. Insulin resistance was demonstrated during the hyperinsulinaemic clamp in the NIDDM patients with a decrease in the major peripheral pathways of intracellular glucose metabolism (oxidation, storage and muscle glycogen synthase activity), but not in the pathway of non-oxidative glycolytic flux which was not completely suppressed during insulin infusion in the NIDDM patients (0.55 +/- 0.15 mg.kg FFM-1.min-1; p < 0.05 vs 0; control subjects: 0.17 +/- 0.29; NS vs 0). Thus, these data also indicate that the defect(s) of peripheral (skeletal muscle) glucose processing in NIDDM goes beyond the site of glucose transport across the cell membrane.