Obesity-induced insulin resistance in human skeletal muscle is characterised by defective activation of p42/p44 MAP kinase

Insulin resistance (IR), an impaired cellular, tissue and whole body response to insulin, is a major pathophysiological defect of type 2 diabetes mellitus. Although IR is closely associated with obesity, the identity of the molecular defect(s) underlying obesity-induced IR in skeletal muscle remains controversial; reduced post-receptor signalling of the insulin receptor substrate 1 (IRS1) adaptor protein and downstream effectors such as protein kinase B (PKB) have previously been implicated. We examined expression and/or activation of a number of components of the insulin-signalling cascade in skeletal muscle of 22 healthy young men (with body mass index (BMI) range, 20–37 kg/m2). Whole body insulin sensitivity (M value) and body composition was determined by the hyperinsulinaemic (40 mU. min−1.m−2.), euglycaemic clamp and by dual energy X-ray absorptiometry (DEXA) respectively. Skeletal muscle (vastus lateralis) biopsies were taken before and after one hour of hyperinsulinaemia and the muscle insulin signalling proteins examined by western blot and immunoprecipitation assay. There was a strong inverse relationship between M-value and BMI. The most striking abnormality was significantly reduced insulin-induced activation of p42/44 MAP kinase, measured by specific assay, in the volunteers with poor insulin sensitivity. However, there was no relationship between individuals' BMI or M-value and protein expression/phosphorylation of IRS1, PKB, or p42/44 MAP kinase protein, under basal or hyperinsulinaemic conditions. In the few individuals with poor insulin sensitivity but preserved p42/44 MAP kinase activation, other signalling defects were evident. These findings implicate defective p42/44 MAP kinase signalling as a potential contributor to obesity-related IR in a non-diabetic population, although clearly multiple signalling defects underlie obesity associated IR

Developing a Simplified Consent Form for Biobanking

BACKGROUND: Consent forms have lengthened over time and become harder for participants to understand. We sought to demonstrate the feasibility of creating a simplified consent form for biobanking that comprises the minimum information necessary to meet ethical and regulatory requirements. We then gathered preliminary data concerning its content from hypothetical biobank participants. METHODOLOGY/PRINCIPAL FINDINGS: We followed basic principles of plain-language writing and incorporated into a 2-page form (not including the signature page) those elements of information required by federal regulations and recommended by best practice guidelines for biobanking. We then recruited diabetes patients from community-based practices and randomized half (n = 56) to read the 2-page form, first on paper and then a second time on a tablet computer. Participants were encouraged to use "More information" buttons on the electronic version whenever they had questions or desired further information. These buttons led to a series of "Frequently Asked Questions" (FAQs) that contained additional detailed information. Participants were asked to identify specific sentences in the FAQs they thought would be important if they were considering taking part in a biorepository. On average, participants identified 7 FAQ sentences as important (mean 6.6, SD 14.7, range: 0-71). No one sentence was highlighted by a majority of participants; further, 34 (60.7%) participants did not highlight any FAQ sentences. CONCLUSIONS: Our preliminary findings suggest that our 2-page form contains the information that most prospective participants identify as important. Combining simplified forms with supplemental material for those participants who desire more information could help minimize consent form length and complexity, allowing the most substantively material information to be better highlighted and enabling potential participants to read the form and ask questions more effectively

Decreased insulin-stimulated brown adipose tissue glucose uptake after short-term exercise training in healthy middle-aged men

Aims: To test the hypothesis that high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) improve brown adipose tissue (BAT) insulin sensitivity.Participants and methods: Healthy middle-aged men (n = 18, age 47 years [95% confidence interval {CI} 49, 43], body mass index 25.3 kg/m(2) [95% CI 24.1-26.3], peak oxygen uptake (VO2peak) 34.8 mL/kg/min [95% CI 32.1, 37.4]) were recruited and randomized into six HIIT or MICT sessions within 2 weeks. Insulin-stimulated glucose uptake was measured using 2-[F-18] flouro-2-deoxy-D-glucose positron-emission tomography in BAT, skeletal muscle, and abdominal and femoral subcutaneous and visceral white adipose tissue (WAT) depots before and after the training interventions.Results: Training improved VO2peak (P =.0005), insulin-stimulated glucose uptake into the quadriceps femoris muscle (P =.0009) and femoral subcutaneous WAT (P =.02) but not into BAT, with no difference between the training modes. Using pre-intervention BAT glucose uptake, we next stratified subjects into high BAT (> 2.9 mu mol/100 g/min; n = 6) or low BAT (< 2.9 mu mol/100 g/min; n = 12) groups. Interestingly, training decreased insulin-stimulated BAT glucose uptake in the high BAT group (4.0 [2.8, 5.5] vs 2.5 [1.7, 3.6]; training*BAT, P =.02), whereas there was no effect of training in the low BAT group (1.5 [1.2, 1.9] vs 1.6 [1.2, 2.0] mu mol/100 g/min). Participants in the high BAT group had lower levels of inflammatory markers compared with those in the low BAT group.Conclusions: Participants with functionally active BAT have an improved metabolic profile compared with those with low BAT activity. Short-term exercise training decreased insulin-stimulated BAT glucose uptake in participants with active BAT, suggesting that training does not work as a potent stimulus for BAT activation

Impact of time since last caloric intake on blood glucose levels

Blood glucose (BG) is usually measured after a caloric restriction of at least 8 h; however evidence-based recommendations for the duration of a fasting status are missing. Here we analyze the effect of fasting duration on levels of BG to determine the minimal fasting duration to achieve comparable BG levels to conventional fasting measurements. We used data of a cross-sectional study on primary care patients, performed in October 2005. We included 28,024 individuals (age-range 18–99 years; 63% women) without known diabetes mellitus and without missing data for BG and fasting status. We computed general linear models, adjusting for age, sex, time of blood withdrawal, systolic blood pressure, waist circumference, total- and HDL-cholesterol, physical activity, smoking, intake of beta-blocker and alcohol. We tested the intra-individual variability with respect to fasting status. Overall, the mean BG differed only slightly between individuals fasting ≥8 h and those fasting <8 h (men: 5.1 ± 0.8 mmol/L versus 5.2 ± 1.2 mmol/L; women: 4.9 ± 0.7 mmol/L, 5.0 ± 1.0 mmol/L). After 3 h of fasting differences of BG diminished in men to −0.08 mmol/L (95%-CI: −0.15; −0.01 mmol/L), in women to −0.07 mmol/L (−0.12; −0.03 mmol/L) compared to individuals fasting ≥8 h. Noteworthy, age, time of day of blood withdrawal, physical activity, and intake of hard liquor influenced BG levels considerably. Our data challenge the necessity for a fasting duration of ≥8 h when measuring blood glucose, suggesting a random sampling or a fasting duration of 3 h as sufficient. Rather, our study indicates that essentially more effort on the assessment of additional external/internal factors on BG levels is necessary

Effects of Meal Frequency on Metabolic Profiles and Substrate Partitioning in Lean Healthy Males

The daily number of meals has an effect on postprandial glucose and insulin responses, which may affect substrate partitioning and thus weight control. This study investigated the effects of meal frequency on 24 h profiles of metabolic markers and substrate partitioning.Twelve (BMI:21.6 ± 0.6 kg/m(2)) healthy male subjects stayed after 3 days of food intake and physical activity standardization 2 × 36 hours in a respiration chamber to measure substrate partitioning. All subjects randomly received two isoenergetic diets with a Low meal Frequency (3 ×; LFr) or a High meal Frequency (14 ×; HFr) consisting of 15 En% protein, 30 En% fat, and 55 En% carbohydrates. Blood was sampled at fixed time points during the day to measure metabolic markers and satiety hormones.Glucose and insulin profiles showed greater fluctuations, but a lower AUC of glucose in the LFr diet compared with the HFr diet. No differences between the frequency diets were observed on fat and carbohydrate oxidation. Though, protein oxidation and RMR (in this case SMR + DIT) were significantly increased in the LFr diet compared with the HFr diet. The LFr diet increased satiety and reduced hunger ratings compared with the HFr diet during the day.The higher rise and subsequently fall of insulin in the LFr diet did not lead to a higher fat oxidation as hypothesized. The LFr diet decreased glucose levels throughout the day (AUC) indicating glycemic improvements. RMR and appetite control increased in the LFr diet, which can be relevant for body weight control on the long term.ClinicalTrials.gov NCT01034293

Impact of physical activity level and dietary fat content on passive overconsumption of energy in non-obese adults

Background: Passive overconsumption is the increase in energy intake driven by the high-fat energy-dense food environment. This can be explained in part because dietary fat has a weaker effect on satiation (i.e. process that terminates feeding). Habitually active individuals show improved satiety (i.e. process involved in post-meal suppression of hunger) but any improvement in satiation is unknown. Here we examined whether habitual physical activity mitigates passive overconsumption through enhanced satiation in response to a high-fat meal. Methods: Twenty-one non-obese individuals with high levels of physical activity (HiPA) and 19 individuals with low levels of physical activity (LoPA) matched for body mass index (mean = 22.8 kg/m2) were recruited. Passive overconsumption was assessed by comparing ad libitum energy intake from covertly manipulated high-fat (HFAT; 50% fat) or high-carbohydrate (HCHO; 70% carbohydrate) meals in a randomized crossover design. Habitual physical activity was assessed using SenseWear accelerometers (SWA). Body composition, resting metabolic rate, eating behaviour traits, fasting appetite-related peptides and hedonic food reward were also measured. Results: In the whole sample, passive overconsumption was observed with greater energy intake at HFAT compared to HCHO (p  0.05). SWA confirmed that HiPA were more active than LoPA (p  0.05 for all). Conclusions: Non-obese individuals with high or low physical activity levels but matched for BMI showed similar susceptibility to passive overconsumption when consuming an ad libitum high-fat compared to a high-carbohydrate meal. This occurred despite increased total daily energy expenditure and improved body composition in HiPA. Greater differences in body composition and/or physical activity levels may be required to impact on satiation

E4orf1: A Novel Ligand That Improves Glucose Disposal in Cell Culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance(IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 ‘requires’ E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as ‘sufficient’ to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras–the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large(Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif(PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs

Persistent Gastric Colonization with Burkholderia pseudomallei and Dissemination from the Gastrointestinal Tract following Mucosal Inoculation of Mice

Melioidosis is a disease of humans caused by opportunistic infection with the soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as an acute, overwhelming infection or as a chronic, recurrent infection. At present, it is not clear where B. pseudomallei resides in the mammalian host during the chronic, recurrent phase of infection. To address this question, we developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei infection and investigated sites of bacterial persistence over 60 days. Sensitive culture techniques and selective media were used to quantitate bacterial burden in major organs, including the gastrointestinal (GI) tract. We found that the GI tract was the primary site of bacterial persistence during the chronic infection phase, and was the only site from which the organism could be consistently cultured during a 60-day infection period. The organism could be repeatedly recovered from all levels of the GI tract, and chronic infection was accompanied by sustained low-level fecal shedding. The stomach was identified as the primary site of GI colonization as determined by fluorescent in situ hybridization. Organisms in the stomach were associated with the gastric mucosal surface, and the propensity to colonize the gastric mucosa was observed with 4 different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were present at low numbers within luminal contents in the small and large intestine and cecum relative to the stomach. Notably, inflammatory lesions were not detected in any GI tissue examined in chronically-infected mice. Only low-dose oral or intranasal inoculation led to GI colonization and development of chronic infection of the spleen and liver. Thus, we concluded that in a mouse model of melioidosis B. pseudomallei preferentially colonizes the stomach following oral inoculation, and that the chronically colonized GI tract likely serves as a reservoir for dissemination of infection to extra-intestinal sites