Early Stages Of Insulin Resistance Research Articles

Branched-chain amino acid supplementation drives dynamic changes in gut microbiota without impairing glucose and lipid homeostasis at the different stages of insulin resistance in mice on a high-fat diet

ObjectiveThe potential role of dietary branched-chain amino acids on circulating branched-chain amino acid levels and their relationship with metabolic health are complex, and the literature is inconsistent. We aimed to explore the dynamic effects of branched-chain amino acid supplementation on glucose and lipid homeostasis at different stages of insulin resistance in high-fat diet–fed mice. MethodsMale C57BL/6J mice were fed with a normal chow diet, high-fat diet, or high-fat diet supplemented with 100% branched-chain amino acids for 12 or 24 wk. Metabolic parameters and gut microbiota profiling were performed at these two time points. ResultsHigh-fat diet feeding caused varying degrees of branched-chain amino acid metabolic disorders in two different stages of insulin resistance. Supplementing with branched-chain amino acids further exacerbated branched-chain amino acid accumulation in the early stage of insulin resistance (12 wk), while adding branched-chain amino acids did not further elevate branched-chain amino acid levels in the hyperglycemia and hyperinsulinemia stage (24 wk). Compared with the high-fat diet group, branched-chain amino acid supplementation did not affect body weight; liver total cholesterol and triacylglycerol levels; and serum glucose, insulin, total cholesterol, triacylglycerol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels as well as glucose tolerance at these two time points but triggered dynamic changes in the gut bacterial diversity and gut microbiota composition and abundance, especially in the genus associated with obesity and related metabolic disorders. ConclusionDietary branched-chain amino acid supplementation drives dynamic changes in circulating branched-chain amino acid levels and gut microbiome without subsequent effects on glucose and lipid homeostasis in high-fat diet–induced obese mice within the parameters of our study.

Chronic supplementation of branched chain amino acids and its impact on glucose and hepatic metabolism during insulin sensitivity

Dysfunctional interactions between branched chain amino acids (BCAAs) and facets of glucose and lipid metabolism are central features of insulin resistant tissues. However, it is unclear whether insulin resistance is a prerequisite for the biochemical relationships between BCAAs and macronutrient networks. We hypothesize that BCAAs will enhance glucose and lipid oxidation, lower gluconeogenesis and decrease hepatic de novo lipogenesis to optimize insulin sensitivity. Male mice (C57BL/6NJ) were placed on either a low-fat (LF; 10% fat Kcal) or LF supplemented with 1.5X BCAAs (LB) diet for a period of 12-32 wks. Oxygen consumption and energy expenditure were determined by indirect calorimetry after 12 and 24-wks on the diets. After 32-wks on the diets and a 4-hr fast, LF and LB mice underwent intraperitoneal glucose tolerance test (IPGTT) with the 2g/kg glucose containing 50% [ 13 C 6 ]glucose. Blood glucose was measured every 15-min and blood was collected every 30-min for 2 hours. Enrichment of 13 C from glucose into pyruvate and other metabolic intermediates was determined by gas-chromatography and mass-spectrometry (GC-MS). Following 1-wk of recovery, these LF and LB mice were randomly split into fed and overnight fasted groups. [ 13 C 3 ] pyruvate (20 mg) was injected intraperitoneally to determine the rate of incorporation of pyruvate into glucose via. gluconeogenesis, utilizing GC-MS. Hepatic gene expression in fed and fasted livers were profiled using qPCR. Supplementation of BCAAs for 24-wks increased energy expenditure and oxygen consumption in the LB mice vs. their LF counterparts (p≤0.05). Additionally, IPGTT revealed improved insulin sensitivity in LB mice vs. their LF counterparts (p<0.05). Dilution of [ 13 C 6 ]glucose was lower in the LB plasma by the 90-min time point of the glucose tolerance test (% enrichment; LB; 16.42±0.74 vs. LF; 18.06±0.54, p=0.09), while the LB blood glucose levels during IPGTT remained lower, indicative of higher rates of glucose oxidation in LB mice. Further, following the pyruvate tolerance test, incorporation of [ 13 C 3 ]pyruvate into glucose tended to be higher in the fasted BCAA supplemented group (% enrichment; LB; 11.79±0.23 vs. LF; 10.58±0.53, p=0.07). Basal levels of tricarboxylic acid (TCA) cycle intermediates in plasma were significantly different between LF vs. LB mice (p<0.05). Fed-to-fasted fold induction of genes involved in lipid oxidation ( Lcad, P para) were higher in the LB livers vs. their LF counterparts (p≤0.05). On the contrary, fed-to-fasted suppression of lipogenic gene expression (A cc, F as) was greater in the LB livers vs. their LF counterparts (p<0.05). In summary, our results indicate that chronic BCAA supplementation to insulin sensitive mice positively impacts aspects of glucose and lipid metabolism. The ability of BCAAs to modulate macronutrient metabolism could have beneficial effects in alleviating metabolic dysfunction during early stages of insulin resistance and fatty liver disease. National Institute of Health R01DK112865 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Circulating microRNA levels differ in the early stages of insulin resistance in prepubertal children with obesity

AimsThe increasing prevalence of childhood obesity escalates the risk for related complications. Circulating microRNAs (miRNAs) have been suggested as good predictive markers of insulin resistance in those with obesity. The aim was to identify a circulating miRNA profile that reflects insulin resistance in prepubertal children with obesity. Material and methodsPlasma miRNAs were measured in prepubertal children (n = 63, 5–9 years) using TaqMan Advanced miRNA Human Serum/Plasma plates and then were validated by RT-qPCR. Subjects were divided into normal weight (n = 20, NW) and overweight or obese (n = 43, OW/OB) groups according to their BMI z-scores. The OW/OB group was further subdivided into insulin sensitive or metabolically healthy obese (n = 26, MHO) and insulin resistant or metabolically unhealthy obese (n = 17, MUO) according to HOMA-IR. Key findingsWhile no differences were observed in the fasting plasma glucose levels, serum insulin levels were significantly elevated in the OW/OB compared to the NW group. Of 188 screened miRNAs, eleven were differentially expressed between the NW and OW/OB groups. Validation confirmed increased circulating levels of miR-146a-5p and miR-18a-5p in the OW/OB group, which correlated with BMI z-score. Interestingly, miR-146a-5p was also correlated with HOMA-IR index. While only miR-18a-5p was upregulated in the OW/OB children, independently of their degree of insulin sensitivity, miR-146-5p, miR-423-3p and miR-152-3p were associated with insulin resistance. SignificanceThe present study provides evidence of molecular alterations that occur early in life in prepubertal obesity. These alterations may potentially be crucial for targeted prevention or prompt precision therapeutic development and subsequent interventions.

Programmed cell death 5 improves skeletal muscle insulin resistance by inhibiting IRS-1 ubiquitination through stabilization of MDM2

AimsInsulin resistance is defined as the decreased sensitivity of tissues and organs to insulin and it is the main pathological basis of metabolic syndrome. PDCD5 is widely expressed in tissues including skeletal muscle and liver, but its exact function and the role in insulin resistance has not been studied. The present study is to explore the effect of PDCD5 on insulin resistance in skeletal muscle, the largest target organ of insulin, and its mechanism. Materials and methodsMice were fed with high-fat diet to establish obesity model. C2C12 myoblasts differentiated into myotubes and then were treated with palmitate to induce insulin resistance. Gain-of-function and loss-of-function experiments were performed by infecting C2C12 with adenovirus containing PDCD5 cDNA or PDCD5 shRNA. Key findingsPDCD5 protein was first increased and then decreased in the skeletal muscle from high-fat diet induced obese mice and consistently in palmitate induced insulin resistance C2C12 myotubes. Overexpression of PDCD5 in C2C12 cells did not affect the sensitivity to insulin but inhibited the palmitate induced insulin resistance, while knockdown of PDCD5 aggravated the insulin resistance. Mechanistically, PDCD5 interacted with ubiquitin ligase MDM2; overexpression of PDCD5 decreased MDM2 protein level, inhibited the increased interaction of MDM2 with IRS-1 and the degradation of IRS-1 by palmitate stimulation. SignificancePDCD5 is upregulated during the early stage of insulin resistance in skeletal muscle. The increased PDCD5 inhibits IRS-1 ubiquitination, increases the stability of IRS-1 by interacting with and degrading MDM2, thus providing a protective effect on insulin resistance in skeletal muscle.

Determining insulin sensitivity from glucose tolerance tests in Iberian and landrace pigs.

As insulin sensitivity may help to explain divergences in growth and body composition between native and modern breeds, metabolic responses to glucose infusion were measured using an intra-arterial glucose tolerance test (IAGTT). Iberian (n = 4) and Landrace (n = 5) barrows (47.0 ± 1.2 kg body weight (BW)), fitted with a permanent carotid artery catheter were injected with glucose (500 mg/kg BW) and blood samples collected at -10, 0, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120 and 180 min following glucose infusion. Plasma samples were analysed for insulin, glucose, lactate, triglycerides, cholesterol, creatinine, albumin and urea. Insulin sensitivity indices were calculated and analysed. Mean plasma glucose, creatinine and cholesterol concentrations were lower (P < 0.01) in Iberian (14, 68 and 22%, respectively) than in Landrace pigs during the IAGTT. However, mean plasma insulin, lactate, triglycerides and urea concentrations were greater (P < 0.001) in Iberian (50, 35, 18 and 23%, respectively) than in Landrace pigs. Iberian pigs had larger area under the curve (AUC) of insulin (P < 0.05) or tended to a greater AUC of lactate (P < 0.10), and a smaller (P < 0.05) AUC for glucose 0-60 min compared with Landrace pigs. Indices for estimating insulin sensitivity in fasting conditions indicated improved β-cell function in Iberian compared with Landrace pigs, but no difference (P > 0.10) in calculated insulin sensitivity index was found after IAGTT between breeds. A time response (P < 0.05) was obtained for insulin, glucose and lactate so that maximum concentration was achieved at 10 and 15 min post-infusion for insulin (Iberian and Landrace pigs, respectively), immediately post-infusion for glucose, and 20 min post-infusion for lactate, decreasing thereafter until basal levels. There was no time effect for the rest of metabolites evaluated. In conclusion, growing Iberian pigs challenged with an IAGTT showed changes in biochemical parameters and insulin response that may indicate an early stage of insulin resistance.

Relationship between insulin resistance, coronary plaque, and clinical outcomes in patients with acute coronary syndromes: an analysis from the PROSPECT study

BackgroundWe investigated the association of insulin resistance (IR) with coronary plaque morphology and the risk of cardiovascular events in patients enrolled in the Providing Regional Observations to Study Predictors of Events in Coronary Tree (PROSPECT) study.MethodsPatients with acute coronary syndromes (ACS) were divided based on DM status. Non-DM patients were further stratified according to homeostasis-model-assessment IR (HOMA-IR) index as insulin sensitive (IS; HOMA-IR ≤ 2), likely-IR (LIR; 2 < HOMA-IR < 5), or diabetic-IR (DIR; HOMA-IR ≥ 5). Coronary plaque characteristics were investigated by intravascular ultrasound. The primary endpoint was major adverse cardiac events (MACE); a composite of cardiac death, cardiac arrest, myocardial infarction, and rehospitalization for unstable/progressive angina.ResultsAmong non-diabetic patients, 109 patients (21.5%) were categorized as LIR, and 65 patients (12.8%) as DIR. Patients with DIR or DM had significantly higher rates of echolucent plaque compared with LIR and IS. In addition, DIR and DM were independently associated with increased risk of MACE compared with IS (adjusted hazard ratio [aHR] 2.29, 95% confidence interval [CI] 1.22–4.29, p = 0.01 and aHR 2.12, 95% CI 1.19–3.75, p = 0.009, respectively).ConclusionsIR is common among patients with ACS. DM and advanced but not early stages of IR are independently associated with increased risk of adverse cardiovascular events.Trial Registration ClinicalTrials.gov Identifier: NCT00180466.

PSVIII-40 Late-Breaking Abstract: Variability in body composition is associated with insulin sensitivity in growing-finishing pigs

Abstract Growing pigs’ body composition variation can be associated with differences in insulin sensitivity given the insulin anabolic effect on protein and lipid synthesis. The objective of this study was to elucidate this association by relating the individual insulin response to the oral glucose tolerance test (OGTT) with the body composition of growing pigs. Thirty 95 kg jugular vein catheterized pigs received an oral dose of 1.75 g of glucose/kg of BW after 18 hours of fasting. Blood samples were collected at -20, -10, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min following glucose ingestion. Insulin sensitivity indexes were calculated and analyzed. Body lipids (LB, %) and protein (PB, %) composition were estimated by dual X-ray densitometry. Association between body composition and insulin sensitivity were studied by using partial least squares and correlations. Average LB and PB were 19.7% (CV = 7.6 %) and 16.2% (CV = 2.2%), respectively. Basal insulin blood concentration and area-under-the-curve (AUC) CV (51.9 % and 26.9 %, respectively) were larger than those for basal glucose and AUC (5.52 and 5.48 %, respectively). Additionally, insulin sensitivity (%S), steady-state beta cell function (%B), and insulin resistance (HOMA-IR) estimated with the Homeostasis Model Assessment (HOMA 2) and whole-body insulin sensitivity index (ISI) were highly variable between pigs which CV ranged from 30.1 % to 54.5 %. These results can indicate an early stage of insulin resistance in an important part of the studied pig population. LB and PB were affected by insulin sensitivity indexes (P &amp;lt; 0.05) which accounted, respectively, for 48% and 44% of the observed variation. In conclusion, lower insulin sensitivity was associated with higher body fat in growing pigs raised under similar conditions.

The metabolic footprint of compromised insulin sensitivity under fasting and hyperinsulinemic-euglycemic clamp conditions in an Arab population

Metabolic pathways that are corrupted at early stages of insulin resistance (IR) remain elusive. This study investigates changes in body metabolism in clinically healthy and otherwise asymptomatic subjects that may become apparent already under compromised insulin sensitivity (IS) and prior to IR. 47 clinically healthy Arab male subjects with a broad range of IS, determined by hyperinsulinemic-euglycemic clamp (HIEC), were investigated. Untargeted metabolomics and complex lipidomics were conducted on serum samples collected under fasting and HIEC conditions. Linear models were used to identify associations between metabolites concentrations and IS levels. Among 1896 identified metabolites, 551 showed significant differences between fasting and HIEC, reflecting the metabolic switch in energy utilization. At fasting, 336 metabolites, predominantly di- and tri-acylglycerols, showed significant differences between subjects with low and high levels of IS. Changes in amino acid, carbohydrate and fatty acid metabolism in response to insulin were impaired in subjects with low IS. Association of altered mannose and amino acids with IS was also replicated in an independent cohort of T2D patients. We identified metabolic phenotypes that characterize clinically healthy Arab subjects with low levels of IS at their fasting state. Our study is providing further insights into the metabolic pathways that precede IR.

Effects of Fish Oil and Grape Seed Extract Combination on Hepatic Endogenous Antioxidants and Bioactive Lipids in Diet-Induced Early Stages of Insulin Resistance in Rats.

Diacylglycerols (DAG) and ceramides have been suggested as early predictors of insulin resistance. This study was aimed to examine the combined effects of fish oil (FO) and grape seed extract (GSE) on hepatic endogenous antioxidants, DAG and ceramides in diet-induced early stages of insulin resistance. Thirty-five rats were fed one of the following diets: (1) a standard diet (STD group), (2) a high-fat high-sucrose diet (HFHS group), (3) an HFHS diet enriched with FO (FO group), (4) an HFHS diet enriched with GSE (GSE group) or (5) an HFHS diet enriched with FO and GSE (FO + GSE group). In the liver, endogenous antioxidants were measured using spectrophotometric and fluorometric techniques, and non-targeted lipidomics was conducted for the assessment of DAG and ceramides. After 24 weeks, the FO + GSE group showed increased glutathione peroxidase activity, as well as monounsaturated fatty acid and polyunsaturated fatty acid-containing DAG, and long-chain fatty acid-containing ceramides abundances compared to the STD group. The FO and GSE combination induced similar activation of the antioxidant system and bioactive lipid accumulation in the liver than the HFHS diet without supplementation. In addition, the FO and GSE combination increased the abundances of polyunsaturated fatty acid-containing DAG in the liver.

Zinc, copper, sirtuin 1 concentration, and glucose metabolism parameters in the blood of women with polycystic ovary syndrome

The aim of this study was to assess concentration of zinc (Zn), copper (Cu), and sirtuin 1 (SIRT1) in serum of women with polycystic ovary syndrome (PCOS) and their relationships with glucose metabolism parameters. The study included 76 women with PCOS aged between 17 and 39 years old. The blood was collected according to the routine procedure during the follicular phase. Zn and Cu concentrations were performed by flame atomic absorption spectrometry (FAAS). Glucose concentration was estimated by colorimetric methods. Insulin (INs) concentration was determined by enzyme-linked immunosorbent assay. SIRT1 concentration was determined using commercial test. We found higher Cu concentration and Cu/Zn value in the serum of women with PCOS with overweight/obesity. Early stage of insulin resistance (IR) in the group of women with PCOS affected on higher Cu concentration and Cu/Zn value. However, increased value of body mass index, waist-hip ratio and homeostatic model assessment of insulin resistance (HOMA-IR) was not associated with changes in Zn and SIRT1 concentration. Further disturbances in glucose metabolic parameters in the blood of women with PCOS were intensified by overweight/obesity and IR. Overweight/obesity and IR in the women with PCOS increase disorders in Cu homeostasis and glucose metabolism parameters.

Effects of prolonged type 2 diabetes on mitochondrial function in cerebral blood vessels.

One of the major characteristics of hyperglycemic states such as type 2 diabetes is increased reactive oxygen species (ROS) generation. Since mitochondria are a major source of ROS, it is vital to understand the involvement of these organelles in the pathogenesis of ROS-mediated conditions. Therefore, we investigated mitochondrial function and ROS production in cerebral blood vessels of 21-wk-old Zucker diabetic fatty obese rats and their lean controls. We have previously shown that in the early stages of insulin resistance, and short periods of type 2 diabetes mellitus, only mild differences exist in mitochondrial function. In the present study, we examined mitochondrial respiration, mitochondrial protein expression, and ROS production in large-surface cerebral arteries. We used 21-wk-old animals exposed to peak glucose levels for 7 wk and compared them with our previous studies on younger diabetic animals. We found that the same segments of mitochondrial respiration (basal respiration and proton leak) were diminished in diabetic groups as they were in younger diabetic animals. Levels of rattin, a rat humanin analog, tended to decrease in the diabetic group but did not reach statistical significance (P = 0.08). Other mitochondrial proteins were unaffected, which might indicate the existence of compensatory mechanisms with extension of this relatively mild form of diabetes. Superoxide levels were significantly higher in large cerebral vessels of diabetic animals compared with the control group. In conclusion, prolonged dietary diabetes leads to stabilization, rather than deterioration, of metabolic status in the cerebral circulation, despite continued overproduction of ROS.NEW & NOTEWORTHY We have characterized for the first time the dynamics of mitochondrial function during the progression of type 2 diabetes mellitus with regard to mitochondrial respiration, protein expression, and reactive oxygen species production. In addition, this is the first measurement of rattin levels in the cerebral vasculature, which could potentially lead to novel treatment options.

Obesity-induced insulin resistance and macrophage infiltration of the adipose tissue: A vicious cycle.

Obesity-induced insulin resistance and macrophage infiltration of the adipose tissue: A vicious cycle.

Leucine Supplementation Differently Modulates Branched-Chain Amino Acid Catabolism, Mitochondrial Function and Metabolic Profiles at the Different Stage of Insulin Resistance in Rats on High-Fat Diet.

The available findings concerning the association between branched-chain amino acids (BCAAs)—particularly leucine—and insulin resistance are conflicting. BCAAs have been proposed to elicit different or even opposite effects, depending on the prevalence of catabolic and anabolic states. We tested the hypothesis that leucine supplementation may exert different effects at different stages of insulin resistance, to provide mechanistic insights into the role of leucine in the progression of insulin resistance. Male Sprague-Dawley rats were fed a normal chow diet, high-fat diet (HFD), HFD supplemented with 1.5% leucine, or HFD with a 20% calorie restriction for 24 or 32 weeks. Leucine supplementation led to abnormal catabolism of BCAA and the incompletely oxidized lipid species that contributed to mitochondrial dysfunction in skeletal muscle in HFD-fed rats in the early stage of insulin resistance (24 weeks). However, leucine supplementation induced no remarkable alternations in BCAA catabolism, but did enhance mitochondrial biogenesis with a concomitant improvement in lipid oxidation and mitochondrial function during the hyperglycaemia stage (32 weeks). These findings suggest that leucine trigger different effects on metabolic signatures at different stages of insulin resistance, and the overall metabolic status of the organisms should be carefully considered to potentiate the benefits of leucine.

Pressor recovery after acute stress is impaired in high fructose-fed Lean Zucker rats.

Insulin resistance is a powerful predictor of cardiovascular disease; however, the mechanistic link remains unclear. This study aims to determine if early cardiovascular changes associated with short‐term fructose feeding in the absence of obesity manifest as abnormal blood pressure control. Metabolic dysfunction was induced in Lean Zucker rats by short‐term high‐fructose feeding. Rats were implanted with telemetry devices for the measurement of mean arterial blood pressure (MAP) and subjected to air jet stress at 5 and 8 weeks after feeding. Additional animals were catheterized under anesthesia for the determination of MAP and blood flow responses in the hind limb and mesenteric vascular beds to intravenous injection of isoproterenol (0.001–0.5 μm), a β‐adrenergic agonist. Metabolic dysfunction in high‐fructose rats was not accompanied by changes in 24‐h MAP. Yet, animals fed a high‐fructose diet for 8 weeks exhibited a marked impairment in blood pressure recovery after air‐jet stress. Dose‐dependent decreases in MAP and peripheral blood flow in response to isoproterenol treatment were significantly attenuated in high‐fructose rats. These data suggest that impaired blood pressure recovery to acute mental stress precedes the onset of hypertension in the early stages of insulin resistance. Further, blunted responses to isoproterenol implicate β 2‐adrenergic sensitivity as a possible mechanism responsible for altered blood pressure control after short‐term high‐fructose feeding.

MicroRNA-194 Modulates Glucose Metabolism and Its Skeletal Muscle Expression Is Reduced in Diabetes.

BackgroundThe regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated.MethodsMicroarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy.ResultsTwenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation.ConclusionsType 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.

Early impairment of coronary microvascular perfusion capacity in rats on a high fat diet.

BackgroundIt remains to be established if, and to what extent, the coronary microcirculation becomes compromised during the development of obesity and insulin resistance. Recent studies suggest that changes in endothelial glycocalyx properties contribute to microvascular dysfunction under (pre-)diabetic conditions. Accordingly, early effects of diet-induced obesity on myocardial perfusion and function were studied in rats under baseline and hyperaemic conditions.MethodsRats were fed a high fat diet (HFD) for 6 weeks and myocardial microvascular perfusion was determined using first-pass perfusion MRI before and after adenosine infusion. The effect of HFD on microcirculatory properties was also assessed by sidestream darkfield (SDF) imaging of the gastrocnemius muscle.ResultsHFD-fed rats developed central obesity and insulin sensitivity was reduced as evidenced by the marked reduction in insulin-induced phosphorylation of Akt in both cardiac and gastrocnemius muscle. Early diet-induced obesity did not lead to hypertension or cardiac hypertrophic remodeling. In chow-fed, control rats a robust increase in cardiac microvascular perfusion was observed upon adenosine infusion (+40 %; p < 0.05). In contrast, the adenosine response was abrogated in rats on a HFD (+8 %; N.S.). HFD neither resulted in rarefaction or loss of glycocalyx integrity in skeletal muscle, nor reduced staining intensity of the glycocalyx of cardiac capillaries.ConclusionsAlterations in coronary microcirculatory function as assessed by first-pass perfusion MRI represent one of the earliest obesity-related cardiac adaptations that can be assessed non-invasively. In this early stage of insulin resistance, disturbances in glycocalyx barrier properties appeared not to contribute to the observed changes in coronary microvascular function.

Optimization and validation of a chiral GC–MS method for the determination of free d-amino acids ratio in human urine: Application to a Gestational Diabetes Mellitus study

Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. It is affecting approximately up to 14% of all pregnancies with an increasing tendency. GDM has been related to relevant short-term and long-term health complications for both mother and offspring. Recent studies strongly emphasized the role of several essential amino acids in the pathogenesis of obesity and highlighted their strong correlation with insulin resistance, but there are no references related to modifications in d-AAs in biological fluids. As d-AA elimination proceeds mainly by renal excretion, urine was the selected sample to evaluate the alterations in free d-AAs ratio in a GDM study.Only 1mL of first void urine or standard solution was required for purification, by using a Discovery DSC-SCX SPE cartridge (500mg/3mL) and derivatization into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomeric separation was carried out by GC–MS on a Chirasil-l-Val N-propionyl-l-valine-tert-butylamide polysiloxane fused-silica capillary column (25m×0.25mm I.D., 0.12μm film thickness, Agilent Technologies, Waldbronn, Germany), under programmed temperature elution. Detection was performed with an ion trap mass analyzer, operating in the full scan mode in the m/z 50–350 range. 14 pairs of derivatives of d-and l-AAs were separated.The steps of sample preparation, derivatization and GC–MS conditions were optimized for both urine and standards. Several conditions affecting the SPE procedure, such as sorbent mass/volume ratio of the cartridge, sample dilution and pH, were optimized. Volume of reagents and solvents and reaction temperature and time were also tested for the derivatization. Regarding the GC–MS parameters, split ratio, temperature program and mass range were optimized.The final method was validated in terms of linearity, sensitivity, accuracy and precision for d-Ala, d-Pro, d-Ser, d-Met, d-Phe, d-Glu, d-Orn and d-Lys. Identification of AAs in urine samples was based on retention time and mass spectra.Urine from 20 women with GDM and 20 pregnant women with normal glucose tolerance (after 2-h 75-g oral glucose tolerance test), matched according to the week of gestation and age (22–28 week of gestation and age 24–37 years), were enrolled into the study. %d-Relative amounts were determined for Ala, Val, Thr, Ser, Leu, Asx (Asp+Asn), Glx (Glu+Gln), Met, Phe, Tyr, Orn and Lys. Statistically significant differences (p<0.05) were observed only for d-Phe and higher values were found in the GDM group. It is possible that d-Phe could be involved in metabolic/signaling pathways to compensate early stages of insulin resistance, although further work is necessary to confirm this hypothesis.

Plasma Lactate Levels Increase during Hyperinsulinemic Euglycemic Clamp and Oral Glucose Tolerance Test.

Insulin resistance, which plays a central role in the pathogenesis of type 2 diabetes (T2D), is an early indicator that heralds the occurrence of T2D. It is imperative to understand the metabolic changes that occur at the cellular level in the early stages of insulin resistance. The objective of this study was to determine the pattern of circulating lactate levels during oral glucose tolerance test (OGTT) and hyperinsulinemic euglycemic clamp (HIEC) study in normal nondiabetic subjects. Lactate and glycerol were determined every 30 minutes during OGTT and HIEC on 22 participants. Lactate progressively increased throughout the HIEC study period (P < 0.001). Participants with BMI < 30 had significantly higher mean M-values compared to those with BMI ≥ 30 at baseline (P < 0.05). This trend also continued throughout the OGTT. In addition, those with impaired glucose tolerance test (IGT) had significantly higher mean lactate levels compared to those with normal glucose tolerance (P < 0.001). In conclusion, we found that lactate increased during HIEC study, which is a state of hyperinsulinemia similar to the metabolic milieu seen during the early stages in the development of T2D.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

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Urinary Sodium Excretion Decreases In the Early Stages of Insulin Resistance In Model of Metabolic Syndrome Melissa E. Williams1, Jacqueline N. Minas1, Ruben Rodriguez1, Daisuke Nakano2, Akira Nishiyama2, and Rudy M. Ortiz11Department of Molecular &amp; Cellular Biology, School of Natural Science, University of California Merced 2Department of Pharmacology, Kagawa Medical University, Kagawa, Japan Type II diabetes is associated with abnormal activation of the renin‐angiotensin aldosterone system and impaired sodium (Na+) regulation contributing to volume dependent hypertension. However, the mechanisms contributing to elevated systolic blood pressure (SBP) during the development of insulin resistance (IR) are not well defined. To examine the effects of the onset of IR to impaired UNaV and SBP, three groups of rats were studied for 6 weeks (n=6/group); 1) non‐IR, Long Evans Tokushima Otsuka (LETO), 2) untreated, IR Otsuka Long Evans Tokushima Fatty (OLETF), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d for 3 weeks). Systolic blood pressure (SBP), urine Na+ excretion (UNaV), and insulin resistance index (IRI) were measured at 9 and 15 weeks of age. At 9 weeks of age, the OLETF group exhibited a 43% increase in IRI compared to the LETO group. However, no differences in SBP and UNaV between LETO and OLETF were observed. At 15 weeks of age, the OLETFs experienced a 54% increase in IRI, accompanied by a 15% (145 mmHg) increase in mean SBP at 15 weeks, which was attributed to the 46% decrease in mean UNaV. This suggests that IR causes impaired renal Na+ regulation, contributing to the onset of hypertension. Conversely, ARB treatment in OLETFs reduced SBP by 25% and increased UNaV by 50%, as well as a 24% decrease in IRI. This indicates that inappropriate activation of AT1 contributes to IR‐associated hypertension observed in OLETF.