Pharmacokinetic and pharmacodynamic properties of once-weekly insulin icodec in individuals with type 1 diabetes.

AIMS: To investigate the pharmacokinetic/pharmacodynamic properties of once-weekly insulin icodec in individuals with type 1 diabetes (T1D). MATERIALS AND METHODS: In this randomized, open-label, two-period crossover trial, 66 individuals with T1D (age 18-64 years; glycated haemoglobin ≤75 mmol/mol [≤ 9%]) were to receive once-weekly icodec (8 weeks) and once-daily insulin glargine U100 (2 weeks) at individualized fixed equimolar total weekly doses established during up to 10 weeks' run-in with glargine U100 titrated to pre-breakfast plasma glucose (PG) of 4.4-7.2 mmol/L (80-130 mg/dL). Insulin aspart was used as bolus insulin. Blood sampling for icodec pharmacokinetics was performed from the first icodec dose until 35 days after the last dose. The glucose infusion rate at steady state was assessed in glucose clamps (target 6.7 mmol/L [120 mg/dL]) at 16-52 h and 138-168 h after the last icodec dose and 0-24 h after the last glargine U100 dose. Icodec pharmacodynamics during 1 week were predicted by pharmacokinetic-pharmacodynamic modelling. Hypoglycaemia was recorded during the treatment periods based on self-measured PG. RESULTS: Icodec reached pharmacokinetic steady state on average within 2-3 weeks. At steady state, model-predicted daily proportions of glucose infusion rate during the 1-week dosing interval were 14.3%, 19.6%, 18.3%, 15.7%, 13.1%, 10.6% and 8.4%, respectively. Rates and duration of Level 2 hypoglycaemic episodes (PG <3.0 mmol/L [54 mg/dL]) were 32.8 versus 23.9 episodes per participant-year of exposure and 33 ± 25 versus 30 ± 18 min (mean ± SD) for icodec versus glargine U100. CONCLUSIONS: The pharmacokinetic/pharmacodynamic properties of icodec suggest its potential to provide basal coverage in a basal-bolus insulin regimen in people with T1D.

Pharmacokinetic and pharmacodynamic properties of once-weekly insulin icodec in individuals with type 2 diabetes.

AIMS: To characterize the pharmacokinetic and pharmacodynamic properties of once-weekly insulin icodec in type 2 diabetes (T2D). MATERIALS AND METHODS: In an open-label trial, 46 individuals with T2D (18-75 years; body mass index 18.0-38.0 kg/m2 ; glycated haemoglobin ≤75 mmol/mol [≤9%]; basal insulin-treated) received subcutaneous once-weekly icodec for ≥8 weeks at individualized doses, aiming at a pre-breakfast plasma glucose concentration of 4.4 to 7.0 mmol/L (80-126 mg/dL) on the last three mornings of each weekly dosing interval. Frequent blood sampling to assess total serum icodec concentration (ie, albumin-bound and unbound) occurred from first icodec dose until 35 days after last dose. Icodec trough concentrations following initiation of once-weekly dosing were predicted by pharmacokinetic modelling. During the final 3 weeks of icodec treatment, while at steady state, the icodec glucose-lowering effect was assessed in three glucose clamps (target 7.5 mmol/L [135 mg/dL]): 0 to 36, 40 to 64 and 144 to 168 h post-dose, thus covering the initial, middle and last part of the 1-week dosing interval. Glucose-lowering effect during a complete dosing interval was predicted by pharmacokinetic-pharmacodynamic modelling. RESULTS: Model-predicted icodec steady state was attained after 3 to 4 weeks. At steady state, model-predicted daily proportions of glucose-lowering effect on days 1 to 7 of the 1-week dosing interval were 14.1%, 16.1%, 15.8%, 15.0%, 14.0%, 13.0% and 12.0%, respectively. Icodec duration of action was at least 1 week in all participants. Once-weekly icodec was overall safe and well tolerated in the current trial. CONCLUSIONS: The pharmacokinetic and pharmacodynamic characteristics of icodec in individuals with T2D support its potential as a once-weekly basal insulin.

Epigenome- and Transcriptome-wide Changes in Muscle Stem Cells from Low Birth Weight Men.

Background: Being born with low birth weight (LBW) is a risk factor for muscle insulin resistance and type 2 diabetes (T2D), which may be mediated by epigenetic mechanisms programmed by the intrauterine environment. Epigenetic mechanisms exert their prime effects in developing cells. We hypothesized that muscle insulin resistance in LBW subjects may be due to early differential epigenomic and transcriptomic alterations in their immature muscle progenitor cells.Results: Muscle progenitor cells were obtained from 23 healthy young adult men born at term with LBW, and 15 BMI-matched normal birth weight (NBW) controls. The cells were subsequently cultured and differentiated into myotubes. DNA and RNA were harvested before and after differentiation for genome-wide DNA methylation and RNA expression measurements.After correcting for multiple comparisons (q ≤ 0.05), 56 CpG sites were found to be significantly, differentially methylated in myoblasts from LBW compared with NBW men, of which the top five gene-annotated CpG sites (SKI, ARMCX3, NR5A2, NEUROG, ESRRG) previously have been associated to regulation of cholesterol, fatty acid and glucose metabolism and muscle development or hypertrophy. LBW men displayed markedly decreased myotube gene expression levels of the AMPK-repressing tyrosine kinase gene FYN and the histone deacetylase gene HDAC7. Silencing of FYN and HDAC7 was associated with impaired myotube formation, which for HDAC7 reduced muscle glucose uptake.Conclusions: The data provides evidence of impaired muscle development predisposing LBW individuals to T2D is linked to and potentially caused by distinct DNA methylation and transcriptional changes including down regulation of HDAC7 and FYN in their immature myoblast stem cells.

PPARG Pro12Ala Ala carriers exhibit greater improvements in peripheral insulin sensitivity in response to 12 weeks of aerobic exercise training.

The Ala allele of PPARG Pro12Ala ( rs1801282 ) is associated with greater improvements to the glucose metabolism in exercise studies, but whether this extends to peripheral insulin sensitivity is unknown. Our objective was to investigate the effect of PPARG Pro12Ala on exercise-induced changes in peripheral insulin sensitivity. A total of 124 (91 Pro homozygotes and 33 Ala carriers) previously physically inactive healthy young men and women with overweight or class 1 obesity who completed a 12 wk aerobic exercise intervention were included in the analysis. All participants underwent a hyperinsulinemic euglycemic clamp before and after the 12 wk intervention. The prescribed exercise frequency was 5-7 days/wk, and the exercise energy expenditure was 2,100 4,200 kcal/wk for men and 1,600 kcal/wk for women. Insulin sensitivity improved significantly in both genotype groups. However, Ala carriers had a 1.13-fold (95% confidence interval 1.01; 1.26, P = 0.032) greater improvement in insulin sensitivity from baseline compared with Pro homozygotes. Our data support that PPARG Pro12Ala modifies the effect of aerobic exercise on peripheral insulin sensitivity.

TCF7L2 Expression Is Regulated by Cell Differentiation and Overfeeding in Human Adipose Tissue.

Aim: The TCF7L2 gene variant rs7903146 has the largest effect on type 2 diabetes risk reported in genome-wide association studies, however its role in adipose tissue development and function is unknown. We investigate the association between gene variant rs7903146 and metabolic parameters and examine in vitro and ex vivo gene expression of TCF7L2 in human adipose tissue and progenitor cells from two independent populations of young healthy men with increased risk of type 2 diabetes due to low birth weight (LBW). Design: Adipose tissue biopsies were excised from 40 healthy young men with low and normal birth weights (NBW) after a control and 5-day high-fat overfeeding diet. In another cohort including 13 LBW and 13 NBW men, adipocyte progenitor cells were isolated and cultivated. Transcriptome-wide expression was performed on RNA extracted from biopsies or cell cultures. Results: Diet-induced peripheral insulin resistance is more pronounced in carriers of the T-risk allele rs7903146, whereas no association with hepatic insulin resistance was shown. TCF7L2 expression increased during adipogenesis in isolated preadipocytes from both LBW and NBW men (p < 0.001) and correlated positively with markers of progenitor cell proliferation and maturation capacity. In the mature adipose tissue, LBW men had lower expression of TCF7L2 compared to NBW men at baseline (p = 0.03) and TCF7L2 expression was suppressed by short-term overfeeding in NBW men (p = 0.005). Conclusions: The results suggest a regulation of TCF7L2 expression during adipogenesis and in mature adipose tissue upon overfeeding, and further that young men exposed to an adverse intrauterine environment have reduced mature adipose tissue TCF7L2 expression.

Epigenetic programming of adipose-derived stem cells in low birthweight individuals.

AIMS/HYPOTHESIS: Low birthweight (LBW) is associated with dysfunctions of adipose tissue and metabolic disease in adult life. We hypothesised that altered epigenetic and transcriptional regulation of adipose-derived stem cells (ADSCs) could play a role in programming adipose tissue dysfunction in LBW individuals. METHODS: ADSCs were isolated from the subcutaneous adipose tissue of 13 normal birthweight (NBW) and 13 LBW adult men. The adipocytes were cultured in vitro, and genome-wide differences in RNA expression and DNA methylation profiles were analysed in ADSCs and differentiated adipocytes. RESULTS: We demonstrated that ADSCs from LBW individuals exhibit multiple expression changes as well as genome-wide alterations in methylation pattern. Reduced expression of the transcription factor cyclin T2 encoded by CCNT2 may play a key role in orchestrating several of the gene expression changes in ADSCs from LBW individuals. Indeed, silencing of CCNT2 in human adipocytes decreased leptin secretion as well as the mRNA expression of several genes involved in adipogenesis, including MGLL, LIPE, PPARG, LEP and ADIPOQ. Only subtle genome-wide mRNA expression and DNA methylation changes were seen in mature cultured adipocytes from LBW individuals. CONCLUSIONS/INTERPRETATION: Epigenetic and transcriptional changes in LBW individuals are most pronounced in immature ADSCs that in turn may programme physiological characteristics of the mature adipocytes that influence the risk of metabolic diseases. Reduced expression of CCNT2 may play a key role in the developmental programming of adipose tissue.

Genetic and phenotypic correlations between surrogate measures of insulin release obtained from OGTT data.

AIMS/HYPOTHESIS: We examined the extent to which surrogate measures of insulin release have shared genetic causes. METHODS: Genetic and phenotypic correlations were calculated in a family cohort (n = 315) in which beta cell indices were estimated based on fasting and oral glucose-stimulated plasma glucose, serum C-peptide and serum insulin levels. Furthermore, we genotyped a large population-based cohort (n = 6,269) for common genetic variants known to associate with type 2 diabetes, fasting plasma glucose levels or fasting serum insulin levels to examine their association with various indices. RESULTS: We found a notable difference between the phenotypic and genetic correlations for the traits, emphasising that the phenotypic correlation is an insufficient measure of the magnitude of shared genetic impact. In addition, we found that corrected insulin response, insulinogenic index and incAUC for insulin after an oral glucose challenge shared the majority of their genetic backgrounds, with genetic correlations of 0.80-0.99. The BIGTT index for acute insulin response differed slightly more from the latter with genetic correlations of 0.78-0.87. The HOMA for beta cell function was genetically closely related to fasting insulin with a genetic correlation of 0.85. The effects of 82 selected susceptibility single nucleotide polymorphisms on these insulin secretion indices supported our interpretation of the data and added insight into the biological differences between the examined traits. CONCLUSIONS/INTERPRETATION: The level of shared genetic background varies between surrogate measures of insulin release, and this should be considered when designing a genetic association study to best obtain information on various mechanisms of insulin release.

Young men with low birthweight exhibit decreased plasticity of genome-wide muscle DNA methylation by high-fat overfeeding.

AIMS/HYPOTHESIS: The association between low birthweight (LBW) and risk of developing type 2 diabetes may involve epigenetic mechanisms, with skeletal muscle being a prime target tissue. Differential DNA methylation patterns have been observed in single genes in muscle tissue from type 2 diabetic and LBW individuals, and we recently showed multiple DNA methylation changes during short-term high-fat overfeeding in muscle of healthy people. In a randomised crossover study, we analysed genome-wide DNA promoter methylation in skeletal muscle of 17 young LBW men and 23 matched normal birthweight (NBW) men after a control and a 5 day high-fat overfeeding diet. METHODS: DNA methylation was measured using Illumina's Infinium BeadArray covering 27,578 CpG sites representing 14,475 different genes. RESULTS: After correction for multiple comparisons, DNA methylation levels were found to be similar in the LBW and NBW groups during the control diet. Whereas widespread DNA methylation changes were observed in the NBW group in response to high-fat overfeeding, only a few methylation changes were seen in the LBW group (χ(2), p < 0.001). CONCLUSIONS/INTERPRETATION: Our results indicate lower DNA methylation plasticity in skeletal muscle from LBW vs NBW men, potentially contributing to understanding the link between LBW and increased risk of type 2 diabetes.

Genetic, nongenetic and epigenetic risk determinants in developmental programming of type 2 diabetes.

Low birthweight (LBW) individuals and offspring of women with gestational diabetes mellitus (GDM) exhibit increased risk of developing type 2 diabetes (T2D) and associated cardiometabolic traits in adulthood, which for both groups may be mediated by adverse events and developmental changes in fetal life. T2D is a multifactorial disease occurring as a result of complicated interplay between genetic and both prenatal and postnatal nongenetic factors, and it remains unknown to what extent the increased risk of T2D associated with LBW or GDM in the mother may be due to, or confounded by, genetic factors. Indeed, it has been shown that genetic changes influencing risk of diabetes may also be associated with reduced fetal growth as a result of reduced insulin secretion and/or action. Similarly, increased risk of T2D among offspring could be explained by T2D susceptibility genes shared between the mother and her offspring. Epigenetic mechanisms may explain the link between factors operating in fetal life and later risk of developing T2D, but so far convincing evidence is lacking for epigenetic changes as a prime and direct cause of T2D. This review addresses recent literature on the early origins of adult disease hypothesis, with a special emphasis on the role of genetic compared with nongenetic and epigenetic risk determinants and disease mechanisms.

Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 + 2a) phosphorylation.

Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr³°8 on Akt (p-Akt-Thr³°8), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr³°8 (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser47³ or IRS-1-PI3K activity. Furthermore, p-Akt-Thr³°8 and Akt2 activity were negatively associated with NH2-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P < 0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH2-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH2-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH2-terminal dephosphorylation may be the site for "fine-tuning" insulin-mediated GS activation in humans.

FOXO3A-short is a novel regulator of non-oxidative glucose metabolism associated with human longevity.

Intronic single-nucleotide polymorphisms (SNPs) in FOXO3A are associated with human longevity. Currently, it is unclear how these SNPs alter FOXO3A functionality and human physiology, thereby influencing lifespan. Here, we identify a primate-specific FOXO3A transcriptional isoform, FOXO3A-Short (FOXO3A-S), encoding a major longevity-associated SNP, rs9400239 (C or T), within its 5' untranslated region. The FOXO3A-S mRNA is highly expressed in the skeletal muscle and has very limited expression in other tissues. We find that the rs9400239 variant influences the stability and functionality of the primarily nuclear protein(s) encoded by the FOXO3A-S mRNA. Assessment of the relationship between the FOXO3A-S polymorphism and peripheral glucose clearance during insulin infusion (Rd clamp) in a cohort of Danish twins revealed that longevity T-allele carriers have markedly faster peripheral glucose clearance rates than normal lifespan C-allele carriers. In vitro experiments in human myotube cultures utilizing overexpression of each allele showed that the C-allele represses glycolysis independently of PI3K signaling, while overexpression of the T-allele represses glycolysis only in a PI3K-inactive background. Supporting this finding inducible knockdown of the FOXO3A-S C-allele in cultured myotubes increases the glycolytic rate. We conclude that the rs9400239 polymorphism acts as a molecular switch which changes the identity of the FOXO3A-S-derived protein(s), which in turn alters the relationship between FOXO3A-S and insulin/PI3K signaling and glycolytic flux in the skeletal muscle. This critical difference endows carriers of the FOXO3A-S T-allele with consistently higher insulin-stimulated peripheral glucose clearance rates, which may contribute to their longer and healthier lifespans.

Molecular and pharmacological characterization of insulin icodec: a new basal insulin analog designed for once-weekly dosing.

INTRODUCTION: Insulin icodec is a novel, long-acting insulin analog designed to cover basal insulin requirements with once-weekly subcutaneous administration. Here we describe the molecular engineering and the biological and pharmacological properties of insulin icodec. RESEARCH DESIGN AND METHODS: A number of in vitro assays measuring receptor binding, intracellular signaling as well as cellular metabolic and mitogenic responses were used to characterize the biological properties of insulin icodec. To evaluate the pharmacological properties of insulin icodec in individuals with type 2 diabetes, a randomized, double-blind, double-dummy, active-controlled, multiple-dose, dose escalation trial was conducted. RESULTS: The long half-life of insulin icodec was achieved by introducing modifications to the insulin molecule aiming to obtain a safe, albumin-bound circulating depot of insulin icodec, providing protracted insulin action and clearance. Addition of a C20 fatty diacid-containing side chain imparts strong, reversible albumin binding, while three amino acid substitutions (A14E, B16H and B25H) provide molecular stability and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro cell-based studies showed that insulin icodec activates the same dose-dependent IR-mediated signaling and metabolic responses as native human insulin (HI). The affinity of insulin icodec for the insulin-like growth factor-1 receptor was proportionately lower than its binding to the IR, and the in vitro mitogenic effect of insulin icodec in various human cells was low relative to HI. The clinical pharmacology trial in people with type 2 diabetes showed that insulin icodec was well tolerated and has pharmacokinetic/pharmacodynamic properties that are suited for once-weekly dosing, with a mean half-life of 196 hours and close to even distribution of glucose-lowering effect over the entire dosing interval of 1 week. CONCLUSIONS: The molecular modifications introduced into insulin icodec provide a novel basal insulin with biological and pharmacokinetic/pharmacodynamic properties suitable for once-weekly dosing. TRIAL REGISTRATION NUMBER: NCT02964104.

Skeletal muscle enhancer interactions identify genes controlling whole-body metabolism.

Obesity and type 2 diabetes (T2D) are metabolic disorders influenced by lifestyle and genetic factors that are characterized by insulin resistance in skeletal muscle, a prominent site of glucose disposal. Numerous genetic variants have been associated with obesity and T2D, of which the majority are located in non-coding DNA regions. This suggests that most variants mediate their effect by altering the activity of gene-regulatory elements, including enhancers. Here, we map skeletal muscle genomic enhancer elements that are dynamically regulated after exposure to the free fatty acid palmitate or the inflammatory cytokine TNFα. By overlapping enhancer positions with the location of disease-associated genetic variants, and resolving long-range chromatin interactions between enhancers and gene promoters, we identify target genes involved in metabolic dysfunction in skeletal muscle. The majority of these genes also associate with altered whole-body metabolic phenotypes in the murine BXD genetic reference population. Thus, our combined genomic investigations identified genes that are involved in skeletal muscle metabolism.

Genetic and metabolic effects on skeletal muscle AMPK in young and older twins.

The protein complex AMP-activated protein kinase (AMPK) is believed to play an important role in the regulation of skeletal muscle glucose and lipid metabolism. Defects in the AMPK system might therefore be an important factor in the pathogenesis of type 2 diabetes. We aimed to identify genetic and environmental mechanisms involved in the regulation of AMPK expression and activity and to examine the association between AMPK protein levels and activity on the one hand, and glucose and fat metabolism on the other. We investigated skeletal muscle biopsies from 100 young and 82 older mono- and dizygotic nondiabetic twins excised during the basal and insulin-stimulated states of a physiological hyperinsulinemic-euglycemic clamp. AMPKalpha1, -alpha2, and -gamma3 mRNA expression was investigated using real-time PCR, and Western blotting was employed to measure protein levels. Multiple regression analyses indicated that skeletal muscle AMPK mRNA and protein expression as well as activity were regulated by sex, age, obesity, and aerobic capacity. Comparison of intraclass correlations on AMPK measurements from mono- and dizygotic twins suggested that skeletal muscle AMPK expression was under minor genetic influence. AMPKgamma3 protein expression and activity were negatively related to whole body glucose uptake through the nonoxidative metabolic pathway and positively related to phosphorylation of glycogen synthase. Our results suggest that skeletal muscle AMPK expression is under minor genetic control but regulated by age and sex and associated with obesity and aerobic capacity. Furthermore, our results indicate a role for gamma3-containing AMPK complexes in downregulation of insulin-stimulated nonoxidative glucose metabolism possibly through inhibition of glycogen synthase activity.

ADAMTS9 Regulates Skeletal Muscle Insulin Sensitivity Through Extracellular Matrix Alterations.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin ß1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

Plasma ceramide levels are altered in low and normal birth weight men in response to short-term high-fat overfeeding.

Low birth weight (LBW) individuals have an increased risk of developing insulin resistance and type 2 diabetes compared with normal birth weight (NBW) individuals. We hypothesised that LBW individuals exhibit an increased fatty acid flux into lipogenesis in non-adipose tissue with a resulting accumulation of lipotoxic lipids, including ceramides, in the blood. Therefore, we measured fasting plasma levels of 27 ceramides in 18 young, healthy, LBW men and 25 NBW controls after an isocaloric control diet and a 5-day high-fat, high-calorie diet by HPLC-HRMS. LBW men did not show elevated plasma ceramide levels after the control or high-fat, high-calorie diet. An increased fatty acid oxidation rate in these individuals during both diets may limit ceramide synthesis and thereby compensate for a likely increased fatty acid load to non-adipose tissue. Interestingly, LBW and NBW men decreased d18:0-18:1/d18:1-18:0 and d18:1-24:2/d18:2-24:1 levels and increased the d18:0-24:1a level in response to overfeeding. Plasma d18:0-24:1a and total ceramide levels were positively associated with the fasting blood glucose level and endogenous glucose production after the control diet, and the total ceramide level was in addition positively associated with hepatic insulin resistance. Further studies are needed to determine if lipotoxicity contributes to insulin resistance in LBW individuals.

Plasma amino acid levels are elevated in young, healthy low birth weight men exposed to short-term high-fat overfeeding.

Low birth weight (LBW) individuals exhibit a disproportionately increased, incomplete fatty acid oxidation and a decreased glucose oxidation, compared with normal birth weight (NBW) individuals, and furthermore have an increased risk of developing insulin resistance and type 2 diabetes. We hypothesized that changes in amino acid metabolism may occur parallel to alterations in fatty acid and glucose oxidation, and could contribute to insulin resistance. Therefore, we measured fasting plasma levels of 15 individual or pools of amino acids in 18 LBW and 25 NBW men after an isocaloric control diet and after a 5-day high-fat, high-calorie diet. We demonstrated that LBW and NBW men increased plasma alanine levels and decreased valine and leucine/isoleucine levels in response to overfeeding. Also, LBW men had higher alanine, proline, methionine, citrulline, and total amino acid levels after overfeeding compared with NBW men. Alanine and total amino acid levels tended to be negatively associated with the insulin-stimulated glucose uptake after overfeeding. Therefore, the higher amino acid levels in LBW men could be a consequence of their reduction in skeletal muscle insulin sensitivity due to overfeeding with a possible increased skeletal muscle proteolysis and/or could potentially contribute to an impaired insulin sensitivity. Furthermore, the alanine level was negatively associated with the plasma acetylcarnitine level and positively associated with the hepatic glucose production after overfeeding. Thus, the higher alanine level in LBW men could be accompanied by an increased anaplerotic formation of oxaloacetate and thereby an enhanced tricarboxylic acid cycle activity and as well an increased gluconeogenesis.

Plasma acylcarnitine profiling indicates increased fatty acid oxidation relative to tricarboxylic acid cycle capacity in young, healthy low birth weight men.

We hypothesized that an increased, incomplete fatty acid beta-oxidation in mitochondria could be part of the metabolic events leading to insulin resistance and thereby an increased type 2 diabetes risk in low birth weight (LBW) compared with normal birth weight (NBW) individuals. Therefore, we measured fasting plasma levels of 45 acylcarnitine species in 18 LBW and 25 NBW men after an isocaloric control diet and a 5-day high-fat, high-calorie diet. We demonstrated that LBW men had higher C2 and C4-OH levels after the control diet compared with NBW men, indicating an increased fatty acid beta-oxidation relative to the tricarboxylic acid cycle flux. Also, they had higher C6-DC, C10-OH/C8-DC, and total hydroxyl-/dicarboxyl-acylcarnitine levels, which may suggest an increased fatty acid omega-oxidation in the liver. Furthermore, LBW and NBW men decreased several acylcarnitine levels in response to overfeeding, which is likely a result of an upregulation of fatty acid oxidation due to the dietary challenge. Moreover, C10-OH/C8-DC and total hydroxyl-/dicarboxyl-acylcarnitine levels tended to be negatively associated with the serum insulin level, and the total hydroxyl-/dicarboxyl-acylcarnitine level additionally tended to be negatively associated with the hepatic insulin resistance index. This indicates that an increased fatty acid omega-oxidation could be a compensatory mechanism to prevent an accumulation of lipid species that impair insulin signaling.

DNA methylation and gene expression of HIF3A: cross-tissue validation and associations with BMI and insulin resistance.

BACKGROUND: Associations between BMI and DNA methylation of hypoxia-inducible factor 3-alpha (HIF3A) in both blood cells and subcutaneous adipose tissue (SAT) have been reported. In this study, we investigated associations between BMI and HIF3A DNA methylation in the blood and SAT from the same individuals, and whether HIF3A gene expression in SAT and skeletal muscle biopsies showed associations with BMI and insulin resistance. Furthermore, we aimed to investigate gender specificity and heritability of these traits. METHODS: We studied 137 first-degree relatives of type 2 diabetes (T2D) patients from 48 families, from whom we had SAT and muscle biopsies. DNA methylation of four CpG sites in the HIF3A promoter was analyzed in the blood and SAT by pyrosequencing, and HIF3A gene expression was analyzed in SAT and muscle by qPCR. An index of whole-body insulin sensitivity was estimated from oral glucose tolerance tests. RESULTS: BMI was associated with HIF3A methylation at one CpG site in the blood, and there was a positive association between the blood and SAT methylation levels at a different CpG site within the individuals. The SAT methylation level did not correlate with HIF3A gene expression. Interestingly, HIF3A expression in SAT, but not in muscle, associated negatively with BMI and whole-body insulin resistance. We found a significant effect of familiality on HIF3A methylation levels in the blood and HIF3A expression levels in skeletal muscle. CONCLUSIONS: Our findings are in line with the previously reported link between BMI and DNA methylation of HIF3A in the blood. The tissue-specific results of HIF3A gene expression indicate that SAT is the more functional tissue in which a low expression may adversely affect whole-body insulin sensitivity.

Altered DNA methylation and differential expression of genes influencing metabolism and inflammation in adipose tissue from subjects with type 2 diabetes.

Genetics, epigenetics, and environment may together affect the susceptibility for type 2 diabetes (T2D). Our aim was to dissect molecular mechanisms underlying T2D using genome-wide expression and DNA methylation data in adipose tissue from monozygotic twin pairs discordant for T2D and independent case-control cohorts. In adipose tissue from diabetic twins, we found decreased expression of genes involved in oxidative phosphorylation; carbohydrate, amino acid, and lipid metabolism; and increased expression of genes involved in inflammation and glycan degradation. The most differentially expressed genes included ELOVL6, GYS2, FADS1, SPP1 (OPN), CCL18, and IL1RN. We replicated these results in adipose tissue from an independent case-control cohort. Several candidate genes for obesity and T2D (e.g., IRS1 and VEGFA) were differentially expressed in discordant twins. We found a heritable contribution to the genome-wide DNA methylation variability in twins. Differences in methylation between monozygotic twin pairs discordant for T2D were subsequently modest. However, 15,627 sites, representing 7,046 genes including PPARG, KCNQ1, TCF7L2, and IRS1, showed differential DNA methylation in adipose tissue from unrelated subjects with T2D compared with control subjects. A total of 1,410 of these sites also showed differential DNA methylation in the twins discordant for T2D. For the differentially methylated sites, the heritability estimate was 0.28. We also identified copy number variants (CNVs) in monozygotic twin pairs discordant for T2D. Taken together, subjects with T2D exhibit multiple transcriptional and epigenetic changes in adipose tissue relevant to the development of the disease.