Brynjulf Mortensen

Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans.

Obesity is a heritable disorder, with children of obese fathers at higher risk of developing obesity. Environmental factors epigenetically influence somatic tissues, but the contribution of these factors to the establishment of epigenetic patterns in human gametes is unknown. Here, we hypothesized that weight loss remodels the epigenetic signature of spermatozoa in human obesity. Comprehensive profiling of the epigenome of sperm from lean and obese men showed similar histone positioning, but small non-coding RNA expression and DNA methylation patterns were markedly different. In a separate cohort of morbidly obese men, surgery-induced weight loss was associated with a dramatic remodeling of sperm DNA methylation, notably at genetic locations implicated in the central control of appetite. Our data provide evidence that the epigenome of human spermatozoa dynamically changes under environmental pressure and offers insight into how obesity may propagate metabolic dysfunction to the next generation.

Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men

Physical inactivity is a risk factor for insulin resistance. We examined the effect of 9 days of bed rest on basal and insulin-stimulated expression of genes potentially involved in insulin action by applying hypothesis-generating microarray in parallel with candidate gene real-time PCR approaches in 20 healthy young men. Furthermore, we investigated whether bed rest affected DNA methylation in the promoter region of the peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) gene. Subjects were reexamined after 4 wk of retraining. We found that bed rest induced insulin resistance and altered the expression of more than 4,500 genes. These changes were only partly normalized after 4 wk of retraining. Pathway analyses revealed significant downregulation of 34 pathways, predominantly those of genes associated with mitochondrial function, including PPARGC1A. Despite induction of insulin resistance, bed rest resulted in a paradoxically increased response to acute insulin stimulation in the general expression of genes, particularly those involved in inflammation and endoplasmatic reticulum (ER) stress. Furthermore, bed rest changed gene expressions of several insulin resistance and diabetes candidate genes. We also observed a trend toward increased PPARGC1A DNA methylation after bed rest. We conclude that impaired expression of PPARGC1A and other genes involved in mitochondrial function as well as a paradoxically increased response to insulin of genes involved in inflammation and ER stress may contribute to the development of insulin resistance induced by bed rest. Lack of complete normalization of changes after 4 wk of retraining underscores the importance of maintaining a minimum of daily physical activity.

Impaired Leptin Gene Expression and Release in Cultured Preadipocytes Isolated From Individuals Born With Low Birth Weight

Low birth weight (LBW) is associated with increased risk of the development of type 2 diabetes (T2D). The appetite-regulating hormone leptin is released from mature adipocytes, and its production may be decreased in immature preadipocytes from LBW individuals. We recruited 14 men born with LBW and 13 controls born with normal birth weight (NBW). Biopsy samples were obtained from subcutaneous abdominal fat depots, and preadipocytes were isolated and cultured. Gene expression of leptin and selected differentiation markers were analyzed during preadipocyte differentiation, and cell culture media were collected to analyze leptin secretion. DNA methylation of CpG sites in the leptin promoter was measured using pyrosequencing. We found that differentiating preadipocytes from LBW individuals showed reduced leptin gene expression and a corresponding reduced leptin release compared with NBW individuals. Mean DNA methylation of the proximal LEP promoter was increased in LBW compared with NBW individuals. The notion of impaired adipocyte maturation in LBW individuals was supported by a lower mRNA expression of the differentiation markers; fatty acid binding protein 4, peroxisome proliferator–activated receptor γ, and GLUT4. Our findings are consistent with impaired preadipocyte maturation, contributing to an increased risk of the development of T2D in LBW subjects.

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.

Effect of birth weight and 12 weeks of exercise training on exercise-induced AMPK signaling in human skeletal muscle.

Subjects with a low birth weight (LBW) display increased risk of developing type 2 diabetes (T2D). We hypothesized that this is associated with defects in muscle adaptations following acute and regular physical activity, evident by impairments in the exercise-induced activation of AMPK signaling. We investigated 21 LBW and 21 normal birth weight (NBW) subjects during 1 h of acute exercise performed at the same relative workload before and after 12 wk of exercise training. Multiple skeletal muscle biopsies were obtained before and after exercise. Protein levels and phosphorylation status were determined by Western blotting. AMPK activities were measured using activity assays. Protein levels of AMPKα1 and -γ1 were significantly increased, whereas AMPKγ3 levels decreased with training independently of group. The LBW group had higher exercise-induced AMPK Thr(172) phosphorylation before training and higher exercise-induced ACC2 Ser(221) phosphorylation both before and after training compared with NBW. Despite exercise being performed at the same relative intensity (65% of Vo2peak), the acute exercise response on AMPK Thr(172), ACC2 Ser(221), AMPKα2β2γ1, and AMPKα2β2γ3 activities, GS activity, and adenine nucleotides as well as hexokinase II mRNA levels were all reduced after exercise training. Increased exercise-induced muscle AMPK activation and ACC2 Ser(221) phosphorylation in LBW subjects may indicate a more sensitive AMPK system in this population. Long-term exercise training may reduce the need for AMPK to control energy turnover during exercise. Thus, the remaining γ3-associated AMPK activation by acute exercise after exercise training might be sufficient to maintain cellular energy balance.

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

Aims/hypothesisLow 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.MethodsADSCs 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.ResultsWe 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/interpretationEpigenetic 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.

Physical inactivity affects skeletal muscle insulin signaling in a birth weight-dependent manner☆ , ☆☆ ,★

AIMS We investigated whether physical inactivity could unmask defects in insulin and AMPK signaling in low birth weight (LBW) subjects. METHODS Twenty LBW and 20 normal birth weight (NBW) subjects were investigated using the euglycemic-hyperinsulinemic clamp with excision of skeletal muscle biopsies pre and post 9days of bed rest. Employing Western blotting, we investigated skeletal muscle Akt, AS160, GLUT4, and AMPK signaling. RESULTS Peripheral insulin action was similar in the two groups and was decreased to the same extent post bed rest. Insulin and AMPK signaling was unaffected by bed rest in NBW individuals. LBW subjects showed decreased insulin-stimulated Akt phosphorylation and increased AMPK α1 and γ3 protein expression post bed rest. Insulin response of AS160 phosphorylation was lower in LBW subjects both pre and post bed rest. CONCLUSIONS Bed rest-induced insulin resistance is not explained by impaired muscle insulin or AMPK signaling in subjects with or without LBW. Lower muscle insulin signaling in LBW subjects post bed rest despite similar degree of insulin resistance as seen in controls may to some extent support the idea that LBW subjects are at higher risk of developing type 2 diabetes when being physically inactive.

Muscle inflammatory signaling in response to 9 days of physical inactivity in young men with low compared with normal birth weight

OBJECTIVE The molecular mechanisms linking physical inactivity and muscle insulin resistance in humans have been suggested to include increased muscle inflammation, possibly associated with impaired oxidative metabolism. We employed a human bed rest study including 20 young males with normal birth weight (NBW) and 20 with low birth weight (LBW) and increased risk of diabetes. METHODOLOGY The subjects were studied before and after 9 days of bed rest using the euglycemic-hyperinsulinemic clamp and muscle biopsy excision. Muscle inflammatory status was assessed as nuclear factor-κB (NF-κB) activity and mRNA expression of the pro-inflammatory MCP1 (CCL2) and IL6 and the macrophage marker CD68. Furthermore, mRNA expression of genes central to oxidative phosphorylation (OXPHOS) was measured including ATP5O, COX7A1, NDUFB6, and UQCRB. RESULTS At baseline, muscle inflammatory status was similar in NBW and LBW individuals. After bed rest, CD68 expression was increased in LBW (P=0.03) but not in NBW individuals. Furthermore, expression levels of all OXPHOS genes were reduced after bed rest in LBW (P ≤ 0.05) but not in NBW subjects and were negatively correlated with CD68 expression in LBW subjects (P ≤ 0.03 for all correlations). MCP1 expression and NF-κB activity were unaffected by bed rest, and IL6 expression was too low for accurate measurements. None of the inflammatory markers correlated with insulin sensitivity. CONCLUSIONS Although LBW subjects exhibit disproportionately elevated CD68 mRNA expression suggesting macrophage infiltration and reduced OXPHOS gene expression when exposed to bed rest, our data altogether do not support the notion that bed rest-induced (9 days) insulin resistance is caused by increased muscle inflammation.

Impact of rs361072 in the Phosphoinositide 3-Kinase p110β Gene on Whole-Body Glucose Metabolism and Subunit Protein Expression in Skeletal Muscle

OBJECTIVE Phosphoinositide 3-kinase (PI3K) is a major effector in insulin signaling. rs361072, located in the promoter of the gene (PIK3CB) for the p110β subunit, has previously been found to be associated with homeostasis model assessment for insulin resistance (HOMA-IR) in obese subjects. The aim was to investigate the influence of rs361072 on in vivo glucose metabolism, skeletal muscle PI3K subunit protein levels, and type 2 diabetes. RESEARCH DESIGN AND METHODS The functional role of rs361072 was studied in 196 Danish healthy adult twins. Peripheral and hepatic insulin sensitivity was assessed by a euglycemic-hyperinsulinemic clamp. Basal and insulin-stimulated biopsies were taken from the vastus lateralis muscle, and tissue p110β and p85α proteins were measured by Western blotting. The genetic association with type 2 diabetes and quantitative metabolic traits was investigated in 9,316 Danes with glucose tolerance ranging from normal to overt type 2 diabetes. RESULTS While hepatic insulin resistance was similar in the fasting state, carriers of the minor G allele had lower hepatic glucose output (per-allele effect: −16%, Padd = 0.004) during high physiological insulin infusion. rs361072 did not associate with insulin-stimulated peripheral glucose disposal despite a decreased muscle p85α:p110β protein ratio (Padd = 0.03) in G allele carriers. No association with HOMA-IR or type 2 diabetes (odds ratio 1.07, P = 0.5) was identified, and obesity did not interact with rs361072 on these traits. CONCLUSIONS Our study suggests that the minor G allele of PIK3CB rs361072 associates with decreased muscle p85α:p110β ratio and lower hepatic glucose production at high plasma insulin levels. However, no impact on type 2 diabetes prevalence was found.

Metabolic and Transcriptional Changes in Cultured Muscle Stem Cells from Low Birth Weight Subjects

CONTEXT/OBJECTIVE Developmental programming of human muscle stem cells could in part explain why individuals born with low birth weight (LBW) have an increased risk of developing type 2 diabetes (T2D) later in life. We hypothesized that immature muscle stem cell functions including abnormal differentiation potential and metabolic function could link LBW with the risk of developing T2D. Design/Settings/Participants: We recruited 23 young men with LBW and 16 age-matched control subjects with normal birth weight. Biopsies were obtained from vastus lateralis, and muscle stem cells were isolated and cultured into fully differentiated myotubes. MAIN OUTCOME MEASURES We studied glucose uptake, glucose transporters, insulin signaling, key transcriptional markers of myotube maturity, selected site-specific DNA methylation, and mitochondrial gene expression. RESULTS We found reduced glucose uptake as well as decreased levels of glucose transporter-1 and -4 mRNA and of the Akt substrate of 160-kDa mRNA and protein in myotubes from LBW individuals compared with normal birth weight individuals. The myogenic differentiation markers, myogenin and myosin heavy chain 1 and 2, were decreased during late differentiation in LBW myotubes. Additionally, mRNA levels of the peroxisome proliferator-activated receptor-γ coactivator-1α and cytochrome c oxidase polypeptide 7A were reduced in LBW myotubes. Decreased gene expression was not explained by changes in DNA methylation levels. CONCLUSION We demonstrate transcriptional and metabolic alterations in cultured primary satellite cells isolated from LBW individuals after several cell divisions, pointing toward a retained intrinsic defect conserved in these myotubes.