Emerging concepts on the role of epigenetics in the relationships between nutrition and health.

Understanding the physiological and metabolic underpinnings that confer individual differences in responses to diet and diet-related chronic disease is essential to advance the field of nutrition. This includes elucidating the differences in gene expression that are mediated through programming of the genome through epigenetic chromatin modifications. Epigenetic landscapes are influenced by age, genetics, toxins and other environmental factors, including dietary exposures and nutritional status. Epigenetic modifications influence transcription and genome stability are established during development with life-long consequences. They can be inherited from one generation to the next. The covalent modifications of chromatin, which include methylation and acetylation, on DNA nucleotide bases, histone proteins and RNA are derived from intermediates of one-carbon metabolism and central metabolism. They influence key physiological processes throughout life, and together with inherited DNA primary sequence, contribute to responsiveness to environmental stresses, diet and risk for age-related chronic disease. Revealing diet-epigenetic relationships has the potential to transform nutrition science by increasing our fundamental understanding of: (i) the role of nutrients in biological systems, (ii) the resilience of living organisms in responding to environmental perturbations, and (iii) the development of dietary patterns that programme physiology for life-long health. Epigenetics may also enable the classification of individuals with chronic disease for specific dietary management and/or for efficacious diet-pharmaceutical combination therapies. These new emerging concepts at the interface of nutrition and epigenetics were discussed, and future research needs identified by leading experts at the 26th Marabou Symposium entitled 'Nutrition, Epigenetics, Genetics: Impact on Health and Disease'. For a compilation of the general discussion at the marabou symposium, click here http://www.marabousymposium.org/.

Effects of Nordic walking on cardiovascular risk factors in overweight individuals with type 2 diabetes, impaired or normal glucose tolerance.

BACKGROUND: Physical activity remains a valuable prevention for metabolic disease. The effects of Nordic walking on cardiovascular risk factors were determined in overweight individuals with normal or disturbed glucose regulation. METHODS: We included 213 individuals, aged 60 ± 5.3 years and with body mass index (BMI) of 30.2 ± 3.8 kg/m(2); of these, 128 had normal glucose tolerance (NGT), 35 had impaired glucose tolerance (IGT) and 50 had type 2 diabetes mellitus (T2DM). Participants were randomized to unaltered physical activity or to 5 h per week of Nordic walking with poles, for a 4-month period. Dietary habits were unaltered. BMI, waist circumference, blood pressure, glucose tolerance, clinical chemistry, maximal oxygen uptake (peak VO(2)) and self-reported physical activity (questionnaire) were assessed at the time of inclusion and after 4 months. The participants in the exercise-intervention group kept a walking diary. RESULTS: In the NGT exercise group, self-reported physical activity increased markedly, and body weight (-2.0 ± 3.8 kg), BMI (-0.8 ± 1.4 kg/m(2)) and waist circumference (-4.9 ± 4.4 cm) (mean ± SD) decreased. Exercise power output (12.9 ± 9.9 W) and peak VO(2) (2.7 ± 2.8 mL/kg/min) increased in the IGT exercise group. More cardiovascular risk factors were improved after exercise intervention in people with NGT compared with those with IGT or T2DM. Exercise capacity improved significantly in all three groups of participants who reported at least 80% compliance with the scheduled exercise. CONCLUSIONS: Nordic walking improved anthropometric measurements and exercise capacity. However, unsupervised Nordic walking may not provide a sufficient increase in exercise intensity to achieve ultimate health-promoting benefits on the cardiovascular parameters assessed in this study, particularly for those with disturbed glucose regulation.

Specificity of insulin signalling in human skeletal muscle as revealed by small interfering RNA.

Insulin action on metabolically active tissues is a complex process involving positive and negative feedback regulation to control whole body glucose homeostasis. At the cellular level, glucose and lipid metabolism, as well as protein synthesis, are controlled through canonical insulin signalling cascades. The discovery of small interfering RNA (siRNA) allows for the molecular dissection of critical components of the regulation of metabolic and gene regulatory events in insulin-sensitive tissues. The application of siRNA to tissues of human origin allows for the molecular dissection of the mechanism(s) regulating glucose and lipid metabolism. Penetration of the pathways controlling insulin action in human tissue may aid in discovery efforts to develop diabetes prevention and treatment strategies. This review will focus on the use of siRNA to validate critical regulators controlling insulin action in human skeletal muscle, a key organ important for the control of whole body insulin-mediated glucose uptake and metabolism.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

AIM: Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. METHODS: Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. RESULTS: Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser473 and Akt Thr308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser2448 phosphorylation, as well as FOXO1 Ser256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser235/236 phosphorylation, as well as 4E-BP1 Thr37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. CONCLUSION: Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury.

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.

Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects.

The serine/threonine kinase Akt (PKB/Rac) has been implicated as playing a role in the insulin-signaling pathway to glucose transport. Little is known regarding the regulation of Akt kinase activity in insulin-sensitive tissues, such as skeletal muscle, or whether this regulation is altered in insulin-resistant states such as NIDDM. We examined the effect of insulin on Akt kinase activity in skeletal muscle from six NIDDM patients and six healthy subjects. Whole-body insulin sensitivity, assessed by the euglycemic-hyperinsulinemic clamp, was significantly lower in NIDDM subjects (P < 0.001), and this was accompanied by impaired in vitro insulin-stimulated glucose transport in skeletal muscle. In both groups, insulin induced a significant increase in Akt kinase activity, but the response to maximal insulin (60 nmol/l) was markedly reduced in skeletal muscle from NIDDM subjects (66% of control levels, P < 0.01). Impaired Akt kinase activity was not accompanied by decreased protein expression of Akt. Instead, a trend toward increased Akt expression was noted in skeletal muscle from NIDDM subjects (P < 0.1). These parallel defects in insulin-stimulated Akt kinase activity and glucose transport in diabetic skeletal muscle suggest that reduced Akt kinase activity may play a role in the development of insulin resistance in NIDDM.

Uncoupling protein 3 is reduced in skeletal muscle of NIDDM patients.

Two recently described proteins in the mitochondrial uncoupling protein (UCP) family, UCP-2 and UCP-3, have been linked to phenotypes of obesity and NIDDM. We determined the mRNA levels of UCP-2 and UCP-3 in skeletal muscle of NIDDM patients and of healthy control subjects. No difference in the mRNA levels or in the protein expression of UCP-2 was observed between the two groups. In contrast, mRNA levels of UCP-3 were significantly reduced in skeletal muscle of NIDDM patients compared with control subjects. In the NIDDM patients, a positive correlation between UCP-3 expression and whole-body insulin-mediated glucose utilization rate was also noted. These results suggest that UCP-3 regulation may be altered in states of insulin resistance.

Molecular scanning of the insulin receptor gene in syndromes of insulin resistance.

Using the molecular scanning technique of single-stranded conformational polymorphism (SSCP), we have examined the exons encoding the insulin receptor gene in 26 patients with syndromes of insulin resistance. We found 27 variant sequences, 4 of which were mutations that altered an amino acid. One patient with the Rabson-Mendenhall syndrome was homozygous for a mutation in the extracellular alpha-subunit (Ser to Leu323), one type A insulin-resistant patient was heterozygous for Pro to Leu1178, and another type A insulin-resistant patient was heterozygous for a mutation in the COOH-terminus of the receptor (Arg to Gln1351). The previously reported, and probably functionally insignificant, variant Val to Met985 was detected in one patient. No missense or nonsense insulin receptor mutations were found in any patients whose insulin resistance was associated with gross obesity, lipoatrophy, or acromegaloid features. No missense or nonsense mutations were found in subjects with polycystic ovary syndrome or Syndrome X. Putting these findings in the context of other work in this field, we conclude that subjects with leprechaunism or Rabson-Mendenhall syndrome have a high probability of having a missense or nonsense insulin receptor mutation. Nonobese, nondysmorphic, severely insulin-resistant females with hirsutism, acanthosis nigricans, and menstrual disturbance (type A phenotype) have an intermediate probability of having this type of insulin receptor mutation. Although insulin receptor mutations have been occasionally described in other phenotypes of insulin resistance, the frequency of point mutations in the exons of the insulin receptor gene in patients with those phenotypes appears to be low.

Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients.

Cell surface GLUT4 levels in skeletal muscle from nine type 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulin-stimulated cell surface GLUT4 above basal in type 2 diabetic patients were only approximately 10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was approximately 50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in type 2 diabetic patients.

Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients.

We characterized metabolic and mitogenic signaling pathways in isolated skeletal muscle from well-matched type 2 diabetic and control subjects. Time course studies of the insulin receptor, insulin receptor substrate (IRS)-1/2, and phosphatidylinositol (PI) 3-kinase revealed that signal transduction through this pathway was engaged between 4 and 40 min. Insulin-stimulated (0.6-60 nmol/l) tyrosine phosphorylation of the insulin receptor beta-subunit, mitogen-activated protein (MAP) kinase phosphorylation, and glycogen synthase activity were not altered in type 2 diabetic subjects. In contrast, insulin-stimulated tyrosine phosphorylation of IRS-1 and anti-phosphotyrosine-associated PI 3-kinase activity were reduced 40-55% in type 2 diabetic subjects at high insulin concentrations (2.4 and 60 nmol/l, respectively). Impaired glucose transport activity was noted at all insulin concentrations (0.6-60 nmol/l). Aberrant protein expression cannot account for these insulin-signaling defects because expression of insulin receptor, IRS-1, IRS-2, MAP kinase, or glycogen synthase was similar between type 2 diabetic and control subjects. In skeletal muscle from type 2 diabetic subjects, IRS-1 phosphorylation, PI 3-kinase activity, and glucose transport activity were impaired, whereas insulin receptor tyrosine phosphorylation, MAP kinase phosphorylation, and glycogen synthase activity were normal. Impaired insulin signal transduction in skeletal muscle from type 2 diabetic patients may partly account for reduced insulin-stimulated glucose transport; however, additional defects are likely to play a role.

Improved glucose tolerance restores insulin-stimulated Akt kinase activity and glucose transport in skeletal muscle from diabetic Goto-Kakizaki rats.

The serine/threonine kinase Akt (protein kinase B [PKB] or related to A and C protein kinase [RAC]) has recently been implicated to play a role in the signaling pathway to glucose transport. However, little is known concerning the regulation of Akt activity in insulin-sensitive tissues such as skeletal muscle. To explore the role of hyperglycemia on Akt kinase activity in skeletal muscle, normal Wistar rats or Goto-Kakizaki (GK) diabetic rats were treated with phlorizin. Phlorizin treatment normalized fasting blood glucose and significantly improved glucose tolerance (P < 0.001) in GK rats, whereas in Wistar rats, the compound had no effect on glucose homeostasis. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) Akt kinase activity was reduced by 68% (P < 0.01) and glucose transport was decreased by 39% (P < 0.05), compared with Wistar rats. Importantly, the defects at the level of Akt kinase and glucose transport were completely restored by phlorizin treatment. There was no significant difference in Akt kinase protein expression among the three groups. At a submaximal insulin concentration (2.4 nmol/l), activity of Akt kinase and glucose transport were unaltered. In conclusion, improved glucose tolerance in diabetic GK rats by phlorizin treatment fully restored insulin-stimulated activity of Akt kinase and glucose transport. Thus, hyperglycemia may directly contribute to the development of muscle insulin resistance through alterations in insulin action on Akt kinase and glucose transport.

Muscle fiber type-specific defects in insulin signal transduction to glucose transport in diabetic GK rats.

To determine whether defects in the insulin signal transduction pathway to glucose transport occur in a muscle fiber type-specific manner, post-receptor insulin-signaling events were assessed in oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skeletal muscle from Wistar or diabetic GK rats. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) glucose transport was significantly decreased, compared with that of Wistar rats. In EDL muscle from GK rats, maximal insulin-stimulated glucose transport was normal, while the submaximal response was reduced compared with that of Wistar rats. We next treated diabetic GK rats with phlorizin for 4 weeks to determine whether restoration of glycemia would lead to improved insulin signal transduction. Phlorizin treatment of GK rats resulted in full restoration of insulin-stimulated glucose transport in soleus and EDL muscle. In soleus muscle from GK rats, submaximal and maximal insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity were markedly reduced, compared with that of Wistar rats, but only submaximal insulin-stimulated PI 3-kinase was restored after phlorizin treatment. In EDL muscle, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI-3 kinase were not altered between GK and Wistar rats. Maximal insulin-stimulated Akt (protein kinase B) kinase activity is decreased in soleus muscle from GK rats and restored upon normalization of glycemia (Krook et al., Diabetes 46:2100-2114, 1997). Here, we show that in EDL muscle from GK rats, maximal insulin-stimulated Akt kinase activity is also impaired and restored to Wistar rat levels after phlorizin treatment. In conclusion, functional defects in IRS-1 and PI 3-kinase in skeletal muscle from diabetic GK rats are fiber-type-specific, with alterations observed in oxidative, but not glycolytic, muscle. Furthermore, regardless of muscle fiber type, downstream steps to PI 3-kinase (i.e., Akt and glucose transport) are sensitive to changes in the level of glycemia.

Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome.

Polycystic ovary syndrome (PCOS) is a common disorder characterized by chronic anovulation and infertility, hyperandrogenaemia, and frequently insulin resistance. This study investigated whether mutations in the insulin receptor gene could explain the insulin resistance in subjects with PCOS. From a total of 108 women with PCOS, a subgroup of 24 were selected on the criteria of being in the upper quartile for insulin resistance as assessed by fasting serum insulin, insulin area under the curve following 75 g oral glucose tolerance test, and endogenous glucose disposal as a measure of insulin sensitivity. An additional five normal women were also investigated. The entire coding region of the insulin receptor gene, comprising of 22 exons, was amplified by the PCR using genomic DNA and then subjected to single-stranded conformation polymorphism (SSCP) analysis to screen for single-base DNA sequence changes. DNA sequencing revealed that SSCP variants were detected in regions encompassing exons 3, 6-8, 11, 13, 15, 17, and 22. SSCP variants in regions of exons 3, 6, 7, 11, 15 and 22 were caused by nucleotide substitutions within intronic regions flanking the exon. The considerable variation seen in the 5' intron of exon 3 was found to be caused by variation in the number of (ATTT, 8-11) and (TC, 10-13) short sequence repeats. SSCP variants in exons 8 (Asp519, Ala523), 13 (Asn 838), and 17 (Tyr984, His1058) were caused by known silent polymorphisms. Southern blotting experiments excluded major gene deletions, insertions, or rearrangements. We conclude that insulin resistance in subjects with PCOS is not commonly a consequence of missense or nonsense mutations in the insulin receptor gene.

Human antibody response to the major adhesin of Mycoplasma pneumoniae: increase in titers against synthetic peptides in patients with pneumonia.

Peptides corresponding to parts of the P1 protein (major adhesin) of Mycoplasma pneumoniae were synthesized. On the basis of predicted antigenicity, seven sequences containing 17 to 21 amino acids were selected. In addition, one peptide containing a sequence of 13 amino acids shown to be related to cytadherence of M. pneumoniae was included. Serum samples from 56 patients with pneumonia were tested for a rise in titers of specific IgG during infection, using the peptides as coating antigens in ELISA. A titer rise against one or more peptides was observed in 10 out of 13 patients with serological evidence of mycoplasmal etiology. Specific antibodies to two or more peptides were demonstrated in three patients, whereas seven patients responded to one peptide only. In the sera from patients with pneumonia of non-mycoplasmal etiology, no titer rises above the cut-off level were observed. Our results indicate that a combination of four peptides would be possible for use as antigen for serological diagnosis of infections with M. pneumoniae.

Minimal criteria for identification of Moraxella (Branhamella) catarrhalis.

A study was performed which aimed at testing the reliability of our routine diagnostic tests for identification of Moraxella (Branhamella) catarrhalis in clinical samples from the respiratory tract. A preliminary diagnosis of 122 isolates as Moraxella catarrhalis was obtained by using colony morphology and results of Gram stain and oxidase test as the sole diagnostic criteria. By using additional tests we could show that the preliminary diagnosis was incorrect for 21 isolates, which were classified as different Neisseria species. 20 of these were isolated from sputum samples. We propose that at least a test for DNA hydrolysis should be included in the routine procedure for identification of Moraxella catarrhalis in sputum.

IgG response to Mycoplasma pneumoniae in patients with community-acquired pneumonia determined by ELISA.

In a prospective study of community-acquired, radiologically verified pneumonia, a solubilized mycoplasma antigen was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG-antibodies to Mycoplasma pneumoniae in paired sera from 60 patients. All of the 13 patients with a positive complement fixation (CF) test for M. pneumoniae were positive in the ELISA, and 46 out of 47 patients with a negative CF test were negative. The only false positive test was recorded from a patient with a positive CF test for Chlamydia. Specific IgG antibodies were also determined in paired sera from 50 pneumonia patients, all positive in the CF test for M. pneumoniae, and collected over a period of 10 years. Of these 50 patients, 45 were recorded as positive in the ELISA for IgG antibodies to M. pneumoniae. In the prospective as well as in the retrospective study, the time for admission to hospital after onset of disease showed considerable variation (1-14 days), with the consequence that high titers were recorded in the CF as well as in the ELISA in some of the first serum samples. A tendency to earlier detection of significant titers was noted in the CF test as compared to the ELISA.

Human immunoglobulin isotype and IgG subclass response to different antigens of Moraxella catarrhalis.

Enzyme immunoassays were tested for the determination of antibodies of different isotypes and IgG subclasses to Moraxella catarrhalis in human sera. An assay based on an outer membrane protein preparation (OMP) as antigen was compared to assays using whole bacterial cells and a purified lipopolysaccharide preparation. There was a good correlation between the results obtained with the OMP preparation and the whole-cell antigen. In paired sera, optimal sensitivity was obtained by using the OMP preparation as coating antigen and testing for a rise in IgG3 antibodies. However, patients with high levels of antibodies in acute serum had no or only an insignificant antibody response during infection.

Relation between concentrations of metronidazole and Bacteroides spp in faeces of patients with Crohn's disease and healthy individuals.

Nine patients wih Crohn's disease and six healthy individuals were given 400 mg metronidazole twice daily and the concentration was measured in plasma and faeces 6 to 8 h after intake. Quantitative and qualitative bacteriological examinations were performed before and during treatment. The concentrations of metronidazole in plasma varied between 5 and 30 mg/l as measured by liquid chromatography (LC) and between 3 and 20 mg/l as measured by a bioassay (BA) method. There was no significant differences between the results of the two methods in patients but compared with BA the LC values were regularly lower in the healthy subjects. Metronidazole was detected in the faeces of most patients. These findings coincided with a significant reduction of Bacteroides spp in their faeces which was not apparent in healthy individuals in whom metronidazole was not detected in the faeces. Higher faecal metronidazole concentrations were found in patients with active disease and a total colon involvement as compared with the patients with a more quiescent disease and involvement of ileum and ascending colon only.

Exercise-associated differences in an array of proteins involved in signal transduction and glucose transport.

Vastus lateralis muscle biopsies were obtained from endurance-trained (running approximately 50 km/wk) and untrained (no regular physical exercise) men, and the expression of an array of insulin-signaling intermediates was determined. Expression of insulin receptor and insulin receptor substrate-1 and -2 was decreased 44% (P < 0.05), 57% (P < 0.001), and 77% (P < 0.001), respectively, in trained vs. untrained muscle. The downstream signaling target, Akt kinase, was not altered in trained subjects. Components of the mitogenic signaling cascade were also assessed. Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase expression was 190% greater (P < 0.05), whereas p38 mitogen-activated protein kinase expression was 32% lower (P < 0.05), in trained vs. untrained muscle. GLUT-4 protein expression was twofold higher (P < 0.05), and the GLUT-4 vesicle-associated protein, the insulin-regulated aminopeptidase, was increased 4.7-fold (P < 0. 05) in trained muscle. In conclusion, the expression of proteins involved in signal transduction is altered in skeletal muscle from well-trained athletes. Downregulation of early components of the insulin-signaling cascade may occur in response to increased insulin sensitivity associated with endurance training.