Genetic architecture of motives for leisure-time physical activity: a twin study.

The aim of this study was to estimate the contribution of genetic and environmental influences on motives for engaging in leisure-time physical activity. The participants were obtained from the FinnTwin16 study. A modified version of the Recreational Exercise Motivation Measure was used to assess the motives for leisure-time physical activity in 2542 twin individuals (mean age of 34.1 years). Linear structural equation modeling was used to investigate the genetic and environmental influences on motive dimensions. The highest heritability estimates were found for the motive dimensions of "enjoyment" [men 33% (95% CI 23-43%), women 53% (95% CI 45-60%)] and "affiliation" [men 39% (95% CI 0.28-0.49%), women 35% (95% CI 0.25-0.43%)]. The lowest heritability estimates were found for others' expectations [men 13% (95% CI 0.04-0.25%), women 15% (95% CI 0.07-0.24%)]. Unique environmental influences explained the remaining variances, which ranged from 47% to 87%. The heritability estimates for summary variables of intrinsic and extrinsic motives were 36% and 32% for men and 40% and 24% for women, respectively. In conclusion, genetic factors contribute to motives for leisure-time physical activity. However, the genetic effects are, at most, moderate, implying the greater relative role of environmental factors.

The effects of parental education on exercise behavior in childhood and youth: a study in Dutch and Finnish twins.

Twin studies have estimated the relative contribution of genes and the environment to variance in exercise behavior and it is known that parental education positively affects exercise levels. This study investigates the role of parental education as a potential modifier of variance in exercise behavior from age 7 to 18 years. The study is based on large datasets from the Netherlands Twin Register (NTR: N = 24 874 twins; surveys around the ages of 7, 10, 12, 14, 16 and 18 years) and two Finnish twin cohorts (FinnTwin12: N = 4399; 12, 14 and 17 years; FinnTwin16: N = 4648; 16, 17 and 18 years). Regular participation in moderate-to-vigorous exercise activities during leisure time was assessed by survey. Parental education was dichotomized ("both parents with a low education" vs "at least one parent with a high education"). The mean in exercise behavior tended to be higher and the variance tended to be lower in children of high educated parents. Evidence for gene-by-environment interaction was weak. To develop successful interventions that specifically target children of low educated parents, the mechanisms causing the mean and variance differences between the two groups should be better understood.

Pharmacotherapy of obesity: targets and perspectives.

The search for anti-obesity agents has become one of the most exciting areas in drug discovery. Subsequent to an enormous increase in the number of possible molecular targets, the focus has shifted from target identification to target validation. Because important biological functions such as the regulation of energy intake and expenditure are controlled by complex systems, an improved understanding of pathophysiology is a prerequisite for the selection of successful development candidates for the treatment of obesity. Although most of the information on the regulation of energy balance has been obtained from rodents, various monogenic forms of human obesity provide clinical proof of concept for some of these mechanisms. However, it is still not known which are the most promising clinical approaches to lowering body weight and subsequently reducing morbidity and mortality.

Regular exercise behaviour in youth is not related to current body mass index or body mass index at 7-year follow-up.

OBJECTIVE: This population-based study aimed (1) to test the presence of an association between regular voluntary exercise behaviour (EB) that is performed in leisure time and body mass index (BMI) in youth and (2) to investigate the causal nature of this association using a longitudinal design in genetically informative subjects. DESIGN AND METHODS: Both EB and BMI were assessed repeatedly over time in 21 458 twin individuals from the Netherlands Twin Register (47.5% male) - first by parental report (ages 7, 10 and 12) and subsequently through self-report surveys (ages 14, 16 and 18). EB was quantified as weekly metabolic equivalent of task hours. RESULTS: Correlations over time were higher for BMI than for EB (r ≈ 0.70 vs. r ≈ 0.35) across 12 different follow-up periods. Cross-sectionally, regular involvement in EB was not associated with lower BMI in childhood and in genetically identical twin pairs discordant for EB; the exercising twin did not have a lower BMI than the non-exercising twin. Longitudinally, linear and quadratic relationships between EB and BMI were non-significant. Changes in EB over time did not induce opposite changes in BMI. CONCLUSIONS: No consistent association between regular EB and BMI was observed from ages 7 to 18 years.

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin.

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostiridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network.

Family physicians and the risk of suicide in the depressed elderly.

BACKGROUND: Depression is the most frequent psychiatric disorder in the elderly. It is the reason for most suicides in this age group. METHOD: We performed a representative survey in primary care. Two written case vignettes were presented to 170 family physicians in face-to-face interviews which took place in their practices. The case vignettes described either (Case 1) a mildly depressed otherwise healthy old patient or a severely depressed patient (Case 2) with somatic comorbidity. Afterwards the interviewers asked standardized open questions. The physicians were not let into the mental health focus of the study. RESULTS: The response rate was 77.6%. Depression was considered for primary or differential diagnosis by 91.2% of the physicians in Case 1 and by 70% in Case 2 (chi2-test; p < 0.01). For further anamnesis, only 2.4% of the physicians were interested in suicidal ideation of the patient. When directly asked at the end of the interview, 76.9% of the physicians said they would talk about suicide. Those who would not, thought that the patient would communicate suicidal intent himself/herself, or they feared to induce suicide by asking directly. CONCLUSION: Thinking of suicidality and its prevention is not uppermost in the physicians' mind. Therefore, and also with regard to the relatively high rate of depression recognition, we conclude that educational means should not only focus on the recognition and screening of depression, but also on the management--'how to talk about...'--of complex problems like suicide in the elderly, in order to change suicide rates.

Gender differences in the recognition of depression in old age.

OBJECTIVE: The study should answer the question of whether identical symptom presentations of depression in male and female patients leads to similar recognition rates in primary care. METHOD: We performed a survey in primary care. Two written case vignettes were presented to 170 family physicians in a face-to-face interview which took place in their practices. The case vignettes described either a mildly depressed otherwise healthy old patient (case 1) or a severely depressed patient with somatic comorbidity (case 2). For each case different versions with regard to patients' gender were used: in case 1 only the gender of the patient varied; in case 2 both the gender and the anamnesis (stroke/hypothyroidism) varied. Afterwards the interviewers asked standardised open questions. The physicians were not aware of the mental health focus and the gender focus of the study. RESULTS: The study is representative with a response rate of 77.6%. For primary diagnosis, the female versions were given the diagnosis of depression more often. There was a non-significant trend that female physicians considered depression more often. CONCLUSION: The results show that gender-related experience and stereotypes on the physicians' side influence the diagnosis of (old age) depression in primary care. Further studies should elucidate the influence of the physicians' gender on the management of psychiatric disorders.

Abundance and subcellular distribution of GTP-binding proteins in 3T3-L1 cells before and after differentiation to the insulin-sensitive phenotype.

The abundance and the subcellular distribution of GTP-binding proteins was studied in membrane fractions (plasma membranes and low-density microsomes) from 3T3-L1 cells before and after differentiation to the insulin-sensitive phenotype. After differentiation, the abundance of alpha i (alpha subunit of GTP-binding protein Gi), alpha o (alpha subunit of GTP-binding protein G(o)), and of a 47-kDa alpha s (alpha subunit of GTP-binding protein Gs) as detected by immunoblotting with specific antisera was reduced by 10-50% when normalized per membrane protein. In contrast, a 43-kDa alpha s was increased about threefold after differentiation. Furthermore, cholera-toxin-catalyzed ADP-ribosylation of both 43-kDa and 47-kDa alpha s was disproportionately increased ninefold and threefold, respectively, possibly reflecting the increased production of an ADP-ribosylation factor in the differentiated cells. The small GTP-binding protein Ha-ras was reduced by 50%, whereas rab1 and other small GTP-binding proteins tentatively identified as rab-isoforms (ras-homologous gene products from brain) were increased by 100% and 70%, respectively. Since the total protein content of 3T3-L1 cells was increased threefold after differentiation, the observed increase of the 43-kDa alpha s, rab1 and of the other rab isoforms was eightfold, sixfold and fivefold, respectively, when normalized/cell count. With the exception of the rab isoforms, all GTP-binding proteins were predominantly, if not exclusively, located in the plasma membrane; comparable amounts of the rab isoforms were found in plasma membranes and low-density microsomes. Insulin induced the characteristic redistribution of glucose transporters GLUT4 from low-density microsomes to the plasma membranes, but failed to alter the subcellular distribution of any of the GTP-binding proteins investigated. These data suggest that the increase in the abundance of the 43-kDa alpha s subunit and of several rab isoforms might be related to specific functions of the adipocyte-like phenotype, but that none of the investigated guanine-nucleotide-binding regulatory (G)-proteins appears to be tightly associated with the GLUT4.

Comparison of the effects of insulin, PDGF, interleukin-6, and interferon-gamma on glucose transport in 3T3-L1 cells: lack of cross-talk between tyrosine kinase receptors and JAK/STAT pathways.

The effects of insulin, insulin-like growth factor (IGF)-I, platelet-derived growth factor (PDGF), interleukin (IL)-6 and interferon-gamma on 2-deoxyglucose uptake and insulin receptor substrate (IRS)-1 phosphorylation were compared in 3T3-L1 cells at confluence and after differentiation to the adipocyte-like phenotype. Insulin and IGF-I produced the expected stimulation of glucose transport and tyrosine phosphorylation of IRS-1 in both confluent and differentiated cells. In contrast, IL-6 and interferon-gamma failed to stimulate glucose transport or IRS-1 phosphorylation, although a marked stimulation of the JAK/STAT pathways as shown by acute-phase response factor (APRF)/Stat3 or Stat1 activation was observed in fibroblasts (IL-6, interferon-gamma) and adipocytes (IL-6). PDGF-AA and PDGF-BB stimulated glucose transport in confluent, undifferentiated cells to the same extent as insulin (approximately six-fold stimulation), but produced only a small portion of the effect of insulin in differentiated cells. Similarly, mRNA levels and autophosphorylation of PDGF receptors were much lower in differentiated cells than in confluent fibroblasts. In contrast to insulin and IGF-I, PDGF failed to stimulate tyrosine phosphorylation of IRS-1. All effects of insulin, IGF-I, and PDGF on glucose transport were inhibited by Wortmannin; the half-maximally inhibiting concentration (IC50) of Wortmannin was increased by insulin. These data demonstrate distinct signalling potentials of the investigated receptors, and indicate that the IL-6 and interferon-gamma controlled JAK/STAT pathways lack the potential to stimulate glucose transport. IRS-1 does not appear to be involved in the PDGF receptor-mediated effects, whereas activation of phosphatidylinositol (PI) 3-kinase is a crucial event in all pathways leading to stimulation of glucose transport.

Functional analyses of amino acid substitutions Arg883Ser and Asp905Tyr of protein phosphatase-1 G-subunit.

The PPP1R3 gene encoding the G-subunit of protein phosphatase-1 has three polymorphisms in linkage disequilibrium in the Pima Indians: an mRNA-destabilizing element in the 3'-untranslated region (ARE1/ARE2 alleles), Arg883Ser, and Asp905Tyr substitutions. The ARE2 allele, Arg883, and Asp905 variants are associated with insulin resistance and higher prevalence of type 2 diabetes in the Pima Indians. The ARE2 allele is associated with lower PPP1R3 transcript and protein levels in muscle tissue. Here we determined the functional contribution of the amino acid substitutions independent of the ARE alleles to insulin-stimulated glycogen synthesis by adenoviral-mediated gene expression in L6 myotubes. Similar overexpression levels of the G-subunit variants increased glycogen synthase fractional activity in the presence ( approximately 1. 5-fold) of insulin compared to control myotubes transduced with adenovirus encoding beta-galactosidase. The glycogen synthesis rate of myotubes overexpressing the G-subunit variants also increased by approximately 1.7-fold over the control with and without insulin. However, these measures were not significantly different among the variants. This study does not support a role for Arg883 and Asp905 variants independent of the ARE2 allele in the impaired insulin-stimulated glycogen synthesis in the muscle of Pima Indians.

Effect of prenatal glucocorticoids and postnatal nitric oxide inhalation on survival of newborn rats with nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Pulmonary hypertension and pulmonary hypoplasia account for the high mortality rate associated with congenital diaphragmatic hernia (CDH). In animal models of CDH, postnatal nitric oxide (NO) inhalation resulted in significantly better survival rates and antenatal glucocorticoid administration in improved lung compliance. The objective of this study was to evaluate the combined effect of prenatal glucocorticoid administration and postnatal NO inhalation on the survival rate of newborn rats with nitrofen-induced CDH. METHODS: Right-sided CDH was induced by maternal administration of a single oral dose (100 mg, intraperitoneally) of nitrofen on day 11.5 of pregnancy. Dexamethasone (DEX, 0.25 mg/kg) was given in groups III and IV by maternal intraperitoneal injection on day 18.5 and 19.5 of pregnancy. Control animals (groups I and II) received vehicle alone. After spontaneous delivery, the newborn animals were exposed to either NO (80 ppm; groups II and IV) or room air (groups I and III). Vitality (Rat-Score), sO(2) and survival were monitored continuously for 12 hours until animals were killed. Hernia size was estimated as percentage of total thoracic content. RESULTS: Right-sided CDH was observed in 392 of 491 newborn rats (81%). Animals with large hernias (>50%) died within 4 hours after birth, irrespective of treatment. Hernias with less than 50% of the thoracic volume were considered clinically relevant hernias. In this category, 12.5% of animals without treatment (group I) survived compared with 63.6% after NO treatment alone (group II; P <.01). Survival rate after DEX treatment alone (group III) was 69.4% (group III v I; P <.01). In group IV (DEX and NO) 95.2% of the animals survived (group IV v I; P <.001). In contrast to DEX alone, NO administration resulted in significantly better sO(2)(group II and IV) compared with group I (P <.05). CONCLUSION: Combination of prenatal maternal glucocorticoids and postnatal NO inhalation significantly improved survival rate of newborn rats with nitrofen-induced CDH.

Cloning of two novel ADP-ribosylation factor-like proteins and characterization of their differential expression in 3T3-L1 cells.

A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.

Dyrk, a dual specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII.

The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.

Uncoupling protein 3 (UCP3) stimulates glucose uptake in muscle cells through a phosphoinositide 3-kinase-dependent mechanism.

UCP3 is a mitochondrial membrane protein expressed in humans selectively in skeletal muscle. To determine the mechanisms by which UCP3 plays a role in regulating glucose metabolism, we expressed human UCP3 in L6 myotubes by adenovirus-mediated gene transfer and in H(9)C(2) cardiomyoblasts by stable transfection with a tetracycline-repressible UCP3 construct. Expression of UCP3 in L6 myotubes increased 2-deoxyglucose uptake 2-fold and cell surface GLUT4 2.3-fold, thereby reaching maximally insulin-stimulated levels in control myotubes. Wortmannin, LY 294002, or the tyrosine kinase inhibitor genistein abolished the effect of UCP3 on glucose uptake, and wortmannin inhibited UCP3-induced GLUT4 cell surface recruitment. UCP3 overexpression increased phosphotyrosine-associated phosphoinositide 3-kinase (PI3K) activity 2.2-fold compared with control cells (p < 0.05). UCP3 overexpression increased lactate release 1.5- to 2-fold above control cells, indicating increased glucose metabolism. In H(9)C(2) cardiomyoblasts stably transfected with UCP3 under control of a tetracycline-repressible promotor, removal of doxycycline resulted in detectable levels of UCP3 at 12 h and 2.2-fold induction at 7 days compared with 12 h. In parallel, glucose transport increased 1.3- and 2-fold at 12 h and 7 days, respectively, and the stimulation was inhibited by wortmannin or genistein. p85 association with membranes was increased 5.5-fold and phosphotyrosine-associated PI3K activity 3.8-fold. In contrast, overexpression of UCP3 in 3T3-L1 adipocytes did not alter glucose uptake, suggesting tissue-specific effects of human UCP3. Thus, UCP3 stimulates glucose transport and GLUT4 translocation to the cell surface in cardiac and skeletal muscle cells by activating a PI3K dependent pathway.