Metabolites as novel biomarkers for childhood obesity-related traits in Mexican-American children.

BACKGROUND/OBJECTIVES: Although newer approaches have identified several metabolites associated with obesity, there is paucity of such information in paediatric populations, especially among Mexican-Americans (MAs) who are at high risk of obesity. Therefore, we performed a global serum metabolite screening in MA children to identify biomarkers of childhood obesity. METHODS: We selected 15 normal-weight, 13 overweight and 14 obese MA children (6-17 years) and performed global serum metabolite screening using ultra-performance liquid chromatography/quadruple orthogonal acceleration time of flight tandem micro mass spectrometer. Metabolite values were analysed to assess mean differences among groups using one-way analysis of variance, to test for linear trend across groups and to examine Pearson's correlations between them and seven cardiometabolic traits (CMTs): body mass index, waist circumference, systolic blood pressure, diastolic blood pressure, homeostasis model of assessment-insulin resistance, triglycerides and high-density lipoprotein cholesterol. RESULTS: We identified 14 metabolites exhibiting differences between groups as well as linear trend across groups with nominal statistical significance. After adjustment for multiple testing, mean differences and linear trends across groups remained significant (P < 5.9 × 10(-5) ) for L-thyronine, bradykinin and naringenin. Of the examined metabolite-CMT trait pairs, all metabolites except for 2-methylbutyroylcarnitine were nominally associated with two or more CMTs, some exhibiting significance even after accounting for multiple testing (P < 3.6 × 10(-3) ). CONCLUSIONS: To our knowledge, this study - albeit pilot in nature - is the first study to identify these metabolites as novel biomarkers of childhood obesity and its correlates. These findings signify the need for future systematic investigations of metabolic pathways underlying childhood obesity.

Localization of a major susceptibility locus influencing preterm birth.

Preterm birth (PTB) is a complex trait, but little is known regarding its major genetic determinants. The objective of this study is to localize genes that influence susceptibility to PTB in Mexican Americans (MAs), a minority population in the USA, using predominantly microfilmed birth certificate-based data obtained from the San Antonio Family Birth Weight Study. Only 1302 singleton births from 288 families with information on PTB and significant covariates were considered for genetic analysis. PTB is defined as a childbirth that occurs at <37 completed weeks of gestation, and the prevalence of PTB in this sample was 6.4%. An ∼10 cM genetic map was used to conduct a genome-wide linkage analysis using the program SOLAR. The heritability of PTB was high (h(2) ± SE: 0.75 ± 0.20) and significant (P = 4.5 × 10(-5)), after adjusting for the significant effects of birthweight and birth order. We found significant evidence for linkage of PTB (LOD = 3.6; nominal P = 2.3 × 10(-5); empirical P = 1.0 × 10(-5)) on chromosome 18q between markers D18S1364 and D18S541. Several other chromosomal regions (2q, 9p, 16q and 20q) were also potentially linked with PTB. A strong positional candidate gene in the 18q linked region is SERPINB2 or PAI-2, a member of the plasminogen activator system that is associated with various reproductive processes. In conclusion, to our knowledge, perhaps for the first time in MAs or US populations, we have localized a major susceptibility locus for PTB on chromosome 18q21.33-q23.

Chromatin occupancy of transcription factor 7-like 2 (TCF7L2) and its role in hepatic glucose metabolism.

AIMS/HYPOTHESIS: The mechanisms by which transcription factor 7-like 2 (TCF7L2) regulates the pathways that are important in the pathogenesis of type 2 diabetes are unknown. We therefore examined the role of TCF7L2 in hepatic glucose production (HGP) in vitro and characterised the whole-genome chromatin occupancy of TCF7L2 in hepatocytes. METHODS: We investigated the effect of TCF7L2 silencing and overexpression on HGP from gluconeogenic precursors and used chromatin-immunoprecipitation (ChIP) combined with massively parallel DNA sequencing (ChIP-Seq) to investigate the DNA binding patterns of TCF7L2 across the whole genome. RESULTS: Silencing of TCF7L2 induced a marked increase in basal HGP, which was accompanied by significant increases in the expression of the gluconeogenic genes Fbp1, Pck1 and G6pc. Overexpression of Tcf7l2 reversed this phenotype and significantly reduced HGP. TCF7L2 silencing did not affect the half-maximal inhibitory concentration of insulin or metformin, but HGP remained elevated in TCF7L2-silenced cells due to the increased baseline HGP. Using ChIP-Seq, we detected 2,119 binding events across the genome. Pathway analysis demonstrated that diabetes genes were significantly over-represented in the dataset. Our results indicate that TCF7L2 binds directly to multiple genes that are important in regulation of glucose metabolism in the liver, including Pck1, Fbp1, Irs1, Irs2, Akt2, Adipor1, Pdk4 and Cpt1a. CONCLUSIONS/INTERPRETATION: TCF7L2 is an important regulator of HGP in vitro and binds directly to genes that are important in pathways of glucose metabolism in the liver. These data highlight the possibility that TCF7L2 may affect fasting and postprandial hyperglycaemia in carriers of at-risk TCF7L2 genetic polymorphisms.

Pioglitazone stimulates AMP-activated protein kinase signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation in human skeletal muscle in vivo: a randomised trial.

AIMS/HYPOTHESIS: The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle. METHODS: A randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients treated with: (1) pioglitazone (n = 14) or (2) aggressive nutritional therapy (n = 12) to reduce HbA(1c) to levels observed in the pioglitazone-treated group. Participants were assigned randomly to treatment using a table of random numbers. Before and after 6 months, patients reported to the Clinical Research Center of the Texas Diabetes Institute for a vastus lateralis muscle biopsy followed by a 180 min euglycaemic-hyperinsulinaemic (80 mU m(-2) min(-1)) clamp. RESULTS: All patients in the pioglitazone (n = 14) or nutritional therapy (n = 12) group were included in the analysis. Pioglitazone significantly increased plasma adiponectin concentration by 79% and reduced fasting plasma NEFA by 35% (both p < 0.01). Following pioglitazone, insulin-stimulated glucose disposal increased by 30% (p < 0.01), and muscle AMPK and acetyl-CoA carboxylase (ACC) phosphorylation increased by 38% and 53%, respectively (p < 0.05). Pioglitazone increased mRNA levels for adiponectin receptor 1 and 2 genes (ADIPOR1, ADIPOR2), peroxisome proliferator-activated receptor gamma, coactivator 1 gene (PPARGC1) and multiple genes involved in mitochondrial function and fat oxidation. Despite a similar reduction in HbA(1c) and similar improvement in insulin sensitivity with nutritional therapy, there were no significant changes in muscle AMPK and ACC phosphorylation, or the expression of ADIPOR1, ADIPOR2, PPARGC1 and genes involved in mitochondrial function and fat oxidation. No adverse (or unexpected) effects or side effects were reported from the study. CONCLUSIONS/INTERPRETATIONS: Pioglitazone increases plasma adiponectin levels, stimulates muscle AMPK signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation. These changes may represent an important cellular mechanism by which thiazolidinediones improve skeletal muscle insulin sensitivity. TRIAL REGISTRATION: NCT 00816218 FUNDING: This trial was funded by National Institutes of Health Grant DK24092, VA Merit Award, GCRC Grant RR01346, Executive Research Committee Research Award from the University of Texas Health Science Center at San Antonio, American Diabetes Association Junior Faculty Award, American Heart Association National Scientist Development Grant, Takeda Pharmaceuticals North America Grant and Canadian Institute of Health Research Grant.

Metabolism in adipose tissue in response to citalopram and trimipramine treatment--an in situ microdialysis study.

The intake of antidepressants is often accompanied by weight gain. Antidepressants may influence lipid and carbohydrate metabolism that can result in metabolic changes and obesity. We investigated the effect of citalopram and trimipramine on interstitial glycerol, glucose and lactate concentration and blood flow in subcutaneous adipose tissue of obese subjects by means of the microdialysis technique. In addition, the effect of stimulation with norepinephrine on metabolic response was investigated. Each subject was compared to a control subject matched for BMI and age. Each group comprised 10 subjects. Circulating plasma triglyceride concentrations were higher in drug-treated groups. In subcutaneous adipose tissue, microdialysis experiments revealed a higher and prolonged glycerol release in the presence of norepinephrine, but not under basal conditions. In citalopram treated subjects, basal glucose and lactate concentrations were higher compared with controls or with the trimipramine treated group. Local administration of norepinephrine induced a decrease in glucose levels and an increase in lactate levels, but without significant differences between groups. Local adipose tissue blood flow decreased in control groups following norepinephrine application, but remained constant in the antidepressant groups. In conclusion, citalopram and trimipramine affected glucose and lipid metabolism in adipose tissue and resulted in enhanced release of glycerol and free fatty acids into the circulation.

Association between variants in the genes for adiponectin and its receptors with insulin resistance syndrome (IRS)-related phenotypes in Mexican Americans.

AIMS/HYPOTHESIS: The aim of this study was to examine whether genetic variation in ADIPOQ, ADIPOR1 and ADIPOR2 may contribute to increased susceptibility to components of the insulin resistance syndrome (IRS). MATERIALS AND METHODS: We genotyped single-nucleotide polymorphisms (SNPs) in ADIPOQ, ADIPOR1 and ADIPOR2 in Mexican American subjects (N=439) and performed an association analysis of IRS-related traits. RESULTS: Of the eight SNPs examined in the ADIPOQ gene, rs4632532 and rs182052 exhibited significant associations with BMI (p=0.029 and p=0.032), fasting specific insulin (p=0.023 and p=0.026), sum of skin folds (SS) (p=0.0089 and p=0.0084) and homeostasis model assessment of insulin sensitivity (HOMA-%S) (p=0.015 and p=0.016). Two other SNPs, rs266729 and rs2241767, were significantly associated with SS (p=0.036 and p=0.013). SNP rs7539542 of ADIPOR1 was significantly associated with BMI, SS and waist circumference (p=0.025, p=0.047 and p=0.0062). Fourteen of the ADIPOR2 SNPs were found to be significantly (p<0.05) associated with fasting plasma triglyceride concentrations. Four of these SNPs (rs10848569, rs929434, rs3809266 and rs12342) were in high pairwise linkage disequilibrium (r (2)=0.99) and were strongly associated with fasting triglyceride levels (p=0.00029, p=0.00016, p=0.00027 and p=0.00021). Adjusting for the effects of BMI and HOMA-%S on triglyceride concentrations increased significance to p=0.000060 for SNP rs929434. Bayesian quantitative trait nucleotide analysis was used to examine all possible models of gene action. Again, SNP rs929434 provided the strongest statistical evidence of an effect on triglyceride concentrations. CONCLUSIONS/INTERPRETATION: These results provide evidence for association of SNPs in ADIPOQ and its receptors with multiple IRS-related phenotypes. Specifically, several genetic variants in ADIPOR2 were strongly associated with decreased triglyceride levels.

Studies of phosphodiesterase effects on adipose tissue metabolism in obese subjects by the microdialysis technique.

The effect of non-selective (theophylline) inhibition of cyclic AMP breakdown on norepinephrine stimulated lipolysis rate was investigated in subcutaneous adipose tissue of obese subjects. In addition, changes in interstitial glucose and lactate concentration were assessed by means of the microdialysis technique. The interaction of endogenous released insulin and theophylline on adipocyte metabolism was determined. Theophylline and norepinephrine alone increased glycerol outflow significantly. When both agents were perfused in combination, interstitial glycerol concentration increased further. The enhanced glycerol level due to theophylline application was slightly decreased by insulin. In the presence of theophylline, extracellular glucose concentration increased, in contrast to the catecholamine. Norepinephrine decreased interstitial glucose level. When both drugs were added in combination, the level of interstitial glucose increased to about 1 mM, greater than with theophylline alone. With each intervention, lactate was synthesized. Local adipose tissue blood flow was increased by theophylline and theophylline plus norepinephrine. In conclusion, post-receptor mechanisms increased norepinephrine maximal stimulated lipolysis rate in subcutaneous adipose tissue. Glucose uptake was inhibited by the non-specific inhibitor of phosphodiesterase. The effect of insulin on inhibition of lipolysis was modest but sustained in the presence of high theophylline (10(-4) M) concentration. Phosphodiesterase activity may be relatively low in obese subjects in comparison with lean subjects. In lean subjects theophylline caused a transient reversal of the antilipolytic effect of insulin.

Effects of moderate consumption of white wine on weight loss in overweight and obese subjects.

BACKGROUND: Patients on dietary, weight-reducing treatment commonly are advised against alcohol consumption. In light of the widespread use of alcoholic beverages and the well-established benefits of light to moderate alcohol consumption in risk reduction, a revision of dietary treatment recommendations may be warranted. OBJECTIVE: To investigate whether daily consumption of moderate amounts of alcohol influences the effectiveness of an energy-restricted diet in overweight and obese subjects. DESIGN: A prospective randomized clinical trial was conducted, with a 3-months intervention period and two isocaloric dietary regimens containing 6.3 MJ (1500 kcal) each, one with 10% of energy from white wine and one with 10% of energy from grape juice. The trial was performed in obese subjects being recruited from the Obesity Outpatient Clinic at the University Hospital, Ulm, who all habitually consumed moderate amounts of alcohol. Out of 87 patients, 49 were eligible to participate and 40 completed the study (age 48.1+/-11.4 y, BMI 34.2+/-6.4 kg/m(2)). Efficacy parameters were body weight and biomarkers of good health. RESULTS: All subjects achieved significant body weight reduction. Weight loss in the grape juice group and white wine group was 3.75+/-0.46 and 4.73+/-0.53 kg, respectively. Percent body fat, waist circumference, blood pressure, blood glucose, insulin, triglycerides, and cholesterol were reduced. The antioxidant status was unchanged, as were liver enzyme activities and other safety parameters. There were no significant differences between the groups. CONCLUSIONS: An energy-restricted diet is effective in overweight and obese subjects used to drinking moderate amounts of alcohol. A diet with 10% of energy derived from white wine is as effective as an isocaloric diet with 10% of energy derived from grape juice.

Adiponectin receptors gene expression and insulin sensitivity in non-diabetic Mexican Americans with or without a family history of Type 2 diabetes.

AIMS/HYPOTHESIS: The recent discovery of two adiponectin receptors (AdipoR1 and AdipoR2) will improve our understanding of the molecular mechanisms underlying the insulin-sensitising effect of adiponectin. The aim of this study was to determine for the first time whether skeletal muscle AdipoR1 and/or AdipoR2 gene expression levels are associated with insulin resistance. METHODS: Using RT-PCR and northern analysis we measured AdipoR1 and AdipoR2 gene expression in skeletal muscle from healthy Mexican Americans with normal glucose tolerance who had (n=8) or did not have (n=10) a family history of Type 2 diabetes. RESULTS: Gene expression profiling indicated that the AdipoR1 and AdipoR2 isoforms are highly expressed in human skeletal muscle, unlike in mice where AdipoR2 expression was highest in the liver, and AdipoR1 was highest in skeletal muscle. In the study subjects, the expression levels of AdipoR1 (p=0.004) and AdipoR2 (p=0.04), as well as plasma adiponectin concentration (p=0.03) were lower in people with a family history of Type 2 diabetes than in those with no family history of the disease. Importantly, the expression levels of both receptors correlated positively with insulin sensitivity (r=0.64, p=0.004 and r=0.47, p=0.048 respectively). CONCLUSIONS/INTERPRETATION: Collectively, these data indicate that both isoforms of the adiponectin receptor play a role in the insulin-sensitising effect of adiponectin.

In vivo alpha(1)-adrenergic lipolytic activity in subcutaneous adipose tissue of obese subjects.

The role of alpha(1)-adrenoceptors in lipid mobilization and blood flow was investigated in situ using microdialysis of subcutaneous adipose tissue in severely obese subjects. The lipolysis rate was assessed by determination of interstitial glycerol concentration. The alpha(1)-adrenoceptor agonist norfenefrine caused an increase in glycerol level in adipose tissue that was similar to that observed with the physiologic alpha(1,2)-beta(1)-adrenoceptor agonist norepinephrine, whereas the alpha(1)-adrenoceptor antagonist urapidil showed no effect on basal lipolysis rate. However, the enhanced glycerol concentration due to norfenefrine and norepinephrine was suppressed in the presence of urapidil. The beta-adrenoceptor antagonist propranolol showed no effect on norfenefrine-stimulated glycerol outflow. Blood flow was assessed using the ethanol escape technique. Perfusion with norfenefrine decreased blood flow, whereas urapidil enhanced blood flow significantly. Despite the increase in blood flow, the basal interstitial glycerol concentration remained unchanged. Although norfenefrine at high concentrations could inhibit the urapidil-induced increase in blood flow, the norfenefrine-induced glycerol output was not affected. These results demonstrate that alpha(1)-adrenoceptors are involved in regulation of lipolysis rate and microcirculation of adipose tissue. However, the observed changes in local blood flow were not related to glycerol output.

Effects of acipimox on the lipolysis rate in subcutaneous adipose tissue of obese subjects.

BACKGROUND: Acipimox is a hypolipidaemic agent reducing serum concentrations of triglycerides and non-esterified fatty acids. Acipimox may reduce triglyceride synthesis by decreasing non-esterified fatty acid availability from adipocytes, but this effect has yet to be demonstrated in vivo. Lipolysis after acipimox treatment was examined in subcutaneous adipose tissue of severely obese subjects with associated metabolic disorders. METHODS: The microdialysis technique was performed in abdominal subcutaneous adipose tissue of eight hyperinsulinaemic subjects. After oral treatment with acipimox, glycerol concentration was determined as an index of lipolysis rate. Blood flow was assessed by the ethanol escape technique. The rates of release of glycerol from human adipose tissue maximally stimulated by norepinephrine were also investigated in the presence of acipimox. Eight weight- and age-matched subjects served as a control group. RESULTS: Under acipimox treatment, basal glycerol release decreased in subcutaneous adipose tissue, whereas no effect was observed on blood flow. In stimulated adipose tissue acipimox showed no effect. CONCLUSION: In the present study basal glycerol outflow from adipose tissue was inhibited by acipimox. The anti-lipolytic action of the agent may diminish elevated plasma concentrations of free fatty acids in subjects with severe obesity.

Skeletal muscle insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes is associated with decreased insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation.

Normoglycemic subjects with a strong family history of type 2 diabetes are insulin resistant, but the mechanism of insulin resistance in skeletal muscle of such individuals is unknown. The present study was undertaken to determine whether abnormalities in insulin-signaling events are present in normoglycemic, nonobese subjects with a strong family history of type 2 diabetes. Hyperinsulinemic-euglycemic clamps with percutaneous muscle biopsies were performed in eight normoglycemic relatives of type 2 diabetic patients (FH(+)) and eight control subjects who had no family history of diabetes (FH(-)), with each group matched for age, sex, body composition, and ethnicity. The FH(+) group had decreased insulin-stimulated glucose disposal (6.64 +/- 0.52 vs. 8.45 +/- 0.54 mg. kg(-1) fat-free mass. min(-1); P < 0.05 vs. FH(-)). In skeletal muscle, the FH(+) and FH(-) groups had equivalent insulin stimulation of insulin receptor tyrosine phosphorylation. In contrast, the FH(+) group had decreased insulin stimulation of insulin receptor substrate (IRS)-1 tyrosine phosphorylation (0.522 +/- 0.077 vs. 1.328 +/- 0.115 density units; P < 0.01) and association of PI 3-kinase activity with IRS-1 (0.299 +/- 0.053 vs. 0.466 +/- 0.098 activity units; P < 0.05). PI 3-kinase activity was correlated with the glucose disposal rate (r = 0.567, P = 0.02). In five subjects with sufficient biopsy material for further study, phosphorylation of Akt was 0.266 +/- 0.061 vs. 0.404 +/- 0.078 density units (P < 0.10) and glycogen synthase activity was 0.31 +/- 0.06 vs. 0.50 +/- 0.12 ng. min(-1). mg(-1) (P < 0.10) for FH(+) and FH(-) subjects, respectively. Therefore, despite normal insulin receptor phosphorylation, postreceptor signaling was reduced and was correlated with glucose disposal in muscle of individuals with a strong genetic background for type 2 diabetes.

The community prevalence of chronic pelvic pain in women and associated illness behaviour.

BACKGROUND: Chronic pelvic pain has often been described as a major women's health issue, but no information exists on the extent of the problem in the United Kingdom. AIM: To investigate the community prevalence of chronic pelvic pain and its effect on the lives of consulting and non-consulting women. DESIGN OF STUDY: Postal questionnaire survey. SETTING: Women aged 18 to 49 (n = 3916) randomly selected from the Oxfordshire Health Authority Register. METHOD: The questionnaire response rate (adjusted for non-deliveries) was 74% (2304/3106). Chronic pelvic pain was defined as recurrent or constant pelvic pain of at least six months' duration, unrelated to periods, intercourse, or pregnancy. Case subgroups comprised recent consulters, past consulters, and non-consulters. Women who reported dysmenorrhoea alone formed a comparison group. RESULTS: The three-month prevalence of chronic pelvic pain was 24.0% (95% CI = 22.1% to 25.8%). One-third of women reported pain that started more than five years ago. Recent consulters (32% of cases) were most affected by their symptoms in terms of pain severity, use of health care, physical and mental health scores, sleep quality, and pain-related absence from work. Non-consulters (41% of cases) did not differ from women with dysmenorrhoea in terms of symptom-related impairment. Irrespective of consulting behaviour, a high rate of symptom-related anxiety was found in women with chronic pelvic pain (31%) compared with women with dysmenorrhoea (7%). CONCLUSIONS: This study showed a high community prevalence of chronic pelvic pain in women of reproductive age. Cases varied substantially in the degree to which they were affected by their symptoms. The high symptom-related anxiety in these women emphasises the need for more information about chronic pelvic pain and its possible causes.

Chronic pelvic pain in the community--symptoms, investigations, and diagnoses.

OBJECTIVES: This study was undertaken to investigate the overlap between chronic pelvic pain, dysmenorrhea, dyspareunia, irritable bowel syndrome, and genitourinary symptoms in the community and also to examine associated investigations and diagnoses. STUDY DESIGN: A postal questionnaire was used to survey 3916 women aged 18 through 49 randomly selected from the Oxfordshire Health Authority Register. The number of responders was 2304 (74% of 3106 questionnaire recipients). Chronic pelvic pain was described as recurrent or constant pelvic pain of > or =6 months' duration unrelated to periods, intercourse, or pregnancy. Case patients (n = 483) were subgrouped as follows: (1) chronic pelvic pain only, (2) chronic pelvic pain and irritable bowel syndrome, (3) chronic pelvic pain and genitourinary symptoms, and (4) chronic pelvic pain, genitourinary symptoms, and irritable bowel syndrome. RESULTS: Half the women with chronic pelvic pain also had either genitourinary symptoms or irritable bowel syndrome, or both. Prevalences of dysmenorrhea and dyspareunia were higher among women with chronic pelvic pain (81% and 41%, respectively) than among women without chronic pelvic pain (58% and 14%, respectively); rates did not differ among the chronic pelvic pain subgroups. Irritable bowel syndrome and stress were the most common diagnoses received by patients with chronic pelvic pain, but 50% had never received a diagnosis. CONCLUSIONS: There is substantial overlap between chronic pelvic pain and other abdominal symptoms in the community. Despite a high prevalence of chronic pelvic pain, many women have never had the condition diagnosed.

Association of dopamine D2 receptor polymorphisms Ser311Cys and TaqIA with obesity or type 2 diabetes mellitus in Pima Indians.

OBJECTIVES: To investigate whether (a) variants within the dopamine D2 receptor gene (DRD2) are associated with obesity and type 2 diabetes in Pima Indians, and (b) whether variation in this gene could be responsible for previously observed linkage to these phenotypes, at chromosome location 11q23-24, in this population. DESIGN: Two single nucleotide polymorphisms (SNPs), Ser311Cys and TaqIA, within the DRD2 gene were genotyped by allelic discrimination PCR in subjects who had provided evidence of linkage to diabetes and obesity in an autosome-wide scan. SUBJECTS: A total of 1,187 subjects were genotyped, including 947 full heritage Pima Indians (80%). Descriptive statistics for all subjects analyzed, for whom clinical data were available, were (mean+/-s.d.): age at time of last exam = 41 +/- 15 y; birth year=1950 +/- 14; age-sex-adjusted body mass index (BMI; adjusted to a mean age of 35y)=36 +/- 8kg/m2; male = 44%; diabetic = 57%. For full heritage Pimas only: age = 43 +/- 15 y; birth year = 1948 +/- 14; sex- age-adjusted BMI = 36 +/- 8 kg/m2; male = 43%; diabetic = 59%. RESULTS: Neither polymorphism was significantly associated with diabetes in full heritage Pimas. Individuals with a 'CG' genotype at the Ser311Cys SNP had a higher BMI than those with a 'CC' genotype (36.7 vs 35.5 kg/m2, P= 0.04). Linkage analysis of BMI, adjusted for either polymorphism, resulted in LOD scores that were similar to those obtained without adjustment. CONCLUSION: Heterozygotes at the Ser311Cys DRD2 polymorphism had a slightly higher BMI than homozygotes, however neither the Ser311Cys nor the TaqIA polymorphism accounted for the linkage with BMI on chromosome 11 in Pima Indians.

Novel polymorphisms in the neuropeptide-Y Y5 receptor associated with obesity in Pima Indians.

OBJECTIVE: To investigate whether the neuropeptide Y receptor 5 gene (NPY5R) is associated with obesity in humans. DESIGN: The NPY5R gene was screened for polymorphisms by direct sequencing in two groups of Pima Indians, selected for extremes of body mass index (BMI). Genotype frequencies were analyzed for association with BMI extreme. SUBJECTS: Full-heritage Pima Indians, non-diabetic and not first degree relatives. Obese group: 19 M/24 F, BMI = 49+/-7 kg/m2 (mean+/-s.d.) age = 24+/-2 y, lean group: 16 M/16 F, BMI = 23+/-2 kg/m2, age = 27+/-3 y. MEASUREMENTS: Initially, the entire gene (proximal promoter, exon 1A, coding sequence, 5' and 3' UTRs) was sequenced in a subset of 20 individuals. No variants were found in the coding sequence, however three novel single nucleotide polymorphisms were detected in the non-coding regions: (1) a C-->T transition located within the promoter 28 bp upstream of the exon 1A transcription start site; (2) a T-->C transition 94 bp downstream of the stop codon; and (3) a G-->A transition 432 bp downstream of the stop codon. The polymorphisms were then screened in all 75 subjects. RESULTS: The polymorphisms had mean heterozygosities of 0.34-0.50 and were in strong linkage disequilibrium (P<0.001). Genotype frequencies differed significantly in lean and obese Pimas for P2 (P=0.04) and for a triple haplotype (P=0.02, Bonferroni corrected). CONCLUSION: Considering the importance of this gene in regulation of body weight, the association of these polymorphisms with extremes of BMI in Pima Indians indicates that NPY5R, or a locus nearby, may contribute to susceptibility to obesity in this population.

Metformin inhibits catecholamine-stimulated lipolysis in obese, hyperinsulinemic, hypertensive subjects in subcutaneous adipose tissue: an in situ microdialysis study.

AIMS: Metformin has been reported to decrease the plasma concentrations of non-esterified fatty acids in Type 2 diabetic subjects. This study investigated the effects of metformin on basal and catecholamine-stimulated lipolysis in abdominal subcutaneous adipose tissue of obese, hyperinsulinaemic, hypertensive subjects. METHODS: Fourteen subjects with severe obesity (12 female, twomale, age 35.4 +/- 4 years, body mass index 48.2 +/- 2 kg/m2, body fat mass 63.3 +/- 5 kg) were recruited. Glycerol and lactate concentrations were determined in the presence of metformin and after administration of catecholamines using microdialysis. Simultaneously, blood flow was assessed with the ethanol escape method. RESULTS: Glycerol release was lowered by metformin during the 3-h experiment (P<0.01). The lipolytic activity of catecholamines was suppressed when adipose tissue was pre-treated with metformin (P<0.001). Lactate concentration increased after application of metformin (P<0.01) and catecholamines (P<0.001). Blood flow was decreased in the presence of adrenaline (P < 0.01), but this effect was abolished by metformin. CONCLUSIONS: The present data demonstrate the effects of metformin on lipolysis in subcutaneous adipose tissue in vivo. In the large body fat mass of obese subjects, a reduction of lipolysis in adipose tissue may contribute to a decrease of VLDL synthesis in the liver resulting in a lowered plasma triglyceride concentration.

Induction of arginase isoforms in the lung during hyperoxia.

L-Arginine can be metabolized by nitric oxide (NO) synthase (NOS) to produce NO or by arginase to produce urea and L-ornithine. In the liver, arginase (the AI isoform) is a key enzyme in the urea cycle. In extrahepatic organs including the lung, the function of arginase (the AII isoform) is less clear. Because we found that lung AII was upregulated during 100% O2 exposure in preliminary experiments, we sought to characterize expression of the arginase isoforms and inducible NOS and to assess the functions of arginase in hyperoxic lung injury. Male Sprague-Dawley rats were exposed to 100% O2 for 60 h. Protein expression of AI and AII and their cellular distribution were determined. The activities of arginase and NOS were also measured. Expression of arginase was correlated with that of ornithine decarboxylase, a biochemical marker for tissue repair, in a separate group of rats allowed to recover in room air for 48 h. We found by Western blot analyses that both AI and AII proteins were upregulated after 60 h of hyperoxic exposure (403 and 88% increases by densitometry, respectively) and, like ornithine decarboxylase, remained elevated during the recovery phase. Arginase activity increased by 37%. Immunostaining showed that increases in AI and AII were mainly in the peribronchial and perivascular connective tissues. NOS activity was unchanged and inducible NOS was not induced, but the level of nitrogen oxides in the lung decreased by 67%. Our study showed in vivo induction of arginase isoforms during hyperoxia. The strong expression of arginase in the connective tissues suggests that the function of pulmonary arginase may be linked to connective tissue elements, e.g., fibroblasts, during lung injury and recovery.

Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima indians.

The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'-untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.