NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet.

Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.

Glyceollin Transcription Factor GmMYB29A2 Regulates Soybean Resistance to Phytophthora sojae.

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean (Glycine max) that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from P. sojae and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and GmMYB29A2, up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1, GmMYB29A1, and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing GmMYB29A1 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing GmMYB29A2 in Williams 82, a soybean variety that encodes the resistance gene Rps1k, rendered it compatible with race 1 P. sojae, whereas overexpressing GmMYB29A2 rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus, GmMYB29A2 is essential for accumulation of glyceollin I and expression of Phytophthora resistance.

Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver.

Lipid metabolism requires CoA, an essential cofactor found in multiple subcellular compartments, including the peroxisomes. In the liver, CoA levels are dynamically adjusted between the fed and fasted states. Elevated CoA levels in the fasted state are driven by increased synthesis; however, this also correlates with decreased expression of Nudix hydrolase (Nudt)7, the major CoA-degrading enzyme in the liver. Nudt7 resides in the peroxisomes, and we overexpressed this enzyme in mouse livers to determine its effect on the size and composition of the hepatic CoA pool in the fed and fasted states. Nudt7 overexpression did not change total CoA levels, but decreased the concentration of short-chain acyl-CoAs and choloyl-CoA in fasted livers, when endogenous Nudt7 activity was lowest. The effect on these acyl-CoAs correlated with a significant decrease in the hepatic bile acid content and in the rate of peroxisomal fatty acid oxidation, as estimated by targeted and untargeted metabolomics, combined with the measurement of fatty acid oxidation in intact hepatocytes. Identification of the CoA species and metabolic pathways affected by the overexpression on Nudt7 in vivo supports the conclusion that the nutritionally driven modulation of Nudt7 activity could contribute to the regulation of the peroxisomal CoA pool and peroxisomal lipid metabolism.

The NAC family transcription factor GmNAC42-1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean.

BACKGROUND: Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. RESULTS: Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42-1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42-1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42-1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42-1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42-1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. CONCLUSIONS: Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42-1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42-1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.

Genome-wide interrogation of hepatic FXR reveals an asymmetric IR-1 motif and synergy with LRH-1.

We used mouse hepatic chromatin enriched with an FXR antibody and chromatin immunoprecipitation-sequencing (ChIP-seq) to evaluate FXR binding on a genome-wide scale. This identified 1656 FXR-binding sites and 10% were located within 2 kb of a transcription start site which is much higher than predicted by random occurrence. A motif search uncovered a canonical nuclear receptor IR-1 site, consistent with in vitro DNA-binding studies reported previously. A separate nuclear receptor half-site for monomeric receptors such as LRH-1 was co-enriched and FXR activation of four newly identified promoters was significantly augmented by an LRH-1 expression vector in a co-transfection assay. There were 1038 genes located within 20 kb of a peak and a gene set enrichment analysis showed that genes identified by our ChIP-seq analysis are highly correlated with genes activated by an FXR-VP16 adenovirus in primary mouse hepatocytes providing functional relevance to the genome-wide binding study. Gene Ontology analysis showed FXR-binding sites close to many genes in lipid, fatty acid and steroid metabolism. Other broad gene clusters related to metabolism, transport, signaling and glycolysis were also significantly enriched. Thus, FXR may have a much wider role in cellular metabolism than previously appreciated.

Genome-wide analysis of SREBP-1 binding in mouse liver chromatin reveals a preference for promoter proximal binding to a new motif.

Lipid homeostasis in vertebrates is regulated by 3 sterol regulatory element binding protein (SREBP) isoforms. Here, we identify targets of SREBP-1 in mammalian liver using chromatin immunoprecipitation-high-throughput DNA sequencing. Antisera to SREBP-1 were used with liver chromatin from mice fed a high-carbohydrate diet after a fast, which leads to superinduction of hepatic SREBP-1c expression. SREBP-1-DNA complexes were subjected to massive parallel DNA sequencing using the Illumina Genome Analyzer II, resulting in 5.7 million sequence reads. Mapping these reads to the mouse reference genome identified 426 peaks of SREBP-1 binding vs. a control antibody. These binding peaks show a striking enrichment in proximal promoter regions, with 52% located within 1 kb upstream of a transcription start site. A previously undescribed sequence motif (5'-ACTACANNTCCC-3') was present in 76% of the total peaks, and we show that it is a functional SREBP-1 response element. Our analysis also reveals that an Sp1 consensus site is present as a "coregulatory" motif in 50% of the SREBP-1 binding peaks, consistent with previous functional studies. SREBP-1 bound not only to many well-characterized SREBP-1 target genes but to several other previously unknown targets in lipid and carbohydrate metabolism as well as many putative target genes in other diverse biological pathways.

Site-Dependent Lineage Preference of Adipose Stem Cells.

Adult stem cells have unique properties in both proliferation and differentiation preference. In this study, we hypothesized that adipose stem cells have a depot-dependent lineage preference. Four rabbits were used to provide donor-matched adipose stem cells from either subcutaneous adipose tissue (ScAT) or infrapatellar fat pad (IPFP). Proliferation and multi-lineage differentiation were evaluated in adipose stem cells from donor-matched ScAT and IPFP. RNA sequencing (RNA-seq) and proteomics were conducted to uncover potential molecular discrepancy in adipose stem cells and their corresponding matrix microenvironments. We found that stem cells from ScAT exhibited significantly higher proliferation and adipogenic capacity compared to those from donor-matched IPFP while stem cells from IPFP displayed significantly higher chondrogenic potential compared to those from donor-matched ScAT. Our findings are strongly endorsed by supportive data from transcriptome and proteomics analyses, indicating a site-dependent lineage preference of adipose stem cells.

Skeletal muscle reprogramming by breast cancer regardless of treatment history or tumor molecular subtype.

Increased susceptibility to fatigue is a negative predictor of survival commonly experienced by women with breast cancer (BC). Here, we sought to identify molecular changes induced in human skeletal muscle by BC regardless of treatment history or tumor molecular subtype using RNA-sequencing (RNA-seq) and proteomic analyses. Mitochondrial dysfunction was apparent across all molecular subtypes, with the greatest degree of transcriptomic changes occurring in women with HER2/neu-overexpressing tumors, though muscle from patients of all subtypes exhibited similar pathway-level dysregulation. Interestingly, we found no relationship between anticancer treatments and muscle gene expression, suggesting that fatigue is a product of BC per se rather than clinical history. In vitro and in vivo experimentation confirmed the ability of BC cells to alter mitochondrial function and ATP content in muscle. These data suggest that interventions supporting muscle in the presence of BC-induced mitochondrial dysfunction may alleviate fatigue and improve the lives of women with BC.

Educational intervention improves fruit and vegetable intake in young adults with metabolic syndrome components.

The FRUVEDomics study investigates the effect of a diet intervention focused on increasing fruit and vegetable intake on the gut microbiome and cardiovascular health of young adults with/at risk for metabolic syndrome (MetS). It was hypothesized that the recommended diet would result in metabolic and gut microbiome changes. The 9-week dietary intervention adhered to the US Department of Agriculture Dietary Guidelines for Americans and focused on increasing fruit and vegetable intake to equal half of the diet. Seventeen eligible young adults with/or at high risk of MetS consented and completed preintervention and postintervention measurements, including anthropometric, body composition, cardiovascular, complete blood lipid panel, and collection of stool sample for microbial analysis. Participants attended weekly consultations to assess food logs, food receipts, and adherence to the diet. Following intention-to-treat guidelines, all 17 individuals were included in the dietary, clinical, and anthropometric analysis. Fruit and vegetable intake increased from 1.6 to 3.4 cups of fruits and vegetables (P < .001) daily. Total fiber (P = .02) and insoluble fiber (P < .0001) also increased. Clinical laboratory changes included an increase in sodium (P = .0006) and low-density lipoprotein cholesterol (P = .04). In the fecal microbiome, Erysipelotrichaceae (phylum Firmicutes) decreased (log2 fold change: -1.78, P = .01) and Caulobacteraceae (phylum Proteobacteria) increased (log2 fold change = 1.07, P = .01). Implementing a free-living 9-week diet, with intensive education and accountability, gave young adults at high risk for/or diagnosed with MetS the knowledge, skills, and feedback to improve diet. To yield greater impact, a longer diet intervention may be needed in this population.

Human Breast Cancer Xenograft Model Implicates Peroxisome Proliferator-activated Receptor Signaling as Driver of Cancer-induced Muscle Fatigue.

PURPOSE: This study tested the hypothesis that a patient-derived orthotopic xenograft (PDOX) model would recapitulate the common clinical phenomenon of breast cancer-induced skeletal muscle (SkM) fatigue in the absence of muscle wasting. This study additionally sought to identify drivers of this condition to facilitate the development of therapeutic agents for patients with breast cancer experiencing muscle fatigue. EXPERIMENTAL DESIGN: Eight female BC-PDOX-bearing mice were produced via transplantation of tumor tissue from 8 female patients with breast cancer. Individual hind limb muscles from BC-PDOX mice were isolated at euthanasia for RNA-sequencing, gene and protein analyses, and an ex vivo muscle contraction protocol to quantify tumor-induced aberrations in SkM function. Differentially expressed genes (DEG) in the BC-PDOX mice relative to control mice were identified using DESeq2, and multiple bioinformatics platforms were employed to contextualize the DEGs. RESULTS: We found that SkM from BC-PDOX-bearing mice showed greater fatigability than control mice, despite no differences in absolute muscle mass. PPAR, mTOR, IL6, IL1, and several other signaling pathways were implicated in the transcriptional changes observed in the BC-PDOX SkM. Moreover, 3 independent in silico analyses identified PPAR signaling as highly dysregulated in the SkM of both BC-PDOX-bearing mice and human patients with early-stage nonmetastatic breast cancer. CONCLUSIONS: Collectively, these data demonstrate that the BC-PDOX model recapitulates the expected breast cancer-induced SkM fatigue and further identify aberrant PPAR signaling as an integral factor in the pathology of this condition.

Association analysis of Krüppel-like factor 11 variants with type 2 diabetes in Pima Indians.

CONTEXT: Krüppel-like factor 11 (KLF11) is a transcription factor of the zinc finger domain family that has been shown to regulate expression of the insulin gene. An initial study reported that a KLF11 variant predicting a Q62R was associated with type 2 diabetes (T2D) in French Caucasians; however, subsequent studies have failed to identify an association between this variant and T2D in subjects from a similar Northern-European ancestry. OBJECTIVE: We sought to determine whether the Q62R or other variants within KLF11 were associated with T2D in Pima Indians, a population with an extremely high prevalence of this disease. DESIGN, SETTING, AND SUBJECTS: KLF11 was sequenced in 24 Pima Indians to identify potentially novel variants. There were 18 variants genotyped in a family-based sample of 1337 Pima Indians to analyze the linkage disequilibrium pattern of this gene and identify representative variants. Four representative variants were further genotyped in a population-based sample of 3501 full-heritage Pima Indians for association analyses. Among these subjects, 413 had undergone metabolic studies when they were nondiabetic to measure traits that predict T2D. RESULTS: Neither the Q62R nor any other common variant in KLF11 was associated with T2D in the Pima population. In addition, no variant was associated with insulin secretion or insulin-stimulated glucose disposal rate. CONCLUSIONS: Common variation in KLF11 variation does not appear to influence the population-based risk for developing T2D among full-heritage Pima Indians. Thus, KLF11 is unlikely to play a major role in the etiology of T2D among this Native American population.

Dysregulation of metabolic-associated pathways in muscle of breast cancer patients: preclinical evaluation of interleukin-15 targeting fatigue.

BACKGROUND: Breast cancer patients report a perception of increased muscle fatigue, which can persist following surgery and standardized therapies. In a clinical experiment, we tested the hypothesis that pathways regulating skeletal muscle fatigue are down-regulated in skeletal muscle of breast cancer patients and that different muscle gene expression patterns exist between breast tumour subtypes. In a preclinical study, we tested the hypothesis that mammary tumour growth in mice induces skeletal muscle fatigue and that overexpression of the cytokine interleukin-15 (IL-15) can attenuate mammary tumour-induced muscle fatigue. METHODS: Early stage non-metastatic female breast cancer patients (n = 14) and female non-cancer patients (n = 6) provided a muscle biopsy of the pectoralis major muscle during mastectomy, lumpectomy, or breast reconstruction surgeries. The breast cancer patients were diagnosed with either luminal (ER+ /PR+ , n = 6), triple positive (ER+ /PR+ /Her2/neu+ , n = 5), or triple negative (ER- /PR- /Her2/neu- , n = 3) breast tumours and were being treated with curative intent either with neoadjuvant chemotherapy followed by surgery or surgery followed by standard post-operative therapy. Biopsies were used for RNA-sequencing to compare the skeletal muscle gene expression patterns between breast cancer patients and non-cancer patients. The C57BL/6 mouse syngeneic mammary tumour cell line, E0771, was used to induce mammary tumours in immunocompetent mice, and isometric muscle contractile properties and fatigue properties were analysed following 4 weeks of tumour growth. RESULTS: RNA-sequencing and subsequent bioinformatics analyses revealed a dysregulation of canonical pathways involved in oxidative phosphorylation, mitochondrial dysfunction, peroxisome proliferator-activated receptor signalling and activation, and IL-15 signalling and production. In a preclinical mouse model of breast cancer, the rate of muscle fatigue was greater in mice exposed to mammary tumour growth for 4 weeks, and this greater muscle fatigue was attenuated in transgenic mice that overexpressed the cytokine IL-15. CONCLUSIONS: Our data identify novel genes and pathways dysregulated in the muscles of breast cancer patients with early stage non-metastatic disease, with particularly aberrant expression among genes that would predispose these patients to greater muscle fatigue. Furthermore, we demonstrate that IL-15 overexpression can attenuate muscle fatigue associated with mammary tumour growth in a preclinical mouse model of breast cancer. Therefore, we propose that skeletal muscle fatigue is an inherent consequence of breast tumour growth, and this greater fatigue can be targeted therapeutically.

CHRM3 gene variation is associated with decreased acute insulin secretion and increased risk for early-onset type 2 diabetes in Pima Indians.

The muscarinic acetylcholine receptor subtype M3 (CHRM3) gene is expressed in islet beta-cells and has a role in stimulating insulin secretion; therefore, CHRM3 was analyzed as a candidate gene for type 2 diabetes in Pima Indians. Ten variants were genotyped in a family-based sample (n = 1,037), and 1 variant (rs3738435) located in the 5' untranslated region of an alternative transcript was found to be modestly associated with both early-onset type 2 diabetes and the acute insulin response in a small subset of these subjects. To better assess whether this variant has a role in acute insulin secretion, which could affect risk for early-onset type 2 diabetes, rs3738435 was genotyped in a larger group of normal glucose-tolerant Pima Indians who had measures of acute insulin secretion (n = 282) and a larger case-control group of Pima Indians selected for early-onset type 2 diabetes (n = 348 case subjects with age of onset <25 years; n = 392 nondiabetic control subjects aged >45 years). Genotyping in these larger sets of subjects confirmed that the C allele of rs3738435 was associated with a reduced acute insulin response (adjusted P = 0.00006) and was also modestly associated with increased risk of early-onset type 2 diabetes (adjusted P = 0.02).

Variations in peptide YY and Y2 receptor genes are associated with severe obesity in Pima Indian men.

Peptide YY (PYY) and Y2 receptor (Y2R) may be important in the central regulation of body weight and food intake. To determine whether genetic variation in PYY and/or Y2R may contribute to morbid obesity in humans, these genes were sequenced in 83 extremely obese Pima Indians (BMI > or = 50 kg/m2). Sequencing of PYY identified three single nucleotide polymorphisms (SNPs) in the untranslated region. Sequencing of the Y2R coding region identified one missense (Ala172Thr) substitution and two silent substitutions. Eight additional SNPs in the 5' untranslated region of Y2R were identified from public databases. These SNPs were genotyped in 489 full-heritage adult Pimas (362 severely obese and 127 nondiabetic, nonobese subjects), who are not first-degree relatives, for association analysis. The PYY variants were not associated with obesity, whereas four variants from two haplotype blocks in Y2R were marginally associated (P = 0.054-0.067) with obesity. However, if the analysis was restricted to men (n = 167, 100 obese and 67 lean), the PYY variants and two SNPs in Y2R that were in complete linkage disequilibrium were significantly associated with severe obesity (P = 0.001 and P = 0.002, respectively). Our data suggest that the PYY-Y2R pathway may influence body weight through a sex-specific mechanism, but this finding requires confirmation in other populations.

Variants in hepatocyte nuclear factor 4alpha are modestly associated with type 2 diabetes in Pima Indians.

Single nucleotide polymorphisms (SNPs) within the hepatocyte nuclear factor 4alpha (HNF4alpha) gene are associated with type 2 diabetes in Finns and Ashkenazi Jews. Previous studies in both populations have reported linkage to type 2 diabetes near the HNF4alpha locus on chromosome 20q12-13. To investigate whether HNF4alpha is a diabetes susceptibility gene in Pima Indians, a population with the highest reported prevalence of type 2 diabetes but with no evidence for linkage of the disease on chromosome 20q, 19 SNPs across the promoter and coding region of HNF4alpha were genotyped for association analysis. In a group of 1,037 Pima Indians (573 diabetic and 464 nondiabetic subjects), three SNPs in HNF4alpha (rs3212183 and rs2071197 located in introns 3 and 1, respectively, and rs6031558, an extremely rare SNP located in the P2 promoter region) were modestly associated with type 2 diabetes (rs3212183 odds ratio [OR] 1.34 [95% CI 1.07-1.67], P = 0.009; rs2071197 1.34 [1.07-1.66], P = 0.008; and rs6031558 3.18 [1.03-9.84], P = 0.04, adjusted for age, sex, birth year, heritage, and family membership). We conclude that variants in HNF4alpha do not appear to be major determinants for type 2 diabetes in Pima Indians; however, HNF4alpha may have a minor role in type 2 diabetes susceptibility within this Native American population.

A novel missense substitution (Val1483Ile) in the fatty acid synthase gene (FAS) is associated with percentage of body fat and substrate oxidation rates in nondiabetic Pima Indians.

Inhibition of fatty acid synthase (FAS) induces a rapid decline in fat stores in mice, suggesting a role for this enzyme in energy homeostasis. The human FAS gene (FAS) maps to chromosome 17q25, a region previously shown to have suggestive linkage to adiposity in a genome-wide linkage scan for genetic determinants of obesity in Pima Indians. To investigate the potential role of FAS in the pathophysiology of human obesity, the FAS gene was sequenced and 13 single nucleotide polymorphisms (SNPs) were identified. Five representative SNPs were genotyped in 216 full-blooded, nondiabetic Pima Indians for association analyses. A Val1483Ile polymorphism (GTC to ATC; allele frequency of A = 0.10) was associated with percentage of body fat and 24-h substrate oxidation rates measured in a respiratory chamber. Compared with homozygotes for the Val variant, subjects with Ile/x had a lower mean percentage of body fat (30 +/- 1 vs. 33 +/- 1%, P = 0.002; adjusted for age, sex, and family membership) and a lower mean carbohydrate oxidation rate (983 +/- 41 vs. 1,094 +/- 19 kcal/day, P = 0.03), which resulted in a lower mean 24-h respiratory quotient (0.845 +/- 0.01 vs. 0.850 +/- 0.01 kcal/day, P = 0.04; both adjusted for age, sex, family membership, percentage of body fat, and energy balance). Our findings indicate that the Val1483Ile substitution in FAS is protective against obesity in Pima Indians, an effect possibly explained by the role of this gene in the regulation of substrate oxidation.

Childhood predictors of young-onset type 2 diabetes.

OBJECTIVE: Optimal prevention of young-onset type 2 diabetes requires identification of the early-life modifiable risk factors. We aimed to do this using longitudinal data in 1,604 5- to 19-year-old initially nondiabetic American Indians. RESEARCH DESIGN AND METHODS: For type 2 diabetes prediction, we derived an optimally weighted, continuously distributed, standardized multivariate score (zMS) comprising commonly measured metabolic, anthropometric, and vascular traits (i.e., fasting and 2-h glucose, A1C, BMI, waist circumference, fasting insulin, HDL cholesterol, triglycerides, and blood pressures) and compared the predictive power for each feature against zMS. RESULTS: In separate Cox proportional hazard models, adjusted for age, sex, and ethnicity, zMS and each of its component risk factors were associated with incident type 2 diabetes. Stepwise proportional hazards models selected fasting glucose, 2-h glucose, HDL cholesterol, and BMI as independent diabetes predictors; individually, these were weaker predictors than zMS (P < 0.01). However, a parsimonious summary score combining only these variables had predictive power similar to that of zMS (P = 0.33). Although intrauterine diabetes exposure or parental history of young-onset diabetes increased a child's absolute risk of developing diabetes, the magnitude of the diabetes-risk relationships for zMS and the parsimonious score were similar irrespective of familial risk factors. CONCLUSIONS: We have determined the relative value of the features of the metabolic syndrome in childhood for the prediction of subsequent type 2 diabetes. Our findings suggest that strategies targeting obesity, dysregulated glucose homeostasis, and low HDL cholesterol during childhood and adolescence may have the most success in preventing diabetes.

Variants in ARHGEF11, a candidate gene for the linkage to type 2 diabetes on chromosome 1q, are nominally associated with insulin resistance and type 2 diabetes in Pima Indians.

A prior genome-wide linkage scan in Pima Indians indicated a young-onset (aged <45 years) type 2 diabetes susceptibility locus on chromosome 1q21-q23. ARHGEF11, which encodes the Rho guanine nucleotide exchange factor 11, was analyzed as a positional candidate gene for this linkage because this protein may stimulate Rho-dependent signals, such as the insulin signaling cascade. The ARHGEF11 gene, and two adjacent genes NTRK1 and INSRR, were sequenced in 24 Pima Indians who were not first-degree relatives. Sequencing of the coding regions, 5' and 3' untranslated regions and putative promoter regions of these genes, identified 28 variants in ARHGEF11, 11 variants in NTRK1, and 8 variants in INSSR. These 47 variants, as well as 84 additional public database variants within/between these genes, were genotyped for association analysis in the same group of Pima Indians who had participated in the linkage study (n = 1,228). An R1467H in ARHGEF11, and several additional noncoding variants that were in high linkage disequilibrium with this variant, were nominally associated with young-onset type 2 diabetes (P = 0.01; odds ratio 3.39) after adjusting for sex, family membership, and Pima heritage. The risk allele H had a frequency of 0.10. In a subgroup of 262 nondiabetic, full-heritage Pima Indians who had undergone detailed metabolic testing, the risk allele H also was associated with a lower mean insulin-mediated glucose disposal rate and a lower mean nonoxidative glucose storage rate after adjusting for age, sex, nuclear family membership, and percentage of body fat (P < or = 0.01). These findings suggest that variation within ARHGEF11 nominally increases risk of type 2 diabetes, possibly as a result of increased insulin resistance.

A search for variants associated with young-onset type 2 diabetes in American Indians in a 100K genotyping array.

OBJECTIVE: To identify genetic variants in linkage disequilibrium with those conferring diabetes susceptibility, a genome-wide association study for young-onset diabetes was conducted in an American-Indian population. RESEARCH DESIGN AND METHODS: Data come from 300 case subjects with type 2 diabetes with age of onset <25 years and 334 nondiabetic control subjects aged >or=45 years. To provide for tests of within-family association, 121 nondiabetic siblings of case subjects were included along with 140 diabetic siblings of control subjects (172 sibships). Individuals were genotyped on the Affymetrix 100K array, resulting in 80,044 usable single nucleotide polymorphisms (SNPs). SNPs were analyzed for within-family association and for general association in case and control subjects, and these tests were combined by Fisher's method, with priority given to the within-family test. RESULTS: There were more SNPs with low P values than expected theoretically under the global null hypothesis of no association, and 128 SNPs had evidence for association at P < 0.001. The association of these SNPs with diabetes was further investigated in 1,207 diabetic and 1,627 nondiabetic individuals from the population study who were not included in the genome-wide study. SNPs from 10 genomic regions showed evidence for replication at P < 0.05. These included SNPs on chromosome 3 near ZNF659, chromosome 11 near FANCF, chromosome 11 near ZBTB15, and chromosome 12 near SENP1. CONCLUSIONS: These studies suggest several regions where marker alleles are potentially in linkage disequilibrium with variants that confer susceptibility to young-onset type 2 diabetes in American Indians.