Genetic perturbation of key central metabolic genes extends lifespan in Drosophila and affects response to dietary restriction.

There is a connection between nutrient inputs, energy-sensing pathways, lifespan variation and aging. Despite the role of metabolic enzymes in energy homeostasis and their metabolites as nutrient signals, little is known about how their gene expression impacts lifespan. In this report, we use P-element mutagenesis in Drosophila to study the effect on lifespan of reductions in expression of seven central metabolic enzymes, and contrast the effects on normal diet and dietary restriction. The major observation is that for five of seven genes, the reduction of gene expression extends lifespan on one or both diets. Two genes are involved in redox balance, and we observe that lower activity genotypes significantly extend lifespan. The hexokinases also show extension of lifespan with reduced gene activity. Since both affect the ATP/ADP ratio, this connects with the role of AMP-activated protein kinase as an energy sensor in regulating lifespan and mediating caloric restriction. These genes possess significant expression variation in natural populations, and our experimental genotypes span this level of natural activity variation. Our studies link the readout of energy state with the perturbation of the genes of central metabolism and demonstrate their effect on lifespan.

A genome-wide association study for diabetic nephropathy genes in African Americans.

A genome-wide association study was performed using the Affymetrix 6.0 chip to identify genes associated with diabetic nephropathy in African Americans. Association analysis was performed adjusting for admixture in 965 type 2 diabetic African American patients with end-stage renal disease (ESRD) and in 1029 African Americans without type 2 diabetes or kidney disease as controls. The top 724 single nucleotide polymorphisms (SNPs) with evidence of association to diabetic nephropathy were then genotyped in a replication sample of an additional 709 type 2 diabetes-ESRD patients and 690 controls. SNPs with evidence of association in both the original and replication studies were tested in additional African American cohorts consisting of 1246 patients with type 2 diabetes without kidney disease and 1216 with non-diabetic ESRD to differentiate candidate loci for type 2 diabetes-ESRD, type 2 diabetes, and/or all-cause ESRD. Twenty-five SNPs were significantly associated with type 2 diabetes-ESRD in the genome-wide association and initial replication. Although genome-wide significance with type 2 diabetes was not found for any of these 25 SNPs, several genes, including RPS12, LIMK2, and SFI1 are strong candidates for diabetic nephropathy. A combined analysis of all 2890 patients with ESRD showed significant association SNPs in LIMK2 and SFI1 suggesting that they also contribute to all-cause ESRD. Thus, our results suggest that multiple loci underlie susceptibility to kidney disease in African Americans with type 2 diabetes and some may also contribute to all-cause ESRD.

Identification of genetic modifiers of lifespan on a high sugar diet in the Drosophila Genetic Reference Panel.

Genome-wide association studies (GWAS) have become beneficial in identifying genetic variants underlying susceptibility to various complex diseases and conditions, including obesity. Utilizing the Drosophila Genetic Reference Panel (DGRP), we performed a GWAS of lifespan of 193 genetically distinct lines on a high sugar diet (HSD). The DGRP analysis pipeline determined the most significant lifespan associated polymorphisms were within loci of genes involved in: neural processes, behavior, development, and apoptosis, among other functions. Next, based on the relevance to obesity pathology, and the availability of transgenic RNAi lines targeting the genes we identified, whole-body in vivo knockdown of several candidate genes was performed. We utilized the GAL4-UAS binary expression system to independently validate the impacts of these loci on Drosophila lifespan during HSD. These loci were largely confirmed to affect lifespan in that HSD setting, as well as a normal diet setting. However, we also detected unexpected dietary effects of the HSD, including inconsistent diet effects on lifespan relative to a normal diet and a strong downregulation of feeding quantity.

Polymorphisms near SOCS3 are associated with obesity and glucose homeostasis traits in Hispanic Americans from the Insulin Resistance Atherosclerosis Family Study.

The SOCS3 gene product participates in the feedback inhibition of a range of cytokine signals. Most notably, SOCS3 inhibits the functioning of leptin and downstream steps in insulin signaling after being expressed by terminal transcription factors, such as STAT3 and c-fos. The SOCS3 gene is located in the chromosome region 17q24-17q25, previously linked to body mass index (BMI), visceral adipose tissue (VAT), and waist circumference (WAIST) in Hispanic families in the Insulin Resistance Atherosclerosis Family Study (IRASFS). A high density map of 1,536 single nucleotide polymorphisms (SNPs) was constructed to cover a portion of the 17q linkage interval in 1,425 Hispanic subjects from 90 extended families in IRASFS. Analysis of this dense SNP map data revealed evidence of association of rs9914220 (located 10 kb 5' of the SOCS3 gene) with BMI, VAT, and WAIST (P-value ranging from 0.003 to 0.017). Using a tagging SNP approach, rs9914220 and 22 additional SOCS3 SNPs were genotyped for genetic association analysis with measures of adiposity and glucose homeostasis. The adiposity phenotypes utilized in association analyses included BMI, WAIST, waist to hip ratio (WHR), subcutaneous adipose tissue, VAT, and visceral to subcutaneous ratio (VSR). Linkage disequilibrium calculations revealed three haplotype blocks near SOCS3. Haplotype Block 3 (5' of SOCS3) contained SNPs consistently associated with BMI, WAIST, WHR, and VAT (P-values ranging from 2.00 x 10(-4) to 0.036). Haplotype Block 1 contained single-SNPs that were associated with most adiposity traits except for VSR (P-values ranging from 0.002 to 0.047). When trait associated SNPs were included in linkage analyses as covariates, a reduction of VAT LOD score from 1.26 to 0.76 above the SOCS3 locus (110 cM) was observed. Multi-SNP haplotype testing using the quantitative pedigree disequilibrium test was broadly consistent with the single-SNP associations. In conclusion, these results support a role for SOCS3 genetic variants in human obesity.

A high-fat diet impacts memory and gene expression of the head in mated female Drosophila melanogaster.

Obesity predisposes humans to a range of life-threatening comorbidities, including type 2 diabetes and cardiovascular disease. Obesity also aggravates neural pathologies, such as Alzheimer's disease, but this class of comorbidity is less understood. When Drosophila melanogaster (flies) are exposed to high-fat diet (HFD) by supplementing a standard medium with coconut oil, they adopt an obese phenotype of decreased lifespan, increased triglyceride storage, and hindered climbing ability. The latter development has been previously regarded as a potential indicator of neurological decline in fly models of neurodegenerative disease. Our objective was to establish the obesity phenotype in Drosophila and identify a potential correlation, if any, between obesity and neurological decline through behavioral assays and gene expression microarray. We found that mated female w1118 flies exposed to HFD maintained an obese phenotype throughout adult life starting at 7 days, evidenced by increased triglyceride stores, diminished life span, and impeded climbing ability. While climbing ability worsened cumulatively between 7 and 14 days of exposure to HFD, there was no corresponding alteration in triglyceride content. Microarray analysis of the mated female w1118 fly head revealed HFD-induced changes in expression of genes with functions in memory, metabolism, olfaction, mitosis, cell signaling, and motor function. Meanwhile, an Aversive Phototaxis Suppression assay in mated female flies indicated reduced ability to recall an entrained memory 6 h after training. Overall, our results support the suitability of mated female flies for examining connections between diet-induced obesity and nervous or neurobehavioral pathology, and provide many directions for further investigation.

A genome-wide association search for type 2 diabetes genes in African Americans.

African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations.

RGS6 variants are associated with dietary fat intake in Hispanics: the IRAS Family Study.

Recently, a genome-wide association scan was completed in the IRAS (Insulin Resistance Atherosclerosis Study) Family Study (IRASFS) Hispanic-American cohort. Multiple single-nucleotide polymorphisms (SNPs) in the G-protein signaling 6 (RGS6) gene were found to be associated with adiposity phenotypes. RGS6 has shown downstream antagonistic interplay with opioid receptors, targets of fatty/sugary food agonists. The possibility that RGS6 promotes tolerance and tachyphylaxis among the opioid receptor is a plausible pathway for overconsuming fat/sugar-laden food. Therefore, we hypothesized that RGS6 variants are associated with intake of fatty/sugary foods. In 932 Hispanics from San Antonio and San Luis Valley, CO, the following dietary intake variables were assessed using the Block Brief 2000 food frequency questionnaire: total calories, total fat, % calories from fat, % calories from saturated fat, protein, % calories from protein, carbohydrates, % calories from carbohydrates, and daily frequency of servings of fats/oils/sweets. We tested for association between 23 SNPs in RGS6 and dietary intake using a variance components measured genotype approach. All models were adjusted for gender, recruitment site, admixture, BMI, and age. Using an additive genetic model, rs1402064 was associated with higher intake of fats/oils/sweets, total calories, total fat and saturated fat (P = 0.0007, 0.026, 0.023, and 0.024). SNPs rs847330 and rs847354 were associated with higher intake of fats/oils/sweets (P = 0.002 and 0.018), total fat (P = 0.040 and 0.048) and saturated fat (P = 0.044 and 0.041). Finally, rs769148 was associated with higher intake of fats/oils/sweets (P = 0.002). RGS6 is a new candidate gene for adiposity traits that may be associated with a behavioral tendency toward fat-laden food intake.

Functional ENTPD1 polymorphisms in African Americans with diabetes and end-stage renal disease.

OBJECTIVE: The vascular ectonucleotidase ENTPD1 protects against renal injury and modulates glucose homeostasis in mouse models. We sought to determine whether human variation in ENTPD1 influences predisposition to diabetes or diabetic nephropathy. RESEARCH DESIGN AND METHODS: We analyzed ENTPD1 single nucleotide polymorphisms (SNPs) in 363 African American control subjects, 380 subjects with type 2 diabetes and end-stage renal disease (DM-ESRD), and 326 subjects with ESRD unrelated to diabetes (non-DM-ESRD). Using human cell lines, we correlated disease-associated ENTPD1 haplotypes with ENTPD1 gene expression. Finally, we studied consequences of ENTPD1 deletion in a mouse model of type 2 diabetes (db/db). RESULTS: A common ENTPD1 two-SNP haplotype was associated with increased risk for DM-ESRD (P = 0.0027), and an uncommon four-SNP haplotype was associated with protection against DM-ESRD (P = 0.004). These haplotypes correlated with ENTPD1 gene expression levels in human cell lines in vitro. Subjects with high ENTPD1-expressing haplotypes were enriched in the DM-ESRD group. By crossing ENTPD1-null mice with db mice, we show that ENTPD1 deletion has prominent effects on metabolic syndrome traits. Specifically, deletion of ENTPD1 lowered glucose levels in control (db/-) mice with one functional leptin receptor and dramatically lowered weights in db/db mice with no functional leptin receptors. Similar effects were seen in aged ENTPD1-null mice with normal leptin receptors. CONCLUSIONS: ENTPD1 polymorphisms appear to influence susceptibility to type 2 diabetes and/or diabetic nephropathy in African Americans. Studies in human cell lines and in vivo mouse data support a potential role for ENTPD1 genetic variation in susceptibility to type 2 diabetes.

INSIG2 SNPs associated with obesity and glucose homeostasis traits in Hispanics: the IRAS Family Study.

The genome-wide association study by Herbert et al. identified the INSIG2 single-nucleotide polymorphism (SNP) rs7566605 as contributing to increased BMI in ethnically distinct cohorts. The present study sought to further clarify the matter, by testing whether SNPs of INSIG2 influenced quantitative adiposity or glucose homeostasis traits in Hispanics of the Insulin Resistance Atherosclerosis Family Study (IRASFS). Using a tagging SNP approach, rs7566605 and 31 additional SNPs were genotyped in 1,425 IRASFS Hispanics. SNPs were tested for association with six adiposity measures: BMI, waist circumference (WAIST), waist-to-hip ratio (WHR), subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and VAT to SAT ratio (VSR). SNPs were also tested for association with fasting glucose (GFAST), fasting insulin (FINS), and three measures obtained from the frequently sampled intravenous glucose tolerance test: insulin sensitivity (S(I)), acute insulin response (AIR), and disposition index (DI). Most prominent association was observed with direct computed tomography (CT)-measured adiposity phenotypes, including VAT, SAT, and VSR (P values range from 0.007 to 0.044 for rs17586756, rs17047718, rs17047731, rs9308762, rs12623648, and rs11673900). Multiple SNP associations were observed with all glucose homeostasis traits (P values range from 0.001 to 0.031 for rs17047718, rs17047731, rs2161829, rs10490625, rs889904, and rs12623648). Using BMI as a covariate in evaluation of glucose homeostasis traits slightly reduced their association. However, association with adiposity and glucose homeostasis phenotypes is not significant following multiple comparisons adjustment. Trending association after multiple comparisons adjustment remains suggestive of a role for genetic variation of INSIG2 in obesity, but these results require validation.

Genome-wide association study and follow-up analysis of adiposity traits in Hispanic Americans: the IRAS Family Study.

We investigated candidate genomic regions associated with computed tomography (CT)-derived measures of adiposity in Hispanics from the Insulin Resistance Atherosclerosis Study Family Study (IRASFS). In 1,190 Hispanic individuals from 92 families 3 from the San Luis Valley, Colorado and San Antonio, Texas, we measured CT-derived visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and visceral:subcutaneous ratio (VSR). A genome-wide association study (GWAS) was completed using the Illumina HumanHap 300 BeadChip (approximately 317K single-nucleotide polymorphisms (SNPs)) in 229 individuals from the San Antonio site (stage 1). In total, 297 SNPs with evidence for association with VAT, SAT, or VSR, adjusting for age and sex (P<0.001), were genotyped in the remaining 961 Hispanic samples. The entire Hispanic cohort (n=1,190) was then tested for association, adjusting for age, sex, site of recruitment, and admixture estimates (stage 2). In stage 3, additional SNPs were genotyped in four genic regions showing evidence of association in stage 2. Several SNPs were associated in the GWAS (P<1x10(-5)) and were confirmed to be significantly associated in the entire Hispanic cohort (P<0.01), including: rs7543757 for VAT, rs4754373 and rs11212913 for SAT, and rs4541696 and rs4134351 for VSR. Numerous SNPs were associated with multiple adiposity phenotypes. Targeted analysis of four genes whose SNPs were significant in stage 2 suggests candidate genes for influencing the distribution (RGS6) and amount of adiposity (NGEF). Several candidate loci, including RGS6 and NGEF, are associated with CT-derived adipose fat measures in Hispanic Americans in a three-stage genetic association study.