Saraswathy Nair

Amyloid precursor protein expression is upregulated in adipocytes in obesity.

The aim of this study was to determine whether amyloid precursor protein (APP) is expressed in human adipose tissue, dysregulated in obesity, and related to insulin resistance and inflammation. APP expression was examined by microarray expression profiling of subcutaneous abdominal adipocytes (SAC) and cultured preadipocytes from obese and nonobese subjects. Quantitative real‐time PCR (QPCR) was performed to confirm differences in APP expression in SAC and to compare APP expression levels in adipose tissue, adipocytes, and stromal vascular cells (SVCs) from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) specimens. Adipose tissue samples were also examined by western blot and immunofluorescence confocal microscopy. Microarray studies demonstrated that APP mRNA expression levels were higher in SAC (∼2.5‐fold) and preadipocytes (∼1.4) from obese subjects. Real‐time PCR confirmed increased APP expression in SAC in a separate group of obese compared with nonobese subjects (P = 0.02). APP expression correlated to in vivo indices of insulin resistance independently of BMI and with the expression of proinflammatory genes, such as monocyte chemoattractant protein‐1 (MCP‐1) (R = 0.62, P = 0.004), macrophage inflammatory protein‐1α (MIP‐1α) (R = 0.60, P = 0.005), and interleukin‐6 (IL‐6) (R = 0.71, P = 0.0005). Full‐length APP protein was detected in adipocytes by western blotting and APP and its cleavage peptides, Aβ40 and Aβ42, were observed in SAT and VAT by immunofluorescence confocal microscopy. In summary, APP is highly expressed in adipose tissue, upregulated in obesity, and expression levels correlate with insulin resistance and adipocyte cytokine expression levels. These data suggest a possible role for APP and/or Aβ in the development of obesity‐related insulin resistance and adipose tissue inflammation.

Differential Expression of Matrix Metalloproteinase 3 (MMP3) in Preadipocytes/Stromal Vascular Cells From Nonobese Nondiabetic Versus Obese Nondiabetic Pima Indians

Prior microarray studies comparing global gene expression patterns in preadipocytes/stromal vascular cells isolated from nonobese nondiabetic versus obese nondiabetic Pima Indians showed that matrix metalloproteinase 9 (MMP9) is upregulated in obese subjects. The current study targeted analysis of nine additional MMP genes that cluster to a region on chromosome 11q22 that is linked to BMI and percent body fat. Differential-display PCR showed that MMP3 is downregulated in preadipocytes/stromal vascular cells from obese subjects, and real-time PCR showed that MMP3 expression levels are negatively correlated with percent body fat. To determine whether variants within MMP3 are responsible for its altered expression, MMP3 was sequenced, and seven representative variants were genotyped in 1,037 Pima subjects for association analyses. Two variants were associated with both BMI and type 2 diabetes, and two additional variants were associated with type 2 diabetes alone; however, none of these variants were associated with MMP3 expression levels. We propose that the MMP3 pathway is altered in human obesity, but this alteration may be the result of a combination of genetic variation within the MMP3 locus itself, as well as variation in additional factors, either primary or secondary to obesity, that regulate expression of the MMP3 gene.

Host susceptibility to tuberculosis: insights from a longitudinal study of gene expression in diabetes.

Tuberculosis (TB) remains a major global disease, and diabetes, which is documented to increase susceptibility to TB threefold, is also becoming pandemic. This susceptibility has been attracting extensive research interest. The increased risk of TB in diabetes may serve as a unique model to understand host susceptibility to specific pathogens in humans. To examine this rationale, we investigated the expression of reported TB candidate genes in a longitudinal diabetes study. Two genes, HK2 and CD28, emerged as potential culprits in diabetes-increased TB susceptibility.

Decreased expression of ATP6V1H in type 2 diabetes: A pilot report on the diabetes risk study in Mexican Americans

OBJECTIVE Previous studies in mice and humans observed down-regulation of the gene expression of ATP6V1H associated with type 2 diabetes. This study identified prospectively changes in ATP6V1H expression before and after overt diabetes. METHODS Expression of ATP6V1H in peripheral blood was compared pre and post development of diabetes in nine individuals. RESULTS Considerable variation of ATP6V1H mRNA levels was observed between different individuals. However, within each individual the decrease in expression of ATP6V1H with the development of diabetes was highly statistically significant. CONCLUSIONS ATP6V1H may represent a critical molecular mechanism involved in the development of type 2 diabetes and its compilations through its important regulatory effect on vacuolar-ATPase activity.

Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A260/A280 between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A260/A280 ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A260/A280 measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.

Association of a serotonin transporter gene (SLC6A4) 5-HTTLPR polymorphism with body mass index categories but not type 2 diabetes mellitus in Mexicans

The serotonergic system has been hypothesized to contribute to the biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI) categories. We investigate a possible association of 5-HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter gene (SLC6A4) with the development of T2DM and/or higher BMI by analyzing a sample of 138 individuals diagnosed with T2DM and 172 unrelated controls from the Mexican general population. In the total sample genotypes were distributed according to Hardy-Weinberg equilibrium, and S allele frequency was 0.58. There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Mexican population sample (p = 0.12). Nevertheless, logistic regression analysis of the L allele and increased BMI disclosed an association, after adjusting for age, sex and T2DM (p = 0.02, OR 1.74, 95% CI: 1.079–2.808).

Metabolic gene expression changes in the hippocampus of obese epileptic male rats in the pilocarpine model of temporal lobe epilepsy

Chronically epileptic male adult rats in the pilocarpine model of temporal lobe epilepsy (TLE), exhibited gross expansion of abdominal fat mass and significant weight gain several months after induction of status epilepticus (SE) when compared to control rats. We hypothesized that epileptogenesis can induce molecular changes in the hippocampus that may be associated with metabolism. We determined the expression levels of genes Hsd11b1, Nr3c1, Abcc8, Kcnj11, Mc4r, Npy, Lepr, Bdnf, and Drd2 that are involved in regulation of energy metabolism, in the hippocampus of age-matched control and chronic epileptic animals. Taqman-based quantitative real time polymerase chain reaction (qPCR) and the delta-delta cycle threshold (CT) methods were used for the gene expression assays. Gene expression of Hsd11b1 (cortisol generating enzyme) was significantly higher in epileptic versus control rats at 24h and 2 months, after induction of SE. Nr3c1 (glucocorticoid receptor) mRNA levels on the other hand were down-regulated at 24h, 10 days and 2 months, post SE. Abcc8 (Sur1; subunit of ATP-sensitive potassium (K(ATP)) channel) was significantly down-regulated at 10 days post SE. Kcnj11 (Kir6.2; subunit of ATP-sensitive potassium (K(ATP)) channel) was significantly up-regulated at 24h, 1 month and 2 months post SE. Thus, we demonstrated development of obesity and changes in the expression of metabolic genes in the hippocampus during epileptogenesis in male rats in the pilocarpine model of TLE.

Promoter polymorphism of the serotonin transporter gene influences the number of sexual partners and smoking habits in a Mexican Mestizo population.

Medicine and Engineering in Computer Systems Faculty of Matamoros (FMeISC), Autonomous University of Tamaulipas (UAT), Human Genetics Institute, University Center for Health Science (CUCS), University of Guadalajara (UdeG), Clinic Department, Los Altos University Center (CUALTOS), University of Guadalajara (UdeG), Genetic Wing, West Center of Biomedical Research (CIBO), Mexican Social Security Institute (IMSS), Department of Molecular Biology and Genomics, University Center for Health Science (CUCS), University of Guadalajara (UdeG), Molecular Genetics Laboratory, Molecular Medicine Wing, West Center of Biomedical Research (CIBO), Mexican Social Security Institute (IMSS), Immunobiology Laboratory, University Center for Biological and Agricultural Sciences (CUCBA), University of Guadalajara (UdeG), Masters Academic Program in Forensic Sciences and Criminology , UAM Reynosa-Aztlán, Autonomous University of Tamaulipas (UAT), Mexico and Center for Biomedical Studies, University of Texas at Brownsville, USA

Application of DNA microarray to the study of human adipose tissue/cells.

Adipose tissue is increasingly recognized as a metabolically active endocrine organ with multiple functions beyond its lipid storage capability. Various constituents of the tissue, such as mature adipocytes and stromal vascular cells, have distinct functions. For example, they express and secrete different kinds of bioactive molecules collectively called adipokines. Altered adipokine secretion patterns characterize obesity and insulin resistance, which are major risk factors for type 2 diabetes mellitus. The contribution of dysregulated adipokine expression to these diseases may be assembled from transcriptomic profiles of the tissue and/or its cellular constituents. The gene expression profiles may also complement genetic approaches to identify disease susceptibility genes. Here, we describe an application of gene expression profiling using DNA microarrays to study human adipose tissue, adipocytes, and stromal vascular cells.

GEF1 is a ciliary Sec7 GEF of Tetrahymena thermophila

Ciliary guanine nucleotide exchange factors (GEFs) potentially activate G proteins in intraflagellar transport (IFT) cargo release. Several classes of GEFs have been localized to cilia or basal bodies and shown to be functionally important in the prevention of ciliopathies, but ciliary Arl-type Sec 7 related GEFs have not been well characterized. Nair et al. [ 1999] identified a Paramecium ciliary Sec7 GEF, PSec7. In Tetrahymena, Gef1p (GEF1), tentatively identified by PSec7 antibody, possesses ciliary and nuclear targeting sequences and like PSec7 localizes to cilia and macronuclei. Upregulation of GEF1 RNA followed deciliation and subsequent ciliary regrowth. Corresponding to similar Psec7 domains, GEF1domains contain IQ-like motifs and putative PH domains, in addition to GBF/BIG canonical motifs. Genomic analysis identified two additional Tetrahymena GBF/BIG Sec7 family GEFs (GEF2, GEF3), which do not possess ciliary targeting sequences. GEF1 and GEF2 were HA modified to determine cellular localization. Cells transformed to produce appropriately truncated GEF1-HA showed localization to somatic and oral cilia, but not to macronuclei. Subtle defects in ciliary stability and function were detected. GEF2-HA localized near basal bodies but not to cilia. These results indicate that GEF1 is the resident Tetrahymena ciliary protein orthologous to PSec7. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.