Expansion of adipose tissue mass results from increased number and size of adipocyte cells. We hypothesized that subcutaneous abdominal preadipocytes in obese individuals might have an intrinsically higher propensity to differentiate into adipocytes. Thus we investigated the relationship between obesity and the level of in vitro preadipocyte differentiation in Pima Indians. Subcutaneous abdominal stromal vascular fractions containing preadipocytes were cultured from 58 nondiabetic subjects [31 M/27 F, 30 Ϯ 6 yr, body fat 34 Ϯ 8% by dual-energy X-ray absorptiometry (means Ϯ SD)]. The average percentage of preadipocyte differentiation (PDIFF; cell count by microscopy) was 11 Ϯ 11% (range 0.2-51%). PDIFF correlated negatively with percent body fat (r ϭ Ϫ0.35, P ϭ 0.006) and waist circumference (r ϭ Ϫ0.45, P ϭ 0.0004). Multiple regression analysis indicated that waist circumference (P ϭ 0.01), sex (P ϭ 0.01), and percent body fat (P ϭ 0.05) were significant determinants of PDIFF. Molecular characterization of predifferentiated cultured cells was performed by real-time PCR measurements of glucocorticoid receptor-␣ (GR␣), insulin-like growth factor I receptor (IGF-IR), peroxisome proliferator-activated receptor-␥ (PPAR␥), enhancer-binding protein GATA-3, CCAAT/ enhancer-binding protein-␣ undifferentiated protein (CUP/AP-2␣), and endothelial cell-specific marker 2 (ECSM2). The mRNA concentrations of GR␣ correlated with PDIFF (r ϭ 0.29, P ϭ 0.03), but the others did not (IGF-IR, r ϭ 0.003, P ϭ 1.0; PPAR␥, r ϭ Ϫ0.1, P ϭ 0.5; GATA-3, r ϭ 0.02, P ϭ 0.9; CUP/AP-2␣, r ϭ Ϫ0.2, P ϭ 0.1; ECSM2, r ϭ 0.04, P ϭ 0.7). Contrary to our hypothesis, the results may indicate a blunted in vitro differentiation potential of preadipocytes in centrally obese individuals. The lower differentiation potential of preadipocytes in the obese subjects might be due, at least partly, to decreased glucocorticoid receptor expression.

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A b b re v i a t i o n s : DCOH, dimerization cofactor of HNF-1; HNF, hepatocyte nuclear factor; IPF-1, insulin p romoter factor 1; MODY, maturity-onset diabetes of the young; PCR, polymerase chain re a c t i o n .A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances. Mutations in the Genes for Hepatocyt e Nuclear Factor (HNF)-1 , -4 , -1 , and -3 ; the Dimerization Cofactor of HNF-1; and Insulin Promoter Factor 1 Are Not Common Causes of Early-Onset Type 2 Diabetes in Pima Indians O R I G I N A L A R T I C L E O B J E C T I V E -Maturity-onset diabetes of the young (MODY)is a genetically hetero g e n e o u s subtype of type 2 diabetes characterized by an early age at onset and autosomal dominant inheritance. MODY can result from heterozygous mutations in at least five genes. The purpose of this study was to determine whether alterations in known MODY genes and two MODY candidate genes contribute to the development of early-onset type 2 diabetes in Pima Indians.RESEARCH DESIGN AND METHODS -The coding regions of the known MODY genes hepatocyte nuclear factor (HNF)-1 , HNF-4 , HNF-1 , and insulin promoter factor 1 and the coding regions of two MODY candidate genes, HNF-3 and the dimerization cofactor of HNF-1, were sequenced in genomic DNA from Pima Indians. The primary "affected" study population consisted of 46 Pima Indians whose age at onset of type 2 diabetes was 20 years. DNA sequence variants identified in the affected group were then analyzed in a group of 80 " u n a ffected" Pima Indians who were at least 40 years old and had normal glucose tolerance.R E S U LT S -A total of 11 polymorphisms were detected in these genes. However, none of the polymorphisms diff e red in frequency among Pima Indians with an early age at onset of diabetes compared with older Pima Indians with normal glucose tolerance. C O N C L U S I O N S -Mutations in these known E p i d e m i o l o g y / H e a l t h S e r v i c e s / P s y c h o s o c i a l R e s e a r c hDIABETES CARE, VOLUME 23, NUMBER 3, MARCH 2000 303Baier and Associates resistance to ketosis, and absence of islet cell antibodies, including GAD65 antibody (12). The high prevalence and young age at onset of type 2 diabetes in this population suggest that MODY may exist in some of the Pima p e d i g rees. MODY2, however, is unlikely to exist in Pima Indians because a polymorphic dinucleotide repeat marker flanking the glucokinase gene was previously genotyped in 470 Pima sib pairs, and no evidence for linkage between glucokinase and acute insulin secretion or type 2 diabetes was detected (13). In addition, prior sequencing of all of the exons, splice sites, and the liverand -cell-specific promoters of the glucokinase gene in 10 Pima Indians with earlyonset type 2 diabetes revealed several variants, none of which was associated with early-onset diabetes. There f o re, in the pre sent study, we screened the additional known MODY genes and the two HNF-re l a t e d genes (DCOH and HNF-3 ) to determ...

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Insulin resistance, a major predictor of type 2 diabetes mellitus, is genetically inherited in Pima Indians, a population with a high prevalence of the metabolically complex disease. Protein targeting to glycogen/ PPP1R5 has recently been identified as a potential regulator of glycogen synthase, the rate-limiting enzyme of the insulin-induced glycogenesis. The gene is located on chromosome 10q23-24, where there is a suggestive linkage to insulin action in this population, establishing it as a functional and positional candidate gene. In this study, we discovered 2 novel polymorphisms upstream of the 5UTR of the gene, with only one found in Pima Indians, but no polymorphism in the coding sequence. The genotype frequencies of the polymorphism and transcript levels of the gene in skeletal muscle do not correlate with insulin action in the subjects. These results exclude any significant role of protein targeting to glycogen/PPP1R5 in insulin resistance in Pima Indians.

Purpose:To identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS), complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette smoke, in human retinal pigment epithelial cells (ARPE-19) in vitro.Methods:ARPE-19 cells were treated for 24 hours with 200 μM, 100 μM, and 50 μM B(e)p or DMSO (dimethyl sulfoxide)-equivalent concentrations. Some cultures were pre-treated with ROS/RNS inhibitors (NG nitro-L-arginine, inhibits nitric oxide synthase; Apocynin, inhibits NADPH oxidase; Rotenone, inhibits mitochondrial complex I; Antimycin A, inhibits mitochondria complex III) and ROS/RNS levels were measured with a fluorescent H2 DCFDA assay. Multiplex bead arrays were used to measure levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Transforming Growth Factor alpha (TGF-α) and Vascular Endothelial Growth Factor (VEGF). IL-6 levels were also measured by an enzyme-linked immunosorbent assay. Real-time qPCR analyses were performed with primers for C3 (component 3), CFH (inhibits complement activation), CD59 (inhibitor of the complement membrane attack complex (MAC)) and CD55/DAF (accelerates decay of target complement target proteins).Results:The ARPE-19 cultures treated with B(e)p showed significantly increased ROS/RNS levels (P < 0.001), which were then partially reversed by 6 μM Antimycin A (19%, P = 0.03), but not affected by the other ROS/RNS inhibitors. The B(e)p treated cultures demonstrated increased levels of IL-6 (33%; P = 0.016) and GM-CSF (29%; P = 0.0001) compared to DMSO-equivalent controls, while the expression levels for components of the complement pathway (C3, CFH, CD59 and CD55/DAF) were not changed.Conclusion:The cytotoxic effects of B(e)p include elevated ROS/RNS levels along with pro-inflammatory IL-6 and GM-CSF proteins. Blocking the Qi site of cytochrome c reductase (complex III) with Antimycin A led to partial reduction in B(e)p induced ROS production. Our findings suggest that inhibitors for multiple pathways would be necessary to protect the retinal cells from B(e)p induced toxicity.

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