Samir S. Deeb

A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity

The peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that has a pivotal role in adipocyte differentiation and expression of adipocyte-specific genes. The PPARγ1 and γ2 isoforms result from alternative splicing and have ligand-dependent and -independent activation domains. PPARγ2 has an additional 28 amino acids at its amino terminus that renders its ligand-independent activation domain 5-10-fold more effective than that of PPARγ1. Insulin stimulates the ligand-independent activation of PPARγ1 and γ2 (ref. 5), however, obesity and nutritional factors only influence the expression of PPARγ2 in human adipocytes. Here, we report that a relatively common Pro12Ala substitution in PPARγ2 is associated with lower body mass index (BMI; P=0.027; 0.015) and improved insulin sensitivity among middle-aged and elderly Finns. A significant odds ratio (4.35, P=0.028) for the association of the Pro/Pro genotype with type 2 diabetes was observed among Japanese Americans. The PPARγ2 Ala allele showed decreased binding affinity to the cognate promoter element and reduced ability to transactivate responsive promoters. These findings suggest that the PPARγ2 Pro12Ala variant may contribute to the observed variability in BMI and insulin sensitivity in the general population.

The organization, promoter analysis, and expression of the human PPARgamma gene

PPARγ is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARγ cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARγ1 and PPARγ2, were detected in man. The relative expression of human PPARγ was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARγ1 and γ2 mRNA. In all tissues analyzed, PPARγ2 was much less abundant than PPARγ1. Adipose tissue and large intestine have the highest levels of PPARγ mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARγ is barely detectable in muscle. This high level expression of PPARγ in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARγs, the human PPARγs are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARγ gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARγ1 and PPARγ2 mRNAs, which differ at their 5′-ends. PPARγ1 is encoded by eight exons, and PPARγ2 is encoded by seven exons. The 5′-untranslated sequence of PPARγ1 is comprised of exons A1 and A2, whereas that of PPARγ2 plus the additional PPARγ2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARγ1 and γ2. Knowledge of the gene structure will allow screening for PPARγ mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.

PPARalpha and PPARgamma activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene.

Increased activity of lipoprotein lipase (LPL) may explain the hypotriglyceridemic effects of fibrates, thiazolidinediones and fatty acids, which are known activators (and/or ligands) of the various peroxisome proliferator‐activated receptors (PPARs). Treatment with compounds which activate preferentially PPARalpha, such as fenofibrate, induced LPL expression exclusively in rat liver. In contrast, the antidiabetic thiazolidinedione BRL 49653, a high affinity ligand for PPARgamma, had no effect on liver, but induced LPL expression in rat adipose tissue. In the hepatocyte cell line AML‐12, fenofibric acid, but not BRL 49653, induced LPL mRNA, whereas in 3T3‐L1 preadipocytes, the PPARgamma ligand induced LPL mRNA levels much quicker and to a higher extent than fenofibric acid. In both the in vivo and in vitro studies, inducibility by either PPARalpha or gamma activators, correlated with the tissue distribution of the respective PPARs: an adipocyte‐restricted expression of PPARgamma, whereas PPARalpha was expressed predominantly in liver. A sequence element was identified in the human LPL promoter that mediates the functional responsiveness to fibrates and thiazolidinediones. Methylation interference and gel retardation assays demonstrated that a PPARalpha or gamma and the 9‐cis retinoic acid receptor (RXR) heterodimers bind to this sequence −169 TGCCCTTTCCCCC −157. These data provide evidence that transcriptional activation of the LPL gene by fibrates and thiazolidinediones is mediated by PPAR‐RXR heterodimers and contributes significantly to their hypotriglyceridemic effects in vivo. Whereas thiazolidinediones predominantly affect adipocyte LPL production through activation of PPARgamma, fibrates exert their effects mainly in the liver via activation of PPARalpha.

Impaired expression of peroxisome proliferator-activated receptor γ in ulcerative colitis

Abstract Background & Aims: The peroxisome proliferator-activated receptor γ (PPARγ) has been proposed as a key inhibitor of colitis through attenuation of nuclear factor κB (NF-κB) activity. In inflammatory bowel disease, activators of NF-κB, including the bacterial receptor toll-like receptor (TLR)4, are elevated. We aimed to determine the role of bacteria and their signaling effects on PPARγ regulation during inflammatory bowel disease (IBD). Methods: TLR4-transfected Caco-2 cells, germ-free mice, and mice devoid of functional TLR4 ( Lps d /Lps d mice) were assessed for their expression of PPARγ in colonic tissues in the presence or absence of bacteria. This nuclear receptor expression and the polymorphisms of gene also were assessed in patients with Crohns disease (CD) and ulcerative colitis (UC), 2 inflammatory bowel diseases resulting from an abnormal immune response to bacterial antigens. Results: TLR4-transfected Caco-2 cells showed that the TLR4 signaling pathway elevated PPARγ expression and a PPARγ-dependent reporter in an Iκκβ dependent fashion. Murine and human intestinal flora induced PPARγ expression in colonic epithelial cells of control mice. PPARγ expression was significantly higher in the colon of control compared with Lps d /Lps d mice. Although PPARγ levels appeared normal in patients with CD and controls, UC patients displayed a reduced expression of PPARγ confined to colonic epithelial cells, without any mutation in the PPARγ gene. Conclusions: These data showed that the commensal intestinal flora affects the expression of PPARγ and that PPARγ expression is considerably impaired in patients with UC.

Common Variants in the Promoter of the Hepatic Lipase Gene Are Associated With Lower Levels of Hepatic Lipase Activity, Buoyant LDL, and Higher HDL2 Cholesterol

Increased hepatic lipase (HL) activity is associated with small, dense, low density lipoprotein (LDL) and low high density lipoprotein2 (HDL2) cholesterol (-C) levels. A polymorphism in the promoter region of the HL gene (LIPC) is associated with HDL-C levels. To test whether this association is mediated by differences in HL activity between different LIPC promoter genotypes, the LIPC promoter polymorphism at position -250 (G-->A), HL activity, LDL buoyancy, and HDL-C levels were studied in white normolipidemic men and men with coronary artery disease (CAD). The less common A allele (frequency=0.21 and 0.25 in normal and CAD subjects, respectively) was associated with lower HL activity (P<0.005 by ANOVA) and buoyant LDL particles (P</=0.01) in both groups. Normal and CAD subjects heterozygous for the A allele had lower HL activity (by 24% and 29%, respectively) and significantly more buoyant LDL particles. Homozygosity for this allele (AA) was associated with an even lower HL activity in normal (-26%) and CAD (-46%) subjects. The A allele was associated with higher HDL2-C in CAD patients (P=0.007); heterozygotes and homozygotes for the A allele had a 92% and a 140% higher HDL2-C level (P<0.01) than did GG individuals. In a small number of normolipidemic subjects, the same trend in HDL2-C was seen. In a univariate analysis, the LIPC genotype accounted for 20% to 32% of the variance in HL levels among normal subjects and CAD patients, respectively. After adjustment for HL, the association between LIPC genotype and LDL buoyancy was no longer significant, suggesting that the effect of LIPC genotype on LDL buoyancy is mediated by its effects on HL activity. The LIPC A allele was more frequent in Japanese-Americans and African-Americans than in whites. In summary, these results suggest that variants in the LIPC promoter may significantly contribute to the variance in levels of HL activity and consequently, to the prevalence of the atherogenic small, dense, LDL particles and low HDL2-C levels.

Lipoprotein lipase is synthesized by macrophage-derived foam cells in human coronary atherosclerotic plaques.

Lipoprotein lipase (LPL), hydrolyzes the core triglycerides of lipoproteins, thereby playing a role in their maturation. LPL may be important in the metabolic pathways that lead to atherosclerosis, since it is secreted in vitro by both of the predominant cell types of the atherosclerotic plaque, i.e., macrophages and smooth muscle cells. Because of uncertainty concerning the primary cellular source of LPL in atherosclerotic lesions, in situ hybridization assays for LPL mRNA were performed on 12 coronary arteries obtained from six cardiac allograft recipients. Macrophages and smooth muscle cells were identified on adjacent sections with cell-specific antibodies and foam cells were identified morphologically. LPL protein was localized using a polyclonal antibody. LPL mRNA was produced by a proportion of plaque macrophages, particularly macrophage-derived foam cells, but was not detected in association with any intimal or medial smooth muscle cells. These findings were confirmed by combined immunocytochemistry and in situ hybridization on the same tissue sections. LPL protein was detected in association with macrophage-derived foam cells, endothelial cells, adventitial adipocytes, and medial smooth muscle cells, and, to a lesser extent, in intimal smooth muscle cells and media underlying well-developed plaque. These results indicate that macrophage-derived foam cells are the primary source of LPL in atherosclerotic plaques and are consistent with a role for LPL in the pathogenesis of atherosclerosis.

Hepatic lipase and dyslipidemia: interactions among genetic variants, obesity, gender, and diet

Hepatic lipase (HL) plays a central role in LDL and HDL remodeling. High HL activity is associated with small, dense LDL particles and with reduced HDL2 cholesterol levels. HL activity is determined by an HL gene promoter polymorphism, by gender (lower in premenopausal women), and by visceral obesity with insulin resistance. The activity is affected by dietary fat intake and selected medications. There is evidence for an interaction of the HL promoter polymorphism with visceral obesity, dietary fat intake, and with lipid-lowering medications in determining the level of HL activity. The dyslipidemia with high HL activity is a potentially proatherogenic lipoprotein profile in the metabolic syndrome, in Type 2 diabetes, and in familial combined hyperlipidemia.

Insulin receptor substrate-1 variants in non-insulin-dependent diabetes.

Insulin receptor substrate-1 (IRS-1) plays an important role in insulin-stimulated signaling mechanisms. Therefore, we investigated the frequency and clinical significance of variants in the coding region of this gene in patients with non-insulin-dependent diabetes (NIDDM). Initial screening included a population-based sample of 40 Finnish patients with typical NIDDM. Applying single strand conformation polymorphism analysis the following amino acid substitutions were found among the 40 NIDDM patients: Gly818-Arg, Ser892Gly, and Gly971Arg. The first two variants have not been previously reported. Additional samples of 72 patients with NIDDM and 104 healthy control subjects with completely normal oral glucose tolerance test and a negative family history of diabetes were screened. The most common polymorphism was the Gly971Arg substitution which was found in 11 (9.8%) of 112 NIDDM patients and in 9 (8.7%) of 104 control subjects. The Gly818Arg substitution was found in 2 (1.8%) of NIDDM patients and in 2 (1.9%) of control subjects, and the Ser892Gly substitution was found in 3 (2.7%) NIDDM patients and in 1 (1.0%) control subject. The Gly971Arg substitution was not associated with an impairment in insulin secretion capacity (estimated by insulin responses in an oral glucose tolerance test or by the hyperglycemic clamp) or insulin action (estimated by the euglycemic clamp). Of the three amino acid substitutions observed Ser892Gly is the most interesting one since it abolishes one of the potential serine phosphorylation sites (SPGE) which is located immediately NH2-terminal to the only SH2 binding site of growth factor receptor-bound protein (GRB2), and thus could potentially influence some aspects of signal transduction and metabolic response to insulin.

Contribution of Hepatic Lipase, Lipoprotein Lipase, and Cholesteryl Ester Transfer Protein to LDL and HDL Heterogeneity in Healthy Women

Hepatic lipase (HL) and cholesteryl ester transfer protein (CETP) have been independently associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) size in different cohorts. These studies have been conducted mainly in men and in subjects with dyslipidemia. Ours is a comprehensive study of the proposed biochemical determinants (lipoprotein lipase, HL, CETP, and triglycerides) and genetic determinants (HL gene [LIPC] and Taq1B) of small dense LDL (sdLDL) and HDL subspecies in a large cohort of 120 normolipidemic, nondiabetic, premenopausal women. HL (P <0.001) and lipoprotein lipase activities (P =0.006) were independently associated with LDL buoyancy, whereas CETP (P =0.76) and triglycerides (P =0.06) were not. The women with more sdLDL had higher HL activity (P =0.007), lower HDL2 cholesterol (P <0.001), and lower frequency of the HL (LIPC) T allele (P =0.034) than did the women with buoyant LDL. The LIPC variant was associated with HL activity (P <0.001), HDL2 cholesterol (P =0.034), and LDL buoyancy (P =0.03), whereas the Taq1B polymorphism in the CETP gene was associated with CETP mass (P =0.002) and HDL3 cholesterol (P =0.039). These results suggest that HL activity and HL gene promoter polymorphism play a significant role in determining LDL and HDL heterogeneity in healthy women without hypertriglyceridemia. Thus, HL is an important determinant of sdLDL and HDL2 cholesterol in normal physiological states as well as in the pathogenesis of various disease processes.

Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity

The recent discovery that point mutations in the beta/A4 amyloid precursor protein may be the cause of certain forms of familial Alzheimers disease provides strong support for the view that a thorough understanding of the metabolism of this protein may elucidate the pathogenesis of most forms of the disease and thus serve as a basis for rational prevention and therapy. Here we show that overexpression of a portion of the amyloid precursor protein molecule produces at least four distinct fragments of the COOH-terminus of amyloid precursor protein, suggesting altered proteolysis of amyloid precursor protein, and that such overexpression is associated with cytotoxicity. The degree of toxicity in the P19 cell culture model (differentiating mouse embryonal carcinoma cells) is shown to be related to the two larger novel COOH-terminal protein fragments (16 and 14 kilodalton), as well as to levels of expression of these two fragments. The toxicity is manifested in several differentiated cell lineages, including neuronal cells.