👤 C L Harris

To determine the prevalence of 'hypertriglyceridemic waist' (HTGW) in Oji-Cree, to examine its interaction with hepatic nuclear factor-1alpha (HNF1A) in association with type 2 diabetes, and to characterize its putative genetic determinants. The presence or absence of HTGW was determined in 522 Oji-Cree subjects (223 males, 299 females), >or=18 years of age, in whom physical measurements and fasting plasma analyte concentrations were gathered, and a 75-g oral glucose tolerance test was administered, as part of a cross-sectional study. Subjects were genotyped for HNF1A codon 319, angiotensinogen (AGT) codons 174 and 235, G-protein beta3-subunit (GNB3) nucleotide 825, fatty acid-binding protein (FABP2) codon 54, nucleotides -455 and -482 of the apolipoprotein (apo) C-III (APOC3) promoter, and a 5-bp insertion/deletion polymorphism within the 3'-untranslated region of protein phosphatase 1 regulatory subunit 3 (PPP1R3). The unadjusted prevalence of HTGW in Oji-Cree adults was 20.5%, with more males affected than females (27.8 vs 15.1%, P=0.0004). Logistic regression analysis, adjusted for age and gender, showed type 2 diabetes was associated with both HNF1A G319S (odds ratio (OR) 4.85, 95% CI 2.45, 9.58) and HTGW (OR 4.96, 95% CI 2.49, 9.88). When the HNF1A mutation and HTGW were present in combination, the OR for type 2 diabetes was markedly increased (OR 43.2, 95% CI 12.4, 150). In women only, both GNB3 825C>T and FABP2 A54T genotypes were significantly associated with HTGW (OR 2.02, 95% CI 1.01, 4.05 and OR 1.95, 95% CI 1.01, 3.74, respectively). HTGW is prevalent in Oji-Cree, especially in men. The ORs for type 2 diabetes were similar ( approximately 5-fold) for subjects with either the presence of HTGW or the private HNF1A G319S mutation. These two independent risk factors acted synergistically to confer an even greater increased risk of type 2 diabetes. Show less

The prevalence rates of type 2 diabetes (T2DM) and coronary heart disease (CHD) in Ontario Oji-Cree are among the world's highest. Since metabolic syndrome (MetS) increases risk of T2DM and CHD, we characterized prevalence and putative genetic determinants of MetS in Oji-Cree. In 515 adult (> or = 18 years old) and 115 adolescent (< 18 years old) Oji-Cree subjects, using the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria, we determined that 29.9% of Oji-Cree adults, and 43.4% of adults > or = 35 years of age, had MetS. Furthermore, 33.9 and 8.7% of female Oji-Cree adults and adolescents, respectively, had MetS. Increased waist girth and depressed HDL cholesterol were the most prevalent individual MetS components, while increased blood pressure was least prevalent. AGT T174M, GNB3 825C>T, and APOC3 -455T>C genotypes were significantly associated with MetS (P = 0.018, 0.0056, and 0.029, respectively) for female adults, whereas FABP2 A54T genotype was associated with MetS (P = 0.040) for female adolescents. The high MetS prevalence in Oji-Cree adults, especially women, is consistent with their high risk of T2DM and CHD. Functional polymorphisms in three candidate genes for plasma lipoproteins and blood pressure were associated with MetS in adult Oji-Cree. Furthermore, several female adolescents met the adult MetS criteria, suggesting that the genesis of MetS begins in youth, especially among aboriginal females. Show less

Delta-like 4 (Dll4), a membrane-bound ligand for Notch1 and Notch4, is selectively expressed in the developing endothelium and in some tumor endothelium, and it is induced by vascular endothelial growth factor (VEGF)-A and hypoxia. Gene targeting studies have shown that Dll4 is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are currently not defined. In this study, we generated primary human endothelial cells that overexpress Dll4 protein to study Dll4 function and mechanism of action. Human umbilical vein endothelial cells retrovirally transduced with Dll4 displayed reduced proliferative and migratory responses selectively to VEGF-A. Expression of VEGF receptor-2, the principal signaling receptor for VEGF-A in endothelial cells, and coreceptor neuropilin-1 was significantly decreased in Dll4-transduced endothelial cells. Consistent with Dll4 signaling through Notch, expression of HEY2, one of the transcription factors that mediates Notch function, was significantly induced in Dll4-overexpressing endothelial cells. The gamma-secretase inhibitor L-685458 significantly reconstituted endothelial cell proliferation inhibited by immobilized extracellular Dll4 and reconstituted VEGFR2 expression in Dll4-overexpressing endothelial cells. These results identify the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating 2 VEGF receptors expressed on endothelial cells and raise the possibility that Dll4 may be exploited therapeutically to modulate angiogenesis. Show less

Decay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembrane-anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock-out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure. Show less

To establish an accurate molecular model of human branched-chain amino acid (BCAA) metabolism, the distribution, activity, and expression of the first 2 enzymes in the catabolic pathway--branched-chain-amino-acid aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) complex--were determined in human tissues. The same enzyme activities were measured in rat and African green monkey tissues. Overall, the activities of BCAT and BCKD were higher in rat than in human and monkey tissues; nevertheless, the ratio of the 2 activities was similar in most tissues in the 3 species. Total oxidative capacity was concentrated in skeletal muscle and liver (> 70%) with muscle having a higher proportion of the total in humans and monkeys. In humans, brain (10-20%) and kidney (8-13%) may contribute significantly to whole-body BCAA metabolism. Furthermore, in primates the high ratio of transaminase to oxidative capacity in the entire gastrointestinal tract serves to prevent loss of essential BCAA carbon and raises the possibility that the gastrointestinal tract contributes to the plasma branched-chain alpha-keto acid pool. Quantitative polymerase chain reaction was used to examine expression of human branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), the key enzyme that regulates the activity state of the human BCKD complex and human BCAT isoenzymes. To design the primers for the polymerase chain reaction, human BCKDK was cloned. BCKDK message was found in all human tissues tested, with the highest amount in human muscle. As in rats, there was ubiquitous expression of mitochondrial BCAT, whereas mRNA for the cytosolic enzyme was at or below the limit of detection outside the brain. Finally, the role of BCAA in body nitrogen metabolism is discussed. Show less

We hypothesized that common genomic variation that affected the expression and/or function of the products of the APOC3, APOE, FABP2, and PON1 genes would be associated with variation in biochemical phenotypes in a previously unstudied human sample. We determined genotypes of functional genomic variants of APOC3, APOE, FABP2, and PON1 in 509 adult aboriginal Canadians from an isolated community in Northern Ontario. We tested for genotype associations with plasma lipoprotein traits. We found that (1) common variation at nucleotide -455 of the APOC3 promoter was associated with variation in plasma triglycerides (P = .006) and (2) common variation of APOE determining plasma isoforms of apo E was associated with variation in plasma apo B (P = .009). Analysis of subjects classed by APOC3 markers showed that homozygosity for presence of a C at nucleotide -455 and a T at nucleotide -482 was associated with significantly increased plasma triglycerides in both men and women. Furthermore, this allele was approximately twice as frequent in subjects within the highest quartile of plasma triglycerides as in subjects within the lowest quartile. Since the DNA variation detected by the APOC3 markers affects in vitro expression of the gene product, it is possible that the marker itself caused the associations. However, the associations could also have resulted from linkage disequilibrium with other functional variants in APOC3 or the closely linked APOA1 and/or APOA4 genes. Show less

We hypothesized that common genomic variants would be associated with variation in lipoprotein phenotypes in young subjects. We determined genotypes of FABP2, PON, APOC3, and APOE in 188 aboriginal Canadians, aged 9 to 17 years. We found that 13 of 32 possible genotype-phenotype associations were significant: (1) the FABP2 codon 54 genotype was associated with variation in plasma triglycerides (P = .045); (2) the PON codon 192 genotype was associated with variation in plasma total and LDL cholesterol and apoB (P = .0099, P = .0088, and P = .016, respectively); (3) the APOC3 insulin-response-element genotype was associated with variation in plasma triglycerides, HDL cholesterol, apoA-I, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .0014, P = .0069, P = .045, P = .0021, and P = .0081, respectively); and (4) the APOE restriction isotype was associated with variation in plasma LDL cholesterol, apoB, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .025, P = .034, P = .045, and P = .047, respectively). The average young age and relative absence of age-dependent secondary environmental factors could have eased the identification of small genetic effects on lipoprotein phenotypes in this study sample. Show less

Four mitochondrial protein kinases have been cloned. These proteins represent a new family of protein kinases, related by sequence to the bacterial protein kinases but by function to the eukaryotic serine protein kinases. Arg288 is required for recognition by BCKDK of the phosphorylation site on the E1alpha subunit of the BCKDH complex. BCKDK inhibits the dehydrogenase activity of the BCKDH complex by introducing a negative charge into the active-site pocket of the E1 component. Protein starvation of rats induces an increase in the amount of BCKDK bound to the BCKDH complex. This causes inactivation of the BCKDH complex and conserves branched-chain amino acids for protein synthesis in the protein-starved state. Expression of the different PDK isoenzymes is tissue specific, and the different PDK isoenzymes are unique with respect to kinetic parameters for ATP and ADP and sensitivity to allosteric effectors (NADH, NAD+, coenzyme A, acetyl-CoA, pyruvate, and dichloroacetate). Preliminary experiments indicate that an increased amount of PDK2 protein partly explains the increase in PDK activity that occurs in rat liver in response to chemically induced diabetes. Show less