Functional mapping of disease susceptibility loci using cell biology

Antinozzi, Peter A.; Garcia-Diaz, Alejandro; Hu, Chuan; Rothman, James E.

doi:10.1073/pnas.0510521103

Cited by 10 publications

(12 citation statements)

References 48 publications

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“…4) (which is a potent insulin stimulant in INS-1 cells (23,25,47), probably because INS-1 cells possess a plasmalemmal monocarboxylate transporter (46) enabling the rapid uptake of pyruvate into the cells), methyl succinate plus ␤-hydroxybutyrate (Fig. 6) (which together also form a fairly strong stimulant of insulin release in INS-1 cells (23, 47)), and 10 mM BCH plus 10 mM glutamine (which together are a potent stimulant of insulin in INS-1 cells (48) (Fig.…”

Section: Inhibition Of Insulin Release In the Presence Of Nonglucosementioning

confidence: 99%

“…In a study designed to validate a process to scan chromosomal regions in order to identify diabetes susceptibility loci, Antinozzi et al (25) transfected two separate small interfering RNA (siRNAs) 3 targeted against the pyruvate carboxylase mRNA into INS-1 cells and observed about a 60% inhibition of hormone secretion. However, they did not report whether the level of the mRNA encoding pyruvate carboxylase or the activity of the enzyme was lowered by their treatment.…”

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confidence: 99%

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Impaired Anaplerosis and Insulin Secretion in Insulinoma Cells Caused by Small Interfering RNA-mediated Suppression of Pyruvate Carboxylase

Hasan¹,

Longacre²,

Stoker³

et al. 2008

Journal of Biological Chemistry

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Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus ␤-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14 C into lipid from [U-14 C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.Glucose is the most potent physiological insulin secretagogue in the pancreatic beta cell, and it stimulates insulin secretion via its metabolism by aerobic glycolysis. Pyruvate, the terminal product of glycolysis, is metabolized in mitochondria to make ATP to power intracellular processes. However, somewhat surprisingly, in the beta cell, a large amount of glucosederived pyruvate, equal to one-half the total pyruvate entering mitochondrial metabolism, is carboxylated in the pyruvate carboxylase reaction to form oxaloacetate (1-5). The oxaloacetate can combine with pyruvate-derived acetyl-CoA to form citrate enabling the beta cell mitochondrion to increase the rate of synthesis of any citric acid cycle intermediate (anaplerosis) (6 -9). The rate of pyruvate carboxylation correlates with the glucose concentration applied to islets and is thus correlated with the rate of insulin secretion (2). The level of pyruvate carboxylase in the pancreatic islet beta cell is higher than in most body tissues (4, 10 -13) and is equal to the levels in gluconeogenic tissues, such as liver and kidney (4), which possess very high levels of the enzyme. However, the beta cell does not possess the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (11, 14, 15) or fructose-1,6-bisphosphatase (8), and this explains why it ...

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Section: Inhibition Of Insulin Release In the Presence Of Nonglucosementioning

confidence: 99%

mentioning

confidence: 99%

Impaired Anaplerosis and Insulin Secretion in Insulinoma Cells Caused by Small Interfering RNA-mediated Suppression of Pyruvate Carboxylase

Hasan¹,

Longacre²,

Stoker³

et al. 2008

Journal of Biological Chemistry

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“…Laminin (Lm)-111 (α1β1γ1 according to new Lm nomenclature [4]) plays an important role in pancreatic organogenesis [5] and differentiation of early pancreatic cells into insulin-positive beta cells [6,7], but is not present in adult mouse [8] or human pancreas [9]. In contrast, collagen IV has been found to inhibit mouse pancreatic development, while other ECM molecules like fibronectin played no apparent role [6].…”

Section: Introductionmentioning

confidence: 99%

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

et al. 2008

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Aims/hypothesis Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. Methods Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. Results Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which coinvaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm α5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR,

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“…A recent functional mapping across genes in 18p11 suggested the LAMA1 gene to have a role in pancreatic BCF (34).…”

Section: Discussionmentioning

confidence: 99%

Quantitative Linkage Analysis for Pancreatic B-cell Function and Insulin Resistance in a Large Twin Cohort

et al. 2008

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OBJECTIVE-Insulin resistance and disturbed glucose homeostasis are key characteristics of metabolic syndrome, diabetes, and cardiovascular disease. The recent nonlinear computer version of homeostasis model assessment (HOMA)2 provides an appropriate and convenient assessment of glucose metabolism, enabling gene-mapping studies in large population samples.RESEARCH DESIGN AND METHODS-Fasting insulin and glucose concentration were measured in 758 dizygous and 305 monozygous nondiabetic female pairs from the St. Thomas' U.K. adult twin registry (TwinsUK). Insulin resistance (IR) and pancreatic ␤-cell function (BCF) were estimated from this data using the HOMA2 model. RESULTS-Genome-wide variance component linkage analysis using 2,231 genetic markers identified a highly significant quantitative trait locus for BCF on chromosome 10p15 (logarithm of odds [LOD] 6.2, P ϭ 0.0001), a region recently shown to contain a functional variant for type 1 diabetes. Both BCF and IR suggested a pleiotropic effect on 17q25 (univariate LOD 3.2, P ϭ 0.0012, and 2.38, P ϭ 0.0087; bivariate LOD 2.66), and one additional region showed linkage for IR on chromosome 22q11 (LOD 3.2, P ϭ 0.0016), providing replication and refining previous findings for diabetes and associated traits.CONCLUSIONS-To our best knowledge, this is the first genome-wide linkage screen for HOMA2 indexes in a large, healthy female sample. These results suggest that loci involved in control of normal glucose homeostasis among nondiabetic individuals might overlap with those involved in the development of diabetes. Linkage replications in independent studies and across populations provide information on important regions of common but potentially heterogeneous variability that can now be used for targeted positional candidate studies.

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Functional mapping of disease susceptibility loci using cell biology

Cited by 10 publications

References 48 publications

Impaired Anaplerosis and Insulin Secretion in Insulinoma Cells Caused by Small Interfering RNA-mediated Suppression of Pyruvate Carboxylase

Impaired Anaplerosis and Insulin Secretion in Insulinoma Cells Caused by Small Interfering RNA-mediated Suppression of Pyruvate Carboxylase

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

Quantitative Linkage Analysis for Pancreatic B-cell Function and Insulin Resistance in a Large Twin Cohort

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