Standardization of Insulin Secretion from Pancreatic Islets: Validation of a DNA Assay

Hopcroft, D. W.; Mason, Deborah; Scott, Russell S.

doi:10.1055/s-2007-1013606

Cited by 44 publications

(31 citation statements)

References 9 publications

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“…Both samples were treated with RNase A (0.5 mg͞ml) for 1 hr at 37°C and then with proteinase K (Sigma, 0.4 mg͞ml) for 1 hr at 37°C. After adding 20 l of 5 M NaCl and 120 l of isopropanol, the samples were incubated overnight at Ϫ20°C, and the DNA concentrations were measured by the method of Hopcroft et al (21). Fragmented DNA was calculated as 100% ϫ soluble DNA͞(soluble ϩ insoluble DNA).…”

Section: Methodsmentioning

confidence: 99%

Fatty acid-induced β cell apoptosis: A link between obesity and diabetes

Shimabukuro

Zhou

Levi³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

1,085

841

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Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early ␤ cell compensation for insulin resistance (4-fold ␤ cell hyperplasia) followed by decompensation (>50% loss of ␤ cells). In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3-and >7-fold, respectively, compared with lean ZDF controls. Ceramide, a fatty acid-containing messenger in cytokineinduced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets. Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA. DNA laddering rose to 19.6% vs. 4.6% in lean control islets, preceded by an 82% increase in ceramide. C 2 -Ceramide without FFA induced DNA laddering, but fumonisin B 1 , a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets. [ H]Palmitate incorporation in [3 H]ceramide in ZDF islets was twice that of controls, but [ 3 H]palmitate oxidation was 77% less. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering. These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis. In ZDF obesity, ␤ cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.

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Section: Methodsmentioning

confidence: 99%

Fatty acid-induced β cell apoptosis: A link between obesity and diabetes

Shimabukuro

Zhou

Levi³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

1,085

841

View full text Add to dashboard Cite

show abstract

“…TG was extracted with 30 l of tert-butyl alcohol and 20 l of a Triton X-104͞methyl alcohol mixture (1:1 vol͞vol). TG content was measured by the Sigma Triglyceride (GPO-Trinder) kit (8), and DNA content was measured by a method of Hopcroft et al (12).…”

Section: Methodsmentioning

confidence: 99%

Direct antidiabetic effect of leptin through triglyceride depletion of tissues

Shimabukuro

Kanemaru

Chen

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

602

474

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Leptin is currently believed to control body composition largely, if not entirely, via hypothalamic receptors that regulate food intake and thermogenesis. Here we demonstrate direct extraneural effects of leptin to deplete fat content of both adipocytes and nonadipocytes to levels far below those of pairfed controls. In cultured pancreatic islets, leptin lowered triglyceride (TG) content by preventing TG formation from free fatty acids (FFA) and by increasing FFA oxidation. In vivo hyperleptinemia, induced in normal rats by adenovirus gene transfer, depleted TG content in liver, skeletal muscle, and pancreas without increasing plasma FFA or ketones, suggesting intracellular oxidation. In islets of obese Zucker Diabetic Fatty rats with leptin receptor mutations, leptin had no effect in vivo or in vitro. The TG content was Ϸ20 times normal, and esterification capacity was increased 3-to 4-fold. Thus, in rats with normal leptin receptors but not in Zucker Diabetic Fatty rats, nonadipocytes and adipocytes esterify FFA, store them as TG, and later oxidize them intracellularly via an ''indirect pathway'' of intracellular fatty acid metabolism controlled by leptin. By maintaining insulin sensitivity and preventing islet lipotoxicity, this activity of leptin may prevent adipogenic diabetes.

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“…About 100 mg of tissue were homogenized with a Polytron in 4 ml of buffer containing 18 mM Tris ⅐ HCl (pH 7.5), 300 mM D-mannitol, and 5 mM EGTA. DNA was measured by the method of Hopcroft et al (11). Triglyceride (TG) was measured by sigma diagnostic kit.…”

Section: Methodsmentioning

confidence: 99%

Gene expression profile of rat adipose tissue at the onset of high-fat-diet obesity

Pan

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

139

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Morbid obesity is the result of massive expansion of white adipose tissue (WAT) and requires recruitment of adipocyte precursor cells and their supporting infrastructure. To characterize the change in the expression profile of the preexisting WAT at the start of obesity, when adipocyte hypertrophy is present but hyperplasia is still minimal, we employed a cDNA subtraction screen for genes differentially expressed in epididymal fat pads harvested 1 wk after the start of a 60% fat diet. Ninety-six genes were upregulated by at least 50% above the WAT of control rats receiving a 4% fat diet. Of these genes, 30 had not previously been identified. Sixteen of the 96 genes, including leptin, adipocyte complement-related protein 30 kDa, and resistin, were predicted to encode a signal peptide. Ten of the 16 had been previously identified in other tissues and implicated in cell growth, proliferation, differentiation, cell cycle control, and angiogenesis. One was a novel gene. Twenty-nine novel fragments were identified. Thus, at the onset of high-fat-diet-induced obesity in rats, adipose tissue increases its expression of factors previously implicated in the expansion of nonadipocyte tissues and of several uncharacterized novel factors. The only one of these thus far characterized functionally was found to promote lipogenesis.

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Standardization of Insulin Secretion from Pancreatic Islets: Validation of a DNA Assay

Cited by 44 publications

References 9 publications

Fatty acid-induced β cell apoptosis: A link between obesity and diabetes

Fatty acid-induced β cell apoptosis: A link between obesity and diabetes

Direct antidiabetic effect of leptin through triglyceride depletion of tissues

Gene expression profile of rat adipose tissue at the onset of high-fat-diet obesity

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