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Results: 7

1.
FIGURE 6:

FIGURE 6:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

RIP2-Opa1KO islets are defective in normal glucose-stimulated ATP production. (A) Glucose-stimulated ATP production. Isolated islets were incubated with the indicated concentrations of glucose for 5 min. Total ATP amounts were measured and normalized to protein amounts (n = 3). (B) The oxygen consumption rate (OCR) was measured in isolated isolates (n = 3). Glucose and oligomycin were added at the time indicated by the arrow and arrowhead, respectively.

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.
2.
FIGURE 3:

FIGURE 3:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

Opa1 deficiency causes reduced islet size. (A) Immunofluorescence of pancreas sections prepared from adult mice (8–12 wk old) using antibodies to insulin for beta cells (red) and glucagon for alpha cells (green). (B) Distribution of beta cell islet size in adult. Insulin-positive area was determined using ImageJ software (n = 3). (C) The number of islets in pancreas sections was determined to calculate their density (n = 3).

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.
3.
FIGURE 5:

FIGURE 5:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

Deletion of Opa1 causes impaired insulin secretion in mice and in isolated islets. (A) Insulin contents in isolated islets were determined and normalized to total protein amounts (n = 3). (B) Insulin secretion from isolated islets. Isolated islets were incubated with 3 and 11 mM glucose for 1 h. Secreted insulin levels were determined and normalized to total insulin contents (n = 3). (C) Isolated islets were stained with 2 μM Fura 2-AM and stimulated with 3 and 11 mM glucose (n > 4). The 340 nm/380 nm fluorescence ratio was measured under a fluorescence microscope at 8 min after stimulation.

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.
4.
FIGURE 2:

FIGURE 2:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

RIP2-Opa1KO mice show hyperglycemia and impaired insulin responses. (A) Blood glucose levels were measured in mice that were fasted for 14–16 h or randomly fed using a glucose meter (n ≥ 12). (B) Glucose tolerance test. Mice were fasted for 14–16 h and subjected to intraperitoneal injection of glucose (1.5 mg/g body weight). Blood glucose levels were measured at the indicated times (n ≥ 11). (C) Insulin tolerance test. After fasting for 14–16 h, mice were subjected to intraperitoneal injection of insulin (0.5 U/kg body weight). Blood glucose concentrations were determined at different time points (n ≥ 11). (D) Blood insulin levels after intraperitoneal glucose injection. Mice between 8 and 12 wk of age were fasted for 14–16 h and subjected to intraperitoneal injection of glucose (1.5 mg/g body weight). Blood insulin concentrations were determined at the indicated time points (n = 15).

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.
5.
FIGURE 7:

FIGURE 7:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

RIP2-Opa1KO islets contain decreased complex IV level. (A) Isolated islets were subjected to immunoblotting using antibodies against ETC complexes (NDUFB8 for complex I, FeS for complex II, Core2 for complex III, subunits I, IV, and Vb for complex IV, α subunit for complex V, and cytochrome c). Antibodies against Tim23 and actin were used as controls. Band intensity was quantitated and normalized to control samples (n = 3). (B) Activities of complexes I and IV in RIP2-Opa1KO islets were normalized to those in controls (n = 3). (C) mtDNA copy number relative to nuclear genome in isolated islets was determined using quantitative PCR. (D) Southern blotting was performed for mtDNA and nuclear DNA (nDNA). DNA was extracted from control (C) and RIP2-Opa1KO (K) islets. The expected sizes of mtDNA and nDNA are 16.3 and 6.3 kbp, respectively. (E) Isolated islets were solubilized with digitonin and analyzed by density gradient centrifugation, followed by immunoblotting using the indicated antibodies. Migration patterns of ETC components are shown (n = 3). Molecular weights are shown in kilodaltons.

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.
6.
FIGURE 4:

FIGURE 4:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

Beta cell proliferation is defective in RIP2-Opa1KO mice. (A) Immunofluorescence of pancreas sections using antibodies to Ki67 for cell proliferation (green) and insulin for beta cells (red) in newborns (1 wk old) and adults (8–12 wk old). (B) The percentage of beta cells showing Ki67 signals was determined (n ≥ 3). (C) Insulin-positive islet area in newborn and adult mice was measured (n = 3). (D) Ratio of beta cell area to alpha cell area was calculated (n = 3). (E) Sections of pancreas were subjected to TUNEL analysis using the In Situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN). TUNEL signals (green) and immunostaining using anti-insulin antibodies are shown (red). As a positive control, sections were incubated with DNase I according to the manufacturer's instructions. (F) Quantification of islets that contain TUNEL-positive beta cells. Average ± SEM is shown (n = 3). Numbers in parentheses indicate the total number of islets examined. (G) Immunoblotting of islets using antibodies to PARP, caspase-3, and actin.

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.
7.
FIGURE 1:

FIGURE 1:. From: The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells.

Loss of Opa1 results in fragmentation of mitochondria and alternation of cristae structure in pancreatic beta cells. (A) Wild-type MEFs and control islets were analyzed by immunoblotting using Opa1 antibodies. MEFs have been shown to mainly express five Opa1 isoforms (L1, L2, S3, S4, and S5) (Song et al., 2007; Merkwirth et al., 2008). Islets also expressed these five isoforms with different expression levels. Whereas isoform L2 demonstrated the highest levels in MEFs, S5 isoform was dominant in islets. (B) Islets isolated from control and RIP2-Opa1KO (KO) mice were subjected to immunoblotting using antibodies to Opa1. Tim23, a mitochondrial inner membrane protein, was used as a control. (C) EM analysis of control and RIP2-Opa1KO beta cells. Low-magnification images (top) and high-magnification images (bottom) are shown. Arrows indicate cristae junctions in insets. (D) Distribution of mitochondrial length (n = 3; a total of 671 mitochondria for control and 1704 for RIP2-Opa1KO). (E) We measured width of cristae junction, which connects the intermembrane space with the intracristal space, using ImageJ software (National Institutes of Health, Bethesda, MD) (n = 3; a total of 213 cristae for control and 709 for RIP2-Opa1KO). (F) Volume densities of mitochondria were determined using a point-counting method (n = 11).

Zhongyan Zhang, et al. Mol Biol Cell. 2011 July 1;22(13):2235-2245.

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