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- Agardh, Carl-David, et al.
(author)
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Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.
- 2006
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In: Metabolism, Clinical and Experimental. - : Elsevier BV. - 1532-8600. ; 55:7, s. 892-898
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Journal article (peer-reviewed)abstract
- To evaluate the expression and protein levels of antioxidant enzymes in the rat retina exposed to oxidative stress induced by ischemia-reperfusion injury. Retinal ischemia was induced in female Wistar rats by ligation of the optic nerve and vessels behind the left eye bulb, and was followed by reperfusion for 0, 3, 6, or 24 hours. The right eye served as control. RNA and protein were extracted simultaneously from each retina. Expressions of the endogenous antioxidant enzymes glutathione peroxidase (GPx1), catalase (CAT), copper/zinc superoxide dismutase, manganese superoxide dismutase, and the catalytic subunit of glutamylcysteine ligase (GCLc) were analyzed with real-time reverse transcription polymerase chain reaction and related to the endogenous control cyclophilin B. Protein levels were measured with Western blot analysis. During the early phase (0 or 3 hours) of reperfusion, no changes were seen in enzyme expression. After 6 hours, GCLc expression increased by a factor of 1.14 (P =.034), followed by a decline of 0.80 after 24 hours (P =.00004), according to the comparative Ct method. After 24 hours of reperfusion, GPx1 expression increased by a factor of 1.14 (P =.028), and CAT had decreased by 0.82 (P =.022). Expressions of copper/zinc superoxide dismutase and manganese superoxide dismutase showed a tendency toward a decrease by factors of 0.86 (P =.055) and 0.88 (P =.053), respectively, after 24 hours. Protein levels did not differ for any of the antioxidants, regardless of reperfusion time. The slightly increased messenger RNA expression of GPx1 after 24 hours of reperfusion with a concomitant very modest decrease in CAT and GCLc expression and no change in protein levels indicate a very modest, if any, response to oxidative stress generated by ischemia followed by reperfusion in rat retina.
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| 2. |
- Agardh, Elisabet, et al.
(author)
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Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels
- 2006
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In: Metabolism, Clinical and Experimental. - : Elsevier BV. - 1532-8600. ; 55:2, s. 168-174
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Journal article (peer-reviewed)abstract
- The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.
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| 3. |
- Annerbrink, Kristina, 1974, et al.
(author)
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Catechol O-methyltransferase val158-met polymorphism is associated with abdominal obesity and blood pressure in men.
- 2008
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In: Metabolism. - : Elsevier BV. - 0026-0495 .- 1532-8600. ; 57:5, s. 708-711
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Journal article (peer-reviewed)abstract
- Catechol O-methyltransferase (COMT) degrades catecholamines and estrogens, both of which are of known importance for cardiovascular risk factors such as obesity and hypertension. The gene coding for COMT contains a val158-met polymorphism that exerts a considerable influence on enzymatic activity. We hypothesized that this polymorphism might influence risk factors for cardiovascular disease. Deoxyribonucleic acid samples and data regarding blood pressure and anthropometry were collected from 240 Swedish men, all 51 years old. Subjects homozygous for the low-activity allele (met) displayed higher blood pressure, heart rate, waist-to-hip ratio, and abdominal sagittal diameter as compared with heterozygous subjects, who in turn displayed higher blood pressure, heart rate, waist-to-hip ratio, and abdominal sagittal diameter than subjects homozygous for the high-activity allele (val). All measured variables were significantly correlated; however, the associations between COMT val158-met and cardiovascular variables, and the association between COMT val158-met and anthropometry, respectively, were partly independent of each other, as revealed by multiple linear regression.
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| 4. |
- Behre, Carl Johan, 1968, et al.
(author)
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Dissociation between adipose tissue expression and serum levels of adiponectin during and after diet-induced weight loss in obese subjects with and without the metabolic syndrome
- 2007
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In: Metabolism: Clinical and Experimental. - : Elsevier BV. - 0026-0495 .- 1532-8600. ; 56:8, s. 1022-8
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Journal article (peer-reviewed)abstract
- The study aimed to examine if dysmetabolic subjects (MetS+) have lower adiponectin gene expression and lower circulating adiponectin levels than non-dysmetabolic obese subjects (MetS-) at baseline, if adiponectin expression and adiponectin concentration rise more in the dysmetabolic group during weight loss, and if v-SNARE Vti1a (vesicle transport soluble NSF attachment protein receptor vps10p tail interacting 1a) expression increases during the weight loss, as a mechanism for increased adiponectin secretion. Twenty-one obese MetS+ and 19 obese MetS- subjects underwent a very low-energy diet for 16 weeks followed by 2 weeks of refeeding. Abdominal subcutaneous adipose tissue biopsies and blood samples were taken before, during, and after dieting for DNA microarray, reverse transcriptase-polymerase chain reaction, and biochemical analyses. Serum adiponectin was also assessed in a sex- and age-matched healthy, nonobese reference group. Weight decreased by 26.3+/-9.8 kg in the MetS+ group and 28.2+/-8.4 kg in the MetS- group with concomitant reductions in insulin, hemoglobin A1c, and triglycerides that were more pronounced in the MetS+ group. Initially, the MetS+ subjects had lower serum adiponectin, but the differences disappeared at week 8, with a continuous increase in serum adiponectin throughout the study in both groups to a level that was higher than in the reference group. The expression of adiponectin and v-SNARE Vti1a did not differ between the groups or over time. In conclusion, obese subjects with the metabolic syndrome had lower circulating adiponectin than subjects without the syndrome. Weight loss increased serum levels of adiponectin without a parallel increase in adiponectin gene expression. The mechanisms involved in the regulation of adiponectin levels merits further investigation.
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| 5. |
- Behre, Carl Johan, 1968, et al.
(author)
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Serum adiponectin in a population sample of 64-year-old women in relation to glucose tolerance, family history of diabetes, autoimmunity, insulin sensitivity, C-peptide, and inflammation
- 2006
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In: Metabolism: Clinical and Experimental. - : Elsevier BV. - 0026-0495 .- 1532-8600. ; 55:2, s. 188-94
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Journal article (peer-reviewed)abstract
- The aim of the study was to describe serum adiponectin levels in a population-based sample of women with different degrees of glucose tolerance and to examine if the variability in serum adiponectin was explained by family history of diabetes, obesity, insulin resistance, glycemia, and inflammation. Repeated oral glucose tolerance tests were used in a screening procedure of a cohort of 64-year-old women to identify those with diabetes mellitus n = 210) and impaired glucose tolerance (n = 201). A random sample of women with normal glucose tolerance (NGT, n = 186) was also included. The examination included history of first-degree relatives with diabetes, anthropometry, measurement of circulating adiponectin, glutamic acid decarboxylase antibodies, blood glucose, HbA1c, insulin, proinsulin, C-peptide, high-sensitivity C-reactive protein, and homeostasis model assessment. Serum adiponectin concentration was lowest among diabetic women, highest in the random-sample NGT group, and intermediate in the impaired glucose tolerance group. This difference was partly explained by homeostasis model assessment, C-peptide, family history, and high-sensitivity C-reactive protein (R2 = 0.33, P < .001), but obesity and glycemia did not contribute to this variability in serum adiponectin. A family history of diabetes was associated with low serum adiponectin concentration independently of obesity, glycemia, or insulin sensitivity (P = .002). Glutamic acid decarboxylase-positive diabetic women (n = 17) had similar serum adiponectin as the NGT group in spite of hyperglycemia. In conclusion, serum adiponectin was lowered in women with type 2 diabetes mellitus, and this difference could only be partly explained by insulin resistance, insulin secretion, family history of diabetes, and inflammation. Family history of diabetes was independently associated with hypoadiponectinemia. Autoimmune diabetic women did not have low adiponectin levels.
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| 6. |
- Behre, Carl Johan, 1968, et al.
(author)
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The reciprocal association of adipocytokines with insulin resistance and C-reactive protein in clinically healthy men
- 2005
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In: Metabolism. - 0026-0495. ; 54:4, s. 439-44
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Journal article (peer-reviewed)abstract
- In experimental models, adiponectin improves and tumor necrosis factor alpha (TNF- alpha ) impairs insulin action, and the expression of these adipocytokines seems to have a reciprocal regulation. The aim was to examine whether in a cross-sectional study, associations supporting this concept may be found in 58-year-old clinically healthy men, and also the relation to C-reactive protein (CRP). In 102 men, euglycemic hyperinsulinemic clamp was used to assess glucose infusion rate (GIR). Total body fat (dual-energy x-ray absorptiometry), plasma adiponectin (radioimmunoassay), TNF-alpha , and CRP (enzyme-linked immunosorbent assay) were measured. Adiponectin correlated positively to GIR (r=0.33, P<.001) and negatively to total fat mass (r=-0.29, P=.004), whereas TNF- alpha showed reverse associations (r=-0.31, P<.01, and r=0.31, P<.01). Adiponectin and TNF- alpha were negatively correlated (-0.28, P=.006). An interaction term (TNF- alpha /adiponectin ratio) and body fat together explained 31.3% (P<.001) in GIR variability. The odds ratio for having insulin resistance was 9.3 (95% CI, 2.2-38.9) in subjects with TNF-alpha values above and adiponectin levels below the median, as compared to subjects with TNF- alpha values below and adiponectin levels above the median. Total fat and TNF-alpha , but not adiponectin, were significantly associated with log CRP (R2=20%, P<.001). In conclusion, this study in man showed that plasma adiponectin and TNF-alpha were independently and reversely associated with insulin resistance. C-reactive protein levels were related to TNF-alpha and obesity.
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