Forced and voluntary exercise counteract insulin resistance in rats: the role of coping style

There are large individual differences in the success rates of exercise intervention programs aimed at the prevention and treatment of obesity-related disorders. In the present study, we tested the hypothesis that differences in coping style may impact the success rates of these intervention programs. We tested insulin responses before and after voluntary wheel running in both passive (insulin resistant) Roman Low Avoidance (RLA) and proactive (insulin sensitive) Roman High Avoidance (RHA) rats using intravenous glucose tolerance tests (IVGTTs). To control for a potential difference between voluntary and forced exercise, we also included RLA and RHA rats that were subjected to forced running. We found the following: 1) when given the opportunity to run voluntarily in a running wheel, passive RLA rats run more than proactively than RHA rats; 2) voluntary exercise leads to a normalization of insulin responses during an IVGTTs in RLA rats; and 3) there were no behavioral and physiological differences in efficacy between voluntary and forced running. We conclude that exercise, both forced and voluntary, is a successful lifestyle intervention for the treatment of hyperinsulinemia, especially in individuals with a passive coping style.     

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]_M) and the rate of cytosolic Ca²⁺ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca²⁺ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca²⁺ concentration ([Ca²⁺]_M) and, consequently, the Ca²⁺-stimulated increase in [ATP]_M, thus allowing studies of how the increase in [ATP]_M and the cytosolic Ca²⁺ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca²⁺]_M and [ATP]_M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca²⁺ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca²⁺ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca²⁺ removal during stimulation with a Ca²⁺-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca²⁺ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca²⁺ pumps. This work reveals the usefulness of rhodamine-based Ca²⁺ indicators in examining the role of intramitochondrial Ca²⁺ in mitochondrial (patho) physiology. human skin fibroblast; OXPHOS disease; calcium ion extrusion; rhod-2; CGP-37157     

Age-related changes in the ultrastructure of the resting follicle pool in human ovaries

Age-related decline of fertility in women is the result of the decline in both quantity and quality of the resting ovarian follicle pool. The aim of the present study was to determine whether the decline of follicle quality with age is reflected by ultrastructural changes in the resting follicle pool. Ovarian biopsy specimens were obtained by laparoscopy from seven healthy women aged 25-32 yr (young group) and from 11 healthy women aged 38-45 yr (advanced-age group). A total of 182 resting follicles from the young group were compared with 81 resting follicles from the advanced-age group for signs of age-related changes by transmission-electron microscopy. The ooplasmic fraction of vacuoles was increased (P = 0.02), and the fraction of mitochondria decreased (P = 0.005), in the advanced-age group. Also, the density of the mitochondrial matrix (P < 0.001) and the frequency of dilated smooth endoplasmic reticulum (SER; P = 0.001) and Golgi complex (P = 0.02) were increased with age. The frequencies of ruptured mitochondrial membranes (P = 0.001) and dilated SER (P = 0.003) were increased with age in the granulosa cells. Overall follicle-quality scores, which should reflect atretic changes, were not different for the young and advanced-age groups. In conclusion, in resting follicles, the morphological changes with age are different from the changes seen in quality decline by atresia. The morphological changes with age specifically involved the mitochondria, the SER, and the Golgi complex, and they may be the cause of atresia on initiation of follicular growth because of the substantial increase in metabolic requirements.     

Disturbed morphogenesis of cardiac outflow tract and increased rate of aortic arch anomalies in the offspring of diabetic rats

BACKGROUND: Maternal diabetes (MD) is a risk factor for offspring to develop cardiovascular anomalies; this is of growing clinical concern since the number of women in childbearing age with compromised glucose homeostasis is increasing. Hyperglycemia abrogates cardiovascular development in vitro; however, a link to cardiovascular defects in diabetic offspring remains to be investigated. METHODS: We have studied cardiovascular development in offspring of MD rats by examining serial histological sections of GD 12.0-18.0 offspring. Development of pharyngeal arch artery malformations was analyzed and related to intracardiac anomalies. RESULTS: Pharyngeal arch artery and intracardiac defects were present in 27 of 37 MD GD 13.0-18.0 offspring. Early sixth arch arteries showed abrogated arteriogenesis, whereas fourth arch artery defects developed as a result of abnormal remodeling. Morphometrical analysis showed increased apoptosis in regressing artery segments and reduced apoptosis in persisting artery segments. Double outlet right ventricle with infundibular stenosis (tetralogy of Fallot) was predominantly found in combination with sixth artery defects and pulmonary atresia. As confirmed by morphometric analysis and three-dimensional (3D)-reconstructions, outflow tract defects coincided with endocardial cushion hypoplasia. Cases with teratology of Fallot additionally showed a shorter outflow tract. No relation with apoptosis or disturbed neural crest cell migration was found. CONCLUSIONS: Our data uniquely demonstrate mechanistic differences involved in the development of sixth and fourth artery anomalies. Whereas increased apoptosis induces fourth artery anomalies, pulmonary outflow obstruction abrogates sixth artery differentiation independent of apoptosis. The model presented allows analysis of diabetic conditions on cardiovascular development in vivo, essential for elucidating this teratology.     

Ultrastructure of the resting ovarian follicle pool in healthy young women

In humans, follicle quantity and quality decline with age by atresia. In the present study we aimed to describe the quality of the follicle pool through an ultrastructural investigation of resting follicles in young healthy women. From ovarian biopsies of 7 women aged 25-32 yr, 182 small follicles were morphometrically assessed for various signs of atresia. Morphometric variables were analyzed by principal components analysis (PCA) to demonstrate correlations between variables and to construct an objective follicle score. One third of small follicles consisted of primordial follicles. Nucleus:cell ratios remained constant for oocytes and granulosa cells from primordial to primary follicles, suggesting that follicles up to primary stages belong to the resting pool. The distribution of follicle quality scores as derived from PCA showed that most follicles were of good quality and with little signs of atresia. Atresia in resting follicles appears to be a necrotic process, starting in the ooplasma. Early atresia was characterized by increasing numbers of multivesicular bodies and lipid droplets, dilation of smooth endoplasmic reticulum and Golgi, and irregular mitochondria with changed matrix density. In progressive atresia mitochondrial membranes ruptured, oocyte nuclear membranes were indented or ruptured, and the ooplasma showed extensive vacuolarization. The early involvement of mitochondria in this process suggests that damage is induced by oxygen radicals. PCA follicle quality scores can be reliably approximated using a reduced number of seven morphometric variables, which were selected by stepwise forward analysis. The algorithm to calculate these follicle scores is presented.     

In vitro culture of rat pre-antral follicles with emphasis on follicular interactions

The present study was designed to determine whether rat pre-antral follicles can grow under in-vitro conditions. Emphasis is on whether follicular interaction is involved in in-vitro follicle culture, and furthermore its role in follicular development has been assessed. Pre-antral follicles were isolated mechanically from 10-day old rat ovaries. They were divided into small (50 microm < diameter < 100 microm) and large (120 microm < diameter < 200 microm) pre-antral follicles and cultured individually or in groups for 6 days in medium with or without fetal calf serum (FCS). Based on morphological criteria, large pre-antral follicles cultured in groups in serum-free medium had significantly higher survival rates than those cultured individually. In the presence of FCS, no significant difference was detected with respect to the survival. However, the large pre-antral follicles cultured in groups had a significantly greater increase in diameter than those cultured individually. Furthermore, follicles cultured in groups in FCS-containing medium exhibited significantly more follicular cell proliferation than those in serum-free medium, based on DNA measurement. The present culture system (with or without FCS) proved to be insufficient for small pre-antral follicles to stimulate growth comparable to that of large pre-antral follicles. The transmission electron microscopical (TEM) study revealed the ultrastructural differences between follicles cultured in FCS-containing and serum-free media. Taken together, the results suggest that interfollicular factors are involved in follicle development in vitro, which especially at the early folliculogenesis stage plays a positive role in terms of follicular growth as well as survival. The present culture model allows further investigation of factors that regulate early folliculogenesis.     

Paraoxonase-1 and linoleic acid oxidation in familial hypercholesterolemia

Serum paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme that can inhibit low-density lipoprotein (LDL) oxidation in vitro. The role of PON1 in vivo still remains to be clarified. We investigated the effect of PON1 genotype (-107C > T and 192Q > R), concentration, paraoxonase activity, and arylesterase activity on the early phase of lipid peroxidation in plasma samples of 110 patients with heterozygous familial hypercholesterolemia. The degree of lipid oxidation was assessed by quantitation of oxidized-linoleic acid (the most abundant fatty acid present in LDL) using high performance liquid chromatography. We found a significant inverse correlation between paraoxonase activity and the oxidized-linoleic acid concentration (r = -0.22, P = 0.03), independent of baseline linoleic acid levels. These findings support an anti-oxidative role for PON1 in patients with FH, and thus may give insight into the functioning of PON1 in vivo.     

Decreased adiponectin levels in familial combined hyperlipidemia patients contribute to the atherogenic lipid profile

Familial combined hyperlipidemia (FCH) is characterized by increased levels of total cholesterol, triglycerides, and/or apolipoprotein B. Other features of FCH are obesity and insulin resistance. Adiponectin is a secretory product of the adipose tissue. Low levels of adiponectin are associated with insulin resistance and accelerated atherosclerosis. The aim of this study was to determine whether decreased adiponectin levels are associated with FCH and its phenotypes. The study population comprised 644 subjects, including 158 patients with FCH. Serum adiponectin levels were determined using a commercially available ELISA. For both males and females, the mean adiponectin level (microg/ml) was significantly lower in FCH patients [2.0 (1.8-2.2) and 2.5 (2.3-2.8), respectively] compared with normolipidemic relatives [2.3 (2.2-2.5) and 3.1 (2.8-3.3), respectively] and spouses [2.4 (2.1-2.7) and 3.2 (2.8-3.6), respectively]. These differences remain significant after adjusting for waist circumference and insulin resistance. Low adiponectin level in FCH patients was a superior independent predictor of the atherogenic lipid profile, including high triglyceride levels, low HDL-cholesterol levels, and the amount of small, dense LDL present, compared with both obesity and insulin resistance. Low adiponectin levels may contribute to the atherogenic lipid profile in FCH, independent of insulin resistance and obesity, as measured by waist circumference. This finding implies a role of adipose tissue metabolism in the pathophysiology of FCH.     

Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083

Arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis releases only C3-linked arabinose residues from double-substituted xylose residues. A genomic library of B. adolescentis DSM20083 was screened for the presence of the axhD3 gene. Two plasmids were identified containing part of the axhD3 gene. The nucleotide sequences were combined and three open reading frames (ORFs) were found. The first ORF showed high homology with xylanases belonging to family 8 of the glycoside hydrolases and this gene was designated xylA. The second ORF was the axhD3 gene belonging to glycoside hydrolase family 43. The third (partial) ORF coded for a putative carboxylesterase. The axhD3 gene was cloned and expressed in Escherichia coli. Several substrates were employed in the biochemical characterization of recombinant AXHd3. The enzyme showed the highest activity toward wheat arabinoxylan oligosaccharides. In addition, -xylanase from Trichoderma sp. was able to degrade soluble wheat arabinoxylan polymer to a higher extent, after pretreatment with recombinant AXHd3. Arabinoxylan oligosaccharides incubated with a combination of recombinant AXHd3 and an -l-arabinofuranosidase from Aspergillus niger did not result in a higher maximal release of arabinose than incubation with these enzymes separately.