Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes.

Hyperglycemia in diabetes mellitus causes oxidative stress and pericyte depletion from the microvasculature of the brain thus leading to the Blood-Brain Barrier (BBB) disruption. The compromised BBB exposes the brain to circulating substances, resulting in neurotoxicity and neuronal cell death. The decline in pericyte numbers in diabetic mouse brain and pericyte apoptosis in high glucose cultures are caused by excess superoxide produced during enhanced respiration (mitochondrial oxidative metabolism of glucose). Superoxide is precursor to all Reactive Oxygen Species (ROS) which, in turn, cause oxidative stress. The rate of respiration and thus the ROS production is regulated by mitochondrial carbonic anhydrases (mCA) VA and VB, the two isoforms expressed in the mitochondria. Inhibition of both mCA: decreases the oxidative stress and restores the pericyte numbers in diabetic brain; and reduces high glucose-induced respiration, ROS, oxidative stress, and apoptosis in cultured brain pericytes. However, the individual role of the two isoforms has not been established. To investigate the contribution of mCA VA in ROS production and apoptosis, a mCA VA overexpressing brain pericyte cell line was engineered. These cells were exposed to high glucose and analyzed for the changes in ROS and apoptosis. Overexpression of mCA VA significantly increased pericyte ROS and apoptosis. Inhibition of mCA VA with topiramate prevented increases both in glucose-induced ROS and pericyte death. These results demonstrate, for the first time, that mCA VA regulates the rate of pericyte respiration. These findings identify mCA VA as a novel and specific therapeutic target to protect the cerebromicrovascular bed in diabetes.

Antisense against Amyloid-ß Protein Precursor Reverses Memory Deficits and Alters Gene Expression in Neurotropic and Insulin-Signaling Pathways in SAMP8 Mice.

The senescence-accelerated mouse (SAMP8) strain exhibits an age-related decrease in memory accompanied by an increase in hippocampal amyloid-ß protein precursor (AßPP) and amyloid-ß peptide (Aß). We have shown that administration of an antisense oligonucleotide against the Aß region of AßPP (AßPP antisense) reverses the memory deficits. The purpose of this study was to determine the effect of peripheral (IV) administration of AßPP antisense on hippocampal gene expression. The AßPP antisense reversed the memory deficits and altered expression of 944 hippocampal genes. Pathway analysis showed significant gene expression changes in nine pathways. These include the MAPK signaling pathway (p = 0.0078) and the phosphatidylinositol signaling pathway (p = 0.043), which we have previously shown to be altered in SAMP8 mice. The changes in these pathways contributed to significant changes in the neurotropin (p = 0.0083) and insulin signaling (p = 0.015) pathways, which are known to be important in learning and memory. Changes in these pathways were accompanied by phosphorylation changes in the downstream target proteins p70S6K, GSK3ß, ERK, and CREB. These changes in hippocampal gene expression and protein phosphorylation may suggest specific new targets for antisense therapy aimed at improving memory.

SAMP8 mice have altered hippocampal gene expression in long term potentiation, phosphatidylinositol signaling, and endocytosis pathways.

The senescence-accelerated mouse (SAMP8) strain exhibits decreased learning and memory and increased amyloid beta (Aß) peptide accumulation at 12 months. To detect differences in gene expression in SAMP8 mice, we used a control mouse that was a 50% cross between SAMP8 and CD-1 mice and which showed no memory deficits (50% SAMs). We then compared gene expression in the hippocampus of 4- and 12-month-old SAMP8 and control mice using Affymetrix gene arrays. At 12 months, but not at 4 months, pathway analysis revealed significant differences in the long term potentiation (6 genes), phosphatidylinositol signaling (6 genes), and endocytosis (10 genes) pathways. The changes in long term potentiation included mitogen-activated protein kinase (MAPK) signaling (N-ras, cAMP responsive element binding protein [CREB], protein phosphatase inhibitor 1) and Ca-dependent signaling (inositol triphosphate [ITP] receptors 1 and 2 and phospholipase C). Changes in phosphatidylinositol signaling genes suggested altered signaling through phosphatidylinositol-3-kinase, and Western blotting revealed phosphorylation changes in serine/threonine protein kinase AKT and 70S6K. Changes in the endocytosis pathway involved genes related to clathrin-mediated endocytosis (dynamin and clathrin). Endocytosis is required for receptor recycling, is involved in Aß metabolism, and is regulated by phosphatidylinositol signaling. In summary, these studies demonstrate altered gene expression in 3 SAMP8 hippocampal pathways associated with memory formation and consolidation. These pathways might provide new therapeutic targets in addition to targeting Aß metabolism itself.

Higher C-reactive protein and soluble tumor necrosis factor receptor levels are associated with poor physical function and disability: a cross-sectional analysis of a cohort of late middle-aged African Americans.

BACKGROUND: This cohort of "late middle-aged" African Americans has an excess of disability. We aimed to determine associations of circulating cytokine receptors (sTNFR1, sTNFR2, and sIL-6R) and C-reactive protein (CRP) with disability, physical function, and body composition. METHODS: Stratified sampling of two socioeconomically diverse strata of St Louis, Missouri, occurred in 2000-2001. Inclusion criteria were self-reported black or African American race, born 1936-1950 inclusive, and Mini-Mental State Examination score of 16 or greater. In-home evaluations of handgrip strength, lean body mass percentage (LBM%), physical performance, upper and lower body functional limitations (UBFLs and LBFLs), and basic and instrumental activities of daily living (BADLs and IADLs) were collected. Of the 998 participants, 368 had blood sampled at baseline. Serum was stored and assayed in 2006. RESULTS: Absolute risks were LBFLs of 2 or more, 46%; UBFLs of 1 or more, 23.5%; BADLs of 2 or more, 20.6%; and IADLs of 2 or more, 22.5%. Independent of age, sex, and underlying comorbid conditions, higher CRP and sTNFR were associated with poorer physical performance (beta = -1.462, p < .001 and beta = -0.618, p = .003), UBFLs (odds ratio [OR] 2.26, 95% confidence interval [CI] 1.1-4.64 and OR 1.39, 95% CI 0.96-2.02), LBFLs (OR 2.30, 95% CI 1.19-4.45 and OR 1.91, 95% CI 1.26-2.91), BADLs (OR 2.79, 95% CI 1.03-5.96 and OR 1.66, 95% CI 1.11-2.46), and IADLs (OR 2.13, 95% CI 1.03-4.41 and OR 1.43, 95% CI 0.99-2.08). Higher CRP (beta = -3.251, p <.001), sIL-6R (beta = -6.152, p = .013), and lower adiponectin (beta = 2.947, p = .052) were associated with lower LBM%. CONCLUSIONS: Higher CRP and sTNFR are independently associated with disability and physical dysfunction. Higher sIL-6R, CRP, and lower adiponectin associate with lower LBM%.

Predictors of serum testosterone and DHEAS in African-American men.

There are few reported data on biochemical and functional correlates of androgen levels in African-American men. This study aimed at reporting physical and biochemical correlates of serum total testosterone (total T), bioavailable testosterone (BT) and dehydroepiandrosterone-sulphate (DHEAS) levels in community-dwelling, African-American men aged 50-65 years. Home-based physical examinations and health status questionnaires were administered to randomly sampled men. Body composition (dual-energy X-ray absorptiometry), lower limb and hand-grip muscle strength, and neuropsychological functions were assessed. Levels of serum total T, BT, DHEAS, oestradiol (E2), adiponectin, leptin, triglycerides and glucose were measured. Multiple linear regression models were constructed to identify factors independently associated with androgen levels. DHEAS levels declined from age 50 to 65 years (p < 0.0001), but total T and BT levels remained constant. Independent of other associated factors, higher total T levels were associated with lower serum triglyceride levels (beta = -0.142, p = 0.049); higher BT was associated with better performance on the trail-making tests (TMT-B:TMT-A ratio: beta = -0.118, p = 0.024) and higher DHEAS levels were associated with lower adiponectin (beta = -0.293, p = 0.047) and higher mini-mental state examination (MMSE) score (beta = 0.098, p = 0.008). Multiple regression models predicted 21, 18 and 29% of variance in total T, BT and DHEAS, respectively. Higher total T levels were associated with serum metabolic markers, particularly lower triglycerides, whereas higher BT was associated with better cognitive and muscle function and DHEAS with lower adiponectin and higher MMSE scores.

Lower serum DHEAS levels are associated with a higher degree of physical disability and depressive symptoms in middle-aged to older African American women.

BACKGROUND: Changes in androgen levels and associations with chronic disease, physical and neuropsychological function and disability in women over the middle to later years of life are not well understood and have not been extensively studied in African American women. AIMS: The present cross-sectional analysis reports such levels and associations in community dwelling, African American women aged 49-65 years from St. Louis, Missouri. METHODS: A home-based physical examination and a health status questionnaire were administered to randomly sampled women. Body composition (DEXA), lower limb and hand-grip muscle strength, physical and neuropsychological function and disability levels were assessed. Blood was drawn and assayed for total testosterone (T), sex hormone-binding globulin (SHBG), dehydroepiandrosterone-sulfate (DHEAS), oestradiol (E2), adiponectin, leptin, triglycerides, glucose, C-reactive protein (CRP) and cytokine receptors (sIL2r, sIL6r, sTNFr1 and sTNFr2). Multiple linear regression modelling was used to identify the best predictors of testosterone, DHEAS and free androgen index (T/SHBG). RESULTS: Seventy-four percent of women were menopausal and a quarter of these were taking oestrogen therapy. DHEAS and E2 declined between the ages of 49 and 65 years, whereas total T, SHBG and FAI remained stable. Total T and DHEAS levels were strongly correlated. In this population sample there were no independent associations of either total T or FAI with indicators of functional limitations, disability or clinically relevant depressive symptoms. Unlike total T and FAI, lower DHEAS levels were independently associated with both higher IADL scores (indicating a higher degree of physical disability) and higher CESD scores (indicating a higher degree of clinically relevant depressive symptoms). CONCLUSION: There is an age-related decline in serum DHEAS in African American women. Lower DHEAS levels appear to be associated with a higher degree of physical disability and depressive symptoms in this population.

Loss of appendicular muscle mass and loss of muscle strength in young postmenopausal women.

BACKGROUND: Different factors may predict loss of appendicular muscle mass (LossAMM) and loss of muscle strength (LossMS). We investigated the relationship between LossAMM or LossMS and baseline anthropometric measures, lifestyle habits, hormones, lipid profiles, and inflammatory markers in 49 healthy postmenopausal women (54.1 +/- 4.3 years) in a 24-36-month prospective study. METHODS: We measured parameters of lifestyle habits, anthropometry, lipid profiles, and blood levels of testosterone, estrone, estradiol, cortisol, dihydroepiandrostenedione, luteinizing hormone, intact parathyroid hormone (iPTH), 25-hydroxyvitamin D, thyroxine, leptin, adiponectin, C-reactive protein (CRP), and interleukin-6 and interleukin-2 receptors. Percentage of loss per year of isometric knee extensor strength defined LossMS, and percentage of loss of AMM per year (dual x-ray absorptiometry) defined LossAMM. RESULTS: The means (standard deviation) for LossMS and LossAMM were 1.17%/y (2.03) and 0.60%/y (0.74) and did not correlate (r = -0.001; p =.99). LossMS correlated negatively with level of physical activity (r = -0.28), femoral BMD (r = -0.30), alcohol consumption (r = -0.30), and luteinizing hormone (r = -0.32) and positively with estrone (r = 0.29) and iPTH (r = 0.32) (each at p <.05). LossAMM correlated negatively with AMM (r = -0.41; p <.01). Stepwise regression analyses showed that LossMS was significantly predicted by baseline physical activity (beta = -0.39) with an explanation of variation of the model (R(2)) of 6%, body mass index (BMI) (-0.40; 3%), high-density lipoprotein cholesterol (-0.29; 3%), estrone (0.32; 6%), iPTH (0.27; 7%), and interleukin-2 receptor (0.32; 5%). LossAMM was predicted by baseline height (0.56; 47%), body mass index (1.04; 83%), AMM (-0.92; 76%), thyroxine (-0.33; 8%), estrone (-0.61; 30%), and dihydroepiandrostenedione (0.44; 28%). CONCLUSIONS: LossMS and LossAMM in young postmenopausal women were not correlated with one another, and were determined by different factors.

Adiponectin levels in obese and non-obese middle-aged African-American women.

CONTEXT: Adiponectin levels in African-Americans are poorly described. OBJECTIVE: To assess predictors of serum adiponectin levels in obese and non-obese middle-aged African-American women. METHODS: Serum adiponectin, testosterone (T), free androgen index (FAI), estradiol, dehydroepiandrosterone sulfate (DHEAS), leptin, sex hormone binding globulin (SHBG), triglycerides and C-reactive protein (CRP) were measured in 142 obese and 102 non-obese, community-dwelling, African-American women in St. Louis, Missouri. Medical history, physical activity, anthropometry, medications and body composition were assessed. RESULTS: Adiponectin and SHBG levels were lower and leptin and CRP were higher in obese compared to non-obese women (P's < 0.01). Overall, log adiponectin was positively associated with age (R = 0.13) and log SHBG (R = 0.29), and inversely associated with anthropometric measures (R's = -0.17 to -0.36), serum androgens (R's = -0.21 to -0.23), log estradiol (R = -0.21), log leptin (R = -0.15), log triglycerides (R = -0.33) and log CRP (R = -0.29). Overall, multivariate modelling significantly predicted 32% of variation in adiponectin level; the most significant factors were WHR (ß = -1.33), SHBG (ß = 0.23) and triglycerides (ß = -0.34). In non-obese women, the model predicted 27% of variation in adiponectin level; no individual factor was independently associated. In obese women, the model predicted 30% of variation in adiponectin level; the most significant factors were WHR (ß = -1.49), triglycerides (ß = -0.34) and history of stroke (ß = -0.71). CONCLUSIONS: Adiponectin level in African-American women is predicted by WHR, SHBG, and triglycerides; stroke history adds predictive value in obese women.

Leptin and adiponectin levels in middle-aged postmenopausal women: associations with lifestyle habits, hormones, and inflammatory markers--a cross-sectional study.

To investigate the relationships between blood levels of leptin or adiponectin and lifestyle habits, hormones, and inflammatory markers, we measured parameters of alcohol intake, smoking, physical activity, and blood levels of leptin, adiponectin, testosterone, estrone, estradiol, cortisol, dihydroepiandrostenedione, luteinizing hormone, thyroxin, C-reactive protein (CRP), and interleukin 6 and interleukin 2 receptor in 76 healthy middle-aged postmenopausal women. Anthropometric measures and body composition (evaluated by dual-energy x-ray absorptiometry) and lipid profiles were also assessed. By simple regression, leptin correlated positively with fat and lean masses, glucose, triglycerides, low-density lipoprotein cholesterol, and total cholesterol, and negatively with high-density lipoprotein cholesterol. Adioponectin correlated negatively with fat and lean masses and low-density lipoprotein cholesterol, and positively with high-density lipoprotein cholesterol. Leptin concentration was correlated inversely with adiponectin (r = -0.26, P < .05) and positively with CRP (r = 0.56, P < .01). Adiponectin concentration was negatively correlated with time since last alcoholic drink (r = -0.24, P < .05) and CRP (r = -0.27, P < .05) and positively with testosterone level (r = 0.23, P < .05). By multiple regression analysis, leptin concentration was predicted by age (P < .05), testosterone (P < .05), adiponectin (P < .05), CRP (P < .01), and interleukin 6 receptor (P < .01). Adiponectin concentration was predicted by the time since last alcoholic drink (P < .05), testosterone (P < .05), leptin (P < .05), and C-reactive protein (P = .05). Similar results were found when leptin or adiponectin concentration was adjusted for fat mass. These results suggested that levels of leptin and adiponectin in middle-aged postmenopausal women are partially determined by sexual hormones and inflammatory marker levels, and both predicted one another. Moreover, adiponectin level may be modulated by alcohol intake.

Validation of salivary testosterone as a screening test for male hypogonadism.

BACKGROUND: Saliva collection is an easy, non-invasive method to measure hormones. METHODS: Two studies were performed. In the first, a convenience sample of 1,454 males who had submitted saliva for salivary testosterone measurements were studied. In the second study, we intensively studied symptoms and measurements of total testosterone, free testosterone symptoms and measurements of total testosterone, free testosterone and bioavailable testosterone in relationship to salivary testosterone in 127 men. A secondary endpoint was to examine the relationship of salivary testosterone to hypogonadal symptoms in the ADAM and AMS questionnaires. RESULTS: In the first study, we have shown that salivary testosterone, measured in 1,454 males aged 20 to 89 years, declines by 47% over the lifespan. In the second study, salivary testosterone was strongly correlated with bioavailable testosterone (p < 0.000001) calculated free testosterone (p < 0.00001) and total testosterone (p < 0.002). Salivary testosterone was significantly related to hypogonadal symptoms on the St. Louis University ADAM questionnaire and the Aging Male Survey. CONCLUSIONS: These studies support the use of salivary testosterone as an acceptable assay for screening for hypogonadism. Salivary testosterone is not a better assay than other measures to diagnose hypogonadism.

Effect of testosterone on functional recovery in a castrate male rat stroke model.

Both increased and decreased testosterone levels have been reported to correlate with poor outcome after acute ischemic stroke. The present study focused on the role of testosterone during recovery from neurological deficits in a rat focal ischemia model. Castrate male rats were subjected to behavioral tests after 90 min of middle cerebral artery occlusion (MCAO). On day 7 post-MCAO, neurological deficit-matched rats were assigned to a treatment group implanted with subcutaneous testosterone pellets or a control group implanted with sham cholesterol pellets. After 4 weeks post-MCAO, the average infarct volume was not significantly different between the two groups. Rats in the testosterone group demonstrated significantly earlier improvement in neurological deficits and shortened latency of adhesive tape removal compared with the control group as analyzed by Wilcoxon signed ranks test. Walking on parallel bars improved in both groups with a trend towards early recovery observed in the testosterone group. Biased left body swings persisted during the test period in both groups post-MCAO. Serum testosterone was within physiological levels in the treatment group but was not detectable in the control group by radioimmunoassay. GAP-43 and synaptophysin expression did not differ between groups. Less GFAP expression and reactive astrocyte hypertrophy were found around the infarct area in testosterone-treated rats compared with control rats. In conclusion, testosterone replacement post-MCAO accelerated functional recovery in castrate rats, suggesting a potential therapeutic role for testosterone replacement in stroke recovery.

Evaluation of assays available to measure free testosterone.

This study compared the results of various testosterone assays in a cross-sectional study of 50 male subjects age 28 to 90 years. The purpose of the study was to determine the relationship of the various testosterone assays to one another. In addition, we determined week-to-week variability in testosterone and bioavailable testosterone in 16 subjects. The following assays were undertaken: total testosterone (T), free testosterone by equilibrium dialysis (FT(D)), bioavailable testosterone (BT), free testosterone by ultracentrifugation (FT(U)), and direct estimation of serum free T by an analog ligand radioimmunoassay (FT(A)). In addition, using total T and sex hormone-binding globulin (SHBG), we calculated the free androgen index (FAI = T/SHBG) and the free testosterone index (FTI) using the method of Vermeulen. In a second study, we measured the week-to-week variation in T and BT in a group of older males. Lastly, we demonstrated excellent stability of serum stored at -70 degrees C for up to 7 years for T and BT. Correlations of the various assays to increasing age were significant for all assays except total T (r = -.126). The best correlation was found with BT (r = -.744, P <.001). All measures were statistically significantly correlated with FT. The best correlations were FTI (r =.807, P <.007) and BT (r =.670, P <.001). If T was used to classify hypogonadism in comparison to BT, it resulted in misclassification in 42% of cases. In addition, we demonstrated a marked week-to-week variability in T and BT in older men with the T and BT being in the eugonadal range some weeks and hypogonadal on other occasions. This occurred in 8 of 16 men for T and 10 of 16 men for BT. Based on these data, we suggest that the FTI or BT are the most practical methods to determine hypogonadism. There is a need for increased awareness that marked week-to-week variability within a single individual can occur for measurements of both T and BT.