Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.

Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2CLCN2) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2CLCN2) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2pEZ cells. ClC-2 over-expression (Caco-2CLCN2) increased occludin protein level compared to control Caco-2pEZ cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2CLCN2 cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2CLCN2 cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.

Dietary Genistein Rescues Reduced Basal Chloride Secretion in Diabetic Jejunum via Sex-Dependent Mechanisms.

BACKGROUND/AIMS: The goal of this study was to determine the effect of dietary genistein (naturally occurring phytoestrogen) on jejunal secretory function in a clinically relevant model of diabetes and obesity, the leptin-defIcient ob/ob mouse. METHODS: We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from 12-week old male and female ob/ob and lean C57Bl/6J mice fed a genistein diet (600 mg genistein/kg diet) for 4-weeks. Separate segments of jejunum were frozen for western blot determination of key proteins involved in secretory transport. RESULTS: Basal Isc was signifIcantly decreased (by 33%, P<0.05) in ob/ob females versus leans, and genistein-diet reversed this. Similarly, in males, basal Isc was decreased (by 47%, P<0.05) in ob/ob mice versus leans, and genistein-diet reversed this. Inhibition with either clotrimazole (100 µM, bilateral) or ouabain (100 µM, basolateral) was signifIcantly reduced in ob/ob mice compared to leans (P<0.05), and genistein-diet reversed clotrimazole-sensitive inhibition in ob/ob females, and reversed the ouabain-sensitive inhibition in males (indicating sex-dependent mechanisms). Our data suggested that PDE3 levels were dysregulated in ob/ob females and genistein reversed this. Expression of total CFTR (normalized to actin) was signifIcantly decreased ∼80% (P<0.05) in all ob/ob mice compared to leans, and genistein-diet was without effect. Expression of total NKCC1 (normalized to actin) was signifIcantly decreased ∼80% (P<0.05) in ob/ob male mice versus leans, and genistein-diet reversed this. CONCLUSIONS: Our data suggests that the reduced basal jejunal Isc in ob/ob female mice is a consequence of reduced CFTR expression, decreased activities of the basolateral KCa channel and Na+/K+-ATPase, and in male mice reduced basal jejunal Isc is a consequence of reduced CFTR and NKCC1 expression, along with decreased activities of the basolateral KCa channel and Na+/K+-ATPase. Genistein-diet has beneficial effects on basal Isc mediated by sex-dependent mechanisms in diabetic mice: in females via increased KCa-sensitive Isc and in males via increased Na+/K+-ATPase activity and increased NKCC1 expression. Improved understanding of intestinal dysfunctions in the ob/ob jejunum, may allow for the development of novel drug targets to treat obesity and diabetes, and may also be of benefit in CF-related diabetes.

Sex-Dependent Effects of Dietary Genistein on Echocardiographic Profile and Cardiac GLUT4 Signaling in Mice.

This study aimed to determine whether genistein diet resulted in changes in cardiac function, using echocardiography, and expression of key proteins involved in glucose uptake by the myocardium. Intact male and female C57BL/6J mice (aged 4-6 weeks) were fed either 600 mg genistein/kg diet (600 G) or 0 mg genistein/kg diet (0 G) for 4 weeks. Echocardiography data revealed sex-dependent differences in the absence of genistein: compared to females, hearts from males exhibited increased systolic left ventricle internal dimension (LVIDs), producing a decrease in function, expressed as fractional shortening (FS). Genistein diet also induced echocardiographic changes in function: in female hearts, 600G induced a 1.5-fold (P < 0.05) increase in LVIDs, resulting in a significant decrease in FS and whole heart surface area when compared to controls (fed 0 G). Genistein diet increased cardiac GLUT4 protein expression in both males (1.51-fold, P < 0.05) and females (1.76-fold, P < 0.05). However, no effects on the expression of notable intracellular signaling glucose uptake-regulated proteins were observed. Our data indicate that consumption of genistein diet for 4 weeks induces echocardiographic changes in indices of systolic function in females and has beneficial effects on cardiac GLUT4 protein expression in both males and females.

Altered Energy Metabolism Pathways in the Posterior Cingulate in Young Adult Apolipoprotein E ɛ4 Carriers.

The APOE gene, encoding apolipoprotein E, is the primary genetic risk factor for late-onset Alzheimer's disease (AD). Apolipoprotein E ɛ4 allele (APOE4) carriers have alterations in brain structure and function (as measured by brain imaging) even as young adults. Examination of this population is valuable in further identifying details of these functional changes and their association with vulnerability to AD decades later. Previous work demonstrates functional declines in mitochondrial activity in the posterior cingulate cortex, a key region in the default mode network, which appears to be strongly associated with functional changes relevant to AD risk. Here, we demonstrate alterations in the pathways underlying glucose, ketone, and mitochondrial energy metabolism. Young adult APOE4 carriers displayed upregulation of specific glucose (GLUT1 & GLUT3) and monocarboxylate (MCT2) transporters, the glucose metabolism enzyme hexokinase, the SCOT & AACS enzymes involved in ketone metabolism, and complexes I, II, and IV of the mitochondrial electron transport chain. The monocarboxylate transporter (MCT4) was found to be downregulated in APOE4 carriers. These data suggest that widespread dysregulation of energy metabolism in this at-risk population, even decades before possible disease onset. Therefore, these findings support the idea that alterations in brain energy metabolism may contribute significantly to the risk that APOE4 confers for AD.

Genistein stimulates jejunum chloride secretion via an Akt-mediated pathway in intact female mice.

BACKGROUND/AIMS: We have previously shown that daily subcutaneous injections with the naturally occurring phytoestrogen genistein (600 mg genistein/kg body weight/day, 600G) results in a significantly increased basal intestinal chloride, Cl(-), secretion (Isc, a measure of transepithelial secretion) in intact C57BL/6J female mice after 1-week of treatment, compared to controls (DMSO vehicle injected). Removal of endogenous estrogen via ovariectomy (OVX) had no effect on the 600G-mediated increase in basal Isc. METHODS: Given the estrogen-like characteristics of genistein, we compared the effects of daily estradiol (E2) injections (10 mg E2/kg body weight/day, 10E2) on basal Isc in intact and OVX mice. In intact mice, 10E2 was without effect on basal Isc, however, in OVX mice, 10E2 significantly increased basal Isc (mimicked 600G). The goal of the current study was to characterize the intracellular signaling pathways responsible for mediating 600G- or 10E2-stimulated increases in basal Isc in intact female or OVX mice. RESULTS: We measured total protein expression in isolated segments of jejunum using western blot from the following six groups of mice; intact or OVX with; 600G, 10E2 or control. The proteins of interest were: Akt, p-Akt, p-PDK1, p-PTEN, p-c-Raf, p-GSK-3ß, rap-1 and ERK1/2. All blots were normalized to GAPDH levels (n = 6-18/group). CONCLUSION: These data suggest that the presence of the endogenous sex steroid, estrogen, modifies the intracellular signaling pathway required to mediate Cl(-) secretion when the intestine is exposed to exogenous 600G or E2. These studies may have relevance for designing pharmacological tools for women with intestinal chloride secretory dysfunctions.

Effect of genistein on basal jejunal chloride secretion in R117H CF mice is sex and route specific.

Cystic fibrosis (CF) results from the loss or reduction in function of the CFTR (cystic fibrosis transmembrane conductance regulatory protein) chloride channel. The third most common CFTR mutation seen clinically is R117H. Genistein, a naturally occurring phytoestrogen, is known to stimulate CFTR function in vitro. We aimed to determine whether route of administration of genistein could mediate differential effects in R117H male and female CF mice. Mice were fed (4 weeks) or injected subcutaneously (1 week) with the following: genistein 600 mg/kg diet (600Gd); genistein-free diet (0Gd); genistein injection 600 mg/kg body weight (600Gi); dimethyl sulfoxide control (0Gi). In male R117H mice fed 600Gd, basal short circuit current (Isc) was unchanged. In 600Gd-fed female mice, there was a subgroup that demonstrated a significant increase in basal Isc (53.14±7.92 µA/cm(2), n=6, P<0.05) and a subgroup of nonresponders (12.05±6.59 µA/cm(2), n=4), compared to 0Gd controls (29.3±6.5 µA/cm(2), n=7). In R117H mice injected with 600Gi, basal Isc was unchanged in both male and female mice compared to 0Gi controls. Isc was measured in response to the following: the adenylate cyclase activator forskolin (10 µM, bilateral), bumetanide (100 µM, basolateral) to indicate the Cl(-) secretory component, and acetazolamide (100 µM, bilateral) to indicate the HCO3 (-) secretory component; however, there was no effect of genistein (diet or injection) on any of these parameters. Jejunal morphology (ie, villi length, number of goblet cells per villus, crypt depth, and number of goblet cells per crypt) in R117H mice suggested no genistein-mediated difference among the groups. Serum levels of genistein were significantly elevated, compared to respective controls, by either 600Gd (equally elevated in males and females) or 600Gi (elevated more in females versus males). These data suggest a sex-dependent increase in basal Isc of R117H mice and that the increase is also specific for route of administration.

Decreased basal chloride secretion and altered cystic fibrosis transmembrane conductance regulatory protein, Villin, GLUT5 protein expression in jejunum from leptin-deficient mice.

Patients with diabetes and obesity are at increased risk of developing disturbances in intestinal function. In this study, we characterized jejunal function in the clinically relevant leptin-deficient ob/ob mouse, a model of diabetes and obesity. We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from 12-week-old ob/ob and lean C57BL/6J (female and male) mice. The basal Isc was significantly decreased (~30%) in the ob/ob mice (66.5±5.7 µA/cm(2) [n=20]) (P< 0.05) compared with their lean counterparts (95.1±9.1 µA/cm(2) [n=19]). Inhibition with clotrimazole (100 µM, applied bilaterally) was significantly reduced in the ob/ob mice (-7.92%±3.67% [n=15]) (P<0.05) compared with the lean mice (10.44%±7.92% [n=15]), indicating a decreased contribution of Ca(2+)-activated K(+) (KCa) channels in the ob/ob mice. Inhibition with ouabain (100 µM, applied serosally) was significantly reduced in the ob/ob mice (1.40%±3.61%, n=13) (P< 0.05) versus the lean mice (18.93%±3.76% [n=18]), suggesting a potential defect in the Na(+)/K(+)-adenosine triphosphate (ATP)ase pump with leptin-deficiency. Expression of cystic fibrosis transmembrane conductance regulatory protein (CFTR) (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) was significantly decreased ~twofold (P<0.05) in the ob/ob mice compared with the leans, whilst crypt depth was unchanged. Villi length was significantly increased by ~25% (P<0.05) in the ob/ob mice compared with the leans and was associated with an increase in Villin and GLUT5 expression. GLUT2 and SGLT-1 expression were both unchanged. Our data suggests that reduced basal jejunal Isc in ob/ob mice is likely a consequence of reduced CFTR expression and decreased activity of the basolateral KCa channel and Na(+)/K(+)-ATPase. Understanding intestinal dysfunctions in ob/ob jejunum may allow for the development of novel drug targets to treat obesity and diabetes.