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.

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.

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 (I_sc), 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 I_sc was signifIcantly decreased (by 33%, P<0.05) in ob/ob females versus leans, and genistein-diet reversed this. Similarly, in males, basal I_sc 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 I_sc in ob/ob female mice is a consequence of reduced CFTR expression, decreased activities of the basolateral K_Ca channel and Na⁺/K⁺-ATPase, and in male mice reduced basal jejunal I_sc is a consequence of reduced CFTR and NKCC1 expression, along with decreased activities of the basolateral K_Ca channel and Na⁺/K⁺-ATPase. Genistein-diet has beneficial effects on basal I_sc mediated by sex-dependent mechanisms in diabetic mice: in females via increased K_Ca-sensitive I_sc 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.

Background:The sex-dependent effects of chronic exposure to dietary genistein on cardiovascular health are poorly understood. Purpose: This study examined the effects of a genistein-containing diet on cardiovascular plasma markers, aortic morphology, blood pressure, and expression of cardioprotective proteins in male and female mice. Methods: C57BL/6J mice were fed either genistein diet (600 mg genistein/kg diet; 600G) or a genistein free diet (0G) for a period of 2 months. Results: After treatment, male and female mice fed 600G gained significantly less weight than their control counterparts fed 0G. Plasma insulin levels were significantly decreased in males only, whereas no changes in the other plasma markers were observed with 600G regardless of sex. Aortae from genistein-fed male mice demonstrated significant decreases in inner and outer luminal diameters and smooth muscle cell density. In female mice fed 600G, no changes in inner and outer luminal diameters were observed compared to female mice fed 0G, but smooth muscle cell density was significantly increased. Despite these differences in aortic morphology, no changes in arterial blood pressure were noted, regardless of sex or diet with genistein. Expression of cardiac glucose transporter type 4 (GLUT4) was increased in male hearts treated with genistein, while expression of endothelium nitric oxide synthase was significantly increased in females fed 600G compared to controls. However, no differences in inducible nitric oxide synthase protein were observed in all groups studied. Conclusion: Our data indicate that a 2-month diet with genistein results in changes in aortic morphology and expression of cardiac protein and its effects appear to be sex-dependent.