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/cm2 [n=20]) (P< 0.05) compared with their lean counterparts (95.1±9.1 μA/cm2 [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 Ca2+-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.

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.

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 (I_sc, 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 I_sc. 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 I_sc in intact and OVX mice. In intact mice, 10E2 was without effect on basal I_sc, however, in OVX mice, 10E2 significantly increased basal I_sc (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 I_sc 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-3β, 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.

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/cm2, n=6, P<0.05) and a subgroup of nonresponders (12.05±6.59 μA/cm2, n=4), compared to 0Gd controls (29.3±6.5 μA/cm2, 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.

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