Endothelial Estrogen Receptor-α Does Not Protect Against Vascular Stiffness Induced by Western Diet in Female Mice.

Consumption of a diet high in fat and refined carbohydrates (Western diet [WD]) is associated with obesity and insulin resistance, both major risk factors for cardiovascular disease (CVD). In women, obesity and insulin resistance abrogate the protection against CVD likely afforded by estrogen signaling through estrogen receptor (ER)α. Indeed, WD in females results in increased vascular stiffness, which is independently associated with CVD. We tested the hypothesis that loss of ERα signaling in the endothelium exacerbates WD-induced vascular stiffening in female mice. We used a novel model of endothelial cell (EC)-specific ERα knockout (EC-ERαKO), obtained after sequential crossing of the ERα double floxed mice and VE-Cadherin Cre-recombinase mice. Ten-week-old females, EC-ERαKO and aged-matched genopairs were fed either a regular chow diet (control diet) or WD for 8 weeks. Vascular stiffness was measured in vivo by pulse wave velocity and ex vivo in aortic explants by atomic force microscopy. In addition, vascular reactivity was assessed in isolated aortic rings. Initial characterization of the model fed a control diet did not reveal changes in whole-body insulin sensitivity, aortic vasoreactivity, or vascular stiffness in the EC-ERαKO mice. Interestingly, ablation of ERα in ECs reduced WD-induced vascular stiffness and improved endothelial-dependent dilation. In the setting of a WD, endothelial ERα signaling contributes to vascular stiffening in females. The precise mechanisms underlying the detrimental effects of endothelial ERα in the setting of a WD remain to be elucidated.

Prevention of obesity-induced renal injury in male mice by DPP4 inhibition.

Therapies to prevent renal injury in obese hypertensive individuals are being actively sought due to the obesity epidemic arising from the Western diet (WD), which is high in fructose and fat. Recently, activation of the immune system and hyperuricemia, observed with high fructose intake, have been linked to the pathophysiology of hypertension and renal injury. Because dipeptidyl peptidase 4 (DPP4) is a driver of maladaptive T-cell/macrophage responses, renal-protective benefits of DPP4 inhibition in the WD-fed mice were examined. Mice fed a WD for 16 weeks were given the DPP4 inhibitor MK0626 in their diet beginning at 4 weeks of age. WD-fed mice were obese, hypertensive, and insulin-resistant and manifested proteinuria and increased plasma DPP4 activity and uric acid levels. WD-fed mice also had elevated kidney DPP4 activity and monocyte chemoattractant protein-1 and IL-12 levels and suppressed IL-10 levels in the kidney, suggesting macrophage-driven inflammation, glomerular and tubulointerstitial injury. WD-induced increases in DPP4 activation in the plasma and kidney and proteinuria in WD mice were abrogated by MK0626, although blood pressure and systemic insulin sensitivity were not improved. Contemporaneously, MK0626 reduced serum uric acid levels, renal oxidative stress, and IL-12 levels and increased IL-10 levels, suggesting that suppression of DPP4 activity leads to suppression of renal immune/inflammatory injury responses to a WD. Taken together, these results demonstrate that DPP4 inhibition prevents high-fructose/high-fat diet-induced glomerular and tubular injury independent of blood pressure/insulin sensitivity and offers a potentially novel therapy for diabetic and obesity-related kidney disease.

Loss of Estrogen Receptor α Signaling Leads to Insulin Resistance and Obesity in Young and Adult Female Mice.

BACKGROUND/AIMS: There are important sex-related differences in the prevalence of obesity, type 2 diabetes mellitus and cardiovascular disease. Indeed, premenopausal women have a lower prevalence of these conditions relative to age-matched men. Estrogen participates in the modulation of insulin sensitivity, energy balance, and body composition. In this paper, we investigated the impact of estrogen signaling through estrogen receptor α (ERα) on systemic insulin sensitivity and insulin signaling in skeletal muscle. METHODS: In 14- and 30-week-old female ERα knockout (ERαKO) mice and age-matched controls, we assessed insulin sensitivity by a euglycemic-hyperinsulinemic clamp and intraperitoneal glucose tolerance testing. Blood pressure was evaluated by tail cuff and telemetry. We studied ex vivo insulin-stimulated glucose uptake in skeletal muscle tissue, as well as insulin metabolic signaling molecule phosphorylation by immunoblotting and oxidative stress by immunostaining for 3-nitrotyrosine. RESULTS: Body weight was higher in ERαKO mice at 14 and 30 weeks of age. At 30 weeks, intraperitoneal glucose tolerance testing and clamp results demonstrated impaired systemic insulin sensitivity in ERαKO mice. Insulin-stimulated glucose uptake in soleus was lower in ERαKO mice at both ages. The insulin receptor substrate 1/phosphatidylinositol 3-kinase association and the activation of protein kinase B were decreased in ERαKO mice, whereas immunostaining for 3-nitrotyrosine was increased. CONCLUSIONS: Our data demonstrate a critical age-dependent role for estrogen signaling through ERα on whole-body insulin sensitivity and insulin metabolic signaling in skeletal muscle tissue. These findings have potential translational implications for the prevention and management of type 2 diabetes mellitus and cardiovascular disease in women, who are at increased risk for these conditions.

Estradiol attenuates multiple tetrodotoxin-sensitive sodium currents in isolated gonadotropin-releasing hormone neurons.

Secretion from gonadotropin-releasing hormone (GnRH) neurons is necessary for the production of gametes and hormones from the gonads. Subsequently, GnRH release is regulated by steroid feedback. However, the mechanisms by which steroids, specifically estradiol, modulate GnRH secretion are poorly understood. We have previously shown that estradiol administered to the female mouse decreases inward currents in fluorescently labeled GnRH neurons. The purpose of this study was to examine the contribution of sodium currents in the negative feedback action of estradiol. Electrophysiology was performed on GnRH neurons dissociated from young, middle-aged, or old female mice. All mice were ovariectomized; half were estradiol replaced. The amplitude of the sodium current underlying the action potential was significantly decreased in GnRH neurons from young estradiol-treated animals. In addition, in vivo estradiol significantly decreased the transient sodium current amplitude, but prolonged the sodium current inactivation time constant. Estradiol decreased the persistent sodium current amplitude, and induced a significant negative shift in peak current potential. In contrast to results obtained from cells from young reproductive animals, estradiol did not significantly attenuate the sodium current underlying the action potential in cells isolated from middle-aged or old mice. Sodium channels can modulate cell threshold, latency of firing, and action potential characteristics. The reduction of sodium current amplitude by estradiol suggests a negative feedback on GnRH neurons, which could lead to a downregulation of cell excitability and hormone release. The attenuation of estradiol regulation in peripostreproductive and postreproductive animals could lead to dysregulated hormone release with advancing age.

Age affects spontaneous activity and depolarizing afterpotentials in isolated gonadotropin-releasing hormone neurons.

Neuronal activity underlying the pulsatile secretion of GnRH remains poorly understood, as does the endogenous generation of such activity. It is clear that changes at the level of the hypothalamus are taking place during reproductive aging, yet virtually nothing is known about GnRH neuronal physiology in aging and postreproductive animals. In these studies, we performed cell-attached and whole-cell recordings in GnRH-enhanced green fluorescent protein neurons dissociated from young (3 months), middle-aged (10 months), and old (15-18 months) female mice. All mice were ovariectomized; half were estradiol replaced. Neurons from all ages fired spontaneously, most in a short-burst pattern that is characteristic of GnRH neuronal firing. Membrane characteristics were not affected by age. However, firing frequency was significantly reduced in neurons from old animals, as was spike patterning. The amplitude of the depolarizing afterpotential, evoked by a 200-msec current pulse, was significantly smaller in aged animals. In addition, inward whole-cell currents were reduced in estradiol-treated animals, although they were not significantly affected by age. Because depolarizing afterpotentials have been shown to contribute to prolonged discharges of activity after a very brief excitatory input, a decreased depolarizing afterpotential could lead to attenuated pulses in older animals. In addition, decreases in frequency and pattern generation could lead to improper information coding. Therefore, changes in the GnRH neuron during aging could lead to dysregulated activity, potentially resulting in the attenuated LH pulses observed in the transition to reproductive senescence.