Cystamine reduces vascular stiffness in Western diet-fed female mice

Consumption of diets high in fat, sugar and salt (Western diet, WD) is associated with accelerated arterial stiffening, a major independent risk factor for cardiovascular disease (CVD). Obese women are more prone to develop arterial stiffening leading to more frequent and severe CVD compared to men. As tissue transglutaminase (TG2) has been implicated in vascular stiffening, our goal herein was to determine the efficacy of cystamine, a non-specific TG2 inhibitor, at reducing vascular stiffness in female mice chronically fed a WD. Three experimental groups of female mice were created. One was fed regular chow diet (CD) for 43 weeks starting at four weeks of age. The second was fed a WD for the same 43 weeks, whereas a third cohort was fed WD, but also received cystamine (216 mg/kg/d) in the drinking water during the last eight weeks on the diet (WD+C). All vascular stiffness parameters assessed, including aortic pulse wave velocity and the incremental modulus of elasticity of isolated femoral and mesenteric arteries, were significantly increased in WD- vs. CD-fed mice, and reduced in WD+C vs. WD-fed mice. These changes coincided with respectively augmented and diminished vascular wall collagen and F-actin content, with no associated effect in blood pressure. In cultured human vascular smooth muscle cells, cystamine reduced TG2 activity, F-actin/G-actin ratio, collagen compaction capacity and cellular stiffness. We conclude that cystamine treatment represents an effective approach to reduce vascular stiffness in female mice in the setting of WD consumption, likely due to its TG2 inhibitory capacity.

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Abstract P293: Endothelial Sodium Channel Activation Promotes Vascular Stiffness in Obese Female Mice

Hypertension ◽

10.1161/hyp.68.suppl_1.p293 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Annayya Aroor ◽

Francisco I Ramirez-Perez ◽

Guanghong Jia ◽

Javad Habibi ◽

Vincent G DeMarco ◽

...

Keyword(s):

Sodium Channel ◽

Aortic Stiffness ◽

Low Dose ◽

Vascular Stiffness ◽

Obese Women ◽

High Fat ◽

Arterial Stiffening ◽

Female Mice ◽

High Fructose ◽

Endothelial Stiffness

Obesity-associated arterial stiffening is an independent predictor of cardiovascular disease (CVD) events. Although premenopausal non-obese women are protected against CVD, aortic stiffening in obese women is more common than in men. This disproportionate increase in vascular stiffness in obese females may partly explain their loss of sex-related CVD protection. Recent studies have suggested a role for endothelial sodium channel (ENaC) activation in promotion of endothelial stiffness and suppression of flow-(nitric oxide) mediated vasodilation. Increased mineralocorticoid receptor (MR) activation mediated endothelial stiffness is promoted, in part, by ENaC activation. In this regard, we have recently reported increased aortic stiffness, MR and ENaC expression and endothelial dysfunction in female mice fed a high fat and high fructose diet (western diet [WD]). This increase in aortic stiffness was prevented by very low dose MR antagonism. Accordingly, we hypothesized that inhibition of MR-mediated ENaC activation by using a very low dose of the ENaC inhibitor, amiloride, would prevent arterial stiffening and vascular dysfunction in WD-fed female mice. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1mg/kg/day) for 16 weeks. Amiloride significantly attenuated WD-induced increases in aortic stiffness in vivo as measured by pulse wave velocity as well as in vitro endothelial stiffness as measured by atomic force microscopy. Moreover, incubation of aortic explants with very low dose of amiloride (1 μM) inhibited WD-induced aortic stiffness in aorta explants from WD-fed female mice. Amiloride also prevented WD-induced impairment in acetylcholine-induced aortic vasodilatation and flow-mediated dilation in mesenteric arteries. Taken together, these observations support a role for ENaC activation in diet-induced vascular stiffening in obese females.

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

Endocrinology ◽

10.1210/en.2015-1681 ◽

2016 ◽

Vol 157 (4) ◽

pp. 1590-1600 ◽

Cited By ~ 12

Author(s):

Camila Manrique ◽

Guido Lastra ◽

Francisco I. Ramirez-Perez ◽

Dominic Haertling ◽

Vincent G. DeMarco ◽

...

Keyword(s):

Insulin Resistance ◽

Estrogen Receptor ◽

Vascular Reactivity ◽

Control Diet ◽

Western Diet ◽

Vascular Stiffness ◽

Whole Body ◽

Chow Diet ◽

Female Mice ◽

Vascular Stiffening

Abstract 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.

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Knockout of glucose transporter GLUT6 has minimal effects on whole body metabolic physiology in mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00082.2018 ◽

2018 ◽

Vol 315 (2) ◽

pp. E286-E293 ◽

Cited By ~ 10

Author(s):

Frances L. Byrne ◽

Ellen M. Olzomer ◽

Robert Brink ◽

Kyle L. Hoehn

Keyword(s):

Glucose Transporter ◽

Gene Expression Pattern ◽

Western Diet ◽

Whole Body ◽

Chow Diet ◽

Mouse Development ◽

Wild Type Littermate ◽

Female Mice ◽

Transporter Family ◽

Metabolic Physiology

Glucose transporter 6 (GLUT6) is a member of the facilitative glucose transporter family. GLUT6 is upregulated in several cancers but is not widely expressed in normal tissues. Previous studies have shown that GLUT6 knockdown kills endometrial cancer cells that express elevated levels of the protein. However, whether GLUT6 represents a viable anticancer drug target is unclear because the role of GLUT6 in normal metabolic physiology is unknown. Herein we generated GLUT6 knockout mice to determine how loss of GLUT6 affected whole body glucose homeostasis and metabolic physiology. We found that the mouse GLUT6 ( Slc2a6) gene expression pattern was similar to humans with mRNA found primarily in brain and spleen. CRISPR-Cas9-mediated deletion of Slc2a6 did not alter mouse development, growth, or whole body glucose metabolism in male or female mice fed either a chow diet or Western diet. GLUT6 deletion did not impact glucose tolerance or blood glucose and insulin levels in male or female mice fed either diet. However, compared with wild-type littermate controls, GLUT6 null female mice had a relatively minor decrease in fat accumulation when fed Western diet and had a lower respiratory exchange ratio when fed chow diet. Collectively, these data show that GLUT6 is not a major regulator of whole body metabolic physiology; therefore, GLUT6 inhibition may have minimal adverse effects if targeted for cancer therapy.

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Petite Integration Factor 1 (PIF1) helicase deficiency increases weight gain in Western diet-fed female mice without increased inflammatory markers or decreased glucose clearance

10.1101/393355 ◽

2018 ◽

Author(s):

Frances R. Belmonte ◽

Nikolaos Dedousis ◽

Ian Sipula ◽

Nikita A. Desai ◽

Aatur D. Singhi ◽

...

Keyword(s):

Gene Expression ◽

Weight Gain ◽

Hepatic Steatosis ◽

Inflammatory Markers ◽

Western Diet ◽

Metabolic Phenotype ◽

Whole Body ◽

Chow Diet ◽

Female Mice ◽

Glucose Clearance

AbstractPetite Integration Factor 1 (PIF1) is a multifunctional helicase present in nuclei and mitochondria. PIF1 knock out (KO) mice exhibit accelerated weight gain and decreased wheel running on a normal chow diet. In the current study, we investigated whether Pif1 removal alters whole body metabolism in response to weight gain. PIF1 KO and wild type (WT) C57BL/6J mice were fed a Western diet (WD) rich in fat and carbohydrates before evaluation of their metabolic phenotype. Compared with weight gain-resistant WT female mice, WD-fed PIF1 KO females, but not males, showed accelerated adipose deposition, decreased locomotor activity, and reduced whole-body energy expenditure without increased dietary intake. Surprisingly, PIF1 KO females were protected against obesity-induced alterations in fasting blood glucose and glucose clearance. WD-fed PIF1 KO females developed mild hepatic steatosis and associated changes in liver gene expression that were absent in weight-matched, WD-fed female controls, linking hepatic steatosis to Pif1 ablation rather than increased body weight. WD-fed PIF1 KO females also showed decreased gene expression of inflammatory markers in adipose tissue. Collectively, these data separated weight gain from inflammation and impaired glucose homeostasis. They also support a role for Pif1 in weight gain resistance and liver metabolic dysregulation during nutrient stress.

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Abstract P068: Endothelial Mineralocorticoid Receptor Activation Promotes Endothelial And Vascular Stiffening In Western Diet Fed Female Mice Through Sgk1 Mediated Activation Of The Endothelial Sodium Channel

Hypertension ◽

10.1161/hyp.76.suppl_1.p068 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Yan Yang ◽

Zhe Sun ◽

Annayya Aroor ◽

Liping Zhang ◽

Guanghong Jia ◽

...

Keyword(s):

Sodium Channel ◽

Mineralocorticoid Receptor ◽

Sodium Current ◽

Receptor Activation ◽

Western Diet ◽

Vascular Stiffness ◽

Female Mice ◽

Isolated Lung ◽

Vascular Stiffening ◽

Endothelial Stiffness

Over-nutrition/obesity predisposes persons, particularly women, to endothelial dysfunction and vascular stiffening. We have employed a clinically relevant model using female mice fed a high fat and high fructose diet (western diet, WD). These mice display high plasma aldosterone levels, endothelial stiffness and dysfunction and increased mineralocorticoid receptor (MR) expression in the vasculature. One potential mechanism by which MR activation may promote endothelial stiffness is through increased expression and activation of epithelial sodium channel (EnNaC) in endothelial cells (ECs) through mTOR2 mediated activation of serum and glucocorticoid regulated kinase 1(SGK1). In this investigation we observed that WD feeding in female mice for 16 wks caused endothelial (atomic force microscopy (AFM)), and aortic stiffening (PW analysis) in concert with increased expression of EnNaC and SGK1 in the endothelium and EnNaC activation in ECs. Further, amelioration of WD induced EC and vascular stiffness was accomplished by EnNaC inhibition with low dose amiloride (1mg/kg/day in drinking water) over the 16 wks of WD. We then explored the idea that inhibition of SGK1 as well as specific deletion of ECMR and EnNaC decreases vascular EC stiffness accompanied by decreased sodium current in isolated lung ECs. Accordingly, female wild type and ECMR and EnNaC KO mice were fed a WD or control diet (CD) for 16 wks. Aortic and coronary artery EC stiffness, measured ex vivo by AFM, was increased in WD fed mice and this was prevented in ECMR and EnNaC KO models. Both ECMR and EnNaC KO mice fed a WD showed decreased amiloride sensitive sodium current in isolated ECs. Further, in cultured ECs , inhibition of SGK1 by a chemical inhibitor attenuated aldosterone mediated sodium currents. Collectively, these findings support the notion that a WD promotes ECMR mediated increases in SGK1 and associated EnNaC activity in ECs together with increased endothelial and vascular stiffness in females.

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Amiloride Improves Endothelial Function and Reduces Vascular Stiffness in Female Mice Fed a Western Diet

Frontiers in Physiology ◽

10.3389/fphys.2017.00456 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 24

Author(s):

Luis A. Martinez-Lemus ◽

Annayya R. Aroor ◽

Francisco I. Ramirez-Perez ◽

Guanghong Jia ◽

Javad Habibi ◽

...

Keyword(s):

Endothelial Function ◽

Western Diet ◽

Vascular Stiffness ◽

Female Mice

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Sexual Dimorphism in Obesity-Associated Endothelial ENaC Activity and Stiffening in Mice

Endocrinology ◽

10.1210/en.2019-00483 ◽

2019 ◽

Vol 160 (12) ◽

pp. 2918-2928 ◽

Cited By ~ 3

Author(s):

Jaume Padilla ◽

Makenzie L Woodford ◽

Guido Lastra-Gonzalez ◽

Vanesa Martinez-Diaz ◽

Shumpei Fujie ◽

...

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

Sexual Dimorphism ◽

Estrogen Receptor Α ◽

Western Diet ◽

Estrogen Signaling ◽

Cellular Mechanisms ◽

Arterial Stiffening ◽

Female Mice ◽

Vascular Stiffening

Abstract Obesity and insulin resistance stiffen the vasculature, with females appearing to be more adversely affected. As augmented arterial stiffness is an independent predictor of cardiovascular disease (CVD), the increased predisposition of women with obesity and insulin resistance to arterial stiffening may explain their heightened risk for CVD. However, the cellular mechanisms by which females are more vulnerable to arterial stiffening associated with obesity and insulin resistance remain largely unknown. In this study, we provide evidence that female mice are more susceptible to Western diet–induced endothelial cell stiffening compared with age-matched males. Mechanistically, we show that the increased stiffening of the vascular intima in Western diet–fed female mice is accompanied by enhanced epithelial sodium channel (ENaC) activity in endothelial cells (EnNaC). Our data further indicate that: (i) estrogen signaling through estrogen receptor α (ERα) increases EnNaC activity to a larger extent in females compared with males, (ii) estrogen-induced activation of EnNaC is mediated by the serum/glucocorticoid inducible kinase 1 (SGK-1), and (iii) estrogen signaling stiffens endothelial cells when nitric oxide is lacking and this stiffening effect can be reduced with amiloride, an ENaC inhibitor. In aggregate, we demonstrate a sexual dimorphism in obesity-associated endothelial stiffening, whereby females are more vulnerable than males. In females, endothelial stiffening with obesity may be attributed to estrogen signaling through the ERα–SGK-1–EnNaC axis, thus establishing a putative therapeutic target for female obesity-related vascular stiffening.

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Effects of High-Fat and High-Fat/High-Sucrose Diet-Induced Obesity on PVAT Modulation of Vascular Function in Male and Female Mice

Frontiers in Pharmacology ◽

10.3389/fphar.2021.720224 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jamaira A. Victorio ◽

Daniele M. Guizoni ◽

Israelle N. Freitas ◽

Thiago R. Araujo ◽

Ana P. Davel

Keyword(s):

Vascular Function ◽

Vascular Complications ◽

Mesenteric Arteries ◽

Chow Diet ◽

High Fat ◽

Perivascular Adipose Tissue ◽

Male And Female ◽

Female Mice ◽

Diet Induced Obesity ◽

High Sucrose

Increased adiposity in perivascular adipose tissue (PVAT) has been related to vascular dysfunction. High-fat (HF) diet-induced obesity models are often used to analyze the translational impact of obesity, but differences in sex and Western diet type complicate comparisons between studies. The role of PVAT was investigated in small mesenteric arteries (SMAs) of male and female mice fed a HF or a HF plus high-sucrose (HF + HS) diet for 3 or 5 months and compared them to age/sex-matched mice fed a chow diet. Vascular responses of SMAs without (PVAT-) or with PVAT (PVAT+) were evaluated. HF and HF + HS diets increased body weight, adiposity, and fasting glucose and insulin levels without affecting blood pressure and circulating adiponectin levels in both sexes. HF or HF + HS diet impaired PVAT anticontractile effects in SMAs from females but not males. PVAT-mediated endothelial dysfunction in SMAs from female mice after 3 months of a HF + HS diet, whereas in males, this effect was observed only after 5 months of HF + HS diet. However, PVAT did not impact acetylcholine-induced relaxation in SMAs from both sexes fed HF diet. The findings suggest that the addition of sucrose to a HF diet accelerates PVAT dysfunction in both sexes. PVAT dysfunction in response to both diets was observed early in females compared to age-matched males suggesting a susceptibility of the female sex to PVAT-mediated vascular complications in the setting of obesity. The data illustrate the importance of the duration and composition of obesogenic diets for investigating sex-specific treatments and pharmacological targets for obesity-induced vascular complications.

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OR17-06 Transglutaminase 2 Inhibition Reduces Aortic Stiffness in Western Diet-Fed Female Mice

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.416 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Bhavana Chinnakotla ◽

Camila Margarita Manrique Acevedo ◽

Padilla Jaume ◽

Makenzie L Woodford ◽

Annayya R Aroor ◽

...

Keyword(s):

Aortic Stiffness ◽

Weight Control ◽

Ex Vivo ◽

Transglutaminase 2 ◽

Competitive Inhibitor ◽

Tolerance Test ◽

Western Diet ◽

Arterial Stiffening ◽

Female Mice

Abstract Widespread consumption of diets high in fat, sugars and salt (Western diet, WD) is associated arterial stiffening, which is a major independent risk factor for cardiovascular disease (CVD). Notably, while WD feeding increases the risk of CVD in both males and females, the latter are more prone to develop arterial stiffening. However, the mechanisms underlying WD-induced arterial stiffening are poorly understood, particularly in females, and there are currently no specific treatments targeted at vascular stiffening.Tissue transglutaminase 2 (TG2) is an enzyme that mediates the cross-linking and stabilization of extracellular matrix proteins such as collagen, and promotes the polymerization of actin stress fibers of the cytoskeleton. It is ubiquitously expressed and abundantly present in the vasculature. Mounting evidence implicates TG2 activation in the pathogenesis of arterial stiffening and vascular fibrosis. Herein we propose that TG2 activation is central to WD-induced arterial stiffening and sought to determine the efficacy of cystamine (a non-specific competitive inhibitor of TG2) for reducing arterial stiffening in the setting of WD consumption. Accordingly, we fed 20 female mice (4 weeks old) a WD (4.65 kcal/g of food, fat 46% kcals, high-fructose corn syrup 17.5%, sucrose 17.5%, protein 17.6%, salt 1.6%) for 43 weeks. Ten of these mice received cystamine (40 mg/Kg/d in the drinking water) during their last 8 weeks on the WD. Another group of female mice (n=10) fed regular chow was used as reference controls. Aortic stiffness was measured in vivo via ultrasound-based pulse wave velocity and ex vivo by aortic explant atomic force microscopy. Vasomotor responses were assessed in isolated aortic rings via wire myography.Cystamine did not influence glucose homeostasis (intraperitoneal glucose tolerance test) or blood pressure (tail-cuff) (control 77.208±2.229 mm Hg versus WD 77.208±6.077 versus WD+Cystamine 76.297±7.894), but it was associated with increased body weight (control 26.860±2.215 grams versus WD 25.320±2.889 versus WD+Cystamine 33.220±4.848, p<0.05). Notably, cystamine reduced aortic stiffness in WD-fed mice both in vivo and ex vivo such that differences between chow-fed and WD-fed mice were normalized (control 5.294±1.713 versus WD 11.735±5.962 p≤0.05, control 5.294±1.713 versus WD+Cystamine 3.940±0.378 KPa, p<0.05). In addition, WD-induced impairments in endothelium-independent vasorelaxation (i.e. responses to sodium nitroprusside) were restored with cystamine. Collectively, our data show that cystamine reduces aortic stiffness and improves endothelium-independent vasorelaxation in female mice chronically exposed to WD, and that these effects occur despite an increase in weight gain. These findings implicate TG2 as a promising therapeutic target for reducing arterial stiffening in the context of chronic over-nutrition in females.

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