Sex hormones, sex chromosomes, and microbiota: Identification of Akkermansia muciniphila as an estrogen-responsive microbiota.

Microbiota composition is known to be linked to sex. However, separating sex hormones and sex chromosome roles in gut microbial diversity is yet to be determined. To investigate the sex chromosome role independent of sex hormones, we used the four-core genotype mouse model. In this mouse model, males with testes and females with ovaries have XX or XY sex chromosome complement. In gonadectomized four-core genotype mice, we observed a significant decrease in the levels of estradiol (P<0.001) and progesterone (P<0.03) in female and testosterone (P<0.0001) in male mice plasma samples. Independent of sex chromosome complement, microbial α diversity was increased in gonadectomized female but not male mice compared to sex-matched gonad-intact controls. ß diversity analysis showed separation between male (P<0.05) but not female XX and XY mice. Importantly, Akkermansia muciniphila was less abundant in gonadectomized compared to gonadal intact female mice (P<0.0001). In the presence of ß-estradiol, Akkermansia muciniphila growth exponentially increased, providing evidence for the identification of a female sex hormone-responsive bacterium (P<0.001).

Testosterone Deficiency Promotes Arterial Stiffening Independent of Sex Chromosome Complement.

Background: Testosterone plays a vital role in men's health. Lower testosterone level is associated with cardiovascular and cardiometabolic diseases, including inflammation, atherosclerosis, and type 2 diabetes. Testosterone replacement is beneficial or neutral to men's cardiovascular health. Testosterone deficiency is associated with cardiovascular events. Testosterone supplementation to hypogonadal men improves libido, increases muscle strength, and enhances mood. We hypothesized that sex chromosomes (XX and XY) interaction with testosterone plays a role in arterial stiffening. Methods: We used four core genotype male mice to understand the inherent contribution of sex hormones and sex chromosome complement in arterial stiffening. Age-matched mice were either gonadal intact or castrated for eight weeks, followed by an assessment of blood pressure, pulse wave velocity, echocardiography, and ex vivo passive vascular mechanics. Results: Arterial stiffening but not blood pressure was more significant in castrated than testes-intact mice independent of sex chromosome complement. Castrated mice showed a leftward shift in stress-strain curves and carotid wall thinning. Sex chromosome complement (XX) in the absence of testosterone increased collagen deposition in the aorta and Kdm6a gene expression. Conclusion: Testosterone deprivation increases arterial stiffening and vascular wall remodeling. Castration increases Col1α1 in male mice with XX sex chromosome complement. Our study shows decreased aortic contractile genes in castrated mice with XX than XY sex chromosomes.

Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1-7).

Compromised barrier function of colon epithelium with aging is largely due to gut microbial dysbiosis. Recent studies implicate an important role for angiotensin converting enzymes, ACE and ACE2, angiotensins, and the receptors, AT1 receptor (AT1R) and Mas receptor (MasR), in the regulation of colon functions. The present study tested the hypothesis that leaky gut in aging is associated with an imbalance in ACE2/ACE and that the treatment with angiotenisn-(1-7) (Ang-(1-7)) will restore gut barrier integrity and microbiome. Studies were carried out in Young (3-4 months) and old (20-24 months) male mice. Ang-(1-7) was administered by using osmotic pumps. Outcome measures included expressions of ACE, ACE2, AT1R, and MasR, intestinal permeability by using FITC-dextran, and immunohistochemistry of claudin 1 and occludin, and intestinal stem cells (ISCs). ACE2 protein and activity were decreased in Old group while that of ACE were unchanged. Increased intestinal permeability and plasma levels of zonulin-1 in the Old group were normalized by Ang-(1-7). Epithelial disintegrity, reduced number of goblet cells and ISCs in the old group were restored by Ang-(1-7). Expression of claudin 1 and occludin in the aging colon was increased by Ang-(1-7). Infiltration of CD11b+ or F4/80+ inflammatory cells in the old colons were decreased by Ang-(1-7). Gut microbial dysbiosis in aging was evident by decreased richness and altered beta diversity that were reversed by Ang-(1-7) with increased abundance of Lactobacillus or Lachnospiraceae. The present study shows that Ang-(1-7) restores gut barrier integrity and reduces inflammation in the aging colon by restoring the layer of ISCs and by restructuring the gut microbiome.

Reversal of aging-associated increase in myelopoiesis and expression of alarmins by angiotensin-(1-7).

Aging is associated with chronic systemic inflammation largely due to increased myelopoiesis, which in turn increases risk for vascular disease. We have previously shown evidence for the therapeutic potential of Angiotensin-(1-7) (Ang-(1-7)) in reversing vasoreparative dysfunction in aging. This study tested the hypothesis that ischemic vascular repair in aging by Ang-(1-7) involves attenuation of myelopoietic potential in the bone marrow and decreased mobilization of inflammatory cells. Young or Old male mice of age 3-4 and 22-24 months, respectively, received Ang-(1-7) (1 µg/kg/min, s.c.) for four weeks. Myelopoiesis was evaluated in the bone marrow (BM) cells by carrying out the colony forming unit (CFU-GM) assay followed by flow cytometry of monocyte-macrophages. Expression of pro-myelopoietic factors and alarmins in the hematopoietic progenitor-enriched BM cells was evaluated. Hindlimb ischemia (HLI) was induced by femoral ligation, and mobilization of monocytes into the blood stream was determined. Blood flow recovery was monitored by Laser Doppler imaging and infiltration of inflammatory cells was evaluated by immunohistochemistry. BM cells from Old mice generated a higher number of monocytes (Ly6G-CD11b+Ly6Chi) and M1 macrophages (Ly6ChiF4/80+) compared to that of Young, which was reversed by Ang-(1-7). Gene expression of selected myelopoietic factors, alarmins (S100A8, S100A9, S100A14 and HMGb1) and the receptor for alarmins, RAGE, was higher in the Old hematopoietic progenitor-enriched BM cells compared to the Young. Increased expressions of these factors were decreased by Ang-(1-7). Ischemia-induced mobilization of monocytes was higher in Old mice with decreased blood flow recovery and increased infiltration of monocyte-macrophages compared to the Young, all of which were reversed by Ang-(1-7). Enhanced ischemic vascular repair by Ang-(1-7) in aging is largely by decreasing the generation and recruitment of inflammatory monocyte-macrophages to the areas of ischemic injury. This is associated with decreased alarmin signaling in the BM-hematopoietic progenitor cells.

Diets with low n-6:n-3 PUFA ratio protects rats from fructose-induced dyslipidemia and associated hepatic changes: Comparison between 18:3 n-3 and long-chain n-3 PUFA.

In the present study, we investigated the impact of substituting alpha-linolenic acid (ALA) or long-chain n-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) for linoleic acid and hence decreasing n-6:n-3 PUFA ratio on high-fructose diet-induced hypertriglyceridemia and associated hepatic changes. Weanling male Wistar rats were divided into four groups and fed with starch-diet (n-6:n-3 PUFA ratio 215:1) and high-fructose diets with different n-6:n-3 PUFA ratio (215:1, 2:1 with ALA and 5:1 with long-chain n-3 PUFA) for twenty-four weeks. Substitution of linoleic acid with ALA (n-6:n-3 PUFA ratio of 2) or long-chain n-3 PUFA (n-6:n-3 PUFA ratio of 5) protected the rats from fructose-induced dyslipidemia, hepatic oxidative stress and corrected lipogenic and proinflammatory gene expression. Both ALA and long-chain n-3 PUFA supplementation also reversed the fructose-induced upregulation of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) gene, which is involved in the generation of active glucocorticoids in tissues. Although both ALA and LC n-3 PUFA prevented fructose-induced dyslipidemia to a similar extent, compared to ALA, LC n-3 PUFA is more effective in preventing hepatic oxidative stress and inflammation.

Chronic consumption of fructose in combination with trans fatty acids but not with saturated fatty acids induces nonalcoholic steatohepatitis with fibrosis in rats.

PURPOSE: Consumption of Western diet high in fat and fructose has been attributed to the recent epidemic of nonalcoholic fatty liver disease (NAFLD). However, the impact of specific fatty acids on the progression of NAFLD to nonalcoholic steatohepatitis (NASH) is poorly understood. In the present study, we investigated the chronic effects of consumption of fructose in combination with saturated fatty acids (SFA) or trans fatty acids (TFA) on the development of NAFLD. METHODS: Male Sprague-Dawley rats were randomly assigned to six isocaloric starch/high fructose (44% of calories), high fat (39% calories) diet containing either starch-peanut oil, fructose-peanut oil, fructose-palmolein, fructose-clarified butter, fructose-coconut oil or fructose-partially hydrogenated vegetable oil and fed for 24 weeks. Palmolein, clarified butter and coconut oil were used as the source of SFA whereas partially hydrogenated vegetable oil was used as the source of TFA. Peanut oil was used as the reference oil. RESULTS: Long-term feeding of fructose in combination with SFA or TFA induced hepatic steatosis of similar extent associated with upregulation of stearoyl CoA desaturase-1. In contrast, fructose in combination with TFA induced NASH with fibrosis as evidenced by upregulation of hepatic proinflammatory cytokine and fibrogenic gene expression, increased hepatic oxidative stress and adipocytokine imbalance. Histopathological analysis revealed the presence of NASH with fibrosis. Further, peanut oil prevented the development of NAFLD in fructose-fed rats. CONCLUSION: Fructose in combination with TFA caused NASH with fibrosis by inducing oxidative stress and inflammation, whereas, fructose in combination with SFA caused simple steatosis, suggesting that the type of fatty acid is more important for the progression of NAFLD.

Transient Decrease in Circulatory Testosterone and Homocysteine Precedes the Development of Metabolic Syndrome Features in Fructose-Fed Sprague Dawley Rats.

Background. Increased fructose consumption is linked to the development of metabolic syndrome (MS). Here we investigated the time course of development of MS features in high-fructose-fed Sprague Dawley rats along with circulatory testosterone and homocysteine levels. Methods. Rats were divided into control and experimental groups and fed with diets containing 54.5% starch and fructose, respectively, for 4, 12, and 24 weeks. Plasma testosterone and homocysteine levels were measured along with insulin, glucose, and lipids. Body composition, insulin resistance, and hepatic lipids were measured. Results. Increase in hepatic triglyceride content was first observed in metabolic disturbance followed by hypertriglyceridemia and systemic insulin resistance in fructose-fed rats. Hepatic lipids were increased in time-dependent manner by fructose-feeding starting from 4 weeks, but circulatory triglyceride levels were increased after 12 weeks. Fasting insulin and Homeostatis Model Assessment of Insulin Resistance (HOMA-IR) were increased after 12 weeks of fructose-feeding. Decreased visceral adiposity, circulatory testosterone, and homocysteine levels were observed after 4 weeks of fructose-feeding, which were normalized at 12 and 24 weeks. Conclusions. We conclude that transient decrease in circulatory testosterone and homocysteine levels and increased hepatic triglyceride content are the earliest metabolic disturbances that preceded hypertriglyceridemia and insulin resistance in fructose-fed SD rats.