Deciphering the sex bias in housekeeping gene expression in adipose tissue: a comprehensive meta-analysis of transcriptomic studies.

BACKGROUND: As the housekeeping genes (HKG) generally involved in maintaining essential cell functions are typically assumed to exhibit constant expression levels across cell types, they are commonly employed as internal controls in gene expression studies. Nevertheless, HKG may vary gene expression profile according to different variables introducing systematic errors into experimental results. Sex bias can indeed affect expression display, however, up to date, sex has not been typically considered as a biological variable. METHODS: In this study, we evaluate the expression profiles of six classical housekeeping genes (four metabolic: GAPDH, HPRT, PPIA, and UBC, and two ribosomal: 18S and RPL19) to determine expression stability in adipose tissues (AT) of Homo sapiens and Mus musculus and check sex bias and their overall suitability as internal controls. We also assess the expression stability of all genes included in distinct whole-transcriptome microarrays available from the Gene Expression Omnibus database to identify sex-unbiased housekeeping genes (suHKG) suitable for use as internal controls. We perform a novel computational strategy based on meta-analysis techniques to identify any sexual dimorphisms in mRNA expression stability in AT and to properly validate potential candidates. RESULTS: Just above half of the considered studies informed properly about the sex of the human samples, however, not enough female mouse samples were found to be included in this analysis. We found differences in the HKG expression stability in humans between female and male samples, with females presenting greater instability. We propose a suHKG signature including experimentally validated classical HKG like PPIA and RPL19 and novel potential markers for human AT and discarding others like the extensively used 18S gene due to a sex-based variability display in adipose tissue. Orthologs have also been assayed and proposed for mouse WAT suHKG signature. All results generated during this study are readily available by accessing an open web resource ( https://bioinfo.cipf.es/metafun-HKG ) for consultation and reuse in further studies. CONCLUSIONS: This sex-based research proves that certain classical housekeeping genes fail to function adequately as controls when analyzing human adipose tissue considering sex as a variable. We confirm RPL19 and PPIA suitability as sex-unbiased human and mouse housekeeping genes derived from sex-specific expression profiles, and propose new ones such as RPS8 and UBB.

Housekeeping genes (HKG) are involved in the maintenance of essential cellular functions. They usually present constant expression levels and are relevant because of their usefulness as internal controls in gene expression studies. However, HKG can vary the gene expression profile depending on different variables such as sex, introducing errors in the experimental results. In this study, we have performed an exhaustive systematic review and applied a massive analysis of expression data to check which HKG presents this bias and which do not. The results confirm that certain classical HKG do not perform adequately as controls when analyzing human adipose tissue considering sex as a variable. We further confirm the suitability of RPL19 and PPIA as human and mouse HKG without sex bias derived from sex-specific expression profiles, and propose new ones such as RPS8 and UBB. These results will be of great use in upcoming studies where expression data need to be normalized without the inclusion of sex bias.

Dapagliflozin Does Not Modulate Atherosclerosis in Mice with Insulin Resistance.

Type 2 diabetes mellitus (T2DM) increases morbimortality in humans via enhanced susceptibility to cardiovascular disease (CVD). Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are drugs designed for T2DM treatment to diminish hyperglycaemia by reducing up to 90% of renal tube glucose reabsorption. Clinical studies also suggest a beneficial action of SGLT2i in heart failure and CVD independent of its hypoglycaemiant effect. In the present study, we explored the effect of SGLT2i dapagliflozin (DAPA) in the metabolism and atherosclerosis in Apoe-/-Irs2+/- mice, which display accelerated atherosclerosis induced by insulin resistance. DAPA treatment of Apoe-/-Irs2+/- mice, which were fed a high-fat, high-cholesterol diet, failed to modify body weight, plasma glucose or lipid. Carbohydrate metabolism characterisation showed no effect of DAPA in the glucose tolerance test (GTT) despite augmented insulin levels during the test. In fact, decreased C-peptide levels in DAPA-treated mice during the GTT suggested impaired insulin release. Consistent with this, DAPA treatment of Apoe-/-Irs2+/- isolated islets displayed lower glucose-stimulated insulin secretion compared with vehicle-treated islets. Moreover, insulin-signalling experiments showed decreased pAKT activation in DAPA-treated adipose tissue indicating impaired insulin signalling in this tissue. No changes were seen in lesion size, vulnerability or content of macrophages, vascular smooth muscle cells, T cells or collagen. DAPA did not affect circulating inflammatory cells or cytokine levels. Hence, this study indicates that DAPA does not protect against atherosclerosis in insulin-resistant mice in hypercholesterolemic conditions.

Type 1 diabetic mellitus patients with increased atherosclerosis risk display decreased CDKN2A/2B/2BAS gene expression in leukocytes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) patients display increased risk of cardiovascular disease (CVD) and are characterized by a diminished regulatory T (Treg) cell content or function. Previous studies have shown an association between decreased CDKN2A/2B/2BAS gene expression and enhanced CVD. In the present study the potential relationship between CDKN2A/2B/2BAS gene expression, immune cell dysfunction and increased cardiovascular risk in T1DM patients was explored. METHODS: A cross-sectional study was performed in 90 subjects divided into controls and T1DM patients. Circulating leukocyte subpopulations analysis by flow cytometry, expression studies on peripheral blood mononuclear cell by qPCR and western blot and correlation studies were performed in both groups of subjects. RESULTS: Analysis indicated that, consistent with the described T cell dysfunction, T1DM subjects showed decreased circulating CD4+CD25+CD127- Treg cells. In addition, T1DM subjects had lower mRNA levels of the transcription factors FOXP3 and RORC and lower levels of IL2 and IL6 which are involved in Treg and Th17 cell differentiation, respectively. T1DM patients also exhibited decreased mRNA levels of CDKN2A (variant 1 p16Ink4a), CDKN2A (p14Arf, variant 4), CDKN2B (p15Ink4b) and CDKN2BAS compared with controls. Notably, T1DM patients had augmented pro-atherogenic CD14++CD16+-monocytes, which predict cardiovascular acute events and enhanced common carotid intima-media thickness (CC-IMT). CONCLUSIONS: Decreased expression of CDKN2A/2B/2BAS in leukocytes associates with increased CC-IMT atherosclerosis surrogate marker and proatherogenic CD14++CD16+ monocytes in T1DM patients. These results suggest a potential role of CDKN2A/2B/2BAS genes in CVD risk in T1DM.

Sequential testicular atrophy involves changes in cellular proliferation and apoptosis associated with variations in aromatase P450 expression levels in Irs-2-deficient mice.

Insulin receptor substrate 2 (Irs-2) is an intracellular protein susceptible to phosphorylation after activation of the insulin receptor. Its suppression affects testis development and its absence induces peripheral resistance to insulin. The aim of this study was to identify changes induced by the deletion of Irs-2 in the testicular structure and by the altered expression of cytochrome P450 aromatase, a protein necessary for the development and maturation of germ cells. Adult knockout (KO) mice (Irs-2-/- , 6 and 12 weeks old) and age-matched wild-type (WT) mice were used in this study. Immunohistochemistry and Western blot analyses were performed to study proliferation (PCNA), apoptosis (active caspase-3) and P450 aromatase expression in testicular histological sections. Deletion of Irs-2 decreased the number of epithelial cells in the seminiferous tubule and rete testis. Aberrant cells were frequently detected in the epithelia of Irs-2-/- mice, accompanied by variations in spermatogonia, which were shown to exhibit small hyperchromatic nuclei as well as polynuclear and anuclear structures. The amount of cell proliferation was significantly lower in Irs-2-/- mice than in WT mice, whereas apoptotic processes were more common in Irs-2-/- mice. Aromatase P450 reactivity was higher in 6-week-old KO mice than in WT mice of the same age and was even higher at 12 weeks. Our results suggest that Irs-2 is a key element in spermatogenesis because silencing Irs-2 induces the sequential development of testicular atrophy. The effects are observed mainly in germ cells present in the seminiferous tubule, which may be due to changes in cytochrome P450 aromatase expression.

Changes in CDKN2A/2B expression associate with T-cell phenotype modulation in atherosclerosis and type 2 diabetes mellitus.

Previous studies indicate a role of CDKN2A/2B/2BAS genes in atherosclerosis and type 2 diabetes mellitus (T2DM). Progression of these diseases is accompanied by T-cell imbalance and chronic inflammation. Our main objective was to investigate a potential association between CDKN2A/2B/2BAS gene expression and T cell phenotype in T2DM and coronary artery disease (CAD) in humans, and to explore the therapeutic potential of these genes to restore immune cell homeostasis and disease progression. Reduced mRNA levels of CDKN2A (p16Ink4a), CDKN2B (p15Ink4b), and CDKN2BAS were observed in human T2DM and T2DM-CAD subjects compared with controls. Protein levels of p16Ink4a and p15Ink4b were also diminished in T2DM-CAD patients while CDK4 levels, the main target of p16Ink4a and p15Ink4b, were augmented in T2DM and T2DM-CAD subjects. Both patient groups displayed higher activated CD3+CD69+ T cells and proatherogenic CD14++CD16+ monocytes, while CD4+CD25+CD127 regulatory T (Treg cells) cells were decreased. Treatment of primary human lymphocytes with PD0332991, a p16Ink4a/p15Ink4b mimetic drug and a proven CDK4 inhibitor, increased Treg cells and the levels of activated transcription factor phosphoSTAT5. In vivo PD0332991 treatment of atherosclerotic apoE-/- mice and insulin resistant apoE-/-Irs2+/- mice augmented Foxp3-expressing Treg cells and decreased lesion size. Thus, atherosclerosis complications in T2DM associate with altered immune cell homeostasis, diminished CDKN2A/2B/2BAS expression, and increased CDK4 levels. The present study also suggests that the treatment with drugs that mimic CDKN2A/2B genes could potential be considered as a promising therapy to delay atherosclerosis.

Pituitary Aromatase P450 May Be Involved in Maintenance of the Population of Luteinizing Hormone-Positive Pituitary Cells in Mice.

As aromatase P450 is located in several pituitary cells, testosterone can be transformed into 17ß-estradiol in the gland by the enzyme. The possible role of this transformation in pituitary function remains to be elucidated, but some evidence suggests a physiological and pathophysiological role for pituitary aromatase. To determine its relevance in the modulation of pituitary function, mainly associated with reproduction, luteinizing hormone (LH)-positive cells in the hypophysis of female and male aromatase knockout (ArKO) mice were studied. In all LH-positive cells, significant increases in the cellular (p < 0.01) and nuclear (p < 0.05) areas were found in the ArKO mice compared to the wild-type mice. In the ArKO mice, LH-positive cells were more abundant (p < 0.01); they were characterized by a stronger cytoplasmic reaction and the cells were more polygonal and exhibited more short, thick cytoplasmic prolongations than those in the wild-type mice. Moreover, LH-positive cells showed a greater proliferation rate in the ArKO mice compared to the wild-type mice (p < 0.01). These findings suggest that the local production of estradiol mediated by pituitary aromatase is necessary for the regulation of LH-positive gonadotropic cells, exerting an autoparacrine inhibitory regulation. These results could underlie the higher pituitary aromatase expression observed in male versus female mice. Similar effects were found in ArKO male and female mice, suggesting that in both sexes the effects of estrogens on maintenance of the LH-positive pituitary cell population could be related to the local aromatization of testosterone to estradiol inside the hypophysis. Therefore, aromatase could modulate pituitary LH-positive cells in males through local estradiol synthesis.

Hepatic lipase inactivation decreases atherosclerosis in insulin resistance by reducing LIGHT/Lymphotoxin ß-Receptor pathway.

Coexistence of insulin resistance (IR) and metabolic syndrome (MetS) increases the risk of cardiovascular disease (CVD). Genetic studies in diabetes have linked Hepatic Lipase (HL) to an enhanced risk of CVD while others indicate a role of HL in inflammatory cells. Thus, we explored the role of HL on atherosclerosis and inflammation in a mouse model of MetS/IR, (apoE-/-Irs2+/- mice) and in patients with MetS and IR. HL-deficiency in apoE-/-Irs2+/- mice reduced atheroma size, plaque vulnerability, leukocyte infiltration and macrophage proliferation. Compared with apoE-/-Irs2+/-HL+/+ mice, MCP1, TNFα and IL6 plasma levels, pro-inflammatory Ly6Chi monocytes and activated(CD69+)-T lymphocytes were also decreased in apoE-/-Irs2+/-HL-/- mice. The LIGHT (Tumour necrosis factor ligand superfamily member 14, TNFSF14)/Lymphotoxin ß-Receptor(LTß-R) pathway, which is involved in T-cell and macrophage activation, was diminished in plasma and in apoE-/-Irs2+/-HL-/- mouse atheromas. Treatment of apoE-/-Irs2+/-HL-/- mice with LIGHT increased the number of Ly6Chi-monocytes and lesion size. Acutely LIGHT-treated apoE-/- mice displayed enhanced proliferating Ly6Chi-monocytes and increased activation of the mitogen-activated protein kinase p38, suggesting that LIGHT/LTß-R axis might promote atherogenesis by increasing proinflammatory monocytes and proliferation. Notably, MetS-IR subjects with increased atherosclerosis displayed up-regulation of the LIGHT/LTß-R axis, enhanced inflammatory monocytes and augmented HL mRNA expression in circulating leukocytes. Thus, HL-deficiency decreases atherosclerosis in MetS/IR states by reducing inflammation and macrophage proliferation which are partly attributed to reduced LIGHT/LTß-R pathway. These studies identify the LIGHT/LTß-R axis as a main pathway in atherosclerosis and suggest that its inactivation might ameliorate inflammation and macrophage proliferation associated with atherosclerosis burden in MetS/IR.

Relevance of pituitary aromatase and estradiol on the maintenance of the population of prolactin-positive cells in male mice.

In previous studies we demonstrated the expression of aromatase in pituitary cells. This expression is gender related, and is also associated with the presence of prolactinomas. To ascertain the relevance of aromatase in modulating the populations of prolactin-positive pituitary cells an immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out using the pituitary glands of adult male and female aromatase-knockout (ArKO) mice. Additionally has been determined if pituitary aromatase is involved in a gender-linked differentiated regulation of the prolactin-producing pituitary cells. Compared to wild-type mice, the knockout animals of both genders showed a significant decrease (p<0.01) in the cellular and nuclear areas of their prolactin cells, as well as in the percentages of the prolactin-positive cells and the proliferating prolactin cells. Our results suggest that estradiol is responsible for the maintenance of the population of prolactin cell in males and, so as not to disturb the endocrine reproductive environment, estradiol is synthesized inside the pituitary by circulating testosterone via means of aromatase P450, which acts in paracrine way. This new role for pituitary aromatase may well explain the previous findings establishing that the pituitary expression of aromatase is higher in males than in females, and the association between the development of prolactinomas and the increased expression of aromatase in tumours.

Hepatic lipase deficiency produces glucose intolerance, inflammation and hepatic steatosis.

Metabolic syndrome and type 2 diabetes mellitus constitute a major problem to global health, and their incidence is increasing at an alarming rate. Non-alcoholic fatty liver disease, which affects up to 90% of obese people and nearly 70% of the overweight, is commonly associated with MetS characteristics such as obesity, insulin resistance, hypertension and dyslipidemia. In the present study, we demonstrate that hepatic lipase (HL)-inactivation in mice fed with a high-fat, high-cholesterol diet produced dyslipidemia including hypercholesterolemia, hypertriglyceridemia and increased non-esterified fatty acid levels. These changes were accompanied by glucose intolerance, pancreatic and hepatic inflammation and steatosis. In addition, compared with WT mice, HL(-/-) mice exhibited enhanced circulating MCP1 levels, monocytosis and higher percentage of CD4+Th17+ cells. Consistent with increased inflammation, livers from HL(-/-) mice had augmented activation of the stress SAPK/JNK- and p38-pathways compared with the activation levels of the kinases in livers from WT mice. Analysis of HL(-/-) and WT mice fed regular chow diet showed dyslipidemia and glucose intolerance in HL(-/-) mice without any other changes in inflammation or hepatic steatosis. Altogether, these results indicate that dyslipidemia induced by HL-deficiency in combination with a high-fat, high-cholesterol diet promotes hepatic steatosis and inflammation in mice which are, at least in part, mediated by the activation of the stress SAPK/JNK- and p38-pathways. Future studies are warranted to asses the viability of therapeutic strategies based on the modulation of these kinases to reduce hepatic steatosis associated to lipase dysfunction.

Local transformations of androgens into estradiol by aromatase P450 is involved in the regulation of prolactin and the proliferation of pituitary prolactin-positive cells.

In previous studies we demonstrated the immunohistochemical expression of aromatase in pituitary cells. In order to determine whether pituitary aromatase is involved in the paracrine regulation of prolactin-producing pituitary cells and the physiological relevance of pituitary aromatase in the control of these cells, an in vivo and in vitro immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out on the pituitary glands of adult male rats treated with the aromatase antagonist fadrozole. Moreover, we analyzed the expression of mRNA for the enzyme in pituitary cells of male adult rats by in situ hybridization. The aromatase-mRNA was seen to be located in the cytoplasm of 41% of pituitary cells and was well correlated with the immunocytochemical staining. After in vivo treatment with fadrozole, the size (cellular and nuclear areas) of prolactin cells, as well as the percentage of prolactin-positive cells and the percentage of proliferating-prolactin cells, was significantly decreased. Moreover, fadrozole decreased serum prolactin levels. In vitro, treatment with fadrozole plus testosterone induced similar effects on prolactin-positive cells, inhibiting their cellular proliferation. Our results suggest that under physiological conditions aromatase P450 exerts a relevant control over male pituitary prolactin-cells, probably transforming testosterone to estradiol in the pituitary gland.

Insulin resistance aggravates atherosclerosis by reducing vascular smooth muscle cell survival and increasing CX3CL1/CX3CR1 axis.

AIMS: Insulin resistance (IR) is a major risk factor for cardiovascular disease and atherosclerosis. Life-threatening acute events are mainly due to rupture of unstable plaques, and the role of vascular smooth muscle cells (VSMCs) in this process in IR, Type 2 diabetes mellitus, and metabolic syndrome (T2DM/MetS) has not been fully addressed. Therefore, the role of VSMC survival in the generation of unstable plaques in T2DM/MetS and the involvement of inflammatory mediators was investigated. METHODS AND RESULTS: Defective insulin receptor substrate 2 (IRS2)-mediated signalling produced insulin-resistant VSMCs with reduced survival, migration, and higher apoptosis than control cells. Silencing of IRS2 or inhibition of the V-akt murine thymomaviral oncogene homologue kinase (AKT)-extracellular signal-regulated kinase (ERK)-dependent pathway in VSMCs augmented expression of the inflammatory chemokine fractalkine (CX3CL1) and its receptor CX3CR1, previously involved in atheroma plaque vulnerability. Interestingly, treatment of VSMCs with CX3CL1 promoted apoptosis in the presence of other stimuli or when the AKT pathway was blocked. Analysis of a mouse model of IR-MetS and accelerated atherosclerosis, apoE-/-Irs2+/- mice, showed reduced VSMC survival, unstable plaques, and up-regulation of CX3CL1/CX3CR1 axis compared with apoE-/- mice. Human studies showed augmented soluble CX3CL1 plasma levels and CX3CR1 expression in monocytes from IR-MetS subjects compared with controls. A positive correlation between insulin levels, homeostatic model assessment (HOMA) index, carotid atherosclerosis, and CX3CR1 mRNA levels was also found in all patients. CONCLUSION: IR increases plaque vulnerability by augmenting the CX3CL1/CX3CR1 axis, which is mechanistically linked to reduced VSMC survival. Thus, modulation of IRS2-dependent signalling emerges as a potential therapeutic strategy to promote VSMC survival and atheroma plaque stability and to reduce inflammatory mediators in IR-MetS.

The expression of AIB1 correlates with cellular proliferation in human prolactinomas.

Estrogens as well as certain growth factors strongly influence the development and growth of prolactinomas. However, the molecular mechanisms by which extracellular factors trigger prolactinomas are not well known. Amplified in breast cancer 1 (AIB1), also known as steroid receptor co-activator 3 (SRC-3), belongs to the p160/SRC family of nuclear receptor co-activators and is a major co-activator of the estrogen receptor. Here, we report that the estrogen receptor coactivator AIB1 is overexpressed in human prolactinomas and correlates with the detection of aromatase and estrogen receptor α (ERα). Of the 87 pituitary tumors evaluated in women, 56%, corresponding to hyperoprolactinemic women, contained an enriched population of prolactin-positive cells and hence were further classified as prolactinomas. All prolactinomas stained positive for both ERα and AIB1. Moreover, AIB1 sub-cellular distribution was indicative of the cell-cycle status of tumors; the nuclear expression of AIB1 was correlated with proliferative markers whereas the cytoplasmic localization of AIB1 coincided with active caspase-3. Thus, our results demonstrate for the first time that AIB1 is expressed in prolactinomas and suggest its participation in the regulation of proliferation and apoptosis of tumoral cells. Because aromatase expression is also enhanced in these prolactinomas and it is involved in the local production of estradiol, both mechanisms, ER-AIB1 and aromatase could be related.

Expression of aromatase P450 is increased in spontaneous prolactinomas of aged rats.

We have recently reported the presence of aromatase P450 in the rat hypophysis. This enzyme is responsible for the aromatization of testosterone to estradiol. Since the induction of prolactinomas has been demonstrated in the rat following chronic treatment with estradiol, the aim of the present study was to analyze whether a relationship exists between the presence of pituitary aromatase and the appearance of spontaneous prolactinomas in aged rats. Of a series of 90 adenomas studied, 53% showed prolactin immunoreactive cells and were classified as prolactinomas; only 33% of the adenomas were pure prolactinomas and the other 20% were multi-hormonal protactinomas. Moreover, 60% of the adenomas were aromatase-positive tumors. Interestingly, 100% of the pure prolactinomas were aromatase-positive while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Western blotting with anti-aromatase antibodies revealed a 3.8-fold increase in expression of aromatase in pituitary tumors as compared to normal rat pituitary gland. Double immunohistochemical labeling detected the coexistence of prolactin and aromatase P450 in prolactinoma cells. ACTH- and LH-positive adenomas were considered as controls; only multi-hormonal ACTH and LH tumors display aromatase-positive cells and all of these also contained prolactin-positive cells. Our results demonstrate for the first time that aromatase is expressed in pituitary adenomas and that it is related to the functional nature of the tumor, especially in the case of pure prolactinomas, suggesting the possibility that an abnormally high conversion of testosterone into estradiol in pituitary cells may contribute to the genesis of spontaneous prolactinomas in aged rats.