Type 1 diabetic mellitus patientswith increased atherosclerosisriskdisplay 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

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

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

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

Abstract 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

Effects of <i>in vitro</i> treatment with fadrozole on prolactin-positive cells.

<p>Double immunocytochemical staining for PCNA (brown) and prolactin (dark blue-grey) in control dishes a), testosterone-treated dishes b), and testosterone- and fadrozole-treated dishes c). Scale bar: a,b,c: 50 µm. d) Plot showing the decrease in cellular area at the different time-points assayed; from 3 to 12 hours of treatment with testosterone and fadrozole a significant decrease (*p<0.01) was observed. e) The percentage of prolactin-positive cells decreases as from 3 to 12 hours of treatment with testosterone and fadrozole (*p<0.01). f) The percentage of PCNA- and prolactin-positive cells, out of the total number of prolactin-positive cells, decreases significantly from 1 to 12 hours of treatment with testosterone and fadrozole (*p<0.01).</p

Effects of <i>in vivo</i> treatment with fadrozole on the cellular proliferation of prolactin-positive cells.

<p>a) Double labelled immunohistochemistry for prolactin (dark blue-grey) and PCNA (brown). In untreated animals, few prolactin-positive cells are labelled for PCNA (arrow). Scale bar: 12 µm. b) After 5 doses of fadrozole it is uncommon to find cells labelled jointly for PCNA and prolactin (arrow points to PCNA- but not prolactin-positive cells). Scale bar: 12 µm. c) The percentage of prolactin-positive cells decreases significantly with respect to the untreated or control animals after 1 dose (**p<0.01 with respect to untreated animals, and p<0.05 with respect to control animals) or 5 doses (**p<0.01 with respect to untreated and control animals and p<0.05 with respect to 1 dose of the fadrozole treated animals). After 1 dose of saline the percentage decreases with respect to untreated animals (#p<0.05), but not after 5 doses. d) Fadrozole decreases the proliferation of prolactin-positive cells (*p<0.05 with respect to untreated and control animals; **p<0.01 with respect to untreated and control animals and p<0.05 with respect to 1 dose of fadrozole-treated animals).</p

Effects of fadrozole on prolactin-positive cells in the pituitary of male rats.

<p>a) General disposition of prolactin-positive cells in the gland. PD: pars distalis of pituitary gland. Scale bar: 100 µm. b) Irregular and strongly stained prolactin-positive cells (arrows) in untreated male rats. c) Polygonal and weakly stained prolactin-positive cells (arrows) after 5 doses of fadrozole. Scale bar b, c: 12 µm d) Plot showing the <i>in vivo</i> effects of the 1 or 5 doses of fadrozole on the serum levels of prolactin (ng/mL) (*p<0.01 with respect to untreated and control animals, **p<0.01, with respect to untreated and control animals and p<0.05 with respect to 1 dose of fadrozole-treated animals). Morphometric effect of treatment with fadrozole on cellular (e) or nuclear (f) areas (µm²); in both cases fadrozole induces decreases in size (*p<0.01 with respect to untreated and control animals).</p

Expression of aromatase by Western blot.

<p>a) preabsorption and expression in the pituitary, b) positive controls from the ovary of a female adult rat, and from a prolactinoma from a female old rat. Images showing the expression of aromatase in the pituitary of adult male rats. c) Aromatase-positive cells revealed by immunohistochemistry. d) mRNA-aromatase-positive cells detected by in situ hybridization. Negative controls for in situ hybridization: e) absence of the probe and f) sense aromatase probe. Scale bar: c, d: 50 µm.</p