Maternal obesity during pregnancy leads to adipose tissue ER stress in mice via miR-126-mediated reduction in Lunapark.

AIMS/HYPOTHESIS: Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. METHODS: miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic-hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. RESULTS: The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. CONCLUSIONS/INTERPRETATION: Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target.

Paternal selenium deficiency but not supplementation during preconception alters mammary gland development and 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in female rat offspring.

Breast cancer is a global public health problem and accumulating evidence indicates early-life exposures as relevant factors in the disease risk determination. Recent studies have shown that paternal nutrition can influence offspring health including breast cancer risk. Selenium is a micronutrient with essential role in central aspects of embryogenesis, male fertility and cancer and that has been extensively studied as a chemopreventive agent in several breast cancer experimental models. Thus, we designed an animal study to evaluate whether paternal selenium deficiency or supplementation during preconception could affect the female offspring mammary gland development and breast cancer susceptibility. Male Sprague-Dawley rats were fed AIN93-G diet containing 0.15 ppm (control diet), 0.05 ppm (deficient diet) or 1 ppm (supplemented diet) of selenium for 9 weeks and mated with control female rats. Mammary carcinogenesis was induced with 7,12-dimethylbenz[a]anthracene (DMBA) in their female offspring. Paternal selenium deficiency increased the number of terminal end buds, epithelial elongation and cell proliferation in the mammary gland of the female rat offspring and these effects were associated with higher susceptibility to DMBA-induced mammary tumors (increased incidence and higher grade tumors). On the other hand, paternal selenium supplementation did not influence any of these parameters. These results highlight the importance of father's nutrition including selenium status as a relevant factor affecting daughter's breast cancer risk and paternal preconception as a potential developmental stage to start disease preventive strategies.

Developmental programming of type 2 diabetes: early nutrition and epigenetic mechanisms.

PURPOSE OF REVIEW: The environment experienced during critical windows of development can 'programme' long-term health and risk of metabolic diseases such as type 2 diabetes in the offspring. The purpose of this review is to discuss potential epigenetic mechanisms involved in the developmental programming of type 2 diabetes by early nutrition. RECENT FINDINGS: Maternal and more recently paternal nutrition have been shown to play key roles in metabolic programming of the offspring. Although the exact mechanisms are still not clear, epigenetic processes have emerged as playing a plausible role. Epigenetic dysregulation is associated with several components that contribute to type 2 diabetes risk, including altered feeding behaviour, insulin secretion and insulin action. It may also contribute to transgenerational risk transmission. SUMMARY: Epigenetic processes may represent a central underlying mechanism of developmental programming of type 2 diabetes. During embryonic and foetal development, extensive epigenetic remodelling takes place not only in somatic but also in primordial germ cells. Therefore, concerns have been raised that epigenetic dysregulation induced by a suboptimal early environment could programme altered phenotypes not only in the first generation but also in the subsequent ones. Characterizing these altered epigenetic marks has great implications for identifying individuals at an increased disease risk as well as potentially leading to novel preventive and treatment strategies.

The chemopreventive activity of the butyric acid prodrug tributyrin in experimental rat hepatocarcinogenesis is associated with p53 acetylation and activation of the p53 apoptotic signaling pathway.

The reversibility of non-genotoxic phenotypic alterations has been explored in order to develop novel preventive and therapeutic approaches for cancer control. Previously, it has been demonstrated that histone deacetylase (HDAC) inhibitor tributyrin, a butyric acid prodrug, to have chemopreventive effects on rat hepatocarcinogenesis. The goal of this study was to determine molecular mechanisms associated with the chemopreventive activity of tributyrin. Male Wistar rats were allocated randomly to untreated control group and two experimental groups. Rats in the experimental group 1 were treated with maltodextrin (3g/kg body wt), and rats in experimental group 2 were treated with tributyrin (2g/kg body wt) daily for 8 weeks. Two weeks after treatment initiation, rats from experimental groups were subjected to a 'resistant hepatocyte' model of hepatocarcinogenesis. Treatment with tributyrin resulted in lower HDAC activity and Hdac3 and Hdac4 gene expression, and an increase of histone H3 lysine 9 and 18 and histone H4 lysine 16 acetylation as compared with the experimental group 1. In addition to the increase in histone acetylation, tributyrin caused an increase in the acetylation of the nuclear p53 protein. These changes were accompanied by a normalization of the p53-signaling network, particularly by the upregulation of pro-apoptotic genes, and a consequent increase of apoptosis and autophagy in the livers of tributyrin-treated rats. These results indicate that the chemopreventive activity of tributyrin may be related to an increase of histone and p53 acetylation, which could lead to the induction of the p53 apoptotic pathway.

Suppression of hypoxia-inducible factor-1α contributes to the antiangiogenic activity of red propolis polyphenols in human endothelial cells.

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.

Vitamin A and beta-carotene inhibitory effect during 1,2-dimethylhydrazine induced hepatocarcinogenesis potentiated by 5-azacytidine.

5-Azacytidine is being used for reactivation of tumor suppressor genes. However, its administration during DNA repair pontentiates hepatocarcinogenesis. We observed chemopreventive activities by vitamin A and beta-carotene during early hepatocarcinogenesis. Thus, in the present study we evaluated vitamin A and beta-carotene chemopreventive potential during early hepatocarcinogenesis potentiated by 5-azacytidine. Wistar rats received vitamin A (VAA group), beta-carotene (betaCA group) or corn oil (CO and COA groups). After three weeks of treatment, all animals were initiated with 1,2-dimethylhydrazine. Twelve hours later VAA, betaCA and COA groups received a single dose of 5-Azc. Hepatocytes were selected/promoted by 2-acetylaminofluorene and 70% partial hepatectomy. All animals were sacrificed six weeks after initiation. Compared to CO group (without 5-azacytidine), COA group presented higher (p<0.05) nodule multiplicity, larger (p<0.05) gamma-GT positive lesions that occupied a larger (p<0.05) area of liver section. Compared to COA group, VAA group presented decreased (p<0.05) nodule multiplicity while betaCA group tended to present smaller gamma-GT positive lesions and to decrease occupied liver section. These results reinforce vitamin A and beta-carotene chemopreventive potential. Considering that 5-azacytidine potentiates hepatocarcinogenesis, more studies are needed to elucidate the efficacy and safety of this drug for cancer control.

Selenium and Breast Cancer Risk: Focus on Cellular and Molecular Mechanisms.

Selenium (Se) is a micronutrient with promising breast cancer prevention and treatment potential. There is extensive preclinical evidence of Se mammary carcinogenesis inhibition. Evidence from epidemiological studies is, however, unclear and intervention studies are rare. Here, we examine Se chemoprotection, chemoprevention, and chemotherapy effects in breast cancer, focusing on associated cellular and molecular mechanisms. Se exerts its protective actions through multiple mechanisms that involve antioxidant activities, induction of apoptosis, and inhibition of DNA damage, cell proliferation, angiogenesis, and invasion. New aspects of Se actions in breast cancer have emerged such as the impact of genetic polymorphisms on Se metabolism and response, new functions of selenoproteins, epigenetic modulation of gene expression, and long-term influence of early-life exposure on disease risk. Opportunity exists to design interventional studies with Se for breast cancer prevention and treatment taking into consideration these key aspects.

Effects of α-tocopherol supplementation on liver of rats chronically exposed to ethanol.

BACKGROUND/AIMS: Chronic alcoholism is characterized by hepatotoxicity associated with antioxidant and redox status imbalance. Continuous ethanol intake induces free radical synthesis, resulting in the depletion of antioxidants, especially α-tocopherol, which has an important role in lipid peroxidation. This study aimed to evaluate if α-tocopherol supplementation can restore liver phenotype in rats chronically exposed to ethanol. METHODS: α-Tocopherol levels were determined and histologic analysis of liver was performed. Hepatic gene expression was analyzed through oligonucleotide microarray and real-time PCR. RESULTS: Alcohol exposure for 6 weeks did not decrease hepatic α-tocopherol levels; however, both groups exposed to ethanol (supplemented or not with α-tocopherol) displayed fatty liver. The antioxidant supplementation prevented Mallory bodies and inflammatory infiltration, but not apoptosis, in liver of the rats exposed to ethanol. Gene expression analysis showed evidence of adaptive response to chronic alcohol consumption, where antioxidant components were not regulated. Nevertheless, differentially expressed genes reflected the change in cellular homeostasis. CONCLUSION: The hepatic α-tocopherol content was coherent with the antioxidant gene expression in this study. Cells are likely to have adapted and restored their antioxidant status after long-term ethanol exposure, which might be the reason for such conflicting reports concerning α-tocopherol status in chronic alcoholism.

Chemopreventive effects of the dietary histone deacetylase inhibitor tributyrin alone or in combination with vitamin A during the promotion phase of rat hepatocarcinogenesis.

The chemopreventive effects of tributyrin (TB) and vitamin A (VA), alone or in combination, were investigated during the promotion phase of rat hepatocarcinogenesis. Compared to diethylnitrosamine control rats, TB and TB+VA-treated rats, but not VA-treated rats, presented a lower incidence and mean number of hepatocyte nodules and a smaller size of persistent preneoplastic lesions (pPNLs). In addition, TB and TB+VA-treated rats exhibited a higher apoptotic body index in pPNL and remodeling PNL, whereas VA-treated rats presented only a higher apoptotic body index in remodeling PNL. None of the treatments inhibited cell proliferation in PNL. TB and TB+VA-treated rats, but not VA-treated rats, exhibited higher levels of H3K9 acetylation and p21 protein expression. TB and VA-treated rats exhibited increased hepatic concentrations of butyric acid and retinoids, respectively. Compared to normal rats, diethylnitrosamine control animals exhibited lower retinyl palmitate hepatic concentrations. All groups had similar expression levels and exhibited similar unmethylated CRBP-I promoter region in microdissected pPNL, indicating that epigenetic silencing of this gene was not involved in alteration of retinol metabolism in early hepatocarcinogenesis. Data support the effectiveness of TB as a dietary histone deacetylase inhibitor during the promotion phase of hepatocarcinogenesis, which should be considered for chemoprevention combination strategies.