The Involvement of Endogenous Enkephalins in Glucose Homeostasis.

Obesity has nearly tripled since 1975 and is predicted to continue to escalate. The surge in obesity is expected to increase the risk of diabetes type 2, hypertension, coronary artery disease, and stroke. Therefore, it is essential to better understand the mechanisms that regulate energy and glucose homeostasis. The opioid system is implicated in regulating both aspects (hedonic and homeostatic) of food intake. Specifically, in the present study, we investigated the role of endogenous enkephalins in changes in food intake and glucose homeostasis. We used preproenkephalin (ppENK) knockout mice and their wildtype littermates/controls to assess changes in body weight, food intake, and plasma glucose levels when mice were fed a high-fat diet for 16 weeks. Body weight and food intake were measured every week (n = 21-23 mice per genotype), and at the end of the 16-week exposure period, mice were tested using the oral glucose tolerance test (OGTT, n = 9 mice per genotype) and insulin tolerance test (n = 5 mice per genotype). Our results revealed no difference in body weight or food intake between mice of the two genotypes. However, HFD-exposed enkephalin-deficient mice demonstrated impaired OGTT associated with reduced insulin sensitivity compared to their wildtype controls. The impaired insulin sensitivity is possibly due to the development of peripheral insulin resistance. Our results reveal a potential role of enkephalins in the regulation of glucose homeostasis and in the pathophysiology of diabetes type 2.

Menin and PRMT5 suppress GLP1 receptor transcript and PKA-mediated phosphorylation of FOXO1 and CREB.

Menin is a scaffold protein that interacts with several epigenetic mediators to regulate gene transcription, and suppresses pancreatic ß-cell proliferation. Tamoxifen-inducible deletion of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes the protein menin, increases ß-cell mass in multiple murine models of diabetes and ameliorates diabetes. Glucagon-like-peptide-1 (GLP1) is another key physiological modulator of ß-cell mass and glucose homeostasis. However, it is not clearly understood whether menin crosstalks with GLP1 signaling. Here, we show that menin and protein arginine methyltransferase 5 (PRMT5) suppress GLP1 receptor (GLP1R) transcript levels. Notably, a GLP1R agonist induces phosphorylation of forkhead box protein O1 (FOXO1) at S253, and the phosphorylation is mediated by PKA. Interestingly, menin suppresses GLP1-induced and PKA-mediated phosphorylation of both FOXO1 and cAMP response element binding protein (CREB), likely through a protein arginine methyltransferase. Menin-mediated suppression of FOXO1 and CREB phosphorylation increases FOXO1 levels and suppresses CREB target genes, respectively. A small-molecule menin inhibitor reverses menin-mediated suppression of both FOXO1 and CREB phosphorylation. In addition, ex vivo treatment of both mouse and human pancreatic islets with a menin inhibitor increases levels of proliferation marker Ki67. In conclusion, our results suggest that menin and PRMT5 suppress GLP1R transcript levels and PKA-mediated phosphorylation of FOXO1 and CREB, and a menin inhibitor may reverse this suppression to induce ß-cell proliferation.

An Epigenetic Pathway Regulates Sensitivity of Breast Cancer Cells to HER2 Inhibition via FOXO/c-Myc Axis.

Human epidermal growth factor receptor 2 (HER2) is upregulated in a subset of human breast cancers. However, the cancer cells often quickly develop an adaptive response to HER2 kinase inhibitors. We found that an epigenetic pathway involving MLL2 is crucial for growth of HER2(+) cells and MLL2 reduces sensitivity of the cancer cells to a HER2 inhibitor, lapatinib. Lapatinib-induced FOXO transcription factors, normally tumor-suppressing, paradoxically upregulate c-Myc epigenetically in concert with a cascade of MLL2-associating epigenetic regulators to dampen sensitivity of the cancer cells to lapatinib. An epigenetic inhibitor suppressing c-Myc synergizes with lapatinib to suppress cancer growth in vivo, partly by repressing the FOXO/c-Myc axis, unraveling an epigenetically regulated FOXO/c-Myc axis as a potential target to improve therapy.

Menin is required for optimal processing of the microRNA let-7a.

Multiple endocrine neoplasia type I (MEN1) is an inherited syndrome that includes susceptibility to pancreatic islet hyperplasia. This syndrome results from mutations in the MEN1 gene, which encodes menin protein. Menin interacts with several transcription factors, including JunD, and inhibits their activities. However, the precise mechanism by which menin suppresses gene expression is not well understood. Here, we show that menin interacts with arsenite-resistant protein 2 (ARS2), a component of the nuclear RNA CAP-binding complex that is crucial for biogenesis of certain miRNAs including let-7a. The levels of primary-let-7a (pri-let-7a) are not affected by menin; however, the levels of mature let-7a are substantially decreased upon Men1 excision. Let-7a targets, including Insr and Irs2, pro-proliferative genes that are crucial for insulin-mediated signaling, are up-regulated in Men1-excised cells. Inhibition of let-7a using anti-miRNA in wild type cells is sufficient to enhance the expression of insulin receptor substrate 2 (IRS2) to levels observed in Men1-excised cells. Depletion of menin does not affect the expression of Drosha and CBP80, but substantially impairs the processing of pri-miRNA to pre-miRNA. Ars2 knockdown decreased let-7a processing in menin-expressing cells but had little impact on let-7a levels in menin-excised cells. As IRS2 is known to mediate insulin signaling and insulin/mitogen-induced cell proliferation, these findings collectively unravel a novel mechanism whereby menin suppresses cell proliferation, at least partly by promoting the processing of certain miRNAs, including let-7a, leading to suppression of Irs2 expression and insulin signaling.

Astrogliosis in the brain of obese Zucker rat: a model of metabolic syndrome.

Metabolic syndrome (MetS) is a disorder characterized primarily by the development of insulin resistance. Insulin resistance and subsequent hyperinsulinemia, originating from abdominal obesity, increases the risk of cerebrovascular and cardiovascular disease and all-cause mortality. Obesity is probably a risk factor for Alzheimer's disease and vascular dementia and is associated with impaired cognitive function. The obese Zucker rat (OZR) represents a model of type 2 diabetes exhibiting a moderate degree of arterial hypertension and of increased oxidative stress. To clarify the possible relationships between MetS and brain damage, the present study has investigated brain microanatomy in OZRs compared with their littermate controls lean Zucker rats (LZRs). Male OZRs and LZRs of 12 weeks of age were used. Their brain was processed for immunochemical and immunohistochemical analysis of glial fibrillary acidic protein (GFAP). In frontal and parietal cortex of OZRs a significant increase in the number of GFAP immunoreactive astrocytes was observed. Similar findings were found in the hippocampus, where an increased number of GFAP immunoreactive astrocytes were detected in the CA1 and CA3 subfields and dentate gyrus of OZRs compared to the LZRs. These findings indicating the occurrence of brain injury accompanied by astrogliosis in OZRs suggest that these rats, developed as an animal model of type 2 diabetes, may also represent a model for assessing the influence of MetS on brain. The identification of neurodegenerative changes in OZRs may represent the first step for better characterizing neuronal involvement in this model of MetS and possible treatment for countering it.

Exercise reduces oxidative stress but does not alleviate hyperinsulinemia or renal dopamine D1 receptor dysfunction in obese rats.

Impairment of renal dopamine D1 receptor (D1R)-mediated natriuresis is associated with hypertension in humans and animal models, including obese Zucker rats. We have previously reported that treatment of these rats with antioxidants or insulin sensitizers reduced insulin levels and oxidative stress, restored D1R-mediated natriuresis, and reduced blood pressure. Furthermore, the redox-sensitive transcription factor, nuclear factor-κB (NF-κB), has been implicated in impairment of D1R-mediated natriuresis during oxidative stress. In this study, we investigated the effect of exercise on insulin levels, oxidative stress, nuclear translocation of NF-κB, blood pressure, albuminuria, and D1R-mediated natriuresis. The exercise protocol involved treadmill exercise from 3 wk of age for 8 wk. Exercise reduced oxidative stress, nuclear translocation of NF-κB, and albuminuria. However, exercise did not reduce plasma insulin levels or blood pressure. Also, selective D1R agonist (SKF-38393)-mediated increases in sodium excretion and guanosine 5'-O-(3-thiotriphosphate) binding were impaired in obese rats compared with lean rats, and exercise did not restore this defect. We conclude that, while exercise is beneficial in reducing oxidative stress and renal injury, reducing insulin levels may be required to restore D1R-mediated natriuresis in this model of obesity and metabolic syndrome. Furthermore, this study supports previous observations that restoring D1R function contributes to blood pressure reduction in this model.

Mechanisms of oxidative stress-induced increase in salt sensitivity and development of hypertension in Sprague-Dawley rats.

High salt intake produces vascular changes that contribute to the development of hypertension in salt-sensitive individuals. Because reactive oxygen species play a role in the pathogenesis of cardiovascular diseases, we investigated whether oxidative stress contributes to salt-sensitive hypertension. Sprague-Dawley rats were divided in different groups and received tap water (vehicle), 30 mmol/L of l-buthionine sulfoximine ([BSO] an oxidant), high salt ([HS] 1% NaCl), and BSO plus HS without and with antioxidant tempol (1 mmol/L) in drinking water for 12 days. Compared with vehicle, BSO treatment caused oxidative stress and mild increase in blood pressure. Thoracic aortic rings from BSO-treated rats exhibited decreased response to endothelium-independent vasorelaxants. In HS-treated rats, the response to vasoactive agents, as well as blood pressure, was unaffected. Concomitant treatment of rats with BSO and HS produced a marked increase in blood pressure and a decreased response to both endothelium-dependent and endothelium-independent vasorelaxants with an increase in EC(50). Incubation of aortic tissue from BSO-treated rats with sodium nitroprusside showed decreased cGMP accumulation, whereas HS rats had decreased basal NO synthase activity. Tempol decreased oxidative stress, normalized blood pressure, and restored NO signaling and responses to vasoactive compounds in BSO and BSO plus HS rats. We conclude that BSO increases oxidative stress and reduces NO signaling, whereas HS reduces NO levels by decreasing the NO synthase activity. These phenomena collectively result in reduced responsiveness to both endothelium -dependent and endothelium- independent vasorelaxants and may contribute to salt-sensitive hypertension.