Circulating microRNAs and diabetes: potential applications in medical practice.

The explosive increase in the worldwide prevalence of diabetes over recent years has transformed the disease into a major public health concern. While diabetes can be screened for and diagnosed by reliable biological tests based on blood glucose levels, by and large there are no means of detecting at-risk patients or of following diabetic complications. The recent discovery that microRNAs are not only chief intracellular players in many biological processes, including insulin secretion and action, but are also circulating, has put them in the limelight as possible biological markers. Here we discuss the potential role of circulating microRNAs as biomarkers in the context of diabetes and its associated complications.

Modulation of glucose metabolism by the renin-angiotensin-aldosterone system.

The renin-angiotensin-aldosterone system (RAAS) is an enzymatic cascade functioning in a paracrine and autocrine fashion. In animals and humans, RAAS intrinsic to tissues modulates food intake, metabolic rate, adiposity, insulin sensitivity, and insulin secretion. A large array of observations shows that dysregulation of RAAS in the metabolic syndrome favors type 2 diabetes. Remarkably, angiotensin-converting enzyme inhibitors, suppressing the synthesis of angiotensin II (ANG II), and angiotensin receptor blockers, targeting the ANG II type 1 receptor, prevent diabetes in patients with hypertensive or ischemic cardiopathy. These drugs interrupt the negative feedback loop of ANG II on the RAAS cascade, which results in increased production of angiotensins. In addition, they change the tissue expression of RAAS components. Therefore, the concept of a dual axis of RAAS regarding glucose homeostasis has emerged. The RAAS deleterious axis increases the production of inflammatory cytokines and raises oxidative stress, exacerbating the insulin resistance and decreasing insulin secretion. The beneficial axis promotes adipogenesis, blocks the production of inflammatory cytokines, and lowers oxidative stress, thereby improving insulin sensitivity and secretion. Currently, drugs targeting RAAS are not given for the purpose of preventing diabetes in humans. However, we anticipate that in the near future the discovery of novel means to modulate the RAAS beneficial axis will result in a decisive therapeutic breakthrough.

[microRNA and diabetes: tiny things causing huge effects]. / MicroARN et diabète - Petites structures - grands effets.

Soon after their discovery microRNA (miRNA) emerged as central natural regulators of gene expression. Although the complex mechanisms of action and impact of miRNA on development, physiology and disease are still elusive, significant progress has been made in deciphering the roles of some miRNA in insulin secretion and action. Here we examine the close relationship existing between miRNA and glucose metabolism as well as their putative role in the pathogenesis of diabetes and their possible utility as biomarkers of this disease.

How Does Our Knowledge on the Na+/H+ Exchanger NHE1 Obtained by Biochemical and Molecular Analyses Keep up With Its Recent Structure Determination?

Na+/H+ exchangers are membrane transporters conserved in all living systems and therefore are assumed to be amongst the most ancestral molecular devices that equipped the first protocells. Following the cloning and sequencing of its gene, the mammalian NHE1, that regulates pH and volume in all cells, has been thoroughly scrutinized by molecular and biochemical analyses. Those gave a series of crucial clues concerning its topology, dimeric organization, pharmacological profile, regulation, and the role of key amino acids. Recently thanks to cryogenic Electron Microscopy (Cryo-EM) the long-awaited molecular structures have been revealed. With this information in mind we will challenge the robustness of the earlier conclusions and highlight how the new information enriches our understanding of this key cellular player. At the mechanistic level, we will pinpoint how the NHE1 3D structures reveal that the previously identified amino acids and regions are organized to coordinate transported cations, and shape the allosteric transition that makes NHE1 able to sense intracellular pH and be regulated by signaling pathways.

microRNA-375 regulates glucose metabolism-related signaling for insulin secretion.

Enhanced beta cell glycolytic and oxidative metabolism are necessary for glucose-induced insulin secretion. While several microRNAs modulate beta cell homeostasis, miR-375 stands out as it is highly expressed in beta cells where it regulates beta cell function, proliferation and differentiation. As glucose metabolism is central in all aspects of beta cell functioning, we investigated the role of miR-375 in this process using human and rat islets; the latter being an appropriate model for in-depth investigation. We used forced expression and repression of mR-375 in rat and human primary islet cells followed by analysis of insulin secretion and metabolism. Additionally, miR-375 expression and glucose-induced insulin secretion were compared in islets from rats at different developmental ages. We found that overexpressing of miR-375 in rat and human islet cells blunted insulin secretion in response to glucose but not to α-ketoisocaproate or KCl. Further, miR-375 reduced O2 consumption related to glycolysis and pyruvate metabolism, but not in response to α-ketoisocaproate. Concomitantly, lactate production was augmented suggesting that glucose-derived pyruvate is shifted away from mitochondria. Forced miR-375 expression in rat or human islets increased mRNA levels of pyruvate dehydrogenase kinase-4, but decreased those of pyruvate carboxylase and malate dehydrogenase1. Finally, reduced miR-375 expression was associated with maturation of fetal rat beta cells and acquisition of glucose-induced insulin secretion function. Altogether our findings identify miR-375 as an efficacious regulator of beta cell glucose metabolism and of insulin secretion, and could be determinant to functional beta cell developmental maturation.

miR-139 impacts FoxO1 action by decreasing FoxO1 protein in mouse hepatocytes.

FoxO1 is a master regulator of signaling pathways used by growth factors and hormones, including insulin. Its activity is regulated by changes in subcellular localization coupled to post-translational modifications such as phosphorylation, ubiquitination, and acetylation. As microRNAs have emerged as a newly identified means by which cells fine-tune gene expression, we hypothesized that they could regulate FoxO1. Since FoxO1 plays a key role in the liver, we used immortalized neonatal mouse hepatocytes to analyze the effects of potential microRNAs targeting FoxO1. We found that miR-139 targets FoxO1 mRNA directly and reduces the level of the protein without affecting transcript levels. This decrease in FoxO1 protein results in a decrease of its target genes, such as AdQR1, AdQR2 and Mttp. Our findings suggest a novel mode of FoxO1 regulation by which miR-139 could maintain the protein level of FoxO1 to preserve homeostatic regulation of its transcriptional activity in response to environmental stimuli.

Tyrosine phosphorylation of insulin receptor substrates during ischemia/reperfusion-induced apoptosis in rat liver.

BACKGROUND: Phosphoregulation of signal transduction pathways is a complex series of reactions that may modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I/R-induced apoptosis on phosphorylation and activation of signal proteins in tyrosine kinase pathways. MATERIALS AND METHODS: In rats, a segmental normothermic ischemia of the liver was induced for 120 min. Liver apoptosis was determined using terminal deoxynucleotide-transferase-mediated deoxyuridine triphosphate nick end labeling assay, and activity of caspases-3 and -7 was determined by fluorescence. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis. RESULTS: Normothermic I-R resulted in increased in vivo caspases-3 and -7 activity and in liver apoptosis. Shc tyrosine phosphorylation and activation of ERK1/2 were increased after reperfusion, while tyrosine phosphorylation of IRS-1 and activation of PKB/Akt were decreased. CONCLUSIONS: Normothermic liver I-R leads to increased apoptosis and to modifications in protein tyrosine phosphorylation pathways.

Amino acid-induced regulation of hepatocyte growth: possible role of Drosha.

In an adult healthy liver, hepatocytes are in a quiescent stage unless a physical injury, such as ablation, or a toxic attack occur. Indeed, to maintain their crucial organismal homeostatic role, the damaged or remaining hepatocytes will start proliferating to restore their functional mass. One of the limiting conditions for cell proliferation is amino-acid availability, necessary both for the synthesis of proteins important for cell growth and division, and for the activation of the mTOR pathway, known for its considerable role in the regulation of cell proliferation. The overarching aim of our present work was to investigate the role of amino acids in the regulation of the switch between quiescence and growth of adult hepatocytes. To do so we used non-confluent primary adult rat hepatocytes as a model of partially ablated liver. We discovered that the absence of amino acids induces in primary rat hepatocytes the entrance in a quiescence state together with an increase in Drosha protein, which does not involve the mTOR pathway. Conversely, Drosha knockdown allows the hepatocytes, quiescent after amino-acid deprivation, to proliferate again. Further, hepatocyte proliferation appears to be independent of miRNAs, the canonical downstream partners of Drosha. Taken together, our observations reveal an intriguing non-canonical action of Drosha in the control of growth regulation of adult hepatocytes responding to a nutritional strain, and they may help to design novel preventive and/or therapeutic approaches for hepatic failure.

Argonaute-2 is associated to brown adipose tissue activation.

MicroRNAs (miRNAs) are important modulators of thermogenic brown adipose tissue (BAT). They have been involved in its differentiation and hence its functioning. While different regulators of the miRNA machinery have been shown to be essential for BAT differentiation, little is known about their implication in BAT activation. The aim of this work was to evaluate the role of AGO2, the chief miRNA mediator, in BAT activation. We took advantage of two non-genetic models of BAT activation to analyze the miRNA machinery and miRNA expression in BAT. We used principal component analysis (PCA) to obtain an overview of miRNA expression according to the BAT activation state. In vitro, we examined AGO2 expression during brown adipocyte differentiation and activation. Finally, we downregulated AGO2 to reveal its potential role in the thermogenic function of brown adipocytes. PCA analysis allowed to cluster animals on their miRNA signature in active BAT. Moreover, hierarchical clustering showed a positive correlation between global upregulation of miRNA expression and active BAT. Consistently, the miRNA machinery, particularly AGO2, was upregulated in vivo in active BAT and in vitro in mature brown adipocytes. Finally, the partial loss-of-function of AGO2 in mature brown adipocytes is sufficient to lead to a diminished expression of UCP1 associated to a decreased uncoupled respiration. Therefore, our study shows the potential contribution of AGO2 in BAT activation. Since BAT is a calorie-burning tissue these data have a translational potential in terms of therapeutic target in the field of altered fuel homeostasis associated to obesity and diabetes.

The OncoAge Consortium: Linking Aging and Oncology from Bench to Bedside and Back Again.

It is generally accepted that carcinogenesis and aging are two biological processes, which are known to be associated. Notably, the frequency of certain cancers (including lung cancer), increases significantly with the age of patients and there is now a wealth of data showing that multiple mechanisms leading to malignant transformation and to aging are interconnected, defining the so-called common biology of aging and cancer. OncoAge, a consortium launched in 2015, brings together the multidisciplinary expertise of leading public hospital services and academic laboratories to foster the transfer of scientific knowledge rapidly acquired in the fields of cancer biology and aging into innovative medical practice and silver economy development. This is achieved through the development of shared technical platforms (for research on genome stability, (epi)genetics, biobanking, immunology, metabolism, and artificial intelligence), clinical research projects, clinical trials, and education. OncoAge focuses mainly on two pilot pathologies, which benefit from the expertise of several members, namely lung and head and neck cancers. This review outlines the broad strategic directions and key advances of OncoAge and summarizes some of the issues faced by this consortium, as well as the short- and long-term perspectives.

5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside reduces glucose uptake via the inhibition of Na+/H+ exchanger 1 in isolated rat ventricular cardiomyocytes.

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by an increased AMP/ATP ratio. AMPK is now well recognized to induce glucose uptake in skeletal muscle and heart. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is phosphorylated to form the AMP analog ZMP, which activates AMPK. Its effects on glucose transport appear to be tissue specific. The purpose of our study was to examine the effect of AICAR on insulin-induced glucose uptake in adult rat ventricular cardiomyocytes. We studied isolated adult rat ventricular cardiomyocytes treated or not with the AMPK activators AICAR and metformin and, subsequently, with insulin or not. Insulin action was investigated by determining deoxyglucose uptake, insulin receptor substrate-1- or -2-associated phosphatidylinositol 3-kinase activity and protein kinase B (PKB) cascade using antibodies to PKB, glycogen synthase kinase-3, and Akt substrate of 160 kDa. Intracellular pH was evaluated using the fluorescent pH-sensitive dye 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Na(+)/H(+) exchanger 1 (NHE1) activity was assessed using the NH(4)(+) prepulse method. Our key findings are as follows. AICAR and metformin enhance insulin signaling downstream of PKB. Metformin potentiates insulin-induced glucose uptake, but surprisingly, AICAR inhibits both basal and insulin-induced glucose uptake. Moreover, we found that AICAR decreases intracellular pH, via inhibition of NHE1. In conclusion, AMPK potentiates insulin signaling downstream of PKB in isolated cardiac myocytes, consistent with findings in the heart in vivo. Furthermore, AICAR inhibits basal and insulin-induced glucose uptake in isolated cardiac myocytes via the inhibition of NHE1 and the subsequent reduction of intracellular pH. Importantly, AICAR exerts these effects in a manner independent of AMPK activation.

Nutritional and genetic determinants of vitamin B and homocysteine metabolisms in neural tube defects: a multicenter case-control study.

Neural tube defects (NTDs) are severe congenital malformations due to failure of neural tube formation in early pregnancy. The proof that folic acid prevents NTDs raises the question of whether other parts of homocysteine (Hcy) metabolism may affect rates of NTDs. This French case-control study covered: 77 women aged 17-42 years sampled prior to elective abortion for a severe NTDs (cases) and 61 women aged 20-43 years with a normal pregnancy. Plasma and erythrocyte folate, plasma B6, B12 and Hcy were tested as five polymorphisms MTHFR 677 C --> T, MTHFR 1298 A --> C, MTR 2756 A --> G, MTTR 66 A --> G and TCN2 776 C --> G. Cases had significantly lower erythrocyte folate, plasma folate, B12 and B6 concentrations than the controls, and higher Hcy concentration. The odds ratio was 2.15 (95% CI: 1.00-4.59) for women with the MTRR 66 A --> G allele and it was decreased for mothers carrying the MTHFR 1298 A --> C allele. In multivariate analysis, only the erythrocyte folate concentration (P = 0.005) and plasma B6 concentration (P = 0.020) were predictors. Red cell folate is the main determinant of NTDs in France. Folic acid supplement or flour fortification would prevent most cases. Increased consumption of vitamins B12 and B6 could contribute to the prevention of NTDs. Genetic polymorphisms played only a small role. Until folic acid fortification becomes mandatory, all women of reproductive age should consume folic acid in a multivitamin that also contains B12 and B6.

[Nuclear FoxO1 as a bridge between metabolism and mitogenesis]. / Facteur de transcription FoxO1 : une passerelle nucléaire entre métabolisme et mitogenèse.

The transcription factor FoxO1 plays a central role in the metabolic adaptations to fasting. Furthermore, FoxO1 and its homologs encode enzymes buffering ROS (reactive oxygen species) or repairing damaged DNA, and are involved in stalling the ageing process across evolution. Our recent work conducted in primary cultures of rat hepatocytes suggests new mechanisms by which FoxO1 exerts some of these functions. They revealed an unexpected role for FoxO1 in amplifying metabolic, survival, mitogenic and stress signals, and the existence of multiple feed-back loops by which FoxO1 integrates and controls these pathways. Furthermore, these effects were found to be independent of FoxO1 direct binding to DNA.

Contribution of the Long Noncoding RNA H19 to ß-Cell Mass Expansion in Neonatal and Adult Rodents.

Pancreatic ß-cell expansion throughout the neonatal period is essential to generate the appropriate mass of insulin-secreting cells required to maintain blood glucose homeostasis later in life. Hence, defects in this process can predispose to diabetes development during adulthood. Global profiling of transcripts in pancreatic islets of newborn and adult rats revealed that the transcription factor E2F1 controls expression of the long noncoding RNA H19, which is profoundly downregulated during the postnatal period. H19 silencing decreased ß-cell expansion in newborns, whereas its re-expression promoted proliferation of ß-cells in adults via a mechanism involving the microRNA let-7 and the activation of Akt. The offspring of rats fed a low-protein diet during gestation and lactation display a small ß-cell mass and an increased risk of developing diabetes during adulthood. We found that the islets of newborn rats born to dams fed a low-protein diet express lower levels of H19 than those born to dams that did not eat a low-protein diet. Moreover, we observed that H19 expression increases in islets of obese mice under conditions of increased insulin demand. Our data suggest that the long noncoding RNA H19 plays an important role in postnatal ß-cell mass expansion in rats and contributes to the mechanisms compensating for insulin resistance in obesity.

Nuclear forkhead box O1 controls and integrates key signaling pathways in hepatocytes.

Insulin inhibits forkhead O class (FoxO) transcription factors, which down-regulate the expression of genes involved in metabolism, cell cycle arrest, and apoptosis. After being phosphorylated by protein kinase B (PKB) on S253 in its DNA-binding domain, Foxo1 is phosphorylated on T24 and additional sites, which overall triggers its nuclear exclusion. During this process, Foxo1 is thought to retain some transcriptional activity and signaling potential. To evaluate this Foxo1 action, we used a Foxo1-ADA mutant that is constitutively nuclear due to mutation of T24 and S316 to A and harbors a mutation of S253 to D. Adenoviral-mediated expression of Foxo1-ADA in hepatocytes activates PKB and MAPK pathways more than expression of wild-type or of a transactivation domain-deleted mutant (Delta256). PKB activation cannot be accounted for by a Foxo1-mediated increase in upstream signaling components such as insulin receptor substrate 1 or 2 or by Foxo1-mediated down-regulation of Tribbles homolog 3. In contrast, Foxo1-ADA increases p38 activity, and p38 is required for effects of Foxo1 on PKB, at least in part. We propose that Foxo1 turns on a feed-forward loop, relayed by p38 and acting to amplify both PKB activation and Foxo1 inhibition. To conclude, key signaling pathways are activated in hepatocytes through nuclear Foxo1.

Methylglyoxal impairs the insulin signaling pathways independently of the formation of intracellular reactive oxygen species.

Nonenzymatic glycation is increased in diabetes and leads to elevated levels of advanced glycation end products (AGEs), which link hyperglycemia to the induction of insulin resistance. In hyperglycemic conditions, intracellularly formed alpha-ketoaldehydes, such as methylglyoxal, are an essential source of intracellular AGEs, and the abnormal accumulation of methylglyoxal is related to the development of diabetes complications in various tissues and organs. We have previously shown in skeletal muscle that AGEs induce insulin resistance at the level of metabolic responses. Therefore, it was important to extend our work to intermediates of the biosynthetic pathway leading to AGEs. Hence, we asked the question whether the reactive alpha-ketoaldehyde methylglyoxal has deleterious effects on insulin action similar to AGEs. We analyzed the impact of methylglyoxal on insulin-induced signaling in L6 muscle cells. We demonstrate that a short exposure to methylglyoxal induces an inhibition of insulin-stimulated phosphorylation of protein kinase B and extracellular-regulated kinase 1/2, without affecting insulin receptor tyrosine phosphorylation. Importantly, these deleterious effects of methylglyoxal are independent of reactive oxygen species produced by methylglyoxal but appear to be the direct consequence of an impairment of insulin-induced insulin receptor substrate-1 tyrosine phosphorylation subsequent to the binding of methylglyoxal to these proteins. Our data suggest that an increase in intracellular methylglyoxal content hampers a key molecule, thereby leading to inhibition of insulin-induced signaling. By such a mechanism, methylglyoxal may not only induce the debilitating complications of diabetes but may also contribute to the pathophysiology of diabetes in general.

Nonsuppressed Glucagon After Glucose Challenge as a Potential Predictor for Glucose Tolerance.

Glucagon levels are classically suppressed after glucose challenge. It is still not clear as to whether a lack of suppression contributes to hyperglycemia and thus to the development of diabetes. We investigated the association of postchallenge change in glucagon during oral glucose tolerance tests (OGTTs), hypothesizing that higher postchallenge glucagon levels are observed in subjects with impaired glucose tolerance (IGT). Glucagon levels were measured during OGTT in a total of 4,194 individuals without diabetes in three large European cohorts. Longitudinal changes in glucagon suppression were investigated in 50 participants undergoing a lifestyle intervention. Only 66-79% of participants showed suppression of glucagon at 120 min (fold change glucagon120/0 <1) during OGTT, whereas 21-34% presented with increasing glucagon levels (fold change glucagon120/0 ≥1). Participants with nonsuppressed glucagon120 had a lower risk of IGT in all cohorts (odds ratio 0.44-0.53, P < 0.01). They were also leaner and more insulin sensitive and had lower liver fat contents. In the longitudinal study, an increase of fold change glucagon120/0 was associated with an improvement in insulin sensitivity (P = 0.003). We characterize nonsuppressed glucagon120 during the OGTT. Lower glucagon suppression after oral glucose administration is associated with a metabolically healthier phenotype, suggesting that it is not an adverse phenomenon.

Age-Dependent Control of Energy Homeostasis by Brown Adipose Tissue in Progeny Subjected to Maternal Diet-Induced Fetal Programming.

Epidemiological and animal studies show that deleterious maternal environments predispose aging offspring to metabolic disorders and type 2 diabetes. Young progenies in a rat model of maternal low-protein (LP) diet are normoglycemic despite collapsed insulin secretion. However, without further worsening of the insulin secretion defect, glucose homeostasis deteriorates in aging LP descendants. Here we report that normoglycemic and insulinopenic 3-month-old LP progeny shows increased body temperature and energy dissipation in association with enhanced brown adipose tissue (BAT) activity. In addition, it is protected against a cold challenge and high-fat diet (HFD)-induced obesity with associated insulin resistance and hyperglycemia. Surgical BAT ablation in 3-month-old LP offspring normalizes body temperature and causes postprandial hyperglycemia. At 10 months, BAT activity declines in LP progeny with the appearance of reduced protection to HFD-induced obesity; at 18 months, LP progeny displays a BAT activity comparable to control offspring and insulin resistance and hyperglycemia occur. Together our findings identify BAT as a decisive physiological determinant of the onset of metabolic dysregulation in offspring predisposed to altered ß-cell function and hyperglycemia and place it as a critical regulator of fetal programming of adult metabolic disease.

Role of amino acids in insulin signaling in adipocytes and their potential to decrease insulin resistance of adipose tissue.

Recently, our knowledge concerning the role of amino acids in signal transduction in mammals has greatly improved. This significant advance is mainly due to the remarkable discovery that the mammalian target of rapamycin (mTOR) protein kinase, known to be activated in response to a large number of hormones, growth factors and cytokines, is also under the tight control of branched-chain amino acids. Actually, both inputs are necessary to fully activate the mTOR pathway, the main function of which is to increase cell size, via the regulation of translational processes. However, amino acids are able to modulate other biological effects and appear to have unexpected actions, as evidenced by our recent work in rat adipocytes. The aim of this review is to summarize novel findings on the role of mTOR and amino acids in insulin signaling in adipocytes. A possible beneficial impact of the use of amino acids in the treatment of insulin resistance is discussed, and hypotheses about the molecular mechanisms underlying their effect are proposed.

Regulation of hypoxia-inducible factor (HIF)-1 activity and expression of HIF hydroxylases in response to insulin-like growth factor I.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of two subunits (HIF-1alpha and HIF-1beta), initially described as a mediator of adaptive responses to changes in tissue oxygenation, has been shown to be activated in an oxygen-independent manner. In this report, we studied the action of IGF-I on the regulation of HIF-1 in human retinal epithelial cells. We show that IGF-I stimulates HIF-1alpha accumulation, HIF-1alpha nuclear translocation, and HIF-1 activity by regulation of HIF-1alpha expression through a posttranscriptional mechanism. In addition, we demonstrate that IGF-I stimulates HIF-1 activity through phosphatidylinositol-3-kinase/ mammalian target of rapamycin and MAPK-dependent signaling pathways leading to VEGF (vascular endothelial growth factor) mRNA expression. Three human prolyl-hydroxylases PHD-1, -2, and -3 (PHD-containing protein) and an asparaginyl-hydroxylase factor inhibiting HIF-1, which regulate HIF-1alpha stability and HIF-1 activity in response to hypoxia, have been described. Our analysis of their mRNA expression showed a different magnitude and time course of expression pattern in response to insulin and IGF-I compared with CoCl(2). Taken together, our data reveal that growth factors and CoCl(2), which mimics hypoxia, lead to HIF-1 activation and ensuing VEGF expression by different mechanisms. Their joined actions are likely to lead to an important and sustained increase in VEGF action on retinal blood vessels, and hence to have devastating effects on the development of diabetic retinopathy.