Antonio Fernández-Pérez

ESSENTIAL ROLE OF PROTEIN TYROSINE PHOSPHATASE 1B IN OBESITY-INDUCED INFLAMMATION AND PERIPHERAL INSULIN RESISTANCE DURING AGING

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes (T2DM). In this study, we have evaluated the role of PTP1B in the development of aging‐associated obesity, inflammation, and peripheral insulin resistance by assessing metabolic parameters at 3 and 16 months in PTP1B−/− mice maintained on mixed genetic background (C57Bl/6J × 129Sv/J). Whereas fat mass and adipocyte size were increased in wild‐type control mice at 16 months, these parameters did not change with aging in PTP1B−/− mice. Increased levels of pro‐inflammatory cytokines, crown‐like structures, and hypoxia‐inducible factor (HIF)‐1α were observed only in adipose tissue from 16‐month‐old wild‐type mice. Similarly, islet hyperplasia and hyperinsulinemia were observed in wild‐type mice with aging‐associated obesity, but not in PTP1B−/− animals. Leanness in 16‐month‐old PTP1B−/− mice was associated with increased energy expenditure. Whole‐body insulin sensitivity decreased in 16‐month‐old control mice; however, studies with the hyperinsulinemic–euglycemic clamp revealed that PTP1B deficiency prevented this obesity‐related decreased peripheral insulin sensitivity. At a molecular level, PTP1B expression and enzymatic activity were up‐regulated in liver and muscle of 16‐month‐old wild‐type mice as were the activation of stress kinases and the expression of p53. Conversely, insulin receptor‐mediated Akt/Foxo1 signaling was attenuated in these aged control mice. Collectively, these data implicate PTP1B in the development of inflammation and insulin resistance associated with obesity during aging and suggest that inhibition of this phosphatase by therapeutic strategies might protect against age‐dependent T2DM.

DREAM mediates cAMP-dependent, Ca2+-induced stimulation of GFAP gene expression and regulates cortical astrogliogenesis

In the developing mouse brain, once the generation of neurons is mostly completed during the prenatal period, precisely coordinated signals act on competent neural precursors to direct their differentiation into astrocytes, which occurs mostly after birth. Among these signals, those provided by neurotrophic cytokines and bone morphogenetic proteins appear to have a key role in triggering the neurogenic to gliogenic switch and in regulating astrocyte numbers. In addition, we have reported previously that the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) is able to promote astrocyte differentiation of cortical precursors via activation of a cAMP-dependent pathway. Signals acting on progenitor cells of the developing cortex to generate astrocytes activate glial fibrillary acidic protein (GFAP) gene expression, but the transcriptional mechanisms that regulate this activation are unclear. Here, we identify the previously known transcriptional repressor downstream regulatory element antagonist modulator (DREAM) as an activator of GFAP gene expression. We found that DREAM occupies specific sites on the GFAP promoter before and after differentiation is initiated by exposure of cortical progenitor cells to PACAP. PACAP raises intracellular calcium concentration via a mechanism that requires cAMP, and DREAM-mediated transactivation of the GFAP gene requires the integrity of calcium-binding domains. Cortical progenitor cells from dream−/− mice fail to express GFAP in response to PACAP. Moreover, the neonatal cortex of dream−/− mice exhibits a reduced number of astrocytes and increased number of neurons. These results identify the PACAP-cAMP-Ca2+-DREAM cascade as a new pathway to activate GFAP gene expression during astrocyte differentiation.

Differential configurations involving binding of USF transcription factors and Twist1 regulate Alx3 promoter activity in mesenchymal and pancreatic cells.

During embryonic development, the aristaless-type homeodomain protein Alx3 is expressed in the forehead mesenchyme and contributes to the regulation of craniofacial development. In the adult, Alx3 is expressed in pancreatic islets where it participates in the control of glucose homoeostasis. In the present study, we investigated the transcriptional regulation of Alx3 gene expression in these two cell types. We found that the Alx3 promoter contains two E-box regulatory elements, named EB1 and EB2, that provide binding sites for the basic helix-loop-helix transcription factors Twist1, E47, USF (upstream stimulatory factor) 1 and USF2. In primary mouse embryonic mesenchymal cells isolated from the forehead, EB2 is bound by Twist1, whereas EB1 is bound by USF1 and USF2. Integrity of both EB1 and EB2 is required for Twist1-mediated transactivation of the Alx3 promoter, even though Twist1 does not bind to EB1, indicating that binding of USF1 and USF2 to this element is required for Twist1-dependent Alx3 promoter activity. In contrast, in pancreatic islet insulin-producing cells, the integrity of EB2 is not required for proximal promoter activity. The results of the present study indicate that USF1 and USF2 are important regulatory factors for Alx3 gene expression in different cell types, whereas Twist1 contributes to transcriptional transactivation in mesenchymal, but not in pancreatic, cells.

Pituitary adenylate cyclase-activating polypeptide stimulates glial fibrillary acidic protein gene expression in cortical precursor cells by activating Ras and Rap1

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on cortical precursor cells to trigger glial fibrillary acidic protein (GFAP) gene expression and astrocyte differentiation by stimulation of intracellular cAMP production. Here, we show that as expected, PACAP activates cAMP-dependent protein kinase A. However, inhibition of protein kinase A does not prevent PACAP-induced GFAP gene expression or astrocytogenesis. PACAP also activates the small GTPases Rap1 and Ras, but either activation of Rap1 alone by selective stimulation of the guanine nucleotide exchange factor Epac, or expression of a constitutively active form of Ras, do not induce GFAP gene expression. Ras is activated by PACAP in a cAMP-dependent manner, and inhibition of Ras and/or Rap1 decreases PACAP-induced GFAP promoter stimulation. Thus, cAMP-dependent PACAP-induced GFAP expression during astrocytogenesis involves the coordinated activation of both Ras and Rap1, but activation of either one of them in isolation is not sufficient to trigger this response.

Alx3-deficient mice exhibit decreased insulin in beta cells, altered glucose homeostasis and increased apoptosis in pancreatic islets.

Aims/hypothesisHomeodomain transcription factors play an important role in the regulation of pancreatic islet function. In previous studies we determined that aristaless-like homeobox 3 (ALX3) is produced in islet cells, binds to the promoter of the insulin gene and regulates its expression. The purpose of the present study was to investigate the functional role of ALX3 in pancreatic islets and its possible involvement in the regulation of glucose homeostasis in vivo.MethodsAlx3-knockout mice were used. Glucose and insulin tolerance tests were carried out, and serum insulin concentrations were determined. Isolated islets were used to test insulin secretion and gene expression. The pancreatic islets were also studied using both confocal and conventional microscopy.ResultsALX3 deficiency resulted in increased blood glucose levels and impaired glucose tolerance in the presence of normal serum insulin concentrations. Insulin, glucagon and glucokinase expression were reduced in Alx3-null pancreatic islets. Reduced insulin content was reflected by decreased insulin secretion from isolated islets. Alx3-deficient islets also showed increased apoptosis, and morphometric analyses indicated that they were, on average, of smaller size than islets from control mice. ALX3 deficiency resulted in reduced beta cell mass. Finally, mature Alx3-null mice developed age-dependent insulin resistance due to impaired peripheral insulin receptor signalling.Conclusions/interpretationALX3 participates in the regulation of the expression of essential genes for the function of pancreatic islets, and its deficiency alters the regulation of glucose homeostasis in vivo. We suggest that ALX3 constitutes a potential candidate to consider in the aetiopathogenesis of diabetes mellitus.

Role of muscle IL-6 in gender-specific metabolism in mice

The aim of the present work was to further explore the physiological roles of muscle-derived IL-6. Adult-floxed and conditional skeletal muscle IL-6 knock out male and female mice were used to study energy expenditure (indirect calorimetry at rest and during treadmill exercise, and body temperature cycle during the light phase) and energy intake (response to fast/refeeding). We also evaluated the responses to leptin and the activity of the insulin signalling pathway in skeletal muscle and liver by phosphorylation of Akt at Ser 473. The stress response was also studied. Results indicate a relevant role of muscle IL-6 in maintaining energy homeostasis, especially in males. Absence of muscle IL-6 in male mice results in lower core body temperature in the light phase, increased respiratory exchange ratio (RER) both at rest and during exercise, increased expression of TCA cycle marked gene, citrate synthase in muscle, reduced fat storage and decreased body weight and food consumption in response to leptin. In females, muscle IL-6 deficiency increases VO2 and CO2 levels similarly. Also in contrast to males, energy expenditure (EE) measured over 48h reveals a significant elevation in female mice with muscle IL-6 deficiency; moreover, they show a modified response to fasting-refeeding and to restraint stress. The present results contribute to the understanding of the role of muscle IL-6 in male and female mouse metabolism, not only during exercise but also in the basal state and in situations where energy balance is altered.

Epigenetic programming at the Mogat1 locus may link neonatal overnutrition with long-term hepatic steatosis and insulin resistance

Postnatal overfeeding increases the risk of chronic diseases later in life, including obesity, insulin resistance, hepatic steatosis, and type 2 diabetes. Epigenetic mechanisms might underlie the long‐lasting effects associated with early nutrition. Here we aimed to explore the molecular pathways involved in early development of insulin resistance and hepatic steatosis, and we examined the potential contribution of DNA methylation and histone modifications to long‐term programming of metabolic disease. We used a well‐characterized mouse model of neonatal overfeeding and early adiposity by litter size reduction. Neonatal overfeeding led to hepatic insulin resistance very early in life that persisted throughout adulthood despite normalizing food intake. Up‐regulation of monoacylglycerol O‐acyltransferase (Mogat)1conceivably mediates hepatic steatosis and insulin resistance through increasing intracellular diacylglycerol content. Early and sustained deregulation of Mogat1 was associated with a combination of histone modifications that might favor Mogat1expression. In sum, postnatal overfeeding causes extremely rapid derangements of hepatic insulin sensitivity that remain relatively stable until adulthood. Epigenetic mechanisms, particularly histone modifications, could contribute to such long‐lasting effects. Our data suggest that targeting hepatic monoacylglycerol acyltransferase activity during early life might provide a novel strategy to improve hepatic insulin sensitivity and prevent late‐onset insulin resistance and fatty liver disease.—Ramon‐Krauel, M., Pentinat, T., Bloks, V. W., Cebrià, J., Ribo, S., Pérez‐Wienese, R., Vilà, M., Palacios‐Marin, I., Fernández‐Pérez, A., Vallejo, M., Téllez, N., Rodríguez, M. À., Yanes, O., Lerin, C., Díaz, R., Plosch, T., Tietge, U. J. F., Jimenez‐Chillaron, J. C. Epigenetic programming at the Mogat1locus may link neonatal overnutrition with long‐term hepatic steatosis and insulin resistance. FASEB J. 32, 6025–6037 (2018).www.fasebj.org