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© 2021 The Author(s)., [Objective]: Pancreatic β-cell dysfunction is a central feature in the pathogenesis of type 2 diabetes (T2D). Accumulating evidence indicates that β-site APP-cleaving enzyme 2 (BACE2) inhibition exerts a beneficial effect on β-cells in different models of T2D. Thus, targeting BACE2 may represent a potential therapeutic strategy for the treatment of this disease. Here, we aimed to investigate the effects of BACE2 suppression on glucose homeostasis in a model of diet-induced obesity., [Methods]: BACE2 knock-out (BKO) and wild-type (WT) mice were fed with a high-fat diet (HFD) for 2 or 16 weeks. Body weight, food intake, respiratory exchange ratio, locomotor activity, and energy expenditure were determined. Glucose homeostasis was evaluated by glucose and insulin tolerance tests. β-cell proliferation was assessed by Ki67-positive nuclei, and β-cell function was determined by measuring glucose-stimulated insulin secretion. Leptin sensitivity was evaluated by quantifying food intake and body weight after an intraperitoneal leptin injection. Neuropeptide gene expression and insulin signaling in the mediobasal hypothalamus were determined by qPCR and Akt phosphorylation, respectively., [Results]: After 16 weeks of HFD feeding, BKO mice exhibited an exacerbated body weight gain and hyperphagia, in comparison to WT littermates. Glucose tolerance was similar in both groups, whereas HFD-induced hyperinsulinemia, insulin resistance, and β-cell expansion were more pronounced in BKO mice. In turn, leptin-induced food intake inhibition and hypothalamic insulin signaling were impaired in BKO mice, regardless of the diet, in accordance with deregulation of the expression of hypothalamic neuropeptide genes. Importantly, BKO mice already showed increased β-cell proliferation and glucose-stimulated insulin secretion with respect to WT littermates after two weeks of HFD feeding, before the onset of obesity., [Conclusions]: Collectively, these results reveal that BACE2 suppression in an obesogenic setting leads to exacerbated body weight gain, hyperinsulinemia, and insulin resistance. Thus, we conclude that inhibition of BACE2 may aggravate the adverse metabolic effects associated with obesity., This work was supported by projects from the Instituto de Salud Carlos III (PI17/00879 to AN and JMS) and by the Spanish Ministry of Economy and Competitiveness (BFU2017-89336-R to MV), co-funded by the Fondo Europeo de Desarrollo Regional (FEDER; A way to build Europe), and by the CERCA Programme and Generalitat de Catalunya (grant 2014_SGR_520). DDC was supported by Conicyt’s fellowship from the Government of Chile (72170321-6357/2016). SR is a recipient of a Juan de la Cierva Incorporación (IJC2018-037341-I) program from the Spanish Ministry of Science and Innovation. This work was developed at the Center Esther Koplowitz (Barcelona). CIBERDEM (Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas) is an initiative of the Instituto de Salud Carlos III.

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits in islets of type 2 diabetic patients. hIAPP misfolding and aggregation is one of the factors that may lead to β-cell dysfunction and death. Endogenous chaperones are described to be important for the folding and functioning of proteins. Here, we examine the effect of the endoplasmic reticulum chaperone protein disulfide isomerase (PDI) on β-cell dysfunction. Among other chaperones, PDI was found to interact with hIAPP in human islet lysates. Furthermore, intrinsically recovered PDI levels were able to restore the effect of high glucose- and palmitate-induced β-cell dysfunction by increasing 3.9-fold the glucose-stimulated insulin secretion levels and restoring insulin content up to basal control values. Additionally, PDI transduction decreased induced apoptosis by glucolipotoxic conditions. This approach could reveal a new therapeutic target and aid in the development of strategies to improve β-cell dysfunction in type 2 diabetic patients.

Objective This study compares the patterns of visceral (VIS) and subcutaneous (SC) adipose tissue (AT)-derived metabolites from non-obese (BMI 24-26 kg/m2) and obese subjects (BMI > 40 kg/m2) with no major metabolic risk factors other than BMI. Methods SC- and VIS- AT obtained from obese (Ob) and non-obese (NOb) subjects during surgery were incubated to obtain their metabolites. Differences related to obesity or anatomical provenances of AT were assessed using an untargeted metabolomics approach based on gas chromatography-mass spectrometry. Results The overall effect of obesity on the metabolite profile resulted more remarkable than the effect of regional AT. Only the depletion of 2-ketoisocaproic (2-KIC) acid reached statistical significance for the SC-AT alone, although it was observed in both depots. Obesity induced more significant changes in several amino acids levels of the VIS-AT metabolites. On the one hand, higher released levels of glutamine and alanine were detected in the VIS- obese AT, whereas on the other, the VIS- obese AT presented a diminished uptake of essential amino acids (methionine, threonine, lysine), BCAAs, leucine, and serine. Conclusion This study shows that obesity markedly affects the amino acid metabolic signature of the AT before the clinical onset of other significant metabolic alterations aside from BMI.

Objective Persistent inflammation and impaired adipogenesis are frequent features of obesity and underlie the development of its complications. However, the factors behind adipose tissue dysfunction are not completely understood. Previously it was shown that histone demethylase KDM1A is required for adipogenesis. Design and Methods Kdm1a expression was knocked down in 3T3-L1 preadipocytes by siRNA transfection and whole-genome expression profiling was performed by microarray hybridization. The role of NF-κβ and C/EBPβ was analyzed by incubation with the inhibitor parthenolide and by cebpb knockdown, respectively. Results Knockdown of kdm1a or rcor2 in 3T3-L1 preadipocytes results in impaired differentiation and induction of inflammatory gene expression. Enhanced expression of il6 in kdm1a knocked down preadipocytes is associated with increased recruitment of C/EBPβ and the NF-κβ subunit RelA to the il6 promoter. Cebpb knockdown attenuates the induction of il6 expression in kdm1a knocked down cells, whereas simultaneous cebpb knockdown and NF-κβ inhibition abrogates it. Dietary-induced and genetic mouse models of obesity display decreased KDM1A in adipose tissue, and this correlates with increased expression of proinflammatory genes and C/EBPβ. Conclusion KDM1A represses the expression of inflammatory genes in preadipocytes. Dysregulated kdm1a expression in preadipocytes may thus participate in the development of obesity-associated inflammation.

Altres ajuts: FI-DGR 2011 de la Generalitat de Catalunya The senescence-accelerated SAMP8 mouse is considered a useful non-transgenic model for studying aspects of progressive cognitive decline and Alzheimer's disease (AD). Using SAMR1 mice as controls, here we explored the effects of 6 months of voluntary wheel running in 10-month-old female SAMP8 mice. Exercise in SAMP8 mice improved phenotypic features associated with premature aging (i.e., skin color and body tremor) and enhanced vascularization and BDNF gene expression in the hippocampus compared with controls. With the aim of identifying genes involved in brain aging responsive to long-term exercise, we performed whole genome microarray studies in hippocampus from sedentary SAMP8 (P8sed), SAMR1 (R1sed), and exercised SAMP8 (P8run) mice. The genes differentially expressed in P8sed versus R1sed were considered as putative aging markers (i) and those differentially expressed in P8run versus P8sed were considered as genes modulated by exercise (ii). Genes differentially expressed in both comparisons (i and ii) were considered as putative aging genes responsive to physical exercise. We identified 34 genes which met both criteria. Gene ontology analysis revealed that they are mainly involved in functions related to extracellular matrix maintenance. Selected genes were validated by real-time quantitative PCR assays, i.e., collagen type 1 alpha 1 (col1a1), collagen type 1 alpha 2 (col1a2), fibromodulin (fmod), prostaglandin D(2) synthase (ptgds), and aldehyde dehydrogenase (Aldh1a2). As a whole, our study suggests that exercise training during adulthood may prevent or delay gene expression alterations and processes associated with hippocampal aging in at-risk subjects.

Objective : Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling. Methods and Results : Slug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries. Conclusions : Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases. Fil: Coll Bonfill, Núria. Universidad de Barcelona; España Fil: Peinado, Victor I.. Universidad de Barcelona; España. CIBER Enfermedades Respiratorias; España Fil: Pisano, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Párrizas, Marcelina. CIBER Diabetes y Enfermedades Metabólicas Asociadas; España Fil: Blanco, Isabel. Universidad de Barcelona; España Fil: Evers, Maurits. Universitat Regensburg; Alemania Fil: Engelmann, Julia C.. Universitat Regensburg; Alemania Fil: García Lucio, Jessica. Universidad de Barcelona; España Fil: Tura Ceide, Olga. CIBER Enfermedades Respiratorias; España Fil: Meister, Gunter. Universitat Regensburg; Alemania Fil: Barberà, Joan Albert. Universidad de Barcelona; España Fil: Musri, Melina Mara. Universidad de Barcelona; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

Purpose of review Epigenetic regulation plays an essential role in cell differentiation, by allowing the establishment and maintenance of the gene-expression pattern of the mature cell type. Because of its importance in chronic diseases, adipogenesis is one of the best-studied differentiation processes. The hormonal and transcriptional cascades governing the differentiation of the adipocytes are well known, but the role of epigenetic mechanisms is only starting to emerge. In this review, we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade. Recent findings The advent of high-throughput technologies has made possible the exhaustive analysis of the epigenetic phenomenons taking place during adipogenesis. The cooperative recruitment of CCAAT/enhancer-binding protein (C/EBPβ) and other early proadipogenic transcription factors to transcription factor hotspots shortly after induction of adipogenesis is required to establish a transient epigenomic state that then informs the recruitment of the later adipogenic transcription factors peroxisome proliferator-activated receptor (PPARγ) and C/EBPα to their target genes. Summary Epigenetic marks and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism.

Aging is a gradual process during which molecular and cellular processes deteriorate progressively, often leading to such pathological conditions as vascular and metabolic disorders and cognitive decline. Although the mechanisms of aging are not yet fully understood, inflammation, oxidative damage, mitochondrial dysfunction, functional alterations in specific neuronal circuits and a restricted degree of apoptosis are involved. Physical exercise improves the efficiency of the capillary system and increases the oxygen supply to the brain, thus enhancing metabolic activity and oxygen intake in neurons, and increases neurotrophin levels and resistance to stress. Regular exercise and an active lifestyle during adulthood have been associated with reduced risk and protective effects for mild cognitive impairment and Alzheimer's disease. Similarly, studies in animal models show that physical activity has positive physiological and cognitive effects that correlate with changes in transcriptional profiles. According to numerous studies, epigenetic events that include changes in DNA methylation patterns, histone modification and alterations in microRNA profiles seem to be a signature of aging. Hence, insight into the epigenetic mechanisms involved in the aging process and their modulation through lifestyle interventions such as physical exercise might open new avenues for the development of preventive and therapeutic strategies to treat aging-related diseases.

Epigenetic mechanisms, in particular the enzymatic modification of histones, are a crucial element of cell differentiation, a regulated process that allows a precursor cell basically to turn into a different cell type while maintaining the same genetic equipment. We have previously described that the promoters of adipogenic genes display significant levels of dimethylation at the Lys4 of histone H3 (H3K4) in preadipocytes, where these genes are still silenced, thus maintaining the chromatin of the precursor cell in a receptive state. Here, we show that the expression of several histone demethylases and methyltransferases increases during adipogenesis, suggesting an important role for these proteins in this process. Knockdown of the H3K4/K9 demethylase LSD1 results in markedly decreased differentiation of 3T3-L1 preadipocytes. This outcome is associated with decreased H3K4 dimethylation and increased H3K9 dimethylation at the promoter of transcription factor cebpa, whose expression must be induced >200-fold upon stimulation of differentiation. Thus, our data suggest that LSD1 acts to maintain a permissive state of the chromatin in this promoter by opposing the action of a H3K9 methyltransferase. Knockdown of H3K9 methyltransferase SETDB1 produced the opposite results, by decreasing H3K9 dimethylation and increasing H3K4 dimethylation levels at the cebpa promoter and favoring differentiation. These findings indicate that the histone methylation status of adipogenic genes as well as the expression and function of the proteins involved in its maintenance play a crucial role in adipogenesis.

Long considered scarcely more than an uninteresting energy depot, adipose tissue has recently achieved star status. Far from being mere fat droplets, the adipocytes secrete a number of hormones and bioactive peptides, collectively known as adipokines, which participate in the regulation of a variety of functions, from haemostasis to angiogenesis to energy balance. Adipose tissue constitutes a bona-fide endocrine organ whose main dysfunctions, obesity and lipodystrophy, are related to the development of diabetes, hypertension, or dyslipidemia. The renewed interest in this tissue has prompted an escalation in the number of studies focusing on every aspect of the biology of the adipose cell, in the belief that a detailed knowledge of the mechanisms involved in the differentiation and function of adipocytes may contribute new therapeutical approaches to the treatment of such alarming medical problems. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities that together, are responsible for the establishment of the gene expression pattern of mature adipocytes. Although the exquisitely regulated transcription factor cascade controlling adipogenesis has been extensively studied, the role of chromatin and chromatin-modifying proteins has become apparent only in recent times.

Mutations in the human genes encoding hepatocyte nuclear factors (HNF) 1α, 1β, 4α, and IPF1(PDX1/IDX1/STF1) result in pancreatic β cell dysfunction and diabetes mellitus. In hepatocytes, hnf4α controls the transcription of hnf1α , suggesting that this same interaction may operate in β cells and thus account for the common diabetic phenotype. We show that, in pancreatic islet and exocrine cells, hnf4α expression unexpectedly depends on hnf1α . This effect is tissue-specific and mediated through direct occupation by hnf1α of an alternate promoter located 45.6 kb from the previously characterized hnf4α promoter. Hnf1α also exerts direct control of pancreatic-specific expression of hnf4 γ and hnf3γ . Hnf1α dependence of hnf4α , hnf4γ , hnf3γ , and two previously characterized distal targets ( glut2 and pklr ) is established only after differentiated cells arise during pancreatic embryonic development. These studies define an unexpected hierarchical regulatory relationship between two genes involved in human monogenic diabetes in the cells, which are relevant to its pathophysiology. Furthermore, they indicate that hnf1α is an essential component of a transcription factor circuit whose role may be to maintain differentiated functions of pancreatic cells.

Mutations in the gene encoding hepatic nuclear factor 1-alpha (HNF1-alpha) cause a subtype of human diabetes resulting from selective pancreatic beta-cell dysfunction. We have analyzed mice lacking HNF1-alpha to study how this protein controls beta-cell-specific transcription in vivo. We show that HNF1-alpha is essential for the expression of glut2 glucose transporter and L-type pyruvate kinase (pklr) genes in pancreatic insulin-producing cells, whereas in liver, kidney, or duodenum tissue, glut2 and pklr expression is maintained in the absence of HNF1-alpha. HNF1-alpha nevertheless occupies the endogenous glut2 and pklr promoters in both pancreatic islet and liver cells. However, it is indispensable for hyperacetylation of histones in glut2 and pklr promoter nucleosomes in pancreatic islets but not in liver cells, where glut2 and pklr chromatin remains hyperacetylated in the absence of HNF1-alpha. In contrast, the phenylalanine hydroxylase promoter requires HNF1-alpha for transcriptional activity and localized histone hyperacetylation only in liver tissue. Thus, different HNF1-alpha target genes have distinct requirements for HNF1-alpha in either pancreatic beta-cells or liver cells. The results indicate that HNF1-alpha occupies target gene promoters in diverse tissues but plays an obligate role in transcriptional activation only in cellular- and promoter-specific contexts in which it is required to recruit histone acetylase activity. These findings provide genetic evidence based on a live mammalian system to establish that a single activator can be essential to direct nucleosomal hyperacetylation to transcriptional targets.

Human embryonic kidney 293 cells and 293 cells overexpressing different amounts of the adaptor protein Crk-II (ranging from 3- to 10-fold higher levels than the parental cell line) were examined for their ability to undergo apoptosis when maintained in control and serum-free (SF) medium. Parental 293 cells undergo apoptosis only when deprived of serum for prolonged periods of time (24–48 h). On the other hand, 293 cells overexpressing different levels of Crk-II present detectable levels of apoptosis as measured by DNA fragmentation when grown in control medium, with a marked increase when they are deprived of serum for 12–48 h. To determine the pathways involved in Crk-II-induced apoptosis, Crk-II overexpressing cells were transiently transfected with a dominant-negative Ras construct (N17-Ras). Compared to cells transfected with control vectors, the cells overexpressing N17-Ras presented lower levels of apoptosis when maintained in SF-medium. On the other hand, transient transfection of a dominant-negative Raf-1 construct (K375W-Raf-1) did not decrease apoptosis; slightly increasing DNA fragmentation levels were seen. Similar results were obtained when the cells were incubated in the presence of a MEK1 inhibitor. The results presented here suggest that overexpression of Crk-II induces apoptosis via a Ras-dependent, Raf-1/MEK1/ERK-independent pathway.

Insulin and insulin-like growth factor-I (IGF-I) receptors were characterized in glycoprotein fractions prepared by wheat germ agglutinin-agarose affinity chromatography from the ovaries of carp. Insulin-specific overall binding in carp ovaries was 6- to 11-fold lower than IGF-I binding (2.7 +/- 0.48% vs. 22.8 +/- 3.6% per 20 microg glycoprotein). Cold IGF-I displaced radiolabeled IGF-I binding in doses 1000- to 3000-fold lower than cold insulin. On the other hand, cold insulin displaced radiolabeled insulin binding at concentrations 5- to 30-fold lower than cold IGF-I. The alpha-subunit molecular masses of carp insulin and IGF-I receptors were smaller than the alpha-subunit molecular mass of rat insulin receptor (125 and 120 vs. 135 kDa, respectively). Autophosphorylation of carp beta-subunit insulin and IGF-I receptors showed similar molecular masses that did not differ from the molecular mass of rat insulin beta subunit. Receptor tyrosine kinase activity was stimulated in a dose-dependent manner by insulin and IGF-I. Insulin and IGF-I stimulated tyrosine kinase activity and reached a maximum, respectively, of 224 +/- 14% and 279 +/- 7% of basal phosphorylation. Insulin and IGF-I binding characteristics were measured through different stages of follicular development. High specific binding of both peptides in primary oocyte growth (5.6 +/- 0.8% and 50 +/- 10% per 20 microg glycoprotein for insulin and IGF-I, respectively) decreased to a minimum at the end of vitellogenesis, followed by a slight increase later, in the preovulatory stage. The presence of insulin and IGF-I receptors in carp ovaries and the changes in percentage of binding throughout the reproductive cycle suggest that, in carp, the roles of insulin and IGF-I depend on the ovarian maturation stage.

We investigated insulin and insulin-like growth factor-I (IGF-I) receptor-binding and receptor intrinsic tyrosine kinase activity in the brain of carp (Cyprinus carpio) and trout (Salmo trutta fario). Glycoprotein fractions of semi-purified receptors were prepared by WGA-agarose affinity chromatography. Insulin receptors were found in the brains of both fish species investigated. Carp and trout brain preparations bound, respectively (per 50 micrograms glycoprotein), with 6.0 +/- 1.5% and 8.0 +/- 2.0% of 125I-labeled insulin added to the assay. Insulin binding was specific: much higher quantity of IGF-I (EC50 165 +/- 11 nM for carp and 88.0 +/- 6 nM for trout receptors) than insulin (EC50 0.26 +/- 0.04 nM for carp and 0.25 +/- 0.02 nM for trout) was necessary to displace bound insulin tracer. In preparations of brain receptors, IGF-I binding (52.8 +/- 6.5% in carp brain and 55.0 +/- 13.0% in trout brain) surpassed insulin binding several fold. IGF-I bound to the brain receptors with high affinity (Kd for carp was 0.13 +/- 0.06 nM and for trout 0.22 +/- 0.11 nM) and specificity. Although IGF-I binding could be displaced with insulin, EC50 were 660 +/- 51 nM for carp and 1557 +/- 194 nM for trout. Both ligands stimulated phosphorylation of exogenous substrates in a dose-dependent manner. Carp brain receptors were not significantly different from trout receptors with respect to basal phosphotransferase activities (250.0 +/- 50.0 fm P/mg glycoprotein in carp and 330.0 +/- 120.0 fm P/mg glycoprotein in trout). In both species IGF-I caused higher maximal stimulation (308.0 +/- 36.0% and 270.0 +/- 39%, for carp and trout, respectively) than insulin (250.0 +/- 13.0% and 209.0 +/- 6.0%, for carp and trout, respectively).

In the Funding section, the grant number FIS to VIP is listed incorrectly: The correct grant number to VIP is FIS PI13/00836.

Insulin and insulin-like growth factor-1 (IGF-1) receptors were quantified in glycoprotein fractions prepared by wheat germ agglutinin-agarose affinity chromatography from skeletal muscle of several species of salmonid fish and carp. Insulin binding in fish skeletal muscle was lower than insulin binding found in rat. IGF-1-specific binding was two- to sixfold higher than insulin binding (16.0 +/- 3.0 versus 5.8 +/- 0.75%/50 micrograms glycoprotein in brown trout, 15.5 +/- 0.9 versus 2.2 +/- 0.5%/50 micrograms in coho salmon, and 39.7 +/- 7.6 versus 16.0 +/- 3.0%/50 micrograms in carp muscle). Specific IGF-1 binding in fish skeletal muscle presented values similar to those in rat muscle. IGF-1 receptor binding was, in addition, highly specific. Cold IGF-1 displaced radiolabeled IGF-1 binding in doses 100- to 1000-fold lower than cold insulin (ED50 of IGF-1 binding to carp receptor preparations was 0.24 +/- 0.04 nM when displaced with cold IGF-1 and 368 +/- 83 nM when displaced with insulin). On the other hand, cold insulin displaced radiolabeled insulin binding at concentrations 10- to 100-fold lower than cold IGF-1. Receptor tyrosine kinase activity was stimulated over basal values in a dose-dependent manner by both insulin and IGF-1, although IGF-1 was more potent than insulin. Basal rates of phosphorus transferred to the artificial exogenous substrate poly(Glu4:Tyr1) were similar in all salmonid species (200-320 fmol P/micrograms protein) and higher in carp (1840 +/- 300 fmol P/micrograms protein). Maximum percentage of stimulation of tyrosine kinase activity by insulin and IGF-1 was in the same range in all salmonid species and carp (130-200% for insulin, 160-232% for IGF-1). Abundance of IGF-1 receptors in fish skeletal muscle contrasts with the pattern observed in higher vertebrates, in which insulin receptors prevail over IGF-1 receptors.

The protein kinase product of the gene mutated in myotonic dystrophy 1 (DMPK) is reported to play a role in cardiac pathophysiology. To gain insight into the molecular mechanisms modulated by DMPK, we characterize the impact of DMPK ablation in the context of cardiac β-adrenergic function. Our data demonstrate that DMPK knockout mice present altered β-agonist-induced responses and suggest that this is due, at least in part, to a reduced density of β(1)-adrenergic receptors in cardiac plasma membranes.

1. 1. Insulin, glucagon and plasma glucose levels were studied during the post-feeding period in juvenile brown trout (Salmo trutta fario). Three experiments were performed, in June, July and October, with animals weighing 60.0 ± 0.9 g, 102 ± 2.6 g and 177.5 ± 5.9 g respectively, with sequential samplings between 1 and 24 hr after feeding. 2. 2. Plasma insulin levels reached maximum values within 2hr after feeding (range of values, 9.4 ± 1.2 ng/ml to 14.1 ± 0.9 ng/ml). Insulin then started to decrease gradually but remained high for 11 hr. At 24 hr post-feeding insulin levels ranging from 4.0 ± 0.4 ng/ml to 7.6 ± 0.6 ng/ml were observed. No seasonal differences were found in the post-feeding profiles of plasma insulin. 3. 3. Glucagon also increased rapidly but was depressed at 2 or 5 hr (depending on the experiment) simultaneously with high insulin. Glucagon maximum levels (ranging from 1779.1 ± 182.3 pg/ml to 2261.3 ±253.5pg/ml) were observed when insulin decreased, reaching values at 24 hr ranging from 638.2 ± 82.1 pg/ml to 857 ± 65.2 pg/ml. Glucose showed a similar tendency to that of glucagon, with an initial fall and later recovery at 8 or 11 hr.

The transcriptional cascade governing adipogenesis has been thoroughly examined throughout the years. Transcription factors PPARγ and C/EBPα are universally recognized as the master regulators of adipocyte differentiation and together they direct the establishment of the gene expression pattern of mature adipose cells. However, this familiar landscape has been considerably broadened in recent years by the identification of novel factors that participate in the regulation of adipogenesis, either favoring or inhibiting it, through their effects on chromatin. Epigenetic signals and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism. In this review we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade.

Adipogenesis is regulated by a coordinated cascade of sequence-specific transcription factors and coregulators with chromatin-modifying activities that are between them responsible for the establishment of the gene expression pattern of mature adipocytes. Here we examine the histone H3 post-translational modifications occurring at the promoters of key adipogenic genes during adipocyte differentiation. We show that the promoters of apM1, glut4, gpd1, and leptin are enriched in dimethylated histone H3 Lys4 (H3-K4) in 3T3-L1 fibroblasts, where none of these genes are yet expressed. A detailed study of the apM1 locus shows that H3-K4 dimethylation is restricted to the promoter region in undifferentiated cells and associates with RNA polymerase II (pol II) loading. The beginning of apM1 transcription at the early stages of adipogenesis coincides with promoter H3 hyperacetylation and H3-K4 trimethylation. At the coding region, H3 acetylation and dimethylation, as well as pol II binding, are found in cells at later stages of differentiation, when apM1 transcription reaches its maximal peak. This same pattern of histone modifications is detected in mouse primary preadipocytes and adipocytes but not in a related fibroblast cell line that is not committed to an adipocyte fate. Inhibition of H3-K4 methylation by treatment of 3T3-L1 cells with methylthioadenosine results in decreased apM1 gene expression as well as decreased adipogenesis. Taken together, our data indicate that H3-K4 dimethylation and pol II binding to the promoter of key adipogenic genes are distinguishing marks of cells that have undergone determination to a preadipocyte stage.

Background/Aims Selective cyclooxygenase (COX)-2 inhibitors do not adversely affect renal function in experimental cirrhosis. In the current study, we investigated the molecular mechanisms underlying the effects of the selective COX-2 inhibitor, celecoxib, and assessed the influence of albumin on its actions. Methods Rat mesangial cells (RMC) were incubated with celecoxib in the absence or presence of albumin, and levels of selected vasoconstrictor eicosanoids, renin release and α-smooth muscle actin (α-SMA) expression were determined. The effects of celecoxib on PPARγ were assessed in RMC co-transfected with PPARγ and luciferase reporter constructs. Results Under resting conditions, RMC expressed COX-1, COX-2 and 12/15-lipoxygenase and mainly generated prostaglandin (PG)E 2 , thromboxane (TX)B 2 , 12-hydroxyeicosatetraenoic acid (12-HETE) and 8-epi-PGF 2α . Celecoxib, in addition to reducing PGE 2 , significantly decreased 8-epi-PGF 2α formation. In the presence of albumin, celecoxib also reduced TXB 2 and 12-HETE. Albumin per se inhibited PGE 2 as well as renin release. In trans-activation assays, celecoxib acted as a PPARγ agonist whereas albumin inhibited PPARγ as well as 15d-PGJ 2 -induced PPARγ activation. Finally, celecoxib and albumin potentiated the inhibitory effect of 15d-PGJ 2 on α-SMA expression. Conclusions These data provide novel molecular mechanisms of celecoxib and their modulation by albumin, that may be relevant to prevent renal dysfunction in conditions of unbalanced effective blood volume.

Apoptosis or programmed cell death is an essential element in the regulation and behavior of mammalian cells [1, 2]. The process is a genetically controlled response for cells to commit suicide and is orchestrated by mechanisms involving various proteins. The purpose of apoptosis is to kill unwanted cells. This process is important in the developing nervous system where a large excess of redundant neurons die off during the period when synapses are being formed between the neurons and their target cells [3, 4]. Apparently, the surviving neurons represent those that receive survival signals from the target cells. Apoptosis is also important in the nervous system following injury. Traumatic injury to the nervous system initially causes necrosis at the injury site, followed by secondary processes which cause spreading of the damage beyond this initial site of damage [5]. This secondary event is apoptosis, and occurs hours after the initial injury. Following spinal cord injury, for example, the apoptotic cells were found to be oligodendrocytes in the spinal white matter as well as in the fiber tracts, which became demyelinated as a result of apoptosis. The process of apoptosis in this model apparently occurs secondary to the release of cytokines such as tumor necrosis factor and certain interleukins. In a separate study of hypoxic-ischemic injury to the brain in rats, insulin-like growth factor-I (IGF-I) was protective against neuronal cell death [6].

A series of the synthetic protein tyrosine kinase inhibitors known as tyrphostins were studied for their effect on insulin-like growth factor-1 and insulin-stimulated cellular proliferation on NIH-3T3 fibroblasts overexpressing either receptor, as well as for their ability to inhibit ligand-stimulated receptor autophosphorylation and tyrosine kinase activity toward exogenous substrates. Several of the tyrphostins tested demonstrated a dramatic effect by inhibiting hormone-stimulated cell proliferation, with IC50s in the submicromolar range, while being unable to block serum-stimulated cell proliferation. The tyrphostins also inhibited receptor autophosphorylation and tyrosine kinase activity, with a higher IC50, in the micromolar range. Most of the tyrphostins tested presented no clear preference for either receptor, although two of them (AG1024 and AG1034) showed significantly lower IC50s for IGF-1 than for insulin receptors. These results suggest that, in spite of the high homology of the kinase regions of both receptors, it could be possible to design and synthesize small molecules capable of discriminating between them. The synthesis of such specific inhibitors could be an excellent tool to establish the precise signalling mechanisms that distinguish between the different effects of these two hormones.

Differentiated PC12 cells, which become dependent on the presence of growth factors in the media and die by apoptosis after several hours of serum deprivation, were used to test the ability of IGF-1 to regulate the endogenous levels of the death-suppressing protein Bcl-xL, IGF-1 was capable of preventing apoptosis of serum-deprived differentiated PC12 cells at physiological concentrations, with optimal results seen at 10(-8) M. Incubation with the hormone resulted in a significant increase of Bcl-x mRNA after 3-6 h incubation and a doubling of Bcl-xL protein levels by 24 h incubation. Our results show that the protective effect of IGF-1 in PC12 cells is associated with an up-regulation of Bcl-xL mRNA and protein levels.

The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10(-9) M and as early as 4 h. The phosphatidylinositol 3' (PI3')-kinase inhibitors wortmannin (at concentrations of 10(-8) M) and LY294002 (10(-6) M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70S6K) inhibitor rapamycin (10(-8) M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3'-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10(-5) M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.

The effects of insulin titres on regulation of receptor binding were studied in several fish species. Insulin receptors were semi-purified by affinity chromatography (WGA-agarose) from skeletal muscle of carp, brown trout and rainbow trout that had been subjected to increases in insulinemia produced either by arginine injection, food administration, or adaptation to an experimental diet (extruded diet with high-digestibility carbohydrates). Arginine injection provoked acute hyper-insulinemia in both carp and trout. Specific binding of insulin to the skeletal muscle was significantly increased 3 h after injection (from 5.8 ± 0.3 to 9.6 ± 0.9%/10 μ g protein in carp and from 0.8 ± 0.2 to 1.5 ± 0.4%/10 μ g in trout). The same effect was observed in carp liver preparations (from 6.0 ± 0.75 to 9.9 ± 1.25%/10 μ g ). No alterations in tyrosine kinase activity of the receptors were detected in either carp or trout preparations: basal activities of the receptors were maintained ( 3100 ± 200 fmol P/fmol receptors/30 min and 3700 ± 400 fmol P/fmol receptors/30 min, in carp and trout, respectively), as were the percentage of stimulation over basal levels obtained by incubation with insulin ( 227 ± 25% and 160 ± 10% respectively). Food ingestion raised plasma insulin levels more steadily. Specific binding also increased in skeletal muscle preparations, especially in carp (from 5.7 ± 0.3 to 11 ± 1.7%/10 μ g at 4 h and 10 ± 0.7%/10 μ g at 8 h). Tyrosine kinase activity was maintained without significant changes. Rainbow trout adapted for 2 months to an extruded diet presented higher insulin titres and higher glycogen reserves in liver and muscle. Insulin binding to skeletal muscle preparations was also significantly increased (from 0.36 ± 0.02 to 0.77 ± 0.1%/10 μ g ), as was tyrosine kinase activity (from 132 ± 4% to 156 ± 6% , without alterations in the basal activity). Results showed that fish can respond to both acute and maintained increases in insulinemia by increasing the number of insulin receptors. Tyrosine kinase activity, in contrast, is only modified after long-term adaptation.

A study is presented of the binding of insulin and IGF-I to their respective receptors in the heart muscle of carp (Cyprinus carpio), coho salmon (Oncorhynchus kisutch), brown trout (Salmo trutta fario) and Sprague–Dawley rats. Receptor preparations were partially purified by wheat germ agglutinin affinity chromatography. Specific binding of insulin/100 mg cardiac muscle was much lower in fish (from 5·6 to 9·2%) than in rat (52·0 ± 5·0%). In both carp and trout, insulin binding to the cardiac muscle receptor preparation was significantly higher than in the white skeletal muscle (3·4 ± 0·3%, carp and 0·9 ± 0·3%, trout) or in the red skeletal muscle of carp (5·5 ± 0·8%). Specific binding of IGF-1/100 mg fish heart preparation ranged between 44 and 68%, surpassing IGF-I binding in the rat heart (20 ± 6%). The affinity of IGF-I receptors in fish heart (Kd 0·17–0·19 nm) was higher than that in rat heart (Kd 0·40 ± 0·05 nm) or insulin receptors in fish and rat heart preparations (0·25–0·72 nm). The IGF-I receptor binding was highly specific and required at least 100 nm insulin to cause any displacement of the bound ligand. Receptor tyrosine kinase activity could be stimulated in a dose-dependent manner by insulin and IGF-I, although in equimolar doses IGF-I was more potent than insulin. Maximum stimulation of tyrosine kinase activity (210–230%) in fish heart was in the same range as in other piscine tissues (150–260%) or in rat cardiac muscle (200–250%). Journal of Endocrinology (1995) 146, 35–44

Insulin and insulin-like growth factor (IGF-I) receptor binding and tyrosine kinase activity were characterized in cardiac and skeletal muscles of several vertebrates. Specific insulin binding per unit weight of skeletal muscle was clearly higher in pigeon and rat than in ectothermic vertebrates (32 +/- 5 and 25 +/- 2.7%/100 mg initial tissue in pigeon and rat, respectively, vs. 4.4 +/- 0.2%/100 mg in carp samples). Insulin binding clearly predominated over IGF-I binding in skeletal muscle of endotherms (IGF-I binding was 7.7 +/- 0.5%/100 mg in rat). In ectothermic vertebrates the situation was reversed, and IGF-I binding was higher than insulin binding. In cardiac muscle, specific binding of both insulin and especially IGF-I was higher than the values found in skeletal muscle of the same species (IGF-I binding was 60 +/- 4, 103 +/- 2, and 20 +/- 3%/100 mg in carp, turtle, and rat, respectively). The tyrosine kinase activity of insulin and IGF-I receptors of all species studied presented basal phosphotransferase rates (250-1,600 fmol P.micrograms protein-1.30 min-1) and percentage of stimulation (150-520%) with clear differences between species. The present data suggest that insulin and IGF-I binding to skeletal and cardiac muscles change through the vertebrate scale in both quantity and activity.

We characterized the insulin receptors in skeletal muscle from several fish species with different nutritional preferences: brown trout (Salmo trutta fario), gilthead sea bream (Sparus aurata), tilapia (Tilapia mossambica), and carp (Cyprinus carpio), semipurified by affinity chromatography (wheat germ agglutinin-agarose). Total specific binding and number of receptors per unit weight of piscine white skeletal muscle were lower than those values found in mammalian skeletal muscle. The same parameters in carp muscle receptor preparations were severalfold higher than in trout muscle (binding capacity 440 +/- 47 fmol/mg glycoprotein in carp and 82 +/- 23 fmol/mg glycoprotein in trout). Piscine insulin receptors phosphorylated exogenous substrate poly(Glu,Tyr) but less so than mammalian receptors. Tyrosine kinase activity of receptors, calculated as percent of 32P incorporated into substrate in the presence of insulin compared with basal incorporation, was also highest in carp (210 +/- 4%) and lowest in trout (150 +/- 2%). In both trout and carp deprived of food for 15 days, specific binding of insulin decreased. Nevertheless, differences between the two species were retained. Our results demonstrate that particular properties of insulin receptors in fish skeletal muscle may be related to nutritional preferences. This finding coincides with the phenomenon of differential glucose tolerance in fish: carnivorous fish, such as trout, are less tolerant, whereas omnivorous fish, such as carp, readily utilize a carbohydrate-rich diet.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.

TCF7L2 is the susceptibility gene for Type 2 diabetes (T2D) with the largest effect on disease risk that has been discovered to date. However, the mechanisms by which TCF7L2 contributes to the disease remain largely elusive. In addition, epigenetic mechanisms, such as changes in DNA methylation patterns, might have a role in the pathophysiology of T2D. This study aimed to investigate the differences in terms of DNA methylation profile of TCF7L2 promoter gene between type 2 diabetic patients and age- and Body Mass Index (BMI)- matched controls. We included 93 type 2 diabetic patients that were recently diagnosed for T2D and exclusively on diet (without any pharmacological treatment). DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Type 2 diabetic patients were more insulin resistant than their matched controls (mean HOMA IR 2.6 vs 1.8 in controls, P

GIP action in type 2 diabetic (T2D) patients is altered. We hypothesized that methylation changes could be present in GIP receptor of T2D patients. This study aimed to assess the differences in DNA methylation profile of GIPR promoter between T2D patients and age- and Body Mass Index (BMI)-matched controls. We included 93 T2D patients (cases) that were uniquely on diet (without any anti-diabetic pharmacological treatment). We matched one control (with oral glucose tolerance test negative, non diabetic), by age and BMI, for every case. Cytokines and hormones were determined by ELISA. DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Our results showed that T2D patients were more insulin resistant and had a poorer β cell function than their controls. Fasting adiponectin was lower in T2D patients as compared to controls (7.0±3.8 µgr/mL vs. 10.0±4.2 µgr/mL). Levels of IL 12 in serum were almost double in T2D patients (52.8±58.3 pg/mL vs. 29.7±37.4 pg/mL). We found that GIPR promoter was hypomethylated in T2D patients as compared to controls. In addition, HOMA-IR and fasting glucose correlated negatively with mean methylation of GIPR promoter, especially in T2D patients. This case-control study confirms that newly diagnosed, drug-naïve T2D patients are more insulin resistant and have worse β cell function than age- and BMI-matched controls, which is partly related to changes in the insulin-sensitizing metabolites (adiponectin), in the proinflammatory profile (IL12) and we suggest in the methylation pattern of GIPR. Our study provides novel findings on GIPR promoter methylation profile which may improve our ability to understand type 2 diabetes pathogenesis.

The existence of an increased number of Kupffer cells is recognized as critical in the initiation of the inflammatory cascade leading to liver fibrosis. Because 5-lipoxygenase (5-LO) is a key regulator of cell growth and survival, in the current investigation we assessed whether inhibition of the 5-LO pathway would reduce the excessive number of Kupffer cells and attenuate inflammation and fibrosis in experimental liver disease. Kupffer cells were the only liver cell type endowed with a metabolically active 5-LO pathway (i.e., expressed mRNAs for 5-LO, 5-LO-activating protein [FLAP], and leukotriene [LT] C4 synthase and generated LTB4 and cysteinyl-LTs). Both the selective 5-LO inhibitor AA861 and the FLAP inhibitor BAY-X-1005 markedly reduced the number of Kupffer cells in culture. The antiproliferative properties of AA861 and BAY-X-1005 were associated with the occurrence of condensed nuclei, fragmented DNA, and changes in DNA content and cell cycle frequency distribution consistent with an apoptotic process. In vivo, in carbon tetrachloride-treated rats, BAY-X-1005 had a significant antifibrotic effect and reduced liver damage and the hepatic content of hydroxyproline. Together, these findings indicate a novel mechanism by which inactivation of the 5-LO pathway could disrupt the sequence of events leading to liver inflammation and fibrosis.