Phosphatidic acid mediates demyelination in Lpin1 mutant mice

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis.

Malignant lymphoma cells induce tolerance of T cells

Inhibitory receptors are involvedin the induction of T cell dysfunctionand exhaustion in chronic viral infectionsand in tumors. In the presentstudy, we analyzed the expressionpattern of 3 different inhibitory receptors(PD-1, Lag-3, 2B4) in a murine Bcell lymphoma model. Furthermore,we functionally characterized CD8+T cells expressing inhibitory receptorsfor cytokine production and proliferation.Expansion and secretion ofpro-inflammatory cytokines of CD8+T cells from lymphoma-bearing E-myc mice were significantly reducedcompared to the healthy controls.Similarly, expansion and effectorfunction of CD8+ TCR transgenic(p14) Tcells specific for the gp-33 antigenof lymphocytic choriomeningitisvirus (LCMV) was reduced inlymphoma-bearing E-myc mice afteractivation with LCMV. The functionalimpairment of CTL in the presenceof lymphoma was reversible aftertransfer to naive C57BL/6 recipients.In vitro co-culture experimentsrevealed that the proliferation ofanti-CD3-activated CD8+ T cellsfrom WT mice was significantly inhibitedby CD19+ lymphoma cellsfrom E-myc mice, whereas no inhibitionwas observed after co-culturewith normal B cells. Supernatants ofin vitro cultured lymphoma B cellsand blood sera from lymphoma-bearingE-myc mice significantly reducedT cell proliferation in vitro, ascompared to supernatants from normalB cells cultures or sera of healthyanimals. These experiments indicatethat the lymphoma B cells inactivateCTL by a soluble factor. Expressionanalysis of different important immunologicalcytokines revealed that themacrophage migration inhibitory factor(MIF) is selectively overexpressedin malignant B cells. This finding wasconfirmed by analyzing MIF proteinin culture supernatants and in celllysates. Therefore, lymphoma B cellsmay reduce T cell function and suppresslymphoma surveillance by secretionof MIF.

A microRNA profile of human CD8.

BackgroundRecently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8+CD25+ Treg cells and the impact of microRNAs on molecules associated with immune regulation.MethodsWe purified human natural CD8+ Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA `signature¿ for CD8+CD25+FOXP3+CTLA-4+ natural Treg cells. We used the `TargetScan¿ and `miRBase¿ bioinformatics programs to identify potential target sites for these microRNAs in the 3¿-UTR of important Treg cell-associated genes.ResultsThe human CD8+CD25+ natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.ConclusionsWe are examining the biological relevance of this `signature¿ by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer.

Central glucose sensing and the control of energy homeostasis

Glucose is an important signal that regulates glucose and energy homeostasis but its precise physiological role and signaling mechanism in the brain are still uncompletely understood. Over the recent years we have investigated the possibility that central glucose sensing may share functional similarities with glucose sensing by pancreatic beta-cells, in particular a requirement for the expression of the glucose transporter Glut2. Using mice with genetic inactivation of Glut2, but rescued pancreatic beta-cell function by transgenic expression of a glucose transporter, we have established that extrapancreatic glucose sensors are involved: i) in the control of glucagon secretion in response to hypoglycemia, ii) in the control of feeding and iii) of energy expenditure. We have more recently shown that central Glut2-dependent glucose sensors are involved in the regulation of NPY and POMC expression by arcuate nucleus neurons and that the sensitivity to leptin of these neurons is enhanced by Glut2-dependent glucose sensors. Using mice with genetic tagging of Glut2-expressing cells, we determined that the NPY and POMC neurons did not express Glut2 but were connected to Glut2 expressing neurons located most probably outside of the arcuate nucleus. We are now defining the electrophysiological behavior of these Glut2 expressing neurons. Our data provide an initial map of glucose sensing neurons expressing Glut2 and link these neurons with the control of specific physiological function.

Extreme self-monitoring, fear of hypoglycemia and obsessive-compulsive disorder in type 1 diabetes mellitus: when less is better

Background: Pre-existing psychological factors can strongly influence coping with type 1 diabetes mellitus and interfere with self-monitoring. Psychiatric disorders seem to be positively associated with poor metabolic control. We present a case of extreme compulsive blood testing due to obsessive fear of hypoglycemia in an adolescent with type 1 diabetes mellitus. Case report: Type 1 diabetes mellitus (anti GAD-antibodies 2624 U/l, norm < 9.5) was diagnosed in a boy aged 14.3 years [170 cm (+ 0.93 SDS), weight 50.5 kg (+ 0.05 SDS)]. Laboratory work-up showed no evidence for other autoimmune disease. Family and past medical history were unremarkable. Growth and developmental milestones were normal. Insulin-analog based basal-bolus regime was initiated, associated to standard diabetic education. Routine psychological evaluation performed at the onset of diabetes revealed intermittent anxiety and obsessivecompulsive traits. Accordingly, a close psychiatric follow-up was initiated for the patient and his family. An adequate metabolic control (HbA1c drop from >14 to 8%) was achieved within 3 months, attributed to residual -cell function. In the following 6 months, HbA1c rose unexpectedly despite seemingly adequate adaptations of insulin doses. Obsessive fear of hypoglycemia leading to a severe compulsive behavior developed progressively with as many as 68 glycemia measurements per day (mean over 1 week). The patient reported that he could not bear leaving home with glycemia < 15 mmol/l, ending up with school eviction and severe intra-familial conflict. Despite intensive psychiatric outpatient support, HbA1c rose rapidly to >14% with glycemia-testing reaching peaks of 120 tests/day. The situation could only be discontinued through psychiatric hospitalization with intensive behavioral training. As a result, adequate metabolic balance was restored (HbA1c value: 7.1 %) with acceptable 10-15 daily glycemia measurements. Discussion: The association of overt psychiatric disorders to type 1 diabetes mellitus is very rare in the pediatric age group. It can lead to a pathological behavior with uncontrolled diabetes. Such exceptional situations require long-term admissions with specialized psychiatric care. Slow acceptation of a "less is better" principle in glycemia testing and amelioration of metabolic control are difficult to achieve.

A role for the transcriptional repressor ICER in the molecular pathogenesis of type 2 diabetes

AbstractType 2 diabetes (T2D) is a metabolic disease which affects more than 200 millions people worldwide. The progression of this affection reaches nowadays epidemic proportions, owing to the constant augmentation in the frequency of overweight, obesity and sedentary. The pathogenesis of T2D is characterized by reduction in the action of insulin on its target tissues - an alteration referred as insulin resistance - and pancreatic β-cell dysfunction. This latter deterioration is defined by impairment in insulin biosynthesis and secretion, and a loss of β-cell mass by apoptosis. Environmental factors related to T2D, such as chronic elevation in glucose and free fatty acids levels, inflammatory cytokines and pro-atherogenic oxidized low- density lipoproteins (LDL), contribute to the loss of pancreatic β-cell function.In this study, we have demonstrated that the transcription factor Inducible Cyclic AMP Early Repressor (ICER) participates to the progression of both β-cell dysfunction and insulin resistance. The expression of this factor is driven by an alternative promoter and ICER protein represents therefore a truncated product of the Cyclic AMP Response Element Modulator (CREM) family which lacks transactivation domain. Consequently, the transcription factor ICER acts as a passive repressor which reduces expression of genes controlled by the cyclic AMP and Cyclic AMP Response Element Binding protein (CREB) pathway.In insulin-secreting cells, the accumulation of reactive oxygen species caused by environmental factors and notably oxidized LDL - a process known as oxidative stress - induces the transcription factor ICER. This transcriptional repressor hampers the secretory capacity of β-cells by silencing key genes of the exocytotic machinery. In addition, the factor ICER reduces the expression of the scaffold protein Islet Brain 1 (IB 1 ), thereby favouring the activation of the c-Jun N-terminal Kinase (JNK) pathway. This triggering alters in turn insulin biosynthesis and survival capacities of pancreatic β-cells.In the adipose tissue of mice and human subjects suffering from obesity, the transcription factor ICER contributes to the alteration in insulin action. The loss in ICER protein in these tissues induces a constant activation of the CREB pathway and the subsequent expression of the Activating Transcription Factor 3 (ATF3). In turn, this repressor reduces the transcript levels of the glucose transporter GLUT4 and the insulin-sensitizer peptide adiponectin, thereby contributing to the diminution in insulin action.In conclusion, these data shed light on the important role of the transcriptional repressor ICER in the pathogenesis of T2D, which contributes to both alteration in β-cell function and aggravation of insulin resistance. Consequently, a better understanding of the molecular mechanisms responsible for the alterations in ICER levels is required and could lead to develop new therapeutic strategies for the treatment of T2D.RésuméLe diabète de type 2 (DT2) est une maladie métabolique qui affecte plus de 200 millions de personnes dans le monde. La progression de cette affection atteint aujourd'hui des proportions épidémiques imputables à l'augmentation rapide dans les fréquences du surpoids, de l'obésité et de la sédentarité. La pathogenèse du DT2 se caractérise par une diminution de l'action de l'insuline sur ses tissus cibles - un processus nommé insulino-résistance - ainsi qu'une dysfonction des cellules β pancréatiques sécrétrices d'insuline. Cette dernière détérioration se définit par une réduction de la capacité de synthèse et de sécrétion de l'insuline et mène finalement à une perte de la masse de cellules β par apoptose. Des facteurs environnementaux fréquemment associés au DT2, tels l'élévation chronique des taux plasmatiques de glucose et d'acides gras libres, les cytokines pro-inflammatoires et les lipoprotéines de faible densité (LDL) oxydées, contribuent à la perte de fonction des cellules β pancréatiques.Dans cette étude, nous avons démontré que le facteur de transcription « Inducible Cyclic AMP Early Repressor » (ICER) participe à la progression de la dysfonction des cellules β pancréatiques et au développement de Pinsulino-résistance. Son expression étant gouvernée par un promoteur alternatif, la protéine d'ICER représente un produit tronqué de la famille des «Cyclic AMP Response Element Modulator » (CREM), sans domaine de transactivation. Par conséquent, le facteur ICER agit comme un répresseur passif qui réduit l'expression des gènes contrôlés par la voie de l'AMP cyclique et des « Cyclic AMP Response Element Binding protein » (CREB).Dans les cellules sécrétrices d'insuline, l'accumulation de radicaux d'oxygène libres, soutenue par les facteurs environnementaux et notamment les LDL oxydées - un processus appelé stress oxydatif- induit de manière ininterrompue le facteur de transcription ICER. Ainsi activé, ce répresseur transcriptionnel altère la capacité sécrétoire des cellules β en bloquant l'expression de gènes clés de la machinerie d'exocytose. En outre, le facteur ICER favorise l'activation de la cascade de signalisation « c-Jun N- terminal Kinase » (JNK) en réduisant l'expression de la protéine « Islet Brain 1 » (IB1), altérant ainsi les fonctions de biosynthèse de l'insuline et de survie des cellules β pancréatiques.Dans le tissu adipeux des souris et des sujets humains souffrant d'obésité, le facteur de transcription ICER contribue à l'altération de la réponse à l'insuline. La disparition de la protéine ICER dans ces tissus entraîne une activation persistante de la voie de signalisation des CREB et une induction du facteur de transcription « Activating Transcription Factor 3 » (ATF3). A son tour, le répresseur ATF3 inhibe l'expression du transporteur de glucose GLUT4 et du peptide adipocytaire insulino-sensibilisateur adiponectine, contribuant ainsi à la diminution de l'action de l'insuline en conditions d'obésité.En conclusion, à la lumière de ces résultats, le répresseur transcriptionnel ICER apparaît comme un facteur important dans la pathogenèse du DT2, en participant à la perte de fonction des cellules β pancréatiques et à l'aggravation de l'insulino-résistance. Par conséquent, l'étude des mécanismes moléculaires responsables de l'altération des niveaux du facteur ICER pourrait permettre le développement de nouvelles stratégies de traitement du DT2.Résumé didactiqueL'énergie nécessaire au bon fonctionnement de l'organisme est fournie par l'alimentation, notamment sous forme de sucres (glucides). Ceux-ci sont dégradés en glucose, lequel sera distribué aux différents organes par la circulation sanguine. Après un repas, le niveau de glucose sanguin, nommé glycémie, s'élève et favorise la sécrétion d'une hormone appelée insuline par les cellules β du pancréas. L'insuline permet, à son tour, aux organes, tels le foie, les muscles et le tissu adipeux de capter et d'utiliser le glucose ; la glycémie retrouve ainsi son niveau basai.Le diabète de type 2 (DT2) est une maladie métabolique qui affecte plus de 200 millions de personnes dans le monde. Le développement de cette affection est causée par deux processus pathologiques. D'une part, les quantités d'insuline secrétée par les cellules β pancréatiques, ainsi que la survie de ces cellules sont réduites, un phénomène connu sous le nom de dysfonction des cellules β. D'autre part, la sensibilité des tissus à l'insuline se trouve diminuée. Cette dernière altération, l'insulino-résistance, empêche le transport et l'utilisation du glucose par les tissus et mène à une accumulation de ce sucre dans le sang. Cette stagnation de glucose dans le compartiment sanguin est appelée hyperglycémie et favorise l'apparition des complications secondaires du diabète, telles que les maladies cardiovasculaires, l'insuffisance rénale, la cécité et la perte de sensibilité des extrémités.Dans cette étude, nous avons démontré que le facteur ICER qui contrôle spécifiquement l'expression de certains gènes, contribue non seulement à la dysfonction des cellules β, mais aussi au développement de l'insulino-résistance. En effet, dans les cellules β pancréatiques en conditions diabétiques, l'activation du facteur ICER altère la capacité de synthèse et de sécrétion d'insuline et réduit la survie ces cellules.Dans le tissu adipeux des souris et des sujets humains souffrant d'obésité, le facteur ICER contribue à la perte de sensibilité à l'insuline. La disparition d'ICER altère l'expression de la protéine qui capte le glucose, le transoprteur GLUT4, et l'hormone adipocytaire favorisant la sensibilité à l'insuline, nommée adiponectine. Ainsi, la perte d'ICER participe à la réduction de la captation de glucose par le tissue adipeux et au développement de l'insulino-résistance au cours de l'obésité.En conclusion, à la lumière de ces résultats, le facteur ICER apparaît comme un contributeur important à la progression du DT2, en soutenant la dysfonction des cellules β pancréatiques et l'aggravation de l'insulino-résistance. Par conséquent, l'étude des mécanismes responsables de la dérégulation du facteur ICER pourrait permettre le développement de nouvelles stratégies de traitement du DT2.

Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells.

AIMS/HYPOTHESIS: We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. MATERIALS AND METHODS: Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. CONCLUSIONS/INTERPRETATION: These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

Aging preferentially affects molecular pathways implicated in development and disease of myelinating glial cells

The central and peripheral nervous systems are involved in multiple age-dependent neurological deficits that are often attributed to alterations in function of myelinating glial cells. However, the molecular events that underlie the age-related decline of glial cell function are unknown. We used Schwann cells as a model to study biological processes affected in glial cells by aging. We comprehensively profiled gene expression of the Schwann cellrich mouse sciatic nerve throughout life, from day of birth until senescence (840 days of age). We combined the aging data with the microarray transcriptional data obtained using nerves isolated from Schwann cell-specific neuropathy-inducing mutants MPZCre/+/Lpin1fE2−3/fE2−3 , MPZCre/+/ScapfE1/fE1 and Pmp22-null mice. The majority of age related transcripts were also affected in the analyzed mouse models of neuropathy (54.4%) and in development (59.5%) indicating a high level of overlapping in implicated molecular pathways. We observed that compared to peripheral nerve development, dynamically changing expression profiles in aging have opposite (anticorrelated) orientation while they copy the orientation of transcriptional changes observed in analyzed neuropathy models. Subsequent clustering and biological annotation of dynamically changing transcripts revealed that the processes most significantly deregulated in aging include inflammatory/immune response and lipid biosynthesis/metabolism. Importantly, the changes in these pathways were also observed in myelinated oligodendrocyte-rich optic nerves of aged mice, albeit with lower magnitude. This observation suggests that similar biological processes are affected in aging glial cells in central and peripheral nervous systems, however with different dynamics. Our data, which provide the first comprehensive comparison of molecular changes in glial cells in three distinct biological conditions comprising development, aging and disease, provide not only a new inside into the molecular alterations underlying neural system aging but also identify target pathways for potential therapeutic approaches to prevent or delay complications associated with age-related and inherited forms of neuropathies. *Current address: Department of Physiology, UCSF, San Francisco, CA, USA.

Aging preferentially affects molecular pathways implicated in development and disease of myelinating glial cells

The central and peripheral nervous systems are involved in multiple agedependent neurological deficits that are often attributed to alterations in function of myelinating glial cells. However, the molecular events that underlie the age-related decline of glial cell function are unknown. We used Schwann cells as a model to study biological processes affected in glial cells by aging. We comprehensively profiled gene expression of the Schwann cell-rich mouse sciatic nerve throughout life, from day of birth until senescence (840 days of age). We combined the aging data with the microarray transcriptional data obtained using nerves isolated from Schwann cell-specific neuropathy-inducing mutants MPZCre/þ/Lpin1fE2-3/fE2-3, MPZCre/þ/ScapfE1/fE1 and Pmp22-null mice. A majority of age related transcripts were also affected in the analyzed mouse models of neuropathy (54.4%) and in development (59.5%) indicating a high level of overlapping in implicated molecular pathways. We observed that compared to peripheral nerve development, dynamically changing expression profiles in aging have opposite (anticorrelated) orientation while they copy the orientation of transcriptional changes observed in analyzed neuropathy models. Subsequent clustering and biological annotation of dynamically changing transcripts revealed that the processes most significantly deregulated in aging include inflammatory/ immune response and lipid biosynthesis/metabolism. Importantly, the changes in these pathways were also observed in myelinated oligodendrocyte- rich optic nerves of aged mice, albeit with lower magnitude. This observation suggests that similar biological processes are affected in aging glial cells in central and peripheral nervous systems, however with different dynamics. Our data, which provide the first comprehensive comparison of molecular changes in glial cells in three distinct biological conditions comprising development, aging and disease, provide not only a new inside into the molecular alterations underlying neural system aging but also identify target pathways for potential therapeutical approaches to prevent or delay complications associated with age-related and inherited forms of neuropathies.

Adaptations of Natural Killer Cells to Self-MHC Class I.

Natural Killer (NK) cells use germ line encoded receptors to detect diseased host cells. Despite the invariant recognition structures, NK cells have a significant ability to adapt to their surroundings, such as the presence or absence of MHC class I molecules. It has been assumed that this adaptation occurs during NK cell development, but recent findings show that mature NK cells can also adapt to the presence or absence of MHC class I molecules. Here, we summarize how NK cells adjust to changes in the expression of MHC class I molecules. We propose an extension of existing models, in which MHC class I recognition during NK cell development sequentially instructs and maintains NK cell function. The elucidation of the molecular basis of the two effects may identify ways to improve the fitness of NK cells and to prevent the loss of NK cell function due to persistent alterations in their environment.

Impulse oscillometry identifies peripheral airway dysfunction in children with adenosine deaminase deficiency.

Adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) is characterized by impaired T-, B- and NK-cell function. Affected children, in addition to early onset of infections, manifest non-immunologic symptoms including pulmonary dysfunction likely attributable to elevated systemic adenosine levels. Lung disease assessment has primarily employed repetitive radiography and effort-dependent functional studies. Through impulse oscillometry (IOS), which is effort-independent, we prospectively obtained objective measures of lung dysfunction in 10 children with ADA-SCID. These results support the use of IOS in the identification and monitoring of lung function abnormalities in children with primary immunodeficiencies.

Modelagem Molecular: Uma Ferramenta para o Planejamento Racional de Fármacos em Química Medicinal

The molecular basis of modern therapeutics consist in the modulation of cell function by the interaction of microbioactive molecules as drug cells macromolecules structures. Molecular modeling is a computational technique developed to access the chemical structure. This methodology, by means of the molecular similarity and complementary paradigm, is the basis for the computer-assisted drug design universally employed in pharmaceutical research laboratories to obtain more efficient, more selective, and safer drugs. In this work, we discuss some methods for molecular modeling and some approaches to evaluate new bioactive structures in development by our research group.

Differential methylation of TCF7L2 promoter in peripheral blood DNA in newly diagnosed, drug-naïve patients with type 2 diabetes

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<0.001) and had a poorer beta-cell function (mean HOMA B 75.7 vs. 113.6 in controls, P<0.001). Results showed that 59% of the CpGs analyzed in TCF7L2 promoter had significant differences between type 2 diabetic patients and matched controls. In addition, fasting glucose, HOMA-B, HOMA-IR, total cholesterol and LDL-cholesterol correlated with methylation in specific CpG sites of TCF7L2 promoter. After adjustment by age, BMI, gender, physical inactivity, waist circumference, smoking status and diabetes status uniquely fasting glucose, total cholesterol and LDL-cholesterol remained significant. Taken together, newly diagnosed, drug-naïve type 2 diabetic patients display specific epigenetic changes at the TCF7L2 promoter as compared to age- and BMI-matched controls. Methylation in TCF7L2 promoter is further correlated with fasting glucose in peripheral blood DNA, which sheds new light on the role of epigenetic regulation of TCF7L2 in T2D.

On Glucose Metabolism in Patients with the m.3243A>G Mutation

Background: The m.3243A>G mutation in mitochondrial DNA is the most common cause for mitochondrial diabetes. In addition, unexpected deaths related to the m.3243A>G associate with encephalopathy and cardiomyopathy. Failing mitochondrial respiratory chain in neurons, myocytes and beta cells is considered to underlie the multiorgan manifestations of the m.3243A>G. Aims: The primary aim of the study was to characterize the organ-specific glucose metabolism in patients with m.3243A>G and secondly, to study patients with or without signs of diabetes, cardiomyopathy or encephalopathy. The insulin-stimulated glucose metabolism in brain, heart, skeletal muscle, adipose tissue and liver were measured with 2-deoxy-2-[18F]fluoro-α-D-glucose in 15 patients and 14 controls. Brain oxygen metabolism was assessed with [15O]oxygen and insulin secretion was modelled based on oral glucose tolerance test. Results: The glucose oxidation in brain was globally decreased in patients with or without clinical encephalopathy. The insulin-stimulated glucose influx to skeletal muscle and adipose tissue was decreased in patients with or without diabetes as the hepatic glucose metabolism was normal. Impaired beta cell function and myocardial glucose uptake were associated with the high m.3243A>G heteroplasmy. Conclusions: This cross-sectional study suggests that: 1) The ability of insulin to stimulate glucose metabolism in skeletal muscle and adipose tissue is weakened before the beta cell failure results in mitochondrial diabetes. 2) Glucose oxidation defect is detected in otherwise unaffected cerebral regions in patients with the m.3243A>G, thus it likely precedes the clinical encephalopathy. 3) Uneconomical glucose hypometabolism during hyperinsulinemia contributes to the cardiac vulnerability in patients with high m.3243A>G heteroplasmy