Unique features of ß-cell metabolism are lost in type 2 diabetes.

Pancreatic ß cells play an essential role in the control of systemic glucose homeostasis as they sense blood glucose levels and respond by secreting insulin. Upon stimulating glucose uptake in insulin-sensitive tissues post-prandially, this anabolic hormone restores blood glucose levels to pre-prandial levels. Maintaining physiological glucose levels thus relies on proper ß-cell function. To fulfill this highly specialized nutrient sensor role, ß cells have evolved a unique genetic program that shapes its distinct cellular metabolism. In this review, the unique genetic and metabolic features of ß cells will be outlined, including their alterations in type 2 diabetes (T2D). ß cells selectively express a set of genes in a cell type-specific manner; for instance, the glucose activating hexokinase IV enzyme or Glucokinase (GCK), whereas other genes are selectively "disallowed", including lactate dehydrogenase A (LDHA) and monocarboxylate transporter 1 (MCT1). This selective gene program equips ß cells with a unique metabolic apparatus to ensure that nutrient metabolism is coupled to appropriate insulin secretion, thereby avoiding hyperglycemia, as well as life-threatening hypoglycemia. Unlike most cell types, ß cells exhibit specialized bioenergetic features, including supply-driven rather than demand-driven metabolism and a high basal mitochondrial proton leak respiration. The understanding of these unique genetically programmed metabolic features and their alterations that lead to ß-cell dysfunction is crucial for a comprehensive understanding of T2D pathophysiology and the development of innovative therapeutic approaches for T2D patients.

Monoamines' role in islet cell function and type 2 diabetes risk.

The two monoamines serotonin and melatonin have recently been highlighted as potent regulators of islet hormone secretion and overall glucose homeostasis in the body. In fact, dysregulated signaling of both amines are implicated in ß-cell dysfunction and development of type 2 diabetes mellitus (T2DM). Serotonin is a key player in ß-cell physiology and plays a role in expansion of ß-cell mass. Melatonin regulates circadian rhythm and nutrient metabolism and reduces insulin release in human and rodent islets in vitro. Herein, we focus on the role of serotonin and melatonin in islet physiology and the pathophysiology of T2DM. This includes effects on hormone secretion, receptor expression, genetic variants influencing ß-cell function, melatonin treatment, and compounds that alter serotonin availability and signaling.

Reduced Expression Level of Protein Phosphatase PPM1E Serves to Maintain Insulin Secretion in Type 2 Diabetes.

Reversible phosphorylation is an important regulatory mechanism. Regulation of protein phosphorylation in ß-cells has been extensively investigated, but less is known about protein dephosphorylation. To understand the role of protein dephosphorylation in ß-cells and type 2 diabetes (T2D), we first examined mRNA expression of the type 2C family (PP2C) of protein phosphatases in islets from T2D donors. Phosphatase expression overall was changed in T2D, and that of PPM1E was the most markedly downregulated. PPM1E expression correlated inversely with HbA1c. Silencing of PPM1E increased glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells and/or islets from patients with T2D, whereas PPM1E overexpression decreased GSIS. Increased GSIS after PPM1E silencing was associated with decreased oxidative stress, elevated cytosolic Ca2+ levels and ATP to ADP ratio, increased hyperpolarization of the inner mitochondrial membrane, and phosphorylation of CaMKII, AMPK, and acetyl-CoA carboxylase. Silencing of PPM1E, however, did not change insulin content. Increased GSIS, cell viability, and activation of AMPK upon metformin treatment in ß-cells were observed upon PPM1E silencing. Thus, protein dephosphorylation via PPM1E abrogates GSIS. Consequently, reduced PPM1E expression in T2D may be a compensatory response of ß-cells to uphold insulin secretion under metabolic duress. Targeting PPM1E in ß-cells may thus represent a novel therapeutic strategy for treatment of T2D.

Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic ß cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human ß cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing ß cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to ß cell dysfunction in T2D pathophysiology.

The human batokine EPDR1 regulates ß-cell metabolism and function.

OBJECTIVE: Ependymin-Related Protein 1 (EPDR1) was recently identified as a secreted human batokine regulating mitochondrial respiration linked to thermogenesis in brown fat. Despite that EPDR1 is expressed in human pancreatic ß-cells and that glucose-stimulated mitochondrial metabolism is critical for stimulus-secretion coupling in ß-cells, the role of EPDR1 in ß-cell metabolism and function has not been investigated. METHODS: EPDR1 mRNA levels in human pancreatic islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets, EndoC-ßH1 and INS1 832/13 cells were transfected with scramble (control) and EPDR1 siRNAs (EPDR1-KD) or treated with human EPDR1 protein, and glucose-stimulated insulin secretion (GSIS) assessed by ELISA. Mitochondrial metabolism was investigated by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis. RESULTS: EPDR1 mRNA expression was upregulated in human islets from T2D and obese donors and positively correlated to BMI of donors. In T2D donors, EPDR1 mRNA levels negatively correlated with HbA1c and positively correlated with GSIS. EPDR1 silencing in human islets and ß-cell lines reduced GSIS whereas treatment with human EPDR1 protein increased GSIS. Epdr1 silencing in INS1 832/13 cells reduced glucose- and pyruvate- but not K+-stimulated insulin secretion. Metabolomics analysis in Epdr1-KD INS1 832/13 cells suggests diversion of glucose-derived pyruvate to lactate production and decreased malate-aspartate shuttle and the tricarboxylic acid (TCA) cycle activity. The glucose-stimulated rise in mitochondrial respiration and ATP/ADP-ratio was impaired in Epdr1-deficient cells. CONCLUSION: These results suggests that to maintain glucose homeostasis in obese people, upregulation of EPDR1 may improve ß-cell function via channelling glycolysis-derived pyruvate to the mitochondrial TCA cycle.

Ribosomal biogenesis regulator DIMT1 controls ß-cell protein synthesis, mitochondrial function, and insulin secretion.

We previously reported that loss of mitochondrial transcription factor B1 (TFB1M) leads to mitochondrial dysfunction and is involved in the pathogenesis of type 2 diabetes (T2D). Whether defects in ribosomal processing impact mitochondrial function and could play a pathogenetic role in ß-cells and T2D is not known. To this end, we explored expression and the functional role of dimethyladenosine transferase 1 homolog (DIMT1), a homolog of TFB1M and a ribosomal RNA (rRNA) methyltransferase implicated in the control of rRNA. Expression of DIMT1 was increased in human islets from T2D donors and correlated positively with expression of insulin mRNA, but negatively with insulin secretion. We show that silencing of DIMT1 in insulin-secreting cells impacted mitochondrial function, leading to lower expression of mitochondrial OXPHOS proteins, reduced oxygen consumption rate, dissipated mitochondrial membrane potential, and a slower rate of ATP production. In addition, the rate of protein synthesis was retarded upon DIMT1 deficiency. Consequently, we found that DIMT1 deficiency led to perturbed insulin secretion in rodent cell lines and islets, as well as in a human ß-cell line. We observed defects in rRNA processing and reduced interactions between NIN1 (RPN12) binding protein 1 homolog (NOB-1) and pescadillo ribosomal biogenesis factor 1 (PES-1), critical ribosomal subunit RNA proteins, the dysfunction of which may play a part in disturbing protein synthesis in ß-cells. In conclusion, DIMT1 deficiency perturbs protein synthesis, resulting in mitochondrial dysfunction and disrupted insulin secretion, both potential pathogenetic processes in T2D.

MAFA and MAFB regulate exocytosis-related genes in human ß-cells.

AIMS: Reduced expression of exocytotic genes is associated with functional defects in insulin exocytosis contributing to impaired insulin secretion and type 2 diabetes (T2D) development. MAFA and MAFB transcription factors regulate ß-cell physiology, and their gene expression is reduced in T2D ß cells. We investigate if loss of MAFA and MAFB in human ß cells contributes to T2D progression by regulating genes required for insulin exocytosis. METHODS: Three approaches were performed: (1) RNAseq analysis with the focus on exocytosis-related genes in MafA-/- mouse islets, (2) correlational analysis between MAFA, MAFB and exocytosis-related genes in human islets and (3) MAFA and MAFB silencing in human islets and EndoC-ßH1 cells followed by functional in vitro studies. RESULTS: The expression of 30 exocytosis-related genes was significantly downregulated in MafA-/- mouse islets. In human islets, the expression of 29 exocytosis-related genes correlated positively with MAFA and MAFB. Eight exocytosis-related genes were downregulated in MafA-/- mouse islets and positively correlated with MAFA and MAFB in human islets. From this analysis, the expression of RAB3A, STXBP1, UNC13A, VAMP2, NAPA, NSF, STX1A and SYT7 was quantified after acute MAFA or MAFB silencing in EndoC-ßH1 cells and human islets. MAFA and MAFB silencing resulted in impaired insulin secretion and reduced STX1A, SYT7 and STXBP1 (EndoC-ßH1) and STX1A (human islets) mRNA expression. STX1A and STXBP1 protein expression was also impaired in islets from T2D donors which lack MAFA expression. CONCLUSION: Our data indicate that STXBP1 and STX1A are important MAFA/B-regulated exocytosis genes which may contribute to insulin exocytosis defects observed in MAFA-deficient human T2D ß cells.

The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in ß-cells.

The amplification of glucose-stimulated insulin secretion (GSIS) through incretin signaling is critical for maintaining physiological glucose levels. Incretins, like glucagon-like peptide 1 (GLP1), are a target of type 2 diabetes drugs aiming to enhance insulin secretion. Here we show that the protein phosphatase 1 inhibitor protein 1A (PPP1R1A), is expressed in ß-cells and that its expression is reduced in dysfunctional ß-cells lacking MafA and upon acute MafA knock down. MafA is a central regulator of GSIS and ß-cell function. We observed a strong correlation of MAFA and PPP1R1A mRNA levels in human islets, moreover, PPP1R1A mRNA levels were reduced in type 2 diabetic islets and positively correlated with GLP1-mediated GSIS amplification. PPP1R1A silencing in INS1 (832/13) ß-cells impaired GSIS amplification, PKA-target protein phosphorylation, mitochondrial coupling efficiency and also the expression of critical ß-cell marker genes like MafA, Pdx1, NeuroD1 and Pax6. Our results demonstrate that the ß-cell transcription factor MafA is required for PPP1R1A expression and that reduced ß-cell PPP1R1A levels impaired ß-cell function and contributed to ß-cell dedifferentiation during type 2 diabetes. Loss of PPP1R1A in type 2 diabetic ß-cells may explains the unresponsiveness of type 2 diabetic patients to GLP1R-based treatments.

PPARGC1A Gene Promoter Methylation as a Biomarker of Insulin Secretion and Sensitivity in Response to Glucose Challenges.

Methylation in CpG sites of the PPARGC1A gene (encoding PGC1-α) has been associated with adiposity, insulin secretion/sensitivity indexes and type 2 diabetes. We assessed the association between the methylation profile of the PPARGC1A gene promoter gene in leukocytes with insulin secretion/sensitivity indexes in normoglycemic women. A standard oral glucose tolerance test (OGTT) and an abbreviated version of the intravenous glucose tolerance test (IVGTT) were carried out in n = 57 Chilean nondiabetic women with measurements of plasma glucose, insulin, and C-peptide. Bisulfite-treated DNA from leukocytes was evaluated for methylation levels in six CpG sites of the proximal promoter of the PPARGC1A gene by pyrosequencing (positions -816, -783, -652, -617, -521 and -515). A strong correlation between the DNA methylation percentage of different CpG sites of the PPARGC1A promoter in leukocytes was found, suggesting an integrated epigenetic control of this region. We found a positive association between the methylation levels of the CpG site -783 with the insulin sensitivity Matsuda composite index (rho = 0.31; p = 0.02) derived from the OGTT. The CpG hypomethylation in the promoter position -783 of the PPARGC1A gene in leukocytes may represent a biomarker of reduced insulin sensitivity after the ingestion of glucose.

Platelet Serotonin Levels Are Associated with Plasma Soluble Leptin Receptor Concentrations in Normoglycemic Women.

Most peripheral serotonin (5-hydroxytryptamine (5HT)) is synthetized in the gut with platelets being its main circulating reservoir. 5HT is acting as a hormone in key organs to regulate glucose and lipid metabolism. However, the relation between platelet 5HT levels and traits related to glucose homeostasis and lipid metabolism in humans remains poorly explored. The objectives of this study were (a) to assess the association between platelet 5HT levels and plasma concentration of nonesterified fatty acids (NEFAs) and some adipokines including leptin and its soluble leptin receptor (sOb-R), (b) to assess the association between platelet 5HT levels and anthropometric traits and indexes of insulin secretion/sensitivity derived from oral glucose tolerance test (OGTT), and (c) to evaluate changes in platelet 5HT levels in response to OGTT. In a cross-sectional study, 59 normoglycemic women underwent a standard 2-hour OGTT. Plasma leptin, sOb-R, total and high molecular weight adiponectin, TNFα, and MCP1 were determined by immunoassays. Platelet 5HT levels and NEFAs were measured before and after OGTT. The free leptin index was calculated from leptin and sOb-R measurements. Insulin sensitivity indexes derived from OGTT (HOMA-S and Matsuda ISICOMP) and plasma NEFAs (Adipose-IR, Revised QUICKI) were also calculated. Our data show that among metabolic traits, platelet 5HT levels were associated with plasma sOb-R (r = 0.39, p = 0.003, corrected p = 0.018). Platelet 5HT levels were reduced in response to OGTT (779 ± 237 vs.731 ± 217 ng/109 platelets, p = 0.005). In conclusion, platelet 5HT levels are positively associated with plasma sOb-R concentrations and reduced in response to glucose intake possibly indicating a role of peripheral 5HT in leptin-mediated appetite regulation.

Insulin Sensitivity Is Associated with Lipoprotein Lipase (LPL) and Catenin Delta 2 (CTNND2) DNA Methylation in Peripheral White Blood Cells in Non-Diabetic Young Women.

Hyperglycaemia and type 2 diabetes (T2D) are associated with impaired insulin secretion and/or insulin action. Since few studies have addressed the relation between DNA methylation patterns with elaborated surrogates of insulin secretion/sensitivity based on the intravenous glucose tolerance test (IVGTT), the aim of this study was to evaluate the association between DNA methylation and an insulin sensitivity index based on IVGTT (calculated insulin sensitivity index (CSi)) in peripheral white blood cells from 57 non-diabetic female volunteers. The CSi and acute insulin response (AIR) indexes, as well as the disposition index (DI = CSi × AIR), were estimated from abbreviated IVGTT in 49 apparently healthy Chilean women. Methylation levels were assessed using the Illumina Infinium Human Methylation 450k BeadChip. After a statistical probe filtering, the two top CpGs whose methylation was associated with CSi were cg04615668 and cg07263235, located in the catenin delta 2 (CTNND2) and lipoprotein lipase (LPL) genes, respectively. Both CpGs conjointly predicted insulin sensitivity status with an area under the curve of 0.90. Additionally, cg04615668 correlated with homeostasis model assessment insulin-sensitivity (HOMA-S) and AIR, whereas cg07263235 was associated with plasma creatinine and DI. These results add further insights into the epigenetic regulation of insulin sensitivity and associated complications, pointing the CTNND2 and LPL genes as potential underlying epigenetic biomarkers for future risk of insulin-related diseases.

Plasma MOTS-c levels are associated with insulin sensitivity in lean but not in obese individuals.

Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) is a mitochondrial-derived peptide that attenuates weight gain and hyperinsulinemia when administered to high fat-fed mice. MOTS-c is therefore a potential regulator of metabolic homeostasis under conditions of high-energy supply. However, the effect of insulin resistance and obesity on plasma MOTS-c concentration in humans is unknown. To gain insight into MOTS-c regulation, we measured plasma MOTS-c concentration and analyzed its relationship with insulin sensitivity surrogates, in lean and obese humans (n=10 per group). Obese individuals had impaired insulin sensitivity as indicated by low Matsuda and high Homeostatic Model Assessment (HOMA) indexes. Although plasma MOTS-c concentration was similar in lean and obese individuals (0.48±0.16 and 0.52±0.15 ng/mL; p=0.60), it was correlated with HOMA (r=0.53; p<0.05) and Matsuda index (r=-0.46; p<0.05). Notably, when the groups were analyzed separately, the associations remained only in lean individuals. We conclude that plasma MOTS-c concentration is unaltered in human obesity. However, MOTS-c associates positively with insulin resistance mostly in lean individuals, indicating that plasma MOTS-c concentration depends on the metabolic status in this population. Such dependence seems altered when obesity settles. The implications of plasma MOTS-c for human metabolic homeostasis deserve future examination.

Leptin/Adiponectin Ratios Using Either Total Or High-Molecular-Weight Adiponectin as Biomarkers of Systemic Insulin Sensitivity in Normoglycemic Women.

Plasma leptin/adiponectin ratio (LAR) is negatively associated with insulin sensitivity indexes. High-molecular-weight adiponectin (HMWA) was proposed as the most biologically active form of this insulin-sensitizing adipokine. There are no studies assessing the relative merits of leptin/HMWA ratio over LAR as a biomarker of systemic insulin sensitivity. A standard 2-hour oral glucose tolerance test (OGTT; 75 g of glucose) and a short minimal-model intravenous glucose tolerance test (IVGTT; 0.3 g/kg body weight) were performed in 58 Chilean normoglycemic women (age: 27 ± 6.3 years, BMI 23.6 ± 3.2 kg/m2). LAR was negatively associated with HOMA-S (r = -0.49; p < 0.0001), Matsuda-ISICOMP (r = -0.54; p < 0.0001), and the calculated sensitivity index (CSi) derived from IVGTT (r = -0.38; p = 0.007). In comparison to LAR, leptin/HMWA ratio did not increase neither the linear fit (r2) nor the magnitude of association with insulin sensitivity indexes (slope of multiple linear regression). The discriminatory capacity of both ratios to classify insulin-resistant versus insulin-sensitive subjects was similar for HOMA-S (p = 0.84), Matsuda-ISICOMP (p = 0.43), or CSi (p = 0.50). In conclusion, LAR showed consistent negative associations with different systemic insulin sensitivity indexes. The use of HMWA to generate leptin/HMWA ratio did not show any advantage over LAR as a biomarker of systemic insulin sensitivity in normoglycemic women.

Prolonged Activation of the Htr2b Serotonin Receptor Impairs Glucose Stimulated Insulin Secretion and Mitochondrial Function in MIN6 Cells.

AIMS: Pancreatic ß-cells synthesize and release serotonin (5 hydroxytryptamine, 5HT); however, the role of 5HT receptors on glucose stimulated insulin secretion (GSIS) and the mechanisms mediating this function is not fully understood. The aims of this study were to determine the expression profile of 5HT receptors in murine MIN6 ß-cells and to examine the effects of pharmacological activation of 5HT receptor Htr2b on GSIS and mitochondrial function. MATERIALS AND METHODS: mRNA levels of 5HT receptors in MIN6 cells were quantified by RT qPCR. GSIS was assessed in MIN6 cells in response to global serotonergic activation with 5HT and pharmacological Htr2b activation or inhibition with BW723C86 or SB204741, respectively. In response to Htr2b activation also was evaluated the mRNA and protein levels of PGC1α and PPARy by RT-qPCR and western blotting and mitochondrial function by oxygen consumption rate (OCR) and ATP cellular content. RESULTS: We found that mRNA levels of most 5HT receptors were either very low or undetectable in MIN6 cells. By contrast, Htr2b mRNA was present at moderate levels in these cells. Preincubation (6 h) of MIN6 cells with 5HT or BW723C86 reduced GSIS and the effect of 5HT was prevented by SB204741. Preincubation with BW723C86 increased PGC1α and PPARy mRNA and protein levels and decreased mitochondrial respiration and ATP content in MIN6 cells. CONCLUSIONS: Our results indicate that prolonged Htr2b activation in murine ß-cells decreases glucose-stimulated insulin secretion and mitochondrial activity by mechanisms likely dependent on enhanced PGC1α/PPARy expression.

Melanocortin-4 Receptor Gene Variation Is Associated with Eating Behavior in Chilean Adults.

BACKGROUND/AIMS: To evaluate the association between allelic variants of melanocortin receptors -3 and -4 (MC3R and MC4R, respectively) and leptin receptor (LEPR) genes with body mass index (BMI) and eating behavior. METHODS: We selected 344 Chilean adults (57.8% women; age 39.1 ± 6.6 years) with a wide variation in BMI (30.3 ± 6.3 kg/m²). The Three-Factor Eating Questionnaire-R18 that measures uncontrolled eating (UE), emotional eating (EE) and cognitive restraint scores was adapted, validated and assessed for association with BMI. Genotypes were determined by polymerase chain reaction followed by restriction fragment length polymorphism techniques and Taqman assays. RESULTS: Higher EE scores were found in obese vs. non-obese in both men (p = 0.01) and women (p < 0.001). UE scores were significantly associated with BMI only in women (p = 0.002). No significant differences in eating behavior scores or BMI were found by LEPR (rs1137101, rs8179183 and rs1137100 polymorphisms) or MC3R (rs3746619 and rs3827103). Carriers of the C allele for MC4R rs17782313 showed significantly higher scores of UE compared to non-carriers (2.3 ± 0.8 vs. 2.0 ± 0.7; p = 0.02). Additionally, we also report a monogenic case of obesity carrying the pathogenic mutation 449C>T (Thr150Ile) in MC4R gene with no apparent alterations in eating behavior scores. CONCLUSIONS: UE scores were higher in C-allele carriers of MC4R-rs17782313 compared to non-carriers.

Colon cancer cell invasion is promoted by protein kinase CK2 through increase of endothelin-converting enzyme-1c protein stability.

Endothelin-converting enzyme-1c (ECE-1c) is a membrane metalloprotease involved in endothelin-1 synthesis, which has been shown in vitro to have a role in breast, ovary and prostate cancer cell invasion. N-terminal end of ECE-1c displays three putative phosphorylation sites for the protein kinase CK2. We studied whether CK2 phosphorylates N-terminal end of ECE-1c as well as whether this has a role in migration and invasion of colon cancer cells. CK2 phosphorylated the N-terminal end of ECE-1c and this was precluded upon inhibition of CK2. Inhibition also led to diminished protein levels of both endogen ECE-1 or GFP-fused N-terminal end of ECE-1c in 293T embryonic and DLD-1 colon cancer cells, which highlighted the importance of this motif on UPS-dependent ECE-1c degradation. Full-length ECE-1c mutants designed either to mimic or abrogate CK2-phosphorylation displayed increased or decreased migration/invasion of colon cancer cells, respectively. Moreover, ECE-1c overexpression or its silencing with a siRNA led to increased or diminished cell migration/invasion, respectively. Altogether, these data show that CK2-increased ECE-1c protein stability is related to augmented migration and invasion of colon cancer cells, shedding light on a novel mechanism by which CK2 may promote malignant progression of this disease.

[Role of peripheral serotonin in the insulin secretion and glucose homeostasis]. / Papel de la serotonina periférica en la secreción de insulina y la homeostasis de la glucosa.

The most studied roles of serotonin (5-hydroxytryptamine, 5HT) have been related to its action in the Central Nervous System (CNS). However, most of 5HT is produced outside the CNS, mainly in the enterochromaffin cells of the gut. Additionally, other tissues such as the endocrine pancreas, particularly ß-cells, have its own serotonin system able to synthesize, secrete and respond to extracellular 5HT through cell surface receptors subtypes that have been grouped in 7 families (HTR1-7). Interestingly, 5HT is stored in granules and released together with insulin from ß-cells and its biological significance is likely a combination of intra and extracellular actions. The expression of enzymes involved in 5HT synthesis and their receptors varied markedly in ß-pancreatic cells during pregnancy, in parallel with an increase in their insulin secretion potential (probably through the action of Htr3a) and an increase in ß-cell mass (through the action of Htr2b and Htr1d). In addition, it has been suggested that gut-derived 5HT may promote hepatic gluconeogenesis during prolonged fasting through Htr2b receptor. Taken together, these findings suggest that peripheral 5HT plays an important role in the regulation of glucose homeostasis through the differential expression and activation of 5-HT membrane receptors on the surface of hepatocytes, adipocytes and pancreatic ß-cells.

La función más conocida de la serotonina (5-Hidroxitriptamina, 5HT) se refiere a su acción en el Sistema Nervioso Central (SNC). Sin embargo, la mayoría de la 5HT corporal se genera periféricamente, principalmente en las células enterocromafines del intestino. Se ha descrito que la célula -pancreática posee un sistema serotoninérgico propio que le permite sintetizar, almacenar, secretar y responder a la 5HT extracelular a través de sus receptores, de los que se conocen numerosos subtipos agrupados en 7 familias (Htr1-7). Interesantemente, la 5HT se libera conjuntamente con la insulina y sólo recientemente se ha descifrado parte de su significado biológico, que incluiría una compleja combinación de efectos intra y extracelulares que eventualmente podrían jugar un papel en la regulación de la secreción de esta hormona. De forma fisiológica, la expresión de las enzimas involucradas en la síntesis de 5HT y de sus receptores se modifica marcadamente en células durante la gestación, en coincidencia con un incremento en el potencial secretor de insulina (vía la acción del receptor ionotrópico Htr3a) y un aumento en la masa de células (vía la acción de receptores Htr1d y Htr2b). En otros tejidos, se ha sugerido que la 5HT procedente del intestino promueve la gluconeogénesis hepática y la lipólisis en adipocitos durante el ayuno, por medio de su acción sobre el receptor Htr2b. En conjunto, estos hallazgos sugieren que la 5HT periférica podría tener un rol importante en la homeostasis de la glucosa por medio de la expresión y activación diferencial de receptores de superficie en células clave, tales como hepatocitos, adipocitos y células -pancreáticas.

[Mitochondrial DNA heteroplasmy of the m.3243A>G mutation in maternally inherited diabetes and deafness]. / Heteroplasmia de la mutación del ADN mitocondrial m.3243A>G en la diabetes y sordera de herencia materna.

Maternally Inherited Diabetes and Deafness (MIDD) is caused by mutations in mitochondrial DNA (mtDNA), mainly m.3243A>G. Severity, onset and clinical phenotype of MIDD patients are partially determined by the proportion of mutant mitochondrial DNA copies in each cell and tissue (heteroplasmy). The identification of MIDD allows a corred treatment with insulin avoiding drugs that may interfere with mitochondrial electrón chain transpon. We estimated the degree of heteroplasmy of the mutation m.3243A>G from blood, saliva, hair root and a muscle biopsy using quantitative PCR (qPCR) in a femóle adult patient. For this purpose, PCR producís were inserted in a vector creating plasmids with 3243A or G. Mutant and wild-type vectors were mixed in different proportions to créate a calibration curve used to interpólate heteroplasmy percentages with qPCR threshold cycles. The proportions of m.3243A>G heteroplasmy were 62% (muscle), 14% (saliva), 6% (blood leukocytes) and 3% in hair root. Quantitative analysis of heteroplasmy showed marked variations in different tissues (highest in muscle and lowest in blood). Given the relatively high heteroplasmy found in saliva, this type of biológical sample may represent an adequate non-invasive way for assessing the presence of m.3243A>G mutations in epidemiologic studies.

Heteroplasmia de la mutación del ADN mitocondrial m.3243A>G en la diabetes y sordera de herencia materna / Mitochondrial DNA heteroplasmy of the m.3243A>G mutation in maternally inherited diabetes and deafness

Maternally Inherited Diabetes and Deafness (MIDD) is caused by mutations in mitochondrial DNA (mtDNA), mainly m.3243A>G. Severity, onset and clinical phenotype of MIDD patients are partially determined by the proportion ofmutant mitochondrial DNA copies in each cell and tissue (heteroplasmy). The identification ofMIDD allows a corred treatment with insulin avoiding drugs that may interfere with mitochondrial electrón chain transpon. We estimated the degree of heteroplasmy ofthe mutation m.3243A>G from blood, saliva, hair root and a muscle biopsy using quantitative PCR (qPCR) in a femóle adult patient. For this purpose, PCR producís were inserted in a vector creatingplasmids with 3243A or G. Mutant and wild-type vectors were mixed in different proportions to créate a calibration curve used to interpólate heteroplasmy percentages with qPCR threshold cycles. The proportions of m.3243A>G heteroplasmy were 62% (muscle), 14% (saliva), 6% (blood leukocytes) and 3% in hair root. Quantitative analysis of heteroplasmy showed marked variations in different tissues (highest in muscle and lowest in blood). Given the relatively high heteroplasmy found in saliva, this type of biológical sample may represent an adequate non-invasive way for assessing the presence of m.3243A>G mutations in epidemiologic studies.

APOA5 Q97X mutation identified through homozygosity mapping causes severe hypertriglyceridemia in a Chilean consanguineous family.

BACKGROUND: Severe hypertriglyceridemia (HTG) has been linked to defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, a number of severe HTG cases are probably caused by as yet unidentified mutations. Very high triglyceride plasma levels (>112 mmol/L at diagnosis) were found in two sisters of a Chilean consanguineous family, which is strongly suggestive of a recessive highly penetrant mutation. The aim of this study was to determine the genetic locus responsible for the severe HTG in this family. METHODS: We carried out a genome-wide linkage study with nearly 300,000 biallelic markers (Illumina Human CytoSNP-12 panel). Using the homozygosity mapping strategy, we searched for chromosome regions with excess of homozygous genotypes in the affected cases compared to non-affected relatives. RESULTS: A large homozygous segment was found in the long arm of chromosome 11, with more than 2,500 consecutive homozygous SNP shared by the proband with her affected sister, and containing the APOA5/A4/C3/A1 cluster. Direct sequencing of the APOA5 gene revealed a known homozygous nonsense Q97X mutation (p.Gln97Ter) found in both affected sisters but not in non-affected relatives nor in a sample of unrelated controls. CONCLUSION: The Q97X mutation of the APOA5 gene in homozygous status is responsible for the severe hypertriglyceridemia in this family. We have shown that homozygosity mapping correctly pinpointed the genomic region containing the gene responsible for severe hypertriglyceridemia in this consanguineous Chilean family.