RNA sequencing unravels novel L cell constituents and mechanisms of GLP-1 secretion in human gastric bypass-operated intestine.

AIMS/HYPOTHESIS: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. METHODS: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo. RESULTS: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. CONCLUSIONS/INTERPRETATION: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.

Identification and functional analysis of missense mutations in the lecithin cholesterol acyltransferase gene in a Chilean patient with hypoalphalipoproteinemia.

BACKGROUND: Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations. METHODS: An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants. RESULTS: LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband's plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels. CONCLUSION: Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency.

Plasma lactate and leukocyte mitochondrial DNA copy number as biomarkers of insulin sensitivity in non-diabetic women.

High plasma lactate levels have been associated with reduced mitochondrial respiratory capacity and increased type 2 diabetes risk, while mitochondrial DNA (mtDNA) copy number has been proposed as a biomarker of mitochondrial function linked to glucose homeostasis. The aim of this study was to evaluate the association between circulating lactate levels and leukocyte mtDNA copy numbers with insulin secretion/sensitivity indexes in 65 Chilean non-diabetic women. mtDNA copy numbers were measured in leukocytes using qPCR and digital-droplet PCR. A 75-g Oral Glucose Tolerance Test (OGTT) was performed to calculate systemic and tissue-specific insulin sensitivity indexes, as well as insulin secretion surrogates based on plasma c-peptide. An intravenous glucose tolerance test (IVGTT; 0.3 g/kg) was also carried out. Disposition indexes were calculated as the product of insulin secretion × sensitivity. Plasma levels of leptin, adiponectin, TNF-α, MCP-1, and non-esterified fatty acids were also determined. Fasting plasma lactate shows a significant association with a wide range of insulin sensitivity/resistance indexes based on fasting plasma samples (HOMA-S, adipose IR index, Revised-QUICKI, leptin-adiponectin ratio, TyG index, McAuley index and TG-to-HDL-C ratio), as well as OGTT-based measures such as the Matsuda index, the hepatic insulin resistance index, and the disposition index. Fasting plasma lactate was also positively associated with the circulating adipokines TNF-α and MCP-1. We also detected a direct association between fasting plasma lactate with leukocyte mtDNA copy numbers. The above results support the use of fasting plasma lactate, and possibly leukocyte mtDNA copy numbers, as biomarkers of reduced oxidative mitochondrial capacity, decreased hepatic insulin sensitivity, and future diabetes risk.

Loss of MafA and MafB expression promotes islet inflammation.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA-/-MafB+/-, but not MafA-/- islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

MafA Expression Preserves Immune Homeostasis in Human and Mouse Islets.

Type 1 (T1D) and type 2 (T2D) diabetes are triggered by a combination of environmental and/or genetic factors. Maf transcription factors regulate pancreatic beta (ß)-cell function, and have also been implicated in the regulation of immunomodulatory cytokines like interferon-ß (IFNß1). In this study, we assessed MAFA and MAFB co-expression with pro-inflammatory cytokine signaling genes in RNA-seq data from human pancreatic islets. Interestingly, MAFA expression was strongly negatively correlated with cytokine-induced signaling (such as IFNAR1, DDX58) and T1D susceptibility genes (IFIH1), whereas correlation of these genes with MAFB was weaker. In order to evaluate if the loss of MafA altered the immune status of islets, MafA deficient mouse islets (MafA-/-) were assessed for inherent anti-viral response and susceptibility to enterovirus infection. MafA deficient mouse islets had elevated basal levels of Ifnß1, Rig1 (DDX58 in humans), and Mda5 (IFIH1) which resulted in reduced virus propagation in response to coxsackievirus B3 (CVB3) infection. Moreover, an acute knockdown of MafA in ß-cell lines also enhanced Rig1 and Mda5 protein levels. Our results suggest that precise regulation of MAFA levels is critical for islet cell-specific cytokine production, which is a critical parameter for the inflammatory status of pancreatic islets.

Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion.

Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle) glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines). We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS). In conditioned media from human myotubes incubated with/without insulin (100 nmol/L) for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p < 0.05). Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets.

Mitochondrial diabetes and deafness: possible dysfunction of strial marginal cells of the inner ear.

OBJECTIVE: Some patients with the syndrome of mitochondrial diabetes and deafness (MIDD) have a m.3243A>G mutation of the MTTL1 gene encoding transfer ribonucleic acid for the amino acid leucine (tRNALeu(UUR)). One of our MIDD patients inspired us to propose an integrated view on how a single mutation of the mitochondrial deoxyribonucleic acid (DNA) affects both the glucose metabolism and the inner ear physiology. DESIGN: (a) Study of mitochondrial DNA in a patient with MIDD. (b) REVIEW OF THE LITERATURE on the impact of the m.3243A>G mutation on glucose metabolism and on the physiology of the hearing process. SETTINGS: Outpatient diabetes and nutrition department and molecular nutrition laboratory. METHODS: (a) Polymerase chain reaction followed by restriction fragment analysis identified the m.3243A>G mutation. (b) REVIEW OF THE LITERATURE from 1994 to 2009. RESULTS: (a) Molecular study: the m.3243A>G mutation was detected with an appreciable level of heteroplasmy. (b) REVIEW OF THE LITERATURE: the strial marginal cells located near the organ of Corti fulfill two characteristics: they are rich in mitochondria, and their dysfunction may produce neurosensorial deafness by means of a reduction in the potassium ion concentration of the endolymph. CONCLUSIONS: The m.3243A>G mutation not only underlies a dysfunction of the insulin-producing beta cell of the pancreas but also results in a reduction in adenosine triphosphate production of the strial marginal cells of the inner ear, thus diminishing the energy (in the form of potassium ion gradient) needed for the outer hair cells of the organ of Corti to amplify the soundwaves, particularly at high frequencies.