Effect of metformin on plasma metabolite profile in the Copenhagen Insulin and Metformin Therapy (CIMT) trial.

AIM: Metformin is the first-line treatment for Type 2 diabetes. However, not all people benefit from this drug. Our aim was to investigate the effects of metformin on the plasma metabolome and whether the pretreatment metabolite profile can predict HbA1c outcome. METHODS: Post hoc analysis of the Copenhagen Insulin and Metformin Therapy (CIMT) trial, a multicentre study from May 2008 to December 2012, was carried out. We used a non-target method to analyse 87 plasma metabolites in participants with Type 2 diabetes (n = 370) who were randomized in a 1 : 1 ratio to 18 months of metformin or placebo treatment. Metabolites were measured by liquid chromatography-mass spectrometry at baseline and at 18-month follow-up and the data were analysed using a linear mixed-effect model. RESULTS: At baseline, participants who were on metformin before the trial (n = 312) had higher levels of leucine/isoleucine and five lysophosphatidylethanolamines (LPEs), and lower levels of carnitine and valine compared with metformin-naïve participants (n = 58). At follow-up, participants randomized to metformin (n = 188) had elevated levels of leucine/isoleucine and reduced carnitine, tyrosine and valine compared with placebo (n = 182). At baseline, participants on metformin treatment with the highest levels of carnitine C10:1 and leucine/isoleucine had the lowest HbA1c (P-interaction = 0.02 and 0.03, respectively). This association was not significant with HbA1c at follow-up. CONCLUSIONS: Metformin treatment is associated with decreased levels of valine, tyrosine and carnitine, and increased levels of leucine/isoleucine. None of the identified metabolites can predict the HbA1c -lowering effect of metformin. Further studies of the association between metformin, carnitine and leucine/isoleucine are warranted.

Metabolite profiling of obese individuals before and after a one year weight loss program.

OBJECTIVE: We and others have previously characterized changes in circulating metabolite levels following diet-induced weight loss. Our aim was to investigate whether baseline metabolite levels and weight-loss-induced changes in these are predictive of or associated with changes in body mass index (BMI) and metabolic risk traits. METHODS: Serum metabolites were analyzed with gas and liquid chromatography/mass spectrometry in 91 obese individuals at baseline and after participating in a 1 year non-surgical weight loss program.ResultsA total of 137 metabolites were identified and semi-quantified at baseline (BMI 42.7±5.8, mean±s.d.) and at follow-up (BMI 36.3±6.6). Weight-loss-induced modification was observed for levels of 57 metabolites in individuals with ⩾10% weight loss. Lower baseline levels of xylitol was predictive of a greater decrease in BMI (ß=0.06, P<0.01) and ⩾10% weight loss (odds ratio (OR)=0.2, confidence interval (CI)=0.07-0.7, P=0.01). Decreases in levels of isoleucine, leucine, valine and tyrosine were associated with decrease in BMI (ß>0.1, P<0.05) and ⩾10% weight loss (isoleucine: OR=0.08, CI=0.01-0.3, leucine: OR=0.1, CI=0.01-0.6, valine: OR=0.1, CI=0.02-0.5, tyrosine: OR=0.1, CI=0.03-0.6, P<0.02). CONCLUSIONS: Diet-induced weight loss leads to mainly reduced levels of metabolites that are elevated in obese insulin resistant individuals. We identified multiple new associations with metabolic risk factors and validated several previous findings related to weight loss-mediated metabolite changes. Levels of specific metabolites, such as xylitol, may be predictive of the response to non-surgical weight loss already at baseline.

Mitochondrial clearance of calcium facilitated by MICU2 controls insulin secretion.

OBJECTIVE: Transport of Ca2+ into pancreatic ß cell mitochondria facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. Recent establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins allows modification of mitochondrial Ca2+ transport in intact cells. We examined the consequences of deficiency of the accessory protein MICU2 in rat and human insulin-secreting cells and mouse islets. METHODS: siRNA silencing of Micu2 in the INS-1 832/13 and EndoC-ßH1 cell lines was performed; Micu2-/- mice were also studied. Insulin secretion and mechanistic analyses utilizing live confocal imaging to assess mitochondrial function and intracellular Ca2+ dynamics were performed. RESULTS: Silencing of Micu2 abrogated GSIS in the INS-1 832/13 and EndoC-ßH1 cells. The Micu2-/- mice also displayed attenuated GSIS. Mitochondrial Ca2+ uptake declined in MICU2-deficient INS-1 832/13 and EndoC-ßH1 cells in response to high glucose and high K+. MICU2 silencing in INS-1 832/13 cells, presumably through its effects on mitochondrial Ca2+ uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to elevated glucose. Despite the loss of mitochondrial Ca2+ uptake, cytosolic Ca2+ was lower in siMICU2-treated INS-1 832/13 cells in response to high K+. It was hypothesized that Ca2+ accumulated in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca2+ channels, lowering total cytosolic Ca2+. Upon high K+ stimulation, MICU2-silenced cells showed higher and prolonged increases in submembrane Ca2+ levels. CONCLUSIONS: MICU2 plays a critical role in ß cell mitochondrial Ca2+ uptake. ß cell mitochondria sequestered Ca2+ from the submembrane compartment, preventing desensitization of voltage-dependent Ca2+ channels and facilitating GSIS.

Sox5 regulates beta-cell phenotype and is reduced in type 2 diabetes.

Type 2 diabetes (T2D) is characterized by insulin resistance and impaired insulin secretion, but the mechanisms underlying insulin secretion failure are not completely understood. Here, we show that a set of co-expressed genes, which is enriched for genes with islet-selective open chromatin, is associated with T2D. These genes are perturbed in T2D and have a similar expression pattern to that of dedifferentiated islets. We identify Sox5 as a regulator of the module. Sox5 knockdown induces gene expression changes similar to those observed in T2D and diabetic animals and has profound effects on insulin secretion, including reduced depolarization-evoked Ca2+-influx and ß-cell exocytosis. SOX5 overexpression reverses the expression perturbations observed in a mouse model of T2D, increases the expression of key ß-cell genes and improves glucose-stimulated insulin secretion in human islets from donors with T2D. We suggest that human islets in T2D display changes reminiscent of dedifferentiation and highlight SOX5 as a regulator of ß-cell phenotype and function.

Altered serotonin (5-HT) 1D and 2A receptor expression may contribute to defective insulin and glucagon secretion in human type 2 diabetes.

Islet produced 5-hydroxy tryptamine (5-HT) is suggested to regulate islet hormone secretion in a paracrine and autocrine manner in rodents. Hitherto, no studies demonstrate a role for this amine in human islet function, nor is it known if 5-HT signaling is involved in the development of beta cell dysfunction in type 2 diabetes (T2D). To clarify this, we performed a complete transcriptional mapping of 5-HT receptors and processing enzymes in human islets and investigated differential expression of these genes in non-diabetic and T2D human islet donors. We show the expression of fourteen 5-HT receptors as well as processing enzymes involved in the biosynthesis of 5-HT at the mRNA level in human islets. Two 5-HT receptors (HTR1D and HTR2A) were over-expressed in T2D islet donors. Both receptors (5-HT1d and 5-HT2a) were localized to human alpha, beta and delta cells. 5-HT inhibited both insulin and glucagon secretion in non-diabetic islet donors. In islets isolated from T2D donors the amine significantly increased release of insulin in response to glucose. Our results suggest that 5-HT signaling participates in regulation of overall islet hormone secretion in non- diabetic individuals and over-expression of HTR1D and HTR2A may either contribute to islet dysfunction in T2D or arise as a consequence of an already dysfunctional islet.

Ultrasonic-trap-enhanced selectivity in capillary electrophoresis.

We combine ultrasonic trapping and capillary electrophoresis (CE) with the goal to detect ultra-low concentrations of proteins via size-selective separation and enrichment of antibody-coated latex spheres. An 8.5 MHz standing ultrasonic wave is longitudinally coupled into the sub-100- micro m diam capillary of the CE system. Competition between acoustic and viscous forces result in in-flow separation of micro m-diam spheres according to their size. Experiments separating 2.8- and 2.1- micro m-diam fluorescent latex particles, which model a protein-specific immunocomplex/free particle mixture, indicate a potential improvement of the concentration limit of detection of 10(4) compared to current CE systems. Theoretical calculations show room for further improvement.

Molecularly imprinted microparticles for capillary electrochromatographic enantiomer separation of propranolol.

Molecularly imprinted microparticles imprinted against (S)-propranolol were synthesised and studied for use in capillary electrochromatographic separation of propranolol enantiomers. The imprinted microparticles were in the size range of 0.2-0.5 micron as determined by scanning electron microscopy. The microparticles were suspended, in high concentration, in the electrolyte and used to perform enantiomer separation by a partial filling technique.

Approaches to molecular imprinting based selectivity in capillary electrochromatography.

The work done during the past decade in order to adapt molecularly imprinted polymers (MIPs) to the capillary format and subsequently use these highly selective matrices for capillary electrochromatography (CEC) are reviewed in this article. MIPs are prepared utilizing a templated polymer synthesis where the template addresses the selectivity of the resulting polymer. These polymers possess binding characteristics that are comparable to the biological antibodies. Due to the polyclonality of the binding sites in the MIP, the separation result in severe peak broadening and tailing when performed in the isocratic mode. This was seen early in the development of MIPs as selective stationary phases in liquid chromatography (LC). As a mean of decreasing these problems, much effort was put into adapting the MIP to fit in CEC systems, that offers an efficiency that is superior to that in LC. Aiming to increase the efficiency of the MIP-CEC systems, different MIP formats have been developed that can be divided into three conceptually different categories, i.e., the monolithic, the microparticle and the coating. The strive for MIP formats that can be used in small bore capillaries has led to the development of MIP formats applicable to miniaturized systems approaching the chip format. Although prepared in order to perform MIP-CEC mediated separations, these formats can be used in a broad range of applications were the characteristics of the MIP, e.g. stability, selectivity and cost efficiency, could offer an interesting solution to cover the needs.

Molecularly imprinted microparticles for capillary electrochromatography: studies on microparticle synthesis and electrolyte composition.

The use of molecularly imprinted polymer (MIP) microparticles in a partial filling application of capillary electrochromatography (CEC) has previously been shown successful for the enantiomer separation of propranolol. In this investigation, the influence of some important parameters in the preparation protocol, i.e., template to monomer ratio, type of cross-linker and functional monomers, and the effect of separation condition, i.e., organic modifier content, pH and the temperature of the column, on the electrochromatographic behavior of the MIP microparticles were studied. It was found that ethyleneglycol dimethacrylate (EDMA), having two reactive double bonds, was superior in terms CEC performance to trimethylpropane trimethacrylate (TRIM) and pentaerythritol tetraacrylate (PETEA) having three and four double bonds, respectively. The use of weak functional monomers, i.e., monomers lacking a strong interaction with the template, was shown to increase the separation efficiency. It was found that the template to functional monomer ratio had a pronounced influence on the MIP microparticle partial filling CEC performance as well as the size of the obtained microparticles. The use of a partial filling technique realizes the use of a new MIP phase in every new separation as well as the ability of altering the selectivity of the separation column and length of the MIP without the need for column switching.