Comprehensive Profiling by Non-targeted Stable Isotope Tracing Capillary Electrophoresis-Mass Spectrometry: A New Tool Complementing Metabolomic Analyses of Polar Metabolites.

Mass spectrometry (MS) driven metabolomics is a frequently used tool in various areas of life sciences; however, the analysis of polar metabolites is less commonly included. In general, metabolomic analyses lead to the detection of the total amount of all covered metabolites. This is currently a major limitation with respect to metabolites showing high turnover rates, but no changes in their concentration. Such metabolites and pathways could be crucial metabolic nodes (e.g., potential drug targets in cancer metabolism). A stable-isotope tracing capillary electrophoresis-mass spectrometry (CE-MS) metabolomic approach was developed to cover both polar metabolites and isotopologues in a non-targeted way. An in-house developed software enables high throughput processing of complex multidimensional data. The practicability is demonstrated analyzing [U-13 C]-glucose exposed prostate cancer and non-cancer cells. This CE-MS-driven analytical strategy complements polar metabolite profiles through isotopologue labeling patterns, thereby improving not only the metabolomic coverage, but also the understanding of metabolism.

Postprandial Dynamics of Proglucagon Cleavage Products and Their Relation to Metabolic Health.

Introduction: While oral glucose ingestion typically leads to a decrease in circulating glucagon levels, a substantial number of persons display stable or rising glucagon concentrations when assessed by radioimmunoassay (RIA). However, these assays show cross-reactivity to other proglucagon cleavage products. Recently, more specific assays became available, therefore we systematically assessed glucagon and other proglucagon cleavage products and their relation to metabolic health. Research Design and Methods: We used samples from 52 oral glucose tolerance tests (OGTT) that were randomly selected from persons with different categories of glucose tolerance in an extensively phenotyped study cohort. Results: Glucagon concentrations quantified with RIA were non-suppressed at 2 hours of the OGTT in 36% of the samples. Non-suppressors showed lower fasting glucagon levels compared to suppressors (p=0.011). Similar to RIA measurements, ELISA-derived fasting glucagon was lower in non-suppressors (p<0.001). Glucagon 1-61 as well as glicentin and GLP-1 kinetics were significantly different between suppressors and non-suppressors (p=0.004, p=0.002, p=0.008 respectively) with higher concentrations of all three hormones in non-suppressors. Levels of insulin, C-peptide, and free fatty acids were comparable between groups. Non-suppressors were leaner and had lower plasma glucose concentrations (p=0.03 and p=0.047, respectively). Despite comparable liver fat content and insulin sensitivity (p≥0.3), they had lower 2-hour post-challenge glucose (p=0.01). Conclusions: Glucagon 1-61, glicentin and GLP-1 partially account for RIA-derived glucagon measurements due to cross-reactivity of the assay. However, this contribution is small, since the investigated proglucagon cleavage products contribute less than 10% to the variation in RIA measured glucagon. Altered glucagon levels and higher post-challenge incretins are associated with a healthier metabolic phenotype.

Metabolic implications of pancreatic fat accumulation.

Fat accumulation outside subcutaneous adipose tissue often has unfavourable effects on systemic metabolism. In addition to non-alcoholic fatty liver disease, which has received considerable attention, pancreatic fat has become an important area of research throughout the past 10 years. While a number of diagnostic approaches are available to quantify pancreatic fat, multi-echo Dixon MRI is currently the most developed method. Initial studies have shown associations between pancreatic fat and the metabolic syndrome, impaired glucose metabolism and type 2 diabetes mellitus. Pancreatic fat is linked to reduced insulin secretion, at least under specific circumstances such as prediabetes, low BMI and increased genetic risk of type 2 diabetes mellitus. This Review summarizes the possible causes and metabolic consequences of pancreatic fat accumulation. In addition, potential therapeutic approaches for addressing pancreatic fat accumulation are discussed.

Incretin Hypersecretion in Gestational Diabetes Mellitus.

CONTEXT: Incretins are crucial stimulators of insulin secretion following food intake. Data on incretin secretion and action during pregnancy are sparse. OBJECTIVE: The aim of the study was to investigate the incretin response during an oral glucose tolerance test (OGTT) in pregnant women with and without gestational diabetes mellitus (GDM). DESIGN: We analyzed data from the ongoing observational PREG study (NCT04270578). SETTING: The study was conducted at the University Hospital Tübingen. PARTICIPANTS: We examined 167 women (33 with GDM) during gestational week 27 ±â€…2.2. INTERVENTION: Subjects underwent 5-point OGTT with a 75-g glucose load. MAIN OUTCOME MEASURES: We assessed insulin secretion and levels of total glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), glicentin, and glucagon during OGTT. Linear regression was used to analyze the relation of GLP-1 and glucose with insulin secretion and the association of incretin levels on birth outcome. RESULTS: Insulin secretion was significantly lower in women with GDM (P < 0.001). Postload GLP-1 and GIP were ~20% higher in women with GDM (all P < 0.05) independent of age, body mass index, and gestational age. GLP-1 increase was associated with insulin secretion only in GDM, but not in normal glucose tolerance. Postprandial GLP-1 levels were negatively associated with birth weight. CONCLUSIONS: The more pronounced GLP-1 increase in women with GDM could be part of a compensatory mechanism counteracting GLP-1 resistance. Higher GLP-1 levels might be protective against fetal overgrowth.

The TUDID Study - Background and Design of a Prospective Cohort.

Prevalence of both type 1 and type 2 diabetes mellitus is growing worldwide and one major cause for morbidity and mortality. However, not every patient develops diabetes-related complications, but causes for the individual susceptibility are still not fully understood. As a platform to address this, we initiated the TUDID (TUebingen DIabetes Database) study, a prospective, monocentric, observational study that includes adults with diabetes mellitus who are treated in the inpatient clinic of a University Hospital in southern Germany. Besides a thorough clinical examination and extensive laboratory tests (with integrated biobanking), major study focuses are the kidneys, the eyes, the vasculature as well as cognition and mood where standardized investigations for early stages for diabetes complications are performed. Analyses of the data generated by this precise characterization of diabetes-related complications will contribute to our understanding of the development and course of such complications, and thus facilitate the implementation of tailored treatment options that can reduce the risk and severity of diabetes-related complications.

Determinants of hepatic insulin clearance - Results from a Mendelian Randomization study.

AIMS/HYPOTHESIS: Besides insulin resistance, type 2 diabetes associates with decreased hepatic insulin clearance (HIC). We now tested for causal relationship of HIC to liver fat accumulation or features of the metabolic syndrome. METHODS: HIC was derived from oral glucose tolerance tests with the "Oral C-peptide and Insulin Minimal Models" (n = 3311). Liver fat was quantified by magnetic resonance spectroscopy (n = 1211). Mendelian Randomization was performed using established single nucleotide polymorphisms (SNPs; 115 for liver fat, 155 alanine-aminotransferase, 37 insulin sensitivity, 37 insulin secretion, 72 fasting insulin, 5285 BMI, 163 visceral fat, 270 waist circumference, 442 triglycerides, 620 HDL-Cholesterol, 193 C-reactive protein, 53 lipodystrophy-like phenotypes). RESULTS: HIC associated inversely with liver fat (p < 0.003) and insulin sensitivity (p < 0.0001). Both liver fat and HIC were independently associated with insulin sensitivity (p < 0.0001). Neither liver fat nor alanine-aminotransferase were causally linked to HIC, as indicated by Mendelian Randomization (Nliver fat = 1054, NHIC = 2254; Nalanineaminotranferase = 1985, NHIC = 2251). BMI-related SNPs were causally associated with HIC (NBMI = 2772, NHIC = 2259, p < 0.001) but not waist circumference-SNPs (NSNPs-waist circumference = 2751, NHIC = 2280). Genetically determined insulin sensitivity was not causally related to HIC (Ninsulin sensitivity = 2752, NHIC = 2286). C-reactive protein and HDL were causally associated with HIC, with higher C-reactive protein and lower HDL leading to higher HIC (NC-reactive protein = 2660, NHIC = 2240; NHDL = 2694, NHIC = 2275). CONCLUSIONS: This Mendelian Randomization analysis does not support a causal link between hepatic steatosis and HIC. Other components of the metabolic syndrome seem to compensate peripheral hyperinsulinemia by increasing hepatic insulin extraction.

Ectopic fat accumulation in human astrocytes impairs insulin action.

Astrocytes provide neurons with structural support and energy in form of lactate, modulate synaptic transmission, are insulin sensitive and act as gatekeeper for water, ions, glutamate and second messengers. Furthermore, astrocytes are important for glucose sensing, possess neuroendocrine functions and also play an important role in cerebral lipid metabolism. To answer the question, if there is a connection between lipid metabolism and insulin action in human astrocytes, we investigated if storage of ectopic lipids in human astrocytes has an impact on insulin signalling in those cells. Human astrocytes were cultured in the presence of a lipid emulsion, consisting of fatty acids and triglycerides, to induce ectopic lipid storage. After several days, cells were stimulated with insulin and gene expression profiling was performed. In addition, phosphorylation of Akt as well as glycogen synthesis and cell proliferation was assessed. Ectopic lipid storage was detected in human astrocytes after lipid exposure and lipid storage was persistent even when the fat emulsion was removed from the cell culture medium. Chronic exposure to lipids induced profound changes in the gene expression profile, whereby some genes showed a reversible gene expression profile upon removal of fat, and some did not. This included FOXO-dependent expression patterns. Furthermore, insulin-induced phosphorylation of Akt was diminished and also insulin-induced glycogen synthesis and proliferation was impaired in lipid-laden astrocytes. Chronic lipid exposure induces lipid storage in human astrocytes accompanied by insulin resistance. Analyses of the gene expression pattern indicated the potential of a partially reversible gene expression profile. Targeting astrocytic insulin resistance by reducing ectopic lipid load might represent a promising treatment target for insulin resistance of the brain in obesity, diabetes and neurodegeneration.

Suitability of hepatocyte cell lines HepG2, AML12 and THLE-2 for investigation of insulin signalling and hepatokine gene expression.

Immortal hepatocyte cell lines are widely used to elucidate insulin-dependent signalling pathways and regulation of hepatic metabolism, although the often tumorigenic origin might not represent the metabolic state of healthy hepatocytes. We aimed to investigate if murine cell line AML12 and human cell line THLE-2, which are derived from healthy liver cells, are comparable to hepatoma cell line HepG2 for studying acute insulin signalling and expression of gluconeogenic enzymes and hepatokines. Insulin responsiveness of AML12 and THLE-2 cells was impaired when cells were cultured in the recommended growth medium, but comparable with HepG2 cells by using insulin-deficient medium. THLE-2 cells showed low abundance of insulin receptor, while protein levels in HepG2 and AML12 were comparable. AML12 and THLE-2 cells showed only low or non-detectable transcript levels of G6PC and PCK1 Expression of ANGPTL4 was regulated similarly in HepG2 and AML12 cells upon peroxisome proliferator-activated receptor δ activation but only HepG2 cells resemble the in vivo regulation of hepatic ANGPTL4 by cAMP. Composition of the culture medium and protein expression levels of key signalling proteins should be considered when AML12 and THLE-2 are used to study insulin signalling. With regard to gluconeogenesis and hepatokine expression, HepG2 cells appear to be closer to the in vivo situation despite the tumorigenic origin.