Circulating metabolomic markers in association with overall burden of microvascular complications in type 1 diabetes.

INTRODUCTION: Diabetic retinopathy (DR), diabetic kidney disease (DKD) and distal symmetric polyneuropathy (DSPN) share common pathophysiology and pose an additive risk of early mortality. RESEARCH DESIGN AND METHODS: In adults with type 1 diabetes, 49 metabolites previously associated with either DR or DKD were assessed in relation to presence of DSPN. Metabolites overlapping in significance with presence of all three complications were assessed in relation to microvascular burden severity (additive number of complications-ie, presence of DKD±DR±DSPN) using linear regression models. Subsequently, the same metabolites were assessed with progression to endpoints: soft microvascular events (progression in albuminuria grade, ≥30% estimated glomerular filtration rate (eGFR) decline, or any progression in DR grade), hard microvascular events (progression to proliferative DR, chronic kidney failure, or ≥40% eGFR decline), and hard microvascular or macrovascular events (hard microvascular events, cardiovascular events (myocardial infarction, stroke, or arterial interventions), or cardiovascular mortality), using Cox models. All models were adjusted for sex, baseline age, diabetes duration, systolic blood pressure, HbA1c, body mass index, total cholesterol, smoking, and statin treatment. RESULTS: The full cohort investigated consisted of 487 participants. Mean (SD) follow-up was 4.8 (2.9, 5.7) years. Baseline biothesiometry was available in 202 participants, comprising the cross-sectional cohort. Eight metabolites were significantly associated with presence of DR, DKD, and DSPN, and six with additive microvascular burden severity. In the full cohort longitudinal analysis, higher levels of 3,4-dihydroxybutanoic acid (DHBA), 2,4-DHBA, ribonic acid, glycine, and ribitol were associated with development of events in both crude and adjusted models. Adding 3,4-DHBA, ribonic acid, and glycine to a traditional risk factor model improved the discrimination of hard microvascular events. CONCLUSIONS: While prospective studies directly assessing the predictive ability of these markers are needed, our results strengthen the role of clinical metabolomics in relation to risk assessment of diabetic complications in chronic type 1 diabetes.

Low sphingolipid levels predict poor survival in patients with alcohol-related liver disease.

Background & Aims: Alcohol-related hepatitis (AH) and alcohol-related cirrhosis are grave conditions with poor prognoses. Altered hepatic lipid metabolism can impact disease development and varies between different alcohol-related liver diseases. Therefore, we aimed to investigate lipidomics and metabolomics at various stages of alcohol-related liver diseases and their correlation with survival. Methods: Patients with newly diagnosed alcohol-related cirrhosis, who currently used alcohol (ALC-A), stable outpatients with decompensated alcohol-related cirrhosis with at least 8 weeks of alcohol abstinence (ALC), and patients with AH, were compared with each other and with healthy controls (HC). Circulating lipids and metabolites were analysed using HPLC and mass spectrometry. Results: Forty patients with ALC, 95 with ALC-A, 30 with AH, and 42 HC provided plasma. Lipid levels changed according to disease severity, with generally lower levels in AH and cirrhosis than in the HC group; this was most pronounced for AH, followed by ALC-A. Nine out of 10 free fatty acids differed between cirrhosis groups by relative increases of 0.12-0.66 in ALC compared with the ALC-A group (p <0.0005). For metabolomics, total bile acids increased by 19.7, 31.3, and 80.4 in the ALC, ALC-A, and AH groups, respectively, compared with HC (all p <0.0001). Low sphingolipid ([d42:1] and [d41:1]) levels could not predict 180-day mortality (AUC = 0.73, p = 0.95 and AUC = 0.73, p = 0.95) more accurately than the model for end-stage liver disease score (AUC = 0.71), but did predict 90-day mortality (AUC d42:1 = 0.922, AUC d41:1 = 0.893; pd42:1 = 0.005, pd41:1 = 0.007) more accurately than the MELD score AUCMELD = 0.70, pMELD = 0.19). Conclusions: Alcohol-related severe liver disease is characterised by low lipid levels progressing with severity of liver disease, especially low sphingomyelins, which also associate to poor prognoses. Impact and implications: Lipidomics has the potential to diagnose and risk stratify patients with liver diseases. Lipidomics differed between patients with alcohol-related hepatitis and alcohol-related cirrhosis with and without recent alcohol use. Furthermore, lipidomics could predict short-term mortality and might be suitable as a prognostic tool in the future. Clinical Trials Registration: Scientific Ethics Committee of the Capital Region of Denmark, journal no. H-21013476.

Atorvastatin for patients with cirrhosis. A randomized, placebo-controlled trial.

BACKGROUND: Patients with cirrhosis and portal hypertension face a high risk of complications. Besides their anti-inflammatory and antifibrotic effects, statins may reduce portal pressure and thus the risk of complications and mortality. We aimed to investigate the effects of atorvastatin on hospital admissions, mortality, inflammation, and lipidomics in cirrhosis with portal hypertension. METHODS: We performed a double-blinded, randomized, placebo-controlled clinical trial among patients with cirrhosis and portal hypertension. Atorvastatin (10-20 mg/d) was administered for 6 months. We measured splanchnic hemodynamics, analyzed inflammatory markers, and performed lipidomics at baseline and after 6 months. RESULTS: Seventy-eight patients were randomized, with 38 patients allocated to atorvastatin and 40 patients to placebo. Fifty-nine patients completed 6 months of intervention. Comparisons between changes in each group were calculated. Liver-related complications and mortality were similar between the groups. The HVPG and Model for End-stage Liver Disease score did not change between groups (p=0.95 and 0.87, respectively). Atorvastatin decreased 3 of 42 inflammatory markers, CD62-L-selectin, matrix metalloproteinases-2, and TNF-α (p-values: 0.005, 0.011, and 0.023, respectively), while lipidomics was not significantly changed. CONCLUSIONS: In patients with cirrhosis, atorvastatin was safe to use, but did not reduce mortality, the risk of liver-related complications, or the HVPG. Atorvastatin induced minor anti-inflammatory effects and minor effects on lipids during a 6-month treatment period.

Microbial metabolite p-cresol inhibits gut hormone expression and regulates small intestinal transit in mice.

p-cresol is a metabolite produced by microbial metabolism of aromatic amino acid tyrosine. p-cresol and its conjugated forms, p-cresyl sulfate and p-cresyl glucuronide, are uremic toxins that correlate positively with chronic kidney disease and diabetes pathogenesis. However, how p-cresol affects gut hormones is unclear. Here, we expose immortalized GLUTag cells to increasing concentrations of p-cresol and found that p-cresol inhibited Gcg expression and reduced glucagon-like peptide-1 (GLP-1) secretion in vitro. In mice, administration of p-cresol in the drinking water for 2 weeks reduced the transcript levels of Gcg and other gut hormones in the colon; however, it did not affect either fasting or glucose-induced plasma GLP-1 levels. Furthermore, it did not affect glucose tolerance but promoted faster small intestinal transit in mice. Overall, our data suggest that microbial metabolite p-cresol suppresses transcript levels of gut hormones and regulates small intestinal transit in mice.

Sphingolipids Are Depleted in Alcohol-Related Liver Fibrosis.

BACKGROUND & AIMS: Alcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in alcohol-related liver disease (ALD) is unclear. In this biopsy-controlled, prospective, observational study, we characterized the liver and plasma lipidomes in patients with early ALD. METHODS: We performed mass spectrometry-based lipidomics of paired liver and plasma samples from 315 patients with ALD and of plasma from 51 matched healthy controls. We associated lipid levels with histologic fibrosis, inflammation, and steatosis with correction for multiple testing and adjustment for confounders. We further investigated sphingolipid regulation by means of quantitative real-time polymerase chain reaction sequencing of microRNA, prediction of liver-related events, and tested causality with Mendelian randomization. RESULTS: We detected 198 lipids in the liver and 236 lipids in the circulation from 18 lipid classes. Most sphingolipids (sphingomyelins and ceramides) and phosphocholines were co-down-regulated in both liver and plasma, where lower abundance correlated with higher fibrosis stage. Sphingomyelins showed the most pronounced negative correlation to fibrosis, mirrored by negative correlations in both liver and plasma with hepatic inflammation. Reduced sphingomyelins predicted future liver-related events. This seemed to be characteristic of "pure ALD," as sphingomyelin levels were higher in patients with concomitant metabolic syndrome and ALD/nonalcoholic fatty liver disease overlap. Mendelian randomization in FinnGen and UK Biobanks indicated ALD as the cause of low sphingomyelins, and alcohol use disorder did not correlate with genetic susceptibility to low sphingomyelin levels. CONCLUSIONS: Alcohol-related liver fibrosis is characterized by selective and progressive lipid depletion in liver and blood, particularly sphingomyelins, which also associates with progression to liver-related events.

Full activation of thermogenesis in brown adipocytes requires Basigin action.

Exploring mechanisms responsible for brown adipose tissue's (BAT) high metabolic activity is crucial to exploit its energy-dissipating ability for therapeutic purposes. Basigin (Bsg), a multifunctional highly glycosylated transmembrane protein, was recently proposed as one of the 98 critical markers allowing to distinguish 'white' and 'brown' adipocytes, yet its function in thermogenic brown adipocytes is unknown. Here, we report that Bsg is negatively associated with obesity in mice. By contrast, Bsg expression increased in the mature adipocyte fraction of BAT upon cold acclimation. Additionally, Bsg levels were highly induced during brown adipocyte maturation in vitro and were further increased upon ß-adrenergic stimulation in a HIF-1α-dependent manner. siRNA-mediated Bsg gene silencing in cultured brown adipocytes did not impact adipogenesis nor mitochondrial function. However, a significant decrease in mitochondrial respiration, lipolysis and Ucp1 transcription was observed in adipocytes lacking Bsg, when activated by norepinephrine. Furthermore, using gas chromatography/mass spectrometry-time-of-flight analysis to assess the composition of cellular metabolites, we demonstrate that brown adipocytes lacking Bsg have lower levels of intracellular lactate and acetoacetate. Bsg was additionally required to regulate intracellular AcAc and tricarboxylic acid cycle intermediate levels in NE-stimulated adipocytes. Our study highlights the critical role of Bsg in active brown adipocytes, possibly by controlling cellular metabolism.

Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration.

Obesity and diabetes are associated with inflammation and altered plasma levels of several metabolites, which may be involved in disease progression. Some metabolites can activate G protein-coupled receptors (GPCRs) expressed on immune cells where they can modulate metabolic inflammation. Here, we find that 3-hydroxydecanoate is enriched in the circulation of obese individuals with type 2 diabetes (T2D) compared with nondiabetic controls. Administration of 3-hydroxydecanoate to mice promotes immune cell recruitment to adipose tissue, which was associated with adipose inflammation and increased fasting insulin levels. Furthermore, we demonstrate that 3-hydroxydecanoate stimulates migration of primary human and mouse neutrophils, but not monocytes, through GPR84 and Gαi signaling in vitro. Our findings indicate that 3-hydroxydecanoate is a T2D-associated metabolite that increases inflammatory responses and may contribute to the chronic inflammation observed in diabetes.

A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Circulating metabolites and molecular lipid species are associated with future cardiovascular morbidity and mortality in type 1 diabetes.

BACKGROUND: Cardiovascular disease remains the leading cause of mortality in individuals with diabetes and improved understanding of its pathophysiology is needed. We investigated the association of a large panel of metabolites and molecular lipid species with future cardiovascular events in type 1 diabetes. METHODS: The study included 669 individuals with type 1 diabetes. Non-targeted serum metabolomics and lipidomics analyses were performed using mass spectrometry. Data on cardiovascular events (cardiovascular mortality, coronary artery disease, stroke, and peripheral arterial interventions) were obtained from Danish Health registries and analyzed by Cox hazards models. Metabolites and molecular lipid species were analyzed in univariate models adjusted for false discovery rate (FDR). Metabolites and molecular lipid species fulfilling a pFDR < 0.05 were subsequently analyzed in adjusted models including age, sex, hemoglobin A1c, mean arterial pressure, smoking, body mass index, low-density lipoprotein cholesterol, estimated glomerular filtration rate, urinary albumin excretion rate and previous cardiovascular disease. Analyses of molecular lipid species were further adjusted for triglycerides and statin use. RESULTS: Of the included participants, 55% were male and mean age was 55 ± 13 years. Higher 4-hydroxyphenylacetic acid (HR 1.35, CI [1.01-1.80], p = 0.04) and lower threonine (HR 0.81, CI [0.67-0.98] p = 0.03) were associated with development of cardiovascular events (n = 95). In lipidomics analysis, higher levels of three different species, diacyl-phosphatidylcholines (PC)(36:2) (HR 0.82, CI [0.70-0.98], p = 0.02), alkyl-acyl-phosphatidylcholines (PC-O)(34:2) (HR 0.76, CI [0.59-0.98], p = 0.03) and (PC-O)(34:3) (HR 0.75, CI [0.58-0.97], p = 0.03), correlated with lower risk of cardiovascular events, whereas higher sphingomyelin (SM)(34:1) (HR 1.32, CI [1.04-1.68], p = 0.02), was associated with an increased risk. CONCLUSIONS: Circulating metabolites and molecular lipid species were associated with future cardiovascular events in type 1 diabetes. While the causal effect of these biomolecules on the cardiovascular system remains unknown, our findings support that omics-based technologies, although still in an early phase, may have the potential to unravel new pathways and biomarkers in the field of cardiovascular disease in type 1 diabetes.

Cardiovascular Autonomic Neuropathy in Type 1 Diabetes Is Associated With Disturbances in TCA, Lipid, and Glucose Metabolism.

Introduction: Diabetic cardiovascular autonomic neuropathy (CAN) is associated with increased mortality and morbidity. To explore metabolic mechanisms associated with CAN we investigated associations between serum metabolites and CAN in persons with type 1 diabetes (T1D). Materials and Methods: Cardiovascular reflex tests (CARTs) (heart rate response to: deep breathing; lying-to-standing test; and the Valsalva maneuver) were used to diagnose CAN in 302 persons with T1D. More than one pathological CARTs defined the CAN diagnosis. Serum metabolomics and lipidomic profiles were analyzed with two complementary non-targeted mass-spectrometry methods. Cross-sectional associations between metabolites and CAN were assessed by linear regression models adjusted for relevant confounders. Results: Participants were median (IQR) aged 55(49, 63) years, 48% males with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had CAN. A total of 75 metabolites and 106 lipids were analyzed. In crude models, the CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4-deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and the amino acid methionine (p<0.05). Upon adjustment, positive associations with the CAN diagnoses were retained for hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, citric acid, and phenols (P<0.05). Conclusion: Metabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives are associated with the CAN diagnosis in T1D. These pathway may be part of the pathogeneses leading to CAN and may be modifiable risk factors for the complication.

Intravenous nicotinamide riboside elevates mouse skeletal muscle NAD+ without impacting respiratory capacity or insulin sensitivity.

In clinical trials, oral supplementation with nicotinamide riboside (NR) fails to increase muscle mitochondrial respiratory capacity and insulin sensitivity but also does not increase muscle NAD+ levels. This study tests the feasibility of chronically elevating skeletal muscle NAD+ in mice and investigates the putative effects on mitochondrial respiratory capacity, insulin sensitivity, and gene expression. Accordingly, to improve bioavailability to skeletal muscle, we developed an experimental model for administering NR repeatedly through a jugular vein catheter. Mice on a Western diet were treated with various combinations of NR, pterostilbene (PT), and voluntary wheel running, but the metabolic effects of NR and PT treatment were modest. We conclude that the chronic elevation of skeletal muscle NAD+ by the intravenous injection of NR is possible but does not affect muscle respiratory capacity or insulin sensitivity in either sedentary or physically active mice. Our data have implications for NAD+ precursor supplementation regimens.

Comparative analysis of oral and intraperitoneal glucose tolerance tests in mice.

OBJECTIVE: The glucose tolerance test (GTT) is widely used in preclinical research to investigate glucose metabolism, but there is no standardised way to administer glucose. The aim of this study was to directly compare the effect of the route of glucose administration on glucose and insulin kinetics during a GTT in mice. METHODS: A GTT was performed in lean male and female mice and obese male mice and glucose was administered via the oral or intraperitoneal (I.P.) route. Samples were collected frequently during the GTT to provide a full time-course of the insulin and glucose excursions. In another cohort of lean male mice, plasma concentrations of insulin, c-peptide, and incretin hormones were measured at early time points after glucose administration. A stable-isotope labelled GTT (SiGTT) was then performed to delineate the contribution of exogenous and endogenous glucose to glycemia during the GTT, comparing both methods of glucose administration. Finally, we present a method to easily measure insulin from small volumes of blood during a GTT by directly assaying whole-blood insulin using ELISA and show a good concordance between whole-blood and plasma insulin measurements. RESULTS: We report that I.P. glucose administration results in an elevated blood glucose excursion and a largely absent elevation in blood insulin and plasma incretin hormones when compared to oral administration. Utilising stable-isotope labelled glucose, we demonstrate that the difference in glucose excursion between the two routes of administration is mainly due to the lack of suppression of glucose production in I.P. injected mice. Additionally, rates of exogenous glucose appearance into circulation were different between lean and obese mice after I.P., but not after oral glucose administration. CONCLUSION: Reflecting on these data, we suggest that careful consideration be given to the route of glucose administration when planning a GTT procedure in mice and that in most circumstances the oral route of glucose administration should be preferred over the I.P. route to avoid possible artifacts originating from a non-physiological route.

Atlas of exercise metabolism reveals time-dependent signatures of metabolic homeostasis.

Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.

Comprehensive lipidomics reveals phenotypic differences in hepatic lipid turnover in ALD and NAFLD during alcohol intoxication.

BACKGROUND & AIMS: In experimental models, alcohol induces acute changes in lipid metabolism that cause hepatocyte lipoapoptosis and inflammation. Here we study human hepatic lipid turnover during controlled alcohol intoxication. METHODS: We studied 39 participants with 3 distinct hepatic phenotypes: alcohol-related liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), and healthy controls. Alcohol was administrated via nasogastric tube over 30 min. Hepatic and systemic venous blood was sampled simultaneously at 3 time points: baseline, 60, and 180 min after alcohol intervention. Liver biopsies were sampled 240 min after alcohol intervention. We used ultra-high performance liquid chromatography mass spectrometry to measure levels of more than 250 lipid species from the blood and liver samples. RESULTS: After alcohol intervention, the levels of blood free fatty acid (FFA) and lysophosphatidylcholine (LPC) decreased, while triglyceride (TG) increased. FFA was the only lipid class to decrease in NAFLD after alcohol intervention, whereas LPC and FFA decreased and TG increased after intervention in ALD and healthy controls. Fatty acid chain uptake preference in FFAs and LPCs were oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. Hepatic venous blood FFA and LPC levels were lower when compared with systemic venous blood levels throughout the intervention. After alcohol intoxication, liver lipidome in ALD was similar to that in NAFLD. CONCLUSIONS: Alcohol intoxication induces rapid changes in circulating lipids including hepatic turnaround from FFA and LPC, potentially leading to lipoapoptosis and steatohepatitis. TG clearance was suppressed in NAFLD, possibly explaining why alcohol and NAFLD are synergistic risk factors for disease progression. These effects may be central to the pathogenesis of ALD. CLINICAL TRIALS REGISTRATION: The study is registered at Clinicaltrials.gov (NCT03018990). LAY SUMMARY: We report that alcohol induces hepatic extraction of free unsaturated fatty acids and lysophosphatidylcholines, hepatotoxic lipids which have not been previously associated with alcohol-induced liver injury. We also found that individuals with non-alcoholic fatty liver disease have reduced lipid turnover during alcohol intoxication when compared with people with alcohol-related fatty liver disease. This may explain why alcohol is particularly more harmful in people with non-alcoholic fatty liver and why elevated BMI and alcohol have a synergistic effect on the risk of liver-related death.

Ecological Adaptation and Succession of Human Fecal Microbial Communities in an Automated In Vitro Fermentation System.

Longitudinal studies of gut microbiota following specific interventions are vital for understanding how they influence host health. However, robust longitudinal sampling of gut microbiota is a major challenge, which can be addressed using in vitro fermentors hosting complex microbial communities. Here, by employing 16S rRNA gene amplicon sequencing, we investigated the adaptation and succession of human fecal microbial communities in an automated multistage fermentor. We performed two independent experiments using different human donor fecal samples, one configured with two units of three colon compartments each studied for 22 days and another with one unit of two colon compartments studied for 31 days. The fermentor maintained a trend of increasing microbial alpha diversity along colon compartments. Within each experiment, microbial compositions followed compartment-specific trajectories and reached independent stable configurations. While compositions were highly similar between replicate units, they were clearly separated between different experiments, showing that they maintained the individuality of fecal inoculum rather than converging on a fermentor-specific composition. While some fecal amplicon sequence variants (ASVs) were undetected in the fermentor, many ASVs undetected in the fecal samples flourished in vitro. These bloomer ASVs accounted for significant proportions of the population and included prominent health-associated microbes such as Bacteroides fragilis and Akkermansia muciniphila. Turnover in community compositions is likely explained by feed composition and pH, suggesting that these communities can be easily modulated. Our results suggest that in vitro fermentors are promising tools to study complex microbial communities harboring important members of human gut microbiota. IMPORTANCE In vitro fermentors that can host complex gut microbial communities are promising tools to investigate the dynamics of human gut microbiota. In this work, using an automated in vitro gut fermentor consisting of different colon compartments, we investigated the adaptation dynamics of two different human fecal microbial communities over 22 and 31 days. By observing the temporal trends of different community members, we found that many dominant members of the fecal microbiota failed to maintain their dominance in vitro, and some of the low-abundance microbes undetected in the fecal microbiota successfully grew in the in vitro communities. Microbiome compositional changes and blooming could largely be explained by feed composition and pH, suggesting that these communities can be modulated to desired compositions via optimizing culture conditions. Thus, our results open up the possibility of modulating in vitro microbial communities to predefined compositions by optimizing feed composition and culture conditions.

Changes in the lipidome in type 1 diabetes following low carbohydrate diet: Post-hoc analysis of a randomized crossover trial.

Aims: Lipid metabolism might be compromised in type 1 diabetes, and the understanding of lipid physiology is critically important. This study aimed to compare the change in plasma lipid concentrations during carbohydrate dietary changes in individuals with type 1 diabetes and identify links to early-stage dyslipidaemia. We hypothesized that (1) the lipidomic profiles after ingesting low or high carbohydrate diet for 12 weeks would be different; and (2) specific annotated lipid species could have significant associations with metabolic outcomes. Methods: Ten adults with type 1 diabetes (mean ± SD: age 43.6 ± 13.8 years, diabetes duration 24.5 ± 13.4 years, BMI 24.9 ± 2.1 kg/m2, HbA1c 57.6 ± 2.6 mmol/mol) using insulin pumps participated in a randomized 2-period crossover study with a 12-week intervention period of low carbohydrate diet (< 100 g carbohydrates/day) or high carbohydrate diet (> 250 g carbohydrates/day), respectively, separated by a 12-week washout period. A large-scale non-targeted lipidomics was performed with mass spectrometry in fasting plasma samples obtained before and after each diet intervention. Longitudinal lipid levels were analysed using linear mixed-effects models. Results: In total, 289 lipid species were identified from 14 major lipid classes. Comparing the two diets, 11 lipid species belonging to sphingomyelins, phosphatidylcholines and LPC(O-16:0) were changed. All the 11 lipid species were significantly elevated during low carbohydrate diet. Two lipid species were most differentiated between diets, namely SM(d36:1) (ß ± SE: 1.44 ± 0.28, FDR = 0.010) and PC(P-36:4)/PC(O-36:5) (ß ± SE: 1.34 ± 0.25, FDR = 0.009) species. Polyunsaturated PC(35:4) was inversely associated with BMI and positively associated with HDL cholesterol (p < .001). Conclusion: Lipidome-wide outcome analysis of a randomized crossover trial of individuals with type 1 diabetes following a low carbohydrate diet showed an increase in sphingomyelins and phosphatidylcholines which are thought to reduce dyslipidaemia. The polyunsaturated phosphatidylcholine 35:4 was inversely associated with BMI and positively associated with HDL cholesterol (p < .001). Results from this study warrant for more investigation on the long-term effect of single lipid species in type 1 diabetes.

Metabolite Changes After Metabolic Surgery - Associations to Parameters Reflecting Glucose Homeostasis and Lipid Levels.

Aims: To test the hypothesis that adipose tissue gene expression patterns would be affected by metabolic surgery and we aimed to identify genes and metabolic pathways as well as metabolites correlating with metabolic changes following metabolic surgery. Materials and Methods: This observational study was conducted at the Obesity Unit at the Catholic University Hospital of the Sacred Heart in Rome, Italy. Fifteen patients, of which six patients underwent Roux-en-Y gastric bypass and nine patients underwent biliopancreatic diversion, were included. The participants underwent an oral glucose tolerance test and a hyperinsulinemic euglycemic clamp. Small polar metabolites were analyzed with a two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). Gene expression analysis of genes related to metabolism of amino acids and fatty acids were analyzed in subcutaneous adipose tissue. All procedures were performed at study start and at follow-up (after 185.3 ± 72.9 days). Results: Twelve metabolites were significantly changed after metabolic surgery. Six metabolites were identified as 3-indoleacetic acid, 2-hydroxybutyric acid, valine, glutamic acid, 4-hydroxybenzeneacetic acid and alpha-tocopherol. The branched chain amino acids displayed a significant decrease together with a decrease in BCAT1 adipose tissue mRNA levels. Changes in the identified metabolites were associated to changes in lipid, insulin and glucose levels. Conclusions: Our study has identified metabolites and metabolic pathways that are altered by metabolic surgery and may be used as biomarkers for metabolic improvement.

Characterization of plasma lipidomics in adolescent subjects with increased risk for type 1 diabetes in the DiPiS cohort.

INTRODUCTION: Type 1 diabetes (T1D) is caused by the destruction of pancreatic islet beta cells resulting in total loss of insulin production. Recent studies have suggested that the destruction may be interrelated to plasma lipids. OBJECTIVES: Specific lipids have previously been shown to be decreased in children who develop T1D before four years of age. Disturbances of plasma lipids prior to clinical diagnosis of diabetes, if true, may provide a novel way to improve prediction, and monitor disease progression. METHODS: A lipidomic approach was utilized to analyze plasma from 67 healthy adolescent subjects (10-15 years of age) with or without islet autoantibodies but all with increased genetic risk for T1D. The study subjects were enrolled at birth in the Diabetes Prediction in Skåne (DiPiS) study and after 10-15 years of follow-up we performed the present cross-sectional analysis. HLA-DRB345, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 genotypes were determined using next generation sequencing. Lipidomic profiles were determined using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. Lipidomics data were analyzed according to genotype. RESULTS: Variation in levels of several specific phospholipid species were related to level of autoimmunity but not development of T1D. Five glycosylated ceramides were increased in insulin autoantibody (IAA) positive adolescent subjects compared to adolescent subjects without this autoantibody. Additionally, HLA genotypes seemed to influence levels of long chain triacylglycerol (TG). CONCLUSION: Lipidomic profiling of adolescent subjects in high risk of T1D may improve sub-phenotyping in this high risk population.

Circulating Metabolites and Lipids Are Associated to Diabetic Retinopathy in Individuals With Type 1 Diabetes.

Omics-based methods may provide new markers associated to diabetic retinopathy (DR). We investigated a wide omics panel of metabolites and lipids related to DR in type 1 diabetes. Metabolomic analyses were performed using two-dimensional gas chromatography with time-of-flight mass spectrometry and lipidomic analyses using an ultra-high-performance liquid chromatography quadruple time-of-flight mass spectrometry method in 648 individuals with type 1 diabetes. Subjects were subdivided into no DR, mild nonproliferative DR (NPDR), moderate NPDR, proliferative DR, and proliferative DR with fibrosis. End points were any progression of DR, onset of DR, and progression from mild to severe DR tracked from standard ambulatory care and investigated using Cox models. The cohort consisted of 648 participants aged a mean of 54.4 ± 12.8 years, 55.5% were men, and follow-up was 5.1-5.5 years. Cross-sectionally, 2,4-dihydroxybutyric acid (DHBA), 3,4-DHBA, ribonic acid, ribitol, and the triglycerides 50:1 and 50:2 significantly correlated (P < 0.042) to DR stage. Longitudinally, higher 3,4-DHBA was a risk marker for progression of DR (n = 133) after adjustment (P = 0.033). We demonstrated multiple metabolites being positively correlated to a higher grade of DR in type 1 diabetes and several triglycerides being negatively correlated. Furthermore, higher 3,4-DHBA was an independent risk marker for progression of DR; however, confirmation is required.

Lipidomics of human adipose tissue reveals diversity between body areas.

BACKGROUND AND AIMS: Adipose tissue plays a pivotal role in storing excess fat and its composition reflects the history of person's lifestyle and metabolic health. Broad profiling of lipids with mass spectrometry has potential for uncovering new knowledge on the pathology of obesity, metabolic syndrome, diabetes and other related conditions. Here, we developed a lipidomic method for analyzing human subcutaneous adipose biopsies. We applied the method to four body areas to understand the differences in lipid composition between these areas. MATERIALS AND METHODS: Adipose tissue biopsies from 10 participants were analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The sample preparation optimization included the optimization of the lipid extraction, the sample amount and the sample dilution factor to detect lipids in an appropriate concentration range. Lipidomic analyses were performed for adipose tissue collected from the abdomen, breast, thigh and lower back. Differences in lipid levels between tissues were visualized with heatmaps. RESULTS: Lipidomic analysis on human adipose biopsies lead to the identification of 186lipids in 2 mg of sample. Technical variation of the lipid-class specific internal standards were below 5%, thus indicating acceptable repeatability. Triacylglycerols were highly represented in the adipose tissue samples, and lipids from 13 lipid classes were identified. Long polyunsaturated triacylglycerols in higher levels in thigh (q<0.05), when compared with the abdomen, breast and lower back, indicating that the lipidome was area-specific. CONCLUSION: The method presented here is suitable for the analysis of lipid profiles in 2 mg of adipose tissue. The amount of fat across the body is important for health but we argue that also the distribution and the particular profile of the lipidome may be relevant for metabolic outcomes. We suggest that the method presented in this paper could be useful for detecting such aberrations.