Sex differences in adipose insulin resistance are linked to obesity, lipolysis and insulin receptor substrate 1.

BACKGROUND/OBJECTIVE: Insulin resistance is more prominent in men than women. If this involves adipose tissue is unknown and was presently examined. SUBJECTS/METHODS: AdipoIR (in vivo adipose insulin resistance index) was measured in 2344 women and 787 men. In 259 of the women and 54 of the men, insulin induced inhibition of lipolysis (acylglycerol breakdown) and stimulation of lipogenesis (glucose conversion to acylglycerols) were determined in subcutaneous adipocytes; in addition, basal (spontaneous) lipolysis was also determined in the fat cells. In 234 women and 115 men, RNAseq expression of canonical insulin signal genes were measured in subcutaneous adipose tissue. Messenger RNA transcripts of the most discriminant genes were quantified in 175 women and 109 men. RESULTS: Men had higher AdipoIR values than women but only when obesity (body mass index 30 kg/m2 or more) was present (p < 0.0001). The latter sex dimorphism was found among physically active and sedentary people, in those with and without cardiometabolic disease and in people using nicotine or not (p = 0.0003 or less). In obesity, adipocyte insulin sensitivity (half maximum effective hormone concentration) and maximal antilipolytic effect were tenfold and 10% lower, respectively, in men than women (p = 0.005 or less). Basal rate of lipolysis was two times higher in men than women (p > 0.0001). Sensitivity and maximum effect of insulin on lipogenesis were similar in both sexes (p = 0.26 and p = 0.18, respectively). When corrected for multiple comparison only RNAseq expression of insulin receptor substrate 1 (IRS1) was lower in men than women (p < 0.0001). The mRNA transcript for IRS1 was 60% higher in women than men (p < 0.0001). CONCLUSIONS: In obesity, adipose tissue insulin resistance is more pronounced in men than in women. The mechanism involves less efficient insulin-mediated inhibition of adipocyte lipolysis, increased basal rate of lipolysis and decreased adipose expression of a key element of insulin signaling, IRS1.

Risk assessment of nutrients: There must be a threshold for their effects.

Nutrients serve physiological functions in a dose-dependent manner and that needs to be recognized in risk assessment. An example of the consequences of not properly considering this can be seen in a recent assessment by the European Food Safety Authority (EFSA). EFSA concluded in 2022 that the intake of added and free sugars should be "as low as possible in the context of a nutritionally adequate diet". That conclusion of EFSA is based on the effects on two surrogate endpoints for an adverse effect found in randomized controlled trials with high sugars intake levels: fasting glucose and fasting triglycerides. The lowest intake levels in these trials were around 10 energy% and at this intake level there were no adverse effects on the two outcomes. This indicates that the adverse effects of sugars have an observable threshold value for these two endpoints. The most appropriate interpretation from the vast amount of data is that currently no definitive conclusion can be drawn on the tolerable upper intake level for dietary sugars. Therefore, EFSA's own guidance would lead to the conclusion that the available data do not allow the setting of an upper limit for added sugars and hence, that more robust data are required to identify the threshold value for intake of sugars.

The New Nordic Renal Diet Induces a Pronounced Reduction of Urine Acid Excretion and Uremic Toxins in Chronic Kidney Disease Patients (Stage 3 and 4).

OBJECTIVE: Metabolic acidosis and the uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are associated with increased risks of kidney disease progression, muscle catabolism, cardiovascular disease, and mortality in patients with chronic kidney disease (CKD). The New Nordic Renal Diet (NNRD) is a plant-focused meal pattern, with reduced phosphorus and protein content compared to an average Danish diet. Due to a higher amount of plant-based products, we hypothesized that NNRD would reduce renal excretion of acids and uremic toxins. Thus, we evaluated the effects of NNRD on metabolic acidosis and uremic toxins in patients with moderate CKD, stages 3-4. DESIGN AND METHODS: This post hoc analysis is based on a randomized controlled crossover trial comparing 1 week of the NNRD to a control 1-week period of an average Danish diet, in patients with CKD stages 3 and 4. Urine pH and urine excretion of bicarbonate, ammonium, titratable acids, IS, and PCS alongside plasma total CO2 (tCO2) were measured at days 1, 4, and 7 in 18 patients. RESULTS: After 7 days on NNRD 24-hour urine net acid excretion was decreased by 80% (P < .001), 24-hour urine excretion of ammonium and bicarbonate decreased by 34% (P < .001), and increased by 678% (P < .001), respectively, compared with the control period. Plasma tCO2 was increased by 8% (P = .005). Moreover, 24-hour urine excretion of PCS and IS were reduced by 31% (P = .018) and 29% (P < .001), respectively. CONCLUSION: One week of NNRD in patients suffering from moderate CKD effectively improved metabolic acidosis with a marked reduction in urine net acid excretion that included a large increase in urine bicarbonate excretion. In addition, NNRD reduced urinary excretion of the uremic toxins PCS and IS. These results encourage further investigations of the long-term effects of NNRD on renal protection in patients with CKD.

Dietary carbohydrate restriction augments weight loss-induced improvements in glycaemic control and liver fat in individuals with type 2 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: Lifestyle modification and weight loss are cornerstones of type 2 diabetes management. However, carbohydrate restriction may have weight-independent beneficial effects on glycaemic control. This has been difficult to demonstrate because low-carbohydrate diets readily decrease body weight. We hypothesised that carbohydrate restriction enhances the beneficial metabolic effects of weight loss in type 2 diabetes. METHODS: This open-label, parallel RCT included adults with type 2 diabetes, HbA1c 48-97 mmol/mol (6.5-11%), BMI >25 kg/m2, eGFR >30 ml min-1 [1.73 m]-2 and glucose-lowering therapy restricted to metformin or dipeptidyl peptidase-4 inhibitors. Participants were randomised by a third party and assigned to 6 weeks of energy restriction (all foods were provided) aiming at ~6% weight loss with either a carbohydrate-reduced high-protein diet (CRHP, percentage of total energy intake [E%]: CH30/P30/F40) or a conventional diabetes diet (CD, E%: CH50/P17/F33). Fasting blood samples, continuous glucose monitoring and magnetic resonance spectroscopy were used to assess glycaemic control, lipid metabolism and intrahepatic fat. Change in HbA1c was the primary outcome; changes in circulating and intrahepatic triacylglycerol were secondary outcomes. Data were collected at Copenhagen University Hospital (Bispebjerg and Herlev). RESULTS: Seventy-two adults (CD 36, CRHP 36, all white, 38 male sex) with type 2 diabetes (mean duration 8 years, mean HbA1c 57 mmol/mol [7.4%]) and mean BMI of 33 kg/m2 were enrolled, of which 67 (CD 33, CRHP 34) completed the study. Body weight decreased by 5.8 kg (5.9%) in both groups after 6 weeks. Compared with the CD diet, the CRHP diet further reduced HbA1c (mean [95% CI] -1.9 [-3.5, -0.3] mmol/mol [-0.18 (-0.32, -0.03)%], p = 0.018) and diurnal mean glucose (mean [95% CI] -0.8 [-1.2, -0.4] mmol/l, p < 0.001), stabilised glucose excursions by reducing glucose CV (mean [95% CI] -4.1 [-5.9, -2.2]%, p < 0.001), and augmented the reductions in fasting triacylglycerol concentration (by mean [95% CI] -18 [-29, -6]%, p < 0.01) and liver fat content (by mean [95% CI] -26 [-45, 0]%, p = 0.051). However, pancreatic fat content was decreased to a lesser extent by the CRHP than the CD diet (mean [95% CI] 33 [7, 65]%, p = 0.010). Fasting glucose, insulin, HOMA2-IR and cholesterol concentrations (total, LDL and HDL) were reduced significantly and similarly by both diets. CONCLUSIONS/INTERPRETATION: Moderate carbohydrate restriction for 6 weeks modestly improved glycaemic control, and decreased circulating and intrahepatic triacylglycerol levels beyond the effects of weight loss itself compared with a CD diet in individuals with type 2 diabetes. Concurrent differences in protein and fat intakes, and the quality of dietary macronutrients, may have contributed to these results and should be explored in future studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT03814694. FUNDING: The study was funded by Arla Foods amba, The Danish Dairy Research Foundation, and Copenhagen University Hospital Bispebjerg Frederiksberg.

The triglyceride-glucose index as an adiposity marker and a predictor of fat loss induced by a low-calorie diet.

BACKGROUND: This study aimed to investigate the putative role of the triglyceride-glucose index (TyG index) computed as ln[TG (mg/dl) × glucose (mg/dl)/2] and derived proxies as predictors of adiposity and weight loss changes after a low-calorie diet (LCD) intervention. METHODS: A total of 744 adult participants from the multicentre DIOGenes intervention study were prescribed a LCD (800 kcal/day) during 8 weeks. Body composition and fat content at baseline and after 8 weeks were estimated by DEXA/BIA. A multivariate analysis approach was used to estimate the difference in ΔWeight1-2 (kg), ΔBMI1-2 (kg/m2 ) or ΔFat1-2 (%) between the basal value (point 1) and after 8 weeks following a LCD (point 2), respectively. The TyG index at baseline (TyG1 ), after following the LCD for 8 weeks (TyG2 ) or the TyG index differences between both time points (ΔTyG1-2 ) were analysed as predictors of weight and fat changes. RESULTS: TyG1 was associated with ΔWeight1-2 (kg) and ΔBMI1-2 (kg/m2 ), with ß = 0.812 (p = .017) and ß = 0.265 (p = .018), respectively. Also, TyG2  values were inversely related to ΔFat1-2 (%), ß = -1.473 (p = .015). Moreover, ΔTyG1-2 was associated with ΔWeight1-2 (kg) and ΔFat1-2 (%), ß = 0.689 (p = .045) and ß = 1.764 (p = .002), respectively. Furthermore, an association between TyG2 and resistance to fat loss was found (p = .015). CONCLUSION: TyG1 index is a good predictor of weight loss induced by LCD. Moreover, TyG2 was closely related to resistance to fat loss, while ΔTyG1-2  values were positively associated with body fat changes. Therefore, TyG index and derived estimations could be used as markers of individualized responses to energy restriction and a surrogate of body composition outcomes in clinical/epidemiological settings in obesity conditions.

Personalized computational model quantifies heterogeneity in postprandial responses to oral glucose challenge.

Plasma glucose and insulin responses following an oral glucose challenge are representative of glucose tolerance and insulin resistance, key indicators of type 2 diabetes mellitus pathophysiology. A large heterogeneity in individuals' challenge test responses has been shown to underlie the effectiveness of lifestyle intervention. Currently, this heterogeneity is overlooked due to a lack of methods to quantify the interconnected dynamics in the glucose and insulin time-courses. Here, a physiology-based mathematical model of the human glucose-insulin system is personalized to elucidate the heterogeneity in individuals' responses using a large population of overweight/obese individuals (n = 738) from the DIOGenes study. The personalized models are derived from population level models through a systematic parameter selection pipeline that may be generalized to other biological systems. The resulting personalized models showed a 4-5 fold decrease in discrepancy between measurements and model simulation compared to population level. The estimated model parameters capture relevant features of individuals' metabolic health such as gastric emptying, endogenous insulin secretion and insulin dependent glucose disposal into tissues, with the latter also showing a significant association with the Insulinogenic index and the Matsuda insulin sensitivity index, respectively.

Susceptibility of Human Plasma N-glycome to Low-Calorie and Different Weight-Maintenance Diets.

Aberrant plasma protein glycosylation is associated with a wide range of diseases, including diabetes, cardiovascular, and immunological disorders. To investigate plasma protein glycosylation alterations due to weight loss and successive weight-maintenance diets, 1850 glycomes from participants of the Diogenes study were analyzed using Ultra-High-Performance Liquid Chromatography (UHPLC). The Diogenes study is a large dietary intervention study in which participants were subjected to a low-calorie diet (LCD) followed by one of five different weight-maintenance diets in a period of 6 months. The most notable alterations of the plasma glycome were 8 weeks after the subjects engaged in the LCD; a significant increase in low-branched glycan structures, accompanied by a decrease in high-branched glycan structures. After the LCD period, there was also a significant rise in N-glycan structures with antennary fucose. Interestingly, we did not observe significant changes between different diets, and almost all effects we observed immediately after the LCD period were annulled during the weight-maintenance diets period.

The Plasma Oxylipin Signature Provides a Deep Phenotyping of Metabolic Syndrome Complementary to the Clinical Criteria.

Metabolic syndrome (MetS) is a complex condition encompassing a constellation of cardiometabolic abnormalities. Oxylipins are a superfamily of lipid mediators regulating many cardiometabolic functions. Plasma oxylipin signature could provide a new clinical tool to enhance the phenotyping of MetS pathophysiology. A high-throughput validated mass spectrometry method, allowing for the quantitative profiling of over 130 oxylipins, was applied to identify and validate the oxylipin signature of MetS in two independent nested case/control studies involving 476 participants. We identified an oxylipin signature of MetS (coined OxyScore), including 23 oxylipins and having high performances in classification and replicability (cross-validated AUCROC of 89%, 95% CI: 85-93% and 78%, 95% CI: 72-85% in the Discovery and Replication studies, respectively). Correlation analysis and comparison with a classification model incorporating the MetS criteria showed that the oxylipin signature brings consistent and complementary information to the clinical criteria. Being linked with the regulation of various biological processes, the candidate oxylipins provide an integrative phenotyping of MetS regarding the activation and/or negative feedback regulation of crucial molecular pathways. This may help identify patients at higher risk of cardiometabolic diseases. The oxylipin signature of patients with metabolic syndrome enhances MetS phenotyping and may ultimately help to better stratify the risk of cardiometabolic diseases.

Effects of carbohydrate restriction on postprandial glucose metabolism, ß-cell function, gut hormone secretion, and satiety in patients with Type 2 diabetes.

Dietary carbohydrate restriction may improve the phenotype of Type 2 diabetes (T2D) patients. We aimed to investigate 6 wk of carbohydrate restriction on postprandial glucose metabolism, pancreatic α- and ß-cell function, gut hormone secretion, and satiety in T2D patients. Methods In a crossover design, 28 T2D patients (mean HbA1c: 60 mmol/mol) were randomized to 6 wk of carbohydrate-reduced high-protein (CRHP) diet and 6 wk of conventional diabetes (CD) diet (energy-percentage carbohydrate/protein/fat: 30/30/40 vs. 50/17/33). Twenty-four-hour continuous glucose monitoring (CGM) and mixed-meal tests were undertaken and fasting intact proinsulin (IP), 32,33 split proinsulin concentrations (SP), and postprandial insulin secretion rates (ISR), insulinogenic index (IGI), ß-cell sensitivity to glucose (Bup), glucagon, and gut hormones were measured. Gastric emptying was evaluated by postprandial paracetamol concentrations and satiety by visual analog scale ratings. A CRHP diet reduced postprandial glucose area under curve (net AUC) by 60% (P < 0.001), 24 h glucose by 13% (P < 0.001), fasting IP and SP concentrations (both absolute and relative to C-peptide, P < 0.05), and postprandial ISR (24%, P = 0.015), while IGI and Bup improved by 31% and 45% (both P < 0.001). The CRHP diet increased postprandial glucagon net AUC by 235% (P < 0.001), subjective satiety by 18% (P = 0.03), delayed gastric emptying by 15 min (P < 0.001), decreased gastric inhibitory polypeptide net AUC by 29% (P < 0.001), but had no significant effect on glucagon-like-peptide-1, total peptide YY, and cholecystokinin responses. A CRHP diet reduced glucose excursions and improved ß-cell function, including proinsulin processing, and increased subjective satiety in patients with T2D.

Estimating and reporting treatment effects in clinical trials for weight management: using estimands to interpret effects of intercurrent events and missing data.

In the approval process for new weight management therapies, regulators typically require estimates of effect size. Usually, as with other drug evaluations, the placebo-adjusted treatment effect (i.e., the difference between weight losses with pharmacotherapy and placebo, when given as an adjunct to lifestyle intervention) is provided from data in randomized clinical trials (RCTs). At first glance, this may seem appropriate and straightforward. However, weight loss is not a simple direct drug effect, but is also mediated by other factors such as changes in diet and physical activity. Interpreting observed differences between treatment arms in weight management RCTs can be challenging; intercurrent events that occur after treatment initiation may affect the interpretation of results at the end of treatment. Utilizing estimands helps to address these uncertainties and improve transparency in clinical trial reporting by better matching the treatment-effect estimates to the scientific and/or clinical questions of interest. Estimands aim to provide an indication of trial outcomes that might be expected in the same patients under different conditions. This article reviews how intercurrent events during weight management trials can influence placebo-adjusted treatment effects, depending on how they are accounted for and how missing data are handled. The most appropriate method for statistical analysis is also discussed, including assessment of the last observation carried forward approach, and more recent methods, such as multiple imputation and mixed models for repeated measures. The use of each of these approaches, and that of estimands, is discussed in the context of the SCALE phase 3a and 3b RCTs evaluating the effect of liraglutide 3.0 mg for the treatment of obesity.

A fully joint Bayesian quantitative trait locus mapping of human protein abundance in plasma.

Molecular quantitative trait locus (QTL) analyses are increasingly popular to explore the genetic architecture of complex traits, but existing studies do not leverage shared regulatory patterns and suffer from a large multiplicity burden, which hampers the detection of weak signals such as trans associations. Here, we present a fully multivariate proteomic QTL (pQTL) analysis performed with our recently proposed Bayesian method LOCUS on data from two clinical cohorts, with plasma protein levels quantified by mass-spectrometry and aptamer-based assays. Our two-stage study identifies 136 pQTL associations in the first cohort, of which >80% replicate in the second independent cohort and have significant enrichment with functional genomic elements and disease risk loci. Moreover, 78% of the pQTLs whose protein abundance was quantified by both proteomic techniques are confirmed across assays. Our thorough comparisons with standard univariate QTL mapping on (1) these data and (2) synthetic data emulating the real data show how LOCUS borrows strength across correlated protein levels and markers on a genome-wide scale to effectively increase statistical power. Notably, 15% of the pQTLs uncovered by LOCUS would be missed by the univariate approach, including several trans and pleiotropic hits with successful independent validation. Finally, the analysis of extensive clinical data from the two cohorts indicates that the genetically-driven proteins identified by LOCUS are enriched in associations with low-grade inflammation, insulin resistance and dyslipidemia and might therefore act as endophenotypes for metabolic diseases. While considerations on the clinical role of the pQTLs are beyond the scope of our work, these findings generate useful hypotheses to be explored in future research; all results are accessible online from our searchable database. Thanks to its efficient variational Bayes implementation, LOCUS can analyze jointly thousands of traits and millions of markers. Its applicability goes beyond pQTL studies, opening new perspectives for large-scale genome-wide association and QTL analyses. Diet, Obesity and Genes (DiOGenes) trial registration number: NCT00390637.

Egg consumption and cardiovascular risk: a dose-response meta-analysis of prospective cohort studies.

PURPOSE: Cardiovascular disease (CVD) is a leading cause of mortality globally and is strongly influenced by dietary risk factors. The aim was to assess the association between egg consumption and risk of CVD risk/mortality, including coronary heart disease (CHD), stroke, and heart failure. METHODS: MEDLINE, Embase, and Web of Science databases were searched through April 2020 for prospective studies. Two independent reviewers screened and extracted the data through standardized methods. Size effects were calculated as summary relative risks (SRRs) in a dose-response fashion through random-effects meta-analyses. RESULTS: Thirty-nine studies including nearly 2 million individuals and 85,053 CHD, 25,103 stroke, 7536 heart failure, and 147,124 CVD cases were included. The summary analysis including 17 datasets from 14 studies conducted on CVD (incidence and/or mortality) showed that intake of up to six eggs per week is inversely associated with CVD events, when compared to no consumption [for four eggs per week, SRR = 0.95 (95% CI: 0.90; 1.00)]; a decreased risk of CVD incidence was observed for consumption of up to one egg per day [SRR = 0.94 (95% CI: 0.89; 0.99)]. The summary analysis for CHD incidence/mortality including 24 datasets from 16 studies showed a decreased risk up to two eggs per week [(SRR = 0.96 (95% CI: 0.91; 1.00)]. No associations were retrieved with risk of stroke. The summary analysis for heart failure risk including six datasets from four studies showed that intake of one egg per day was associated with increased risk raising for higher intakes compared to no consumption [for 1 egg per day, SRR = 1.15 (95% CI:1.02; 1.30)]. After considering GRADE criteria for strength of the evidence, it was rated low for all outcomes but stroke, for which it was moderate (yet referring to no risk). CONCLUSION: There is no conclusive evidence on the role of egg in CVD risk, despite the fact that higher quality studies are warranted to obtain stronger evidence for a possible protection of CVD associated with moderate weekly egg consumption compared to no intake; equally, future studies may strengthen the evidence for increased heart failure risk associated with high regular egg consumption.

Body weight and metabolic risk factors in patients with type 2 diabetes on a self-selected high-protein low-carbohydrate diet.

PURPOSE: We previously reported beneficial glucoregulatory effects of a fully provided carbohydrate-reduced, high-protein (CRHP) diet in patients with type 2 diabetes mellitus (T2DM) in a crossover 2 × 6-week trial, in which patients maintained their body weight. Here, we investigated physiological changes during an additional 6-month period on a self-selected and self-prepared CRHP diet. METHODS: Twenty-eight patients with T2DM were instructed to consume a CRHP diet (30% of energy from carbohydrate and 30% from protein) for 24 weeks, after an initial 2 × 6-week trial when all food was prepared and provided to them. Patients received dietary advice every 2 weeks. At weeks 0, 6, 12 and 36, they underwent a 3-h intravenous glucose tolerance test, a 4-h mixed meal test, and a 48-h continuous glucose monitoring. Liver, muscle, pancreas, and visceral fat contents were measured by magnetic resonance imaging. RESULTS: During the 24-week self-selected diet period (weeks 12-36), body weight, visceral fat, liver fat, and glycated haemoglobin were maintained at the same levels achieved at the end of the fully provided diet period, and were still lower than at baseline (P < 0.05). Postprandial insulinaemia and insulin secretion were significantly greater (P < 0.05). At week 36, fasting insulin and C-peptide levels increased (P < 0.01) and daily glycaemia decreased further (P < 0.05) when compared with the end of the fully provided diet period. CONCLUSION: Substituting dietary carbohydrate for protein and fat has metabolic benefits in patients with T2DM. These beneficial effects are maintained or augmented over the next 6 months when patients self-select and self-prepare this diet in a dietitian-supported setting. TRIAL REGISTRATION: ClinicalTrials.gov NCT02764021.

Sagittal abdominal diameter and waist circumference appear to be equally good as identifiers of cardiometabolic risk.

BACKGROUND AND AIMS: Body mass index (BMI) and waist circumference (WC) are commonly used markers of cardiometabolic risk. However, sagittal abdominal diameter (SAD) has been proposed as a possibly more sensitive marker of intra-abdominal obesity. We investigated differences in how SAD, WC, and BMI were correlated with cardiometabolic risk markers. METHODS AND RESULTS: This cross-sectional study investigated anthropometric and metabolic baseline measurements of individuals from six trials. Multiple linear regression and (partial) correlation coefficients were used to investigate associations between SAD, WC, and BMI and cardiometabolic risk markers, including components of the metabolic syndrome as well as insulin resistance, blood lipids, and lowgrade inflammation. In total 1516 mostly overweight or obese individuals were included in the study. SAD was significantly more correlated with TG than WC for all studies, and overall increase in correlation was 0.05 (95% CI (0.02; 0.08). SAD was significantly more correlated with the markers TG and DBP 0.11 (95% CI (0.08, 0.14)) and 0.04 (95% CI (0.006, 0.07), respectively compared to BMI across all or most studies. CONCLUSION: This study showed that no single anthropometric indicator was consistently more strongly correlated across all markers of cardiometabolic risk. However, SAD was significantly more strongly correlated with TG than WC and significantly more strongly correlated with DBP and TG than BMI.

Analysis of 1508 Plasma Samples by Capillary-Flow Data-Independent Acquisition Profiles Proteomics of Weight Loss and Maintenance.

Comprehensive, high throughput analysis of the plasma proteome has the potential to enable holistic analysis of the health state of an individual. Based on our own experience and the evaluation of recent large-scale plasma mass spectrometry (MS) based proteomic studies, we identified two outstanding challenges: slow and delicate nano-flow liquid chromatography (LC) and irreproducibility of identification of data-dependent acquisition (DDA). We determined an optimal solution reducing these limitations with robust capillary-flow data-independent acquisition (DIA) MS. This platform can measure 31 plasma proteomes per day. Using this setup, we acquired a large-scale plasma study of the diet, obesity and genes dietary (DiOGenes) comprising 1508 samples. Proving the robustness, the complete acquisition was achieved on a single analytical column. Totally, 565 proteins (459 identified with two or more peptide sequences) were profiled with 74% data set completeness. On average 408 proteins (5246 peptides) were identified per acquisition (319 proteins in 90% of all acquisitions). The workflow reproducibility was assessed using 34 quality control pools acquired at regular intervals, resulting in 92% data set completeness with CVs for protein measurements of 10.9%.The profiles of 20 apolipoproteins could be profiled revealing distinct changes. The weight loss and weight maintenance resulted in sustained effects on low-grade inflammation, as well as steroid hormone and lipid metabolism, indicating beneficial effects. Comparison to other large-scale plasma weight loss studies demonstrated high robustness and quality of biomarker candidates identified. Tracking of nonenzymatic glycation indicated a delayed, slight reduction of glycation in the weight maintenance phase. Using stable-isotope-references, we could directly and absolutely quantify 60 proteins in the DIA.In conclusion, we present herein the first large-scale plasma DIA study and one of the largest clinical research proteomic studies to date. Application of this fast and robust workflow has great potential to advance biomarker discovery in plasma.

Metabolic profiling of tissue-specific insulin resistance in human obesity: results from the Diogenes study and the Maastricht Study.

BACKGROUND: Recent evidence indicates that insulin resistance (IR) in obesity may develop independently in different organs, representing different etiologies toward type 2 diabetes and other cardiometabolic diseases. The aim of this study was to investigate whether IR in the liver and IR in skeletal muscle are associated with distinct metabolic profiles. METHODS: This study includes baseline data from 634 adults with overweight or obesity (BMI ≥ 27 kg/m2) (≤65 years; 63% women) without diabetes of the European Diogenes Study. Hepatic insulin resistance index (HIRI) and muscle insulin sensitivity index (MISI), were derived from a five-point OGTT. At baseline 17 serum metabolites were identified and quantified by nuclear-magnetic-resonance spectroscopy. Linear mixed model analyses (adjusting for center, sex, body mass index (BMI), waist-to-hip ratio) were used to associate HIRI and MISI with these metabolites. In an independent sample of 540 participants without diabetes (BMI ≥ 27 kg/m2; 40-65 years; 46% women) of the Maastricht Study, an observational prospective population-based cohort study, 11 plasma metabolites and a seven-point OGTT were available for validation. RESULTS: Both HIRI and MISI were associated with higher levels of valine, isoleucine, oxo-isovaleric acid, alanine, lactate, and triglycerides, and lower levels of glycine (all p < 0.05). HIRI was also associated with higher levels of leucine, hydroxyisobutyrate, tyrosine, proline, creatine, and n-acetyl and lower levels of acetoacetate and 3-OH-butyrate (all p < 0.05). Except for valine, these results were replicated for all available metabolites in the Maastricht Study. CONCLUSIONS: In persons with obesity without diabetes, both liver and muscle IR show a circulating metabolic profile of elevated (branched-chain) amino acids, lactate, and triglycerides, and lower glycine levels, but only liver IR associates with lower ketone body levels and elevated ketogenic amino acids in circulation, suggestive of decreased ketogenesis. This knowledge might enhance developments of more targeted tissue-specific interventions to prevent progression to more severe disease stages.

Quantification of diacylglycerol and triacylglycerol species in human fecal samples by flow injection Fourier transform mass spectrometry.

The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites provides insight in metabolic interactions between gut microbiota and host organism. In this work, we applied flow injection analysis coupled to Fourier transform mass spectrometry (FIA-FTMS) to identify and quantify lipid species in human fecal samples. Fecal homogenates were subjected to lipid extraction and analyzed by FIA-FTMS. The analysis of different subjects revealed a vast heterogeneity of lipid species abundance. The majority of samples displayed prominent signals of triacylglycerol (TG) and diacylglycerol (DG) species that could be verified by MS2 spectra. Therefore, we focused on the quantification of TG and DG. Method validation included limit of quantification, linearity, evaluation of matrix effects, recovery, and reproducibility. The validation experiments demonstrated the suitability of the method, with exception for approximately 10% of samples, where we observed coefficients of variation higher than 15%. Impaired reproducibility was related to sample inhomogeneity and could not be improved by additional sample preparation steps. Additionally, these experiments demonstrated that compared with aqueous samples, samples containing isopropanol showed higher amounts of DG, presumably due to lysis of bacteria and increased TG lipolysis. These effects were sample-specific and substantiate the high heterogeneity of fecal materials as well as the need for further evaluation of pre-analytic conditions. In summary, FIA-FTMS offers a fast and accurate tool to quantify DG and TG species and is suitable to provide insight into the fecal lipidome and its role in health and disease.

Effects of a highly controlled carbohydrate-reduced high-protein diet on markers of oxidatively generated nucleic acid modifications and inflammation in weight stable participants with type 2 diabetes; a randomized controlled trial.

Carbohydrate-restricted diets are increasingly recognized as options for dietary management of type 2 diabetes mellitus (T2DM). We investigated the effects of a carbohydrate-reduced high-protein (CRHP) and a conventional diabetes (CD) diet on oxidative stress and inflammation in weight stable individuals with T2DM. We hypothesized that the CRHP diet would improve markers of oxidatively generated RNA and DNA modifications as well as inflammatory parameters. Thirty participants with T2DM were randomized to 6 weeks of CRHP or CD dietary treatment (30/50 energy percentage (E%) carbohydrate, 30/17E% protein, 40/33E% fat), followed by a cross-over to the opposite diet for a subsequent 6-week period. All meals were provided during the study and body weight was controlled. Diurnal urine samples were collected after 4 weeks on each diet and oxidatively generated RNA and DNA modifications were measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), respectively. Fasting concentrations of soluble urokinase plasminogen activator receptor, high-sensitivity C-reactive protein, tumor necrosis factor alpha and interleukin-6 were measured before and after 6 weeks of interventions. Compared with the CD diet, the CRHP diet increased 24-hour urinary excretion of 8-oxoGuo by 9.3% (38.6 ± 12.6 vs. 35.3 ± 11.0 nmol/24 h, p = .03), whereas 8-oxodG did not differ between diets (24.0 ± 9.5 vs. 24.8 ± 11.1 nmol/24 h, p = .17). Changes in plasma inflammatory parameters did not differ between CRHP and CD diets, all p ≥ .2. The clinical implications of increased RNA oxidation following a CRHP diet as well as long-term effects of carbohydrate-restriction on markers of oxidatively generated nucleic acid modifications should be a field of future study.

A carbohydrate-reduced high-protein diet improves HbA1c and liver fat content in weight stable participants with type 2 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: Dietary recommendations for treating type 2 diabetes are unclear but a trend towards recommending a diet reduced in carbohydrate content is acknowledged. We compared a carbohydrate-reduced high-protein (CRHP) diet with an iso-energetic conventional diabetes (CD) diet to elucidate the effects on glycaemic control and selected cardiovascular risk markers during 6 weeks of full food provision of each diet. METHODS: The primary outcome of the study was change in HbA1c. Secondary outcomes reported in the present paper include glycaemic variables, ectopic fat content and 24 h blood pressure. Eligibility criteria were: men and women with type 2 diabetes, HbA1c 48-97 mmol/mol (6.5-11%), age >18 years, haemoglobin >6/>7 mmol/l (women/men) and eGFR >30 ml min-1 (1.73 m)-2. Participants were randomised by drawing blinded ballots to 6 + 6 weeks of an iso-energetic CRHP vs CD diet in an open label, crossover design aiming at body weight stability. The CRHP/CD diets contained carbohydrate 30/50 energy per cent (E%), protein 30/17E% and fat 40/33E%, respectively. Participants underwent a meal test at the end of each diet period and glycaemic variables, lipid profiles, 24 h blood pressure and ectopic fat including liver and pancreatic fat content were assessed at baseline and at the end of each diet period. Data were collected at Copenhagen University Hospital, Bispebjerg and Copenhagen University Hospital, Herlev. RESULTS: Twenty-eight participants completed the study. Fourteen participants carried out 6 weeks of the CRHP intervention followed by 6 weeks of the CD intervention, and 14 participants received the dietary interventions in the reverse order. Compared with a CD diet, a CRHP diet reduced the primary outcome of HbA1c (mean ± SEM: -6.2 ± 0.8 mmol/mol (-0.6 ± 0.1%) vs -0.75 ± 1.0 mmol/mol (-0.1 ± 0.1%); p < 0.001). Nine (out of 37) pre-specified secondary outcomes are reported in the present paper, of which five were significantly different between the diets, (p < 0.05); compared with a CD diet, a CRHP diet reduced the secondary outcomes (mean ± SEM or medians [interquartile range]) of fasting plasma glucose (-0.71 ± 0.20 mmol/l vs 0.03 ± 0.23 mmol/l; p < 0.05), postprandial plasma glucose AUC (9.58 ± 0.29 mmol/l × 240 min vs 11.89 ± 0.43 mmol/l × 240 min; p < 0.001) and net AUC (1.25 ± 0.20 mmol/l × 240 min vs 3.10 ± 0.25 mmol/l × 240 min; p < 0.001), hepatic fat content (-2.4% [-7.8% to -1.0%] vs 0.2% [-2.3% to 0.9%]; p < 0.01) and pancreatic fat content (-1.7% [-3.5% to 0.6%] vs 0.5% [-1.0% to 2.0%]; p < 0.05). Changes in other secondary outcomes, i.e. 24 h blood pressure and muscle-, visceral- or subcutaneous adipose tissue, did not differ between diets. CONCLUSIONS/INTERPRETATION: A moderate macronutrient shift by substituting carbohydrates with protein and fat for 6 weeks reduced HbA1c and hepatic fat content in weight stable individuals with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT02764021. FUNDING: The study was funded by grants from Arla Food for Health; the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen; the Department of Clinical Medicine, Aarhus University; the Department of Nutrition, Exercise and Sports, University of Copenhagen; and Copenhagen University Hospital, Bispebjerg.

Overfed but undernourished: recognizing nutritional inadequacies/deficiencies in patients with overweight or obesity.

Overweight and obesity are highly prevalent throughout the world and can adversely affect the nutritional status of individuals. Studies have shown that many people with obesity have inadequate intake of iron, calcium, magnesium, zinc, copper, folate and vitamins A and B12, likely as a result of poor diet quality. Nutritional inadequacies or deficiencies may also occur due to altered pharmacokinetics in the individual with obesity and due to interactions in those with overweight or obesity with various pharmaceuticals. However, it has been demonstrated that the adult population in the United States as a whole is deficient in certain micronutrients as a result of the availability and overconsumption of high-calorie, low-nutrient processed foods. Poor nutrition may contribute to the development of certain chronic conditions, such as type 2 diabetes, which is already more prevalent in those with obesity. Clinicians need to be aware of these gaps, particularly in those individuals with obesity who are undergoing bariatric surgery or taking pharmaceutical products long term to facilitate weight loss. Patients with overweight or obesity likely struggle to achieve a balanced diet and may benefit from consultation with a dietitian. Along with providing recommendations for healthy eating and exercise, supplementation with specific micronutrients or multivitamins should be considered for individuals at the highest risk for or with established deficiencies. Further research is needed to understand the factors underlying nutritional inadequacies in individuals with overweight or obesity, as well as the outcomes of treatment strategies employed to address them.