Dysregulated blood lipids are a major predictor of cardiovascular events. A recent genome-wide association study (GWAS) with five clinically relevant lipid traits in 1.65 million individuals implicated over 770 genomic regions in regulating blood lipid metabolism. To translate these associations into clinical applications, a functional understanding of their roles in lipoprotein metabolism, transport and remodeling (LPmtr) is required. Here, we report the deep molecular fine-mapping of 554 of these lipid risk loci using 168 lipoprotein-related traits and all possible ratios between them in over 273,000 participants of the UK Biobank. We identified new ratio-based markers of pathways shared by multiple LPmtr genes, such as the linoleic acid fraction of the polyunsaturated fatty acid pool to reveal potential causal genes at poorly characterized lipid risk loci, the percentage of esterified cholesterol moieties in LDL particles as a proxy for soluble LDL receptor levels, and the HDL fraction of total lipoprotein particle number as a predictor of incident myocardial infarction. We demonstrate how lipoprotein fine-mapping can generate new hypotheses for drug target development while uncovering new mechanisms relevant to hyperlipidemia. Ratio-driven clustering further implicated miR-148 in TG secretion, linking ER-stress responses at postprandial state to VLDL metabolism via mTORC1, shown through series of integrated cellular assays and mouse studies. Moreover, consistent with its regulatory influence on lipid flux we identify miR-148a a previously unrecognized determinat of Lp(a) levels. Our study implements a novel approach of using metabolomic data to follow-up on genetic evidence from GWAS with clinical traits and generates new insights into the biology of lipoprotein particles, supporting the emerging view that assessing lipoprotein size and composition is essential for the understanding, prevention, and treatment of lipid-related disorders.

Brown adipose tissue (BAT) is a metabolically highly active tissue that dissipates energy stored within its intracellular triglyceride droplets as heat. Others have previously utilized MRI to show that the fat fraction of human supraclavicular BAT (scBAT) decreases upon cold exposure, compared with baseline (i.e., pre-cooling). However, comparisons to a control group that was not exposed to cold are largely lacking. We recently developed a non-invasive dynamic MRI protocol that allows for quantifying scBAT fat fraction changes over time. Here, we aimed to study the effect of cold exposure versus thermoneutrality on fat fraction changes in human scBAT. Ten young (mean age: 21.5 ± 0.7 years), lean (mean BMI: 21.7 ± 0.5 kg/m2), 12 h-fasted volunteers (9 females; 1 male) underwent up to 70 consecutive MRI scans each on two separate study visits in a cross-over design. Participants were exposed to a temperature of 32°C for 10 scans (i.e., ±16 min), which was then either lowered to 18°C (i.e., cold exposure) or was maintained at 32°C (i.e., thermoneutrality). Dynamic fat fraction changes were quantified, and self-reported thermal perception scores were monitored. The fat fraction in scBAT decreased over time upon cold exposure (r = -.222, p < .001). Interestingly however, we also observed a decrease in scBAT fat fraction over time upon thermoneutrality (r = -.212, p < .001). No difference was observed between the two temperature conditions (p = .55), while self-reported thermal perception scores were consistently higher (i.e., colder) upon cold exposure. In the trapezius muscle and the humerus bone as control tissues, the fat fraction was unaffected in both temperature conditions. The fat fraction in subcutaneous white adipose tissue (sWAT) however, also decreased over time upon cold exposure (r = -.270, p < .001) and during thermoneutrality (r = -.190, p < .001), again with no difference (p = .92) between the two temperature conditions. In conclusion, our results show that in 12 h-fasted, healthy individuals cold exposure and thermoneutrality similarly reduce the fat fraction within scBAT and sWAT. While we interpret that the cold exposure was sufficient to induce thermogenesis, we suggest that an increased lipolytic activity within adipocytes, as a consequence of fasting, may be the primary cause of the decreased fat fraction in both sWAT and scBAT in our study. The current study highlights the potential influence of fasting on the fat fraction in scBAT and stresses the importance of inclusion of a thermoneutral control group in studies investigating the BAT-modulating effect of cold exposure.

Lifestyle interventions, such as diet and exercise, are currently the main therapies against metabolic dysfunction-associated steatotic liver disease (MASLD). However, not much is known about the combined impact of fiber and exercise on the modulation of gut-liver axis and MASLD amelioration. Here, we studied the impact of the combination of exercise training and a fiber-rich diet on the amelioration of MASLD. Male APOE*3-Leiden.CETP mice were fed a high-fat high-cholesterol diet with or without the addition of fiber (10% inulin) and exercise trained on a treadmill, or remained sedentary. Exercise training and fiber supplementation reduced fat mass gain and lowered plasma glucose levels. Only the combination treatment, however, induced fat loss and decreased plasma triglyceride and cholesterol levels compared with sedentary control mice. Exercise training with and without the addition of fiber had a similar ameliorating effect on the MASLD score. Only exercise without fiber decreased the hepatic expression of inflammatory markers. Fiber diet was mainly responsible for remodeling the gut microbial composition, with an increase in the relative abundance of the short-chain fatty acid (SCFA)-producing genera Anaerostipes and Muribaculaceae, whereas, surprisingly, exercise training alone and with fiber resulted in the highest increase of SCFA production. Overall, the combination of exercise training and dietary fiber decreases fat mass and improves glucose and lipid homeostasis but does not have an additional synergistic positive effect on liver health compared with exercise training alone.NEW & NOTEWORTHY The combination of dietary fiber intake and exercise training has a synergetic beneficial effect on the metabolic health, resulting in fat loss, lowered blood glucose, and lowered plasma lipid levels in mice with steatotic liver disease. However, fiber supplementation, despite a positive remodulation of the gut-liver axis, does not have an additional positive effect on liver health compared with exercise training alone.

Plasma 1H-NMR metabolomic measures have yielded significant insight into the pathophysiology of cardiometabolic disease, but their inter-related nature complicates causal inference and clinical interpretation. This study aimed to investigate the associations of unrelated 1H-NMR metabolomic profiles with coronary artery disease (CAD) and ischemic stroke (IS). Principal component (PC) analysis was performed on 168 1H-NMR metabolomic measures in 56,712 unrelated European participants from UK Biobank to retrieve uncorrelated PCs, which were used in Cox-proportional hazard models. For each outcome, two-sample Mendelian randomization analyses were then conducted based on three nonoverlapping databases, followed by a meta-analysis. The first six PCs collectively explaining 88% of the total variance were identified. For CAD, results from Cox and Mendelian randomization analyses were generally directionally consistent. The pooled odds ratios (95% CI) for CAD per one-SD increase in genetically influenced PC1 and PC3 (both characterized by distinct apolipoprotein B [ApoB]-associated lipoprotein profiles) were 1.04 (1.03, 1.05) and 0.94 (0.93, 0.96), respectively. Besides, the pooled odds ratio (95% CI) for CAD per one-SD increase in genetically influenced PC4, characterized by simultaneously decreased small HDL and increased large HDL, and independent of ApoB, was 1.05 (1.03, 1.07). For IS, increases of PC3 and PC5 (characterized by increased amino acids) were associated with a lower risk and a higher risk, respectively. This study confirms associations of ApoB-associated lipoprotein profiles with CAD and IS, and highlights the possible existence of an ApoB-independent lipoprotein profile, characterized by a distinctive HDL subparticle distribution, driving CAD.

Background/Objectives: Mounting evidence indicates that the short-chain fatty acid butyrate protects against obesity and associated comorbidities, partially through the induction of adipose tissue thermogenesis. However, the effects of butyrate on white adipose tissue (WAT) browning and its molecular mechanism are still elusive. The objective of this study was to investigate butyrate-induced thermogenesis in white adipose tissue and its underlying mechanism. Methods: We studied the effects of butyrate on diet-induced obesity in the humanized APOE*3-Leiden.CETP transgenic mouse model and explored factors related to white adipose browning. Specifically, mice were challenged with a high-fat diet supplemented with butyrate. Adiposity was measured to assess obesity development. Energy metabolism was detected using an indirect calorimetry system. RNA-seq analysis was conducted to analyze the transcription landscape of WAT and responsible targets. Furthermore, the revealed molecular mechanism was verified in vitro. Results: Butyrate alleviated high-fat diet-induced obesity and promoted energy expenditure accompanied by brown adipose tissue activation and WAT browning. Mechanistically, RNA-seq analysis revealed that butyrate downregulated HDAC9 in WAT. Additionally, butyrate decreased HDAC9 while increasing thermogenesis in vitro. Inhibition of HDAC9 with TMP269 promoted thermogenic gene expression, mimicking the effects of butyrate. Conclusions: Butyrate protects against diet-induced obesity accompanied by decreasing the expression of HDAC9 in white adipose tissue and inducing browning. This study reveals a new mechanism whereby butyrate activates adaptive thermogenesis and provides new insights for the development of weight-loss drugs targeting adipose HDAC9.

Until two decades ago, brown adipose tissue (BAT) was studied primarily as a thermogenic organ of small rodents in the context of cold adaptation. The discovery of functional human BAT has opened new opportunities to understand its physiological role in energy balance and therapeutic applications for metabolic disorders. Significantly, the role of BAT extends far beyond thermogenesis, including glucose and lipid homeostasis, by releasing mediators that communicate with other cells and organs. The field has made major advances by using new model systems, ranging from subcellular studies to clinical trials, which have also led to debates. In this perspective, we identify six fundamental issues that are currently controversial and comprise dichotomous models. Each side presents supporting evidence and, critically, the necessary methods and falsifiable experiments that would resolve the dispute. With this collaborative approach, the field will continue to productively advance the understanding of BAT physiology, appreciate the importance of thermogenic adipocytes as a central area of ongoing research, and realize the therapeutic potential.

AIMS:[D3,G40,K41.C16 diacid]exendin-4 (acyl-ExD3) and [F1,G40,K41.C16 diacid]exendin-4 (acyl-ExF1) are oppositely biased glucagon-like peptide-1 (GLP-1) receptor agonists that preferentially promote β-arrestin recruitment or G protein-induced signalling, respectively. The latter is more favourable in glycaemic control and induces a steeper reduction in body weight in diet-induced obese mice. Here, we compared the effects of G protein-biased agonist acyl-ExF1 to those of β-arrestin-biased agonist acyl-ExD3 on lipid metabolism in hyperlipidaemic mice. MATERIALS AND METHODS:APOE*3-Leiden.CETP mice were treated with saline, acyl-ExD3 or acyl-ExF1 via intraperitoneal injections for 6 weeks or intracerebroventricular infusion for 18 days. Body weight and composition were monitored at regular intervals, as were plasma glucose, triglyceride and cholesterol levels. At endpoint, mice were injected with very low-density lipoprotein (VLDL)-like particles containing glycerol tri[3H]oleate to study triglyceride-derived fatty acid uptake by peripheral tissues including brown and white adipose tissue (BAT and WAT). RESULTS:Upon peripheral treatment, body weight gain was prevented and plasma glucose levels were reduced by acyl-ExF1, but circulating lipids were not affected by either acyl-ExF1 or acyl-ExD3. In contrast, central administration of either agonist strongly reduced plasma triglyceride and cholesterol levels, but did not affect glucose levels. Acyl-ExD3 and acyl-ExF1 increased [3H]oleate uptake by adipose tissue, reaching statistical significance for the uptake by BAT and WAT, respectively, compared to vehicle treatment. CONCLUSION:The oppositely biased GLP-1 receptor agonists acyl-ExD3 and acyl-ExF1 do not differentially affect lipid metabolism in APOE*3-Leiden.CETP mice, while effects on glucose homeostasis and prevention of body weight gain are more pronounced upon peripheral acyl-ExF1 treatment.

The accumulation of cholesterol and other lipids leading to hepatic lipotoxicity drives the progression of metabolic dysfunction-associated steatotic liver (MASL) to metabolic dysfunction-associated steatohepatitis (MASH), the advanced progressive stage of metabolic dysfunction-associated steatotic liver disease (MASLD). For MASH diagnosis, liver biopsy remains the reference standard, despite its invasiveness and limitations. Thus, this study aimed to find blood-derived lipid markers for MASH. We investigated serum samples from 86 patients with histologically characterized MASLD, spanning the disease spectrum (i.e. 62 patients with MASL (Fibrosis grade 0-4) and 24 patients with MASH (Fibrosis grade 2-4) with a balanced distribution of hepatocellular carcinoma) and analyzed sterol composition and lipidome. To identify the presence of MASH, logistic regression was performed on each candidate either in a single or combination with various clinical parameters. Serum levels of desmosterol and phosphatidylcholine are increased in patients with MASH compared to those with MASL. After exclusion of patients using lipid lowering drugs, an increase was also found in serum levels of cholesterol, cholesterol ester, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, and several individual lipid species. The ROC curve of each lipid candidate show the potential use of desmosterol, phosphatidylcholine, and a panel of lipid species in combination with alanine aminotransferase as potential diagnostic markers, characterized by a respective AUROC of 0.79 (95% CI 0.66-0.92), 0.80 (95% CI 0.64-0.97), and 0.91 (95% CI 0.82-1.00). Serum sterol and lipidome markers are characterized by strong AUROC results to distinguish with high accuracy MASH from MASL, potentially paving the way for future MASH biomarker development.

Context The growth hormone (GH) secretagogue receptor, encoded by GHSR, is expressed on somatotrophs of the pituitary gland. Stimulation with its ligand ghrelin, as well as its constitutive activity, enhances GH secretion. Studies in knockout mice suggest that heterozygous loss-of-function of GHSR is associated with decreased GH response to fasting, but patient observations in small case reports have been equivocal.Objective This work aims to establish the phenotype of GHSR haploinsufficiency and its growth response to GH treatment.Methods This case series includes 26 patients with short stature and heterozygous GHSR variants. Pathogenicity was studied in vitro using total protein levels, cell surface expression, and receptor activity in basal, stimulated, and inhibited states.Results Ten different variants were identified, of which 6 were novel. Variants showed either partial or complete loss of function, primarily through loss of constitutive activity. Patients (aged 4.0-15.1 years) had proportionate short stature (height -2.8 +/- 0.5 SDS), failure to thrive with low appetite (n = 4), a mean serum insulin-like growth factor-I (IGF-I) of -1.6 +/- 0.7 SDS, and a normal stimulated GH response. Nine patients received GH treatment, showing a height gain of 0.9 +/- 0.4 SDS after 1 year and 1.5 +/- 0.4 SDS after 2 years (n = 5).Conclusion This study combines phenotypical and functional data in a uniquely large group of children with short stature carrying GHSR variants, and shows their good response to GH treatment. The results strengthen the hypothesis of GHSR's role in GH secretion.

ObjectiveInflammation contributes to the development of type 2 diabetes mellitus (T2DM). While South Asians are more prone to develop T2DM than Europids, the inflammatory phenotype of the South Asian population remains relatively unknown. Therefore, we aimed to investigate potential differences in circulating levels of inflammation-related proteins in South Asians compared with Europids with T2DM.MethodIn this secondary analysis of three randomized controlled trials, relative plasma levels of 73 inflammation-related proteins were measured using an Olink Target Inflammation panel and the serum fibroblast growth factor 21 (FGF21) concentration using an ELISA kit in Dutch South Asians (n = 47) and Dutch Europids (n = 49) with T2DM.ResultsOf the 73 inflammation-related proteins, the relative plasma levels of six proteins were higher (stem cell factor, caspase-8, C–C motif chemokine ligand 28, interferon-gamma, sulfotransferase 1A1 and cystatin D; q-value <0.05), while relative levels of six proteins were lower (FGF21, human fibroblast collagenase, interferon-8, C–C motif chemokine ligand 4, C–X–C motif chemokine ligand 6 and monocyte chemoattractant protein-1; q-value <0.05) in South Asians compared with Europids. Of these, the effect size of FGF21 was the largest, particularly in females. We validated this finding by assessing the FGF21 concentration in serum. The FGF21 concentration was indeed lower in South Asians compared with Europids with T2DM in both males (−42.2%; P < 0.05) and females (−58.5%; P < 0.001).ConclusionRelative plasma levels of 12 inflammation-related proteins differed between South Asians and Europids with T2DM, with a significantly pronounced reduction in FGF21. In addition, the serum FGF21 concentration was significantly lower in South Asian males and females compared with Europids. Whether low FGF21 is an underlying cause or consequence of T2DM in South Asians remains to be determined.Clinical trial registrationClinicalTrials.gov (NCT01761318, registration date 20-12-2012; NCT02660047, registration date 21-03-2018; and NCT03012113, registration date 06-01-2017).

Sepsis is the dysregulated immune response to an infection and is a leading cause of mortality. Low levels of high-density lipoprotein (HDL) cholesterol are associated with increased risk of death from sepsis, and increasing levels of HDL by inhibition of cholesteryl ester transfer protein (CETP) has been shown to decrease mortality in mouse models of sepsis. The objective of this study was to investigate the cellular mechanisms by which CETP inhibition and HDL lead to improved survival during sepsis. We found that HDL inhibits lipopolysaccharide (LPS)-induced activation of IL-1β in a mouse model of sepsis. The activation of IL-1β was dependent on the activity of scavenger receptor class B type 1 (SR-B1), and knockdown of SR-B1 significantly attenuated LPS-induced production of IL-1β in macrophages. Additionally, we found that LPS-induced SR-B1 internalization occurs through the endosome-lysosome pathway, which is also likely responsible for LPS degradation in the macrophages. Furthermore, we revealed that raising HDL by CETP inhibition markedly enhanced HDL-mediated anti-inflammatory effects in response to LPS stimulation, and these effects were not due to CETP itself but rather were HDL-dependent. Finally, we show that pharmacological inhibition of CETP significantly improved endotoxemia-induced mortality by inhibiting IL-1β production in the liver and circulation after LPS injection. Pathologically, CETP inhibition attenuated LPS-induced diffuse alveolar damage and hepatocyte necrosis, which may contribute to the improved mortality in mice treated with the CETP inhibitor anacetrapib. Taken together, our findings uncover a cellular mechanism by which HDL attenuates LPS-induced pro-inflammatory response via SR-B1-mediated LPS degradation.

BACKGROUND:Cannabinoid receptor 1 inhibition poses an effective treatment strategy in obesity, but has a risk of psychiatric side effects. As high-fat diet (HFD)-feeding acutely increases expression of endocannabinoid synthesis enzymes and circulating endocannabinoid levels in mice, here we tested whether inhibition of these enzymes alleviates metabolic disturbances caused by high-fat diet feeding. METHODS:C57BL/6J mice received daily intraperitoneal injections with a NAPE-PLD inhibitor (LEI-401), DAGL inhibitor (DH-376), or vehicle for 1 week while on a HFD. An extra group of vehicle-treated mice was maintained on regular chow diet. RESULTS:Both inhibitors effectively lowered blood and brain levels of endocannabinoids after 1 week of treatment. DH-376 reduced plasma insulin levels compared with vehicle (-53%) already within 2 h after the first dose. In contrast, LEI-401 did not change insulin or glucose levels. Both inhibitors suppressed caloric intake during the first day of treatment (-25% and -21%, respectively). In addition, LEI-401 elevated carbohydrate oxidation. The combined effect was a 2.0 and 1.6 g lower body weight after 24 h, respectively. Nevertheless, after 1 week body weight was no longer different between the HFD-fed groups. Moreover, DH-376 increased brown adipose tissue weight and reduced insulin-stimulated glucose uptake. CONCLUSIONS:DAGL and NAPE-PLD inhibition effectively lower levels of endocannabinoids and related bioactive lipids. Both inhibitors caused a transient reduction in food intake and body weight, but also led to alterations in glucose homeostasis. The long-term effects of endocannabinoid biosynthesis inhibitors on (cardio)metabolic health remains to be investigated.

Exercise is generally beneficial for health but strenuous exercise can have detrimental effects on the gastrointestinal tract. The combination of ischemia and heat shock during exercise is a crucial contributor to intestinal epithelial damage. Growing evidence points towards an important regulatory role of gut microbes in intestinal homeostasis. Here, we characterize and compare the effects of moderate and vigorous exercise training on intestinal epithelial damage, stress response, inflammatory response, and gut microbiota alterations in mice and investigate the mechanisms underlying exercise-induced intestinal injury. Exercise training for six weeks caused heat stress in the intestine, resulting in the disruption of the intestinal epithelial barrier and local inflammation. This was characterized by increased colonic HSP-70 and HSF-1 protein expression, increased epithelial permeability, decreased colonic expression of tight junction proteins ZO-1 and occludin and intestinal morphological changes. Daily moderate exercise training caused hereby more severe injury than vigorous training on alternating days. Furthermore, exercise training altered the gut microbiota profile. The abundance of Lactobacillaceae was reduced, potentially contributing to the deteriorated intestinal status, while the abundance of short-chain fatty acid-producing Lachnospiraceae was increased, especially following vigorous training. This increase in short-chain fatty acid-producing bacteria following vigorous training possibly counteracted the impairment of the intestinal barrier function. In summary, exercise disrupts the intestinal barrier function, with vigorous exercise training with intermittent rest days being less damaging than daily moderate exercise training.

Purpose: Sepsis is one of the leading causes of morbidity and mortality worldwide with approximately 50 million annual cases. There is ongoing debate on the clinical benefit of hydrocortisone in the prevention of death in septic patients. Here we evaluated the association between hydrocortisone treatment and mortality in patients diagnosed with sepsis in a large-scale clinical dataset. Methods: Data from patients between 2008 and 2019 were extracted from the retrospective Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Patients who received hydrocortisone after diagnosis were matched using propensity-score matching with patients who did not, to balance confounding (by indication and contraindication) factors between the groups. 90-day mortality and survivors' length of hospital stay was compared between patients who did or did not receive hydrocortisone. Results: A total of 31,749 septic patients were included in the study (mean age: 67, men: 57.3%, in-hospital mortality: 15.6%). 90-day mortality was higher among the 1802 patients receiving hydrocortisone when compared with the 6348 matched non-users (hazard ratio: 1.35, 95% CI: 1.24-1.47). Hydrocortisone treatment was also associated with increased in-hospital mortality (40.9% vs. 27.6%, p < 0.0001) and prolonged hospital stay in those who survived until discharge (median 12.6 days vs. 10.8 days, p < 0.0001). Stratification for age, gender, ethnicity, occurrence of septic shock, and the need for vasopressor drug administration such as (nor) epinephrine did not reveal sub-population(s) benefiting of hydrocortisone use. Conclusion: Hydrocortisone treatment is associated with increased risk of death as well as prolonged hospital stay in septic patients. Although residual confounding (by indication) cannot be ruled out completely due to the observational nature of the study, the present study suggests clinical implication of hydrocortisone use in patients with sepsis.