The Potential of PIP3 in Enhancing Wound Healing.

Given the role of phosphatidylinositol 3,4,5-trisphosphate (PIP3) in modulating cellular processes such as proliferation, survival, and migration, we hypothesized its potential as a novel therapeutic agent for wound closure enhancement. In this study, PIP3 was examined in its free form or as a complex with cationic starch (Q-starch) as a carrier. The intracellular bioactivity and localization of free PIP3 and the Q-starch/PIP3 complexes were examined. Our results present the capability of Q-starch to form complexes with PIP3, facilitate its cellular membrane internalization, and activate intracellular paths leading to enhanced wound healing. Both free PIP3 and Q-starch/PIP3 complexes enhanced monolayer gap closure in scratch assays and induced amplified collagen production within HaCAT and BJ fibroblast cells. Western blot presented enhanced AKT activation by free or complexed PIP3 in BJ fibroblasts in which endogenous PIP3 production was pharmacologically inhibited. Furthermore, both free PIP3 and Q-starch/PIP3 complexes expedited wound closure in mice, after single or daily dermal injections into the wound margins. Free PIP3 and the Q-starch/PIP3 complexes inherently activated the AKT signaling pathway, which is responsible for crucial wound healing processes such as migration; this was also observed in wound assays in mice. PIP3 was identified as a promising molecule for enhancing wound healing, and its ability to circumvent PI3K inhibition suggests possible implications for chronic wound healing.

Circulating isomiRs May Be Superior Biomarkers Compared to Their Corresponding miRNAs: A Pilot Biomarker Study of Using isomiR-Ome to Detect Coronary Calcium-Based Cardiovascular Risk in Patients with NAFLD.

Circulating miRNAs are increasingly being considered as biomarkers in various medical contexts, but the value of analyzing isomiRs (isoforms of canonical miRNA sequences) has not frequently been assessed. Here we hypothesize that an in-depth analysis of the full circulating miRNA landscape could identify specific isomiRs that are stronger biomarkers, compared to their corresponding miRNA, for identifying increased CV risk in patients with non-alcoholic fatty liver disease (NAFLD)-a clinical unmet need. Plasma miRNAs were sequenced with next-generation sequencing (NGS). Liver fat content was measured with magnetic-resonance spectrometry (MRS); CV risk was determined, beyond using traditional biomarkers, by a CT-based measurement of coronary artery calcium (CAC) score and the calculation of a CAC score-based CV-risk percentile (CAC-CV%). This pilot study included n = 13 patients, age > 45 years, with an MRS-measured liver fat content of ≥5% (wt/wt), and free of overt CVD. NGS identified 1103 miRNAs and 404,022 different isomiRs, of which 280 (25%) and 1418 (0.35%), respectively, passed an abundance threshold. Eighteen (sixteen/two) circulating miRNAs correlated positively/negatively, respectively, with CAC-CV%, nine of which also significantly discriminated between high/low CV risk through ROC-AUC analysis. IsomiR-ome analyses uncovered 67 isomiRs highly correlated (R ≥ 0.55) with CAC-CV%. Specific isomiRs of miRNAs 101-3p, 144-3p, 421, and 484 exhibited stronger associations with CAC-CV% compared to their corresponding miRNA. Additionally, while miRNAs 140-3p, 223-3p, 30e-5p, and 342-3p did not correlate with CAC-CV%, specific isomiRs with altered seed sequences exhibited a strong correlation with coronary atherosclerosis burden. Their predicted isomiRs-specific targets were uniquely enriched (compared to their canonical miRNA sequence) in CV Disease (CVD)-related pathways. Two of the isomiRs exhibited discriminative ROC-AUC, and another two showed a correlation with reverse cholesterol transport from cholesterol-loaded macrophages to ApoB-depleted plasma. In summary, we propose a pipeline for exploring circulating isomiR-ome as an approach to uncover novel and strong CVD biomarkers.

Circulating miRNAs Detect High vs Low Visceral Adipose Tissue Inflammation in Patients Living With Obesity.

CONTEXT: The severity of visceral adipose tissue (VAT) inflammation in individuals with obesity is thought to signify obesity subphenotype(s) associated with higher cardiometabolic risk. Yet, this tissue is not accessible for direct sampling in the nonsurgical patient. OBJECTIVE: We hypothesized that circulating miRNAs (circ-miRs) could serve as biomarkers to distinguish human obesity subgroups with high or low extent of VAT inflammation. METHODS: Discovery and validation cohorts of patients living with obesity undergoing bariatric surgery (n = 35 and 51, respectively) were included. VAT inflammation was classified into low/high based on an expression score derived from the messenger RNA levels of TNFA, IL6, and CCL2 (determined by reverse transcription polymerase chain reaction). Differentially expressed circ-miRs were identified, and their discriminative power to detect low/high VAT inflammation was assessed by receiver operating characteristic-area under the curve (ROC-AUC) analysis. RESULTS: Fifty three out of 263 circ-miRs (20%) were associated with high-VAT inflammation according to Mann-Whitney analysis in the discovery cohort. Of those, 12 (12/53 = 23%) were differentially expressed according to Deseq2, and 6 significantly discriminated between high- and low-VAT inflammation with ROC-AUC greater than 0.8. Of the resulting 5 circ-miRs that were differentially abundant in all 3 statistical approaches, 3 were unaffected by hemolysis and validated in an independent cohort. Circ-miRs 181b-5p, 1306-3p, and 3138 combined with homeostatic model assessment of insulin resistance (HOMA-IR) exhibited ROC-AUC of 0.951 (95% CI, 0.865-1) and 0.808 (95% CI, 0.654-0.963) in the discovery and validation cohorts, respectively, providing strong discriminative power between participants with low- vs high-VAT inflammation. Predicted target genes of these miRNAs are enriched in pathways of insulin and inflammatory signaling, circadian entrainment, and cellular senescence. CONCLUSION: Circ-miRs that identify patients with low- vs high-VAT inflammation constitute a putative tool to improve personalized care of patients with obesity.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Visceral adipocyte size links obesity with dysmetabolism more than fibrosis, and both can be estimated by circulating miRNAs.

OBJECTIVE: In obesity, adipocyte hypertrophy is detrimental to health, but its' interrelation with fibrosis in the visceral adipose tissue (VAT) depot remains unclear. Because VAT is less accessible via biopsy, biomarkers for VAT quality are needed. The authors hypothesized that VAT adipocyte size and fibrosis are interrelated and can be estimated by circulating microRNAs (circ-miRNAs), contributing to subphenotyping obesity. METHODS: Adipocyte size and AT fibrosis were estimated in n = 43 participants (BMI ≥ 30 kg/m2 ). Circ-miRNAs were sequenced (Next Generation Sequencing). RESULTS: Participants with above- versus below-median VAT adipocyte area exhibited metabolic dysfunction but lower total and pericellular fibrosis. VAT adipocyte size remained associated with metabolic dysfunction even when controlling for BMI or VAT fibrosis in the entire cohort, as in matched-pairs subanalyses. Next Generation Sequencing uncovered 22 and 6 circ-miRNAs associated with VAT adipocyte size and fibrosis, respectively, with miRNA-130b-3p common to both analyses. The combination of miRNA-130b-3p + miR-150-5p + high-density lipoprotein cholesterol discriminated among those with large versus small VAT adipocytes (receiver operating characteristic-area under the curve: 0.872 [95% CI: 0.747-0.996]), whereas miRNA-130b-3p + miRNA-15a-5p + high-density lipoprotein cholesterol discriminated among those with low and high fibrosis (receiver operating characteristic-area under the curve: 0.823 [95% CI: 0.676-0.97]). CONCLUSIONS: These findings suggest that VAT adipocyte size and fibrosis are inversely correlated in obesity and can be estimated by distinct circ-miRNAs, providing a potential tool to subphenotype obesity via a liquid biopsy-like approach to assess VAT health in nonsurgical patients.

Vertebrae but not femur marrow fat transiently decreases in response to body weight loss in an 18-month randomized control trial.

BACKGROUND: Increased levels of bone marrow adipose tissue (BMAT) are negatively associated with skeletal health and hematopoiesis. BMAT is known to increase with age; however, the effect of long-term weight loss on BMAT is still unknown. OBJECTIVE: In this study, we examined BMAT response to lifestyle-induced weight loss in 138 participants (mean age 48 y; mean body mass index 31 kg/m2), who participated in the CENTRAL-MRI trial. METHODS: Participants were randomized for dietary intervention of low-fat or low-carb, with or without physical activity. Magnetic resonance imaging (MRI) was used to quantify BMAT and other fat depots at baseline, six and eighteen months of intervention. Blood biomarkers were also measured at the same time points. RESULTS: At baseline, the L3 vertebrae BMAT is positively associated with age, HDL cholesterol, HbA1c and adiponectin; but not with other fat depots or other metabolic markers tested. Following six months of dietary intervention, the L3 BMAT declined by an average of 3.1 %, followed by a return to baseline after eighteen months (p < 0.001 and p = 0.189 compared to baseline, respectively). The decrease of BMAT during the first six months was associated with a decrease in waist circumference, cholesterol, proximal-femur BMAT, and superficial subcutaneous adipose tissue (SAT), as well as with younger age. Nevertheless, BMAT changes did not correlate with changes in other fat depots. CONCLUSIONS: We conclude that physiological weight loss can transiently reduce BMAT in adults, and this effect is more prominent in younger adults. Our findings suggest that BMAT storage and dynamics are largely independent of other fat depots or cardio-metabolic risk markers, highlighting its unique functions.

Visceral Adipose Tissue E2F1-miRNA206/210 Pathway Associates with Type 2 Diabetes in Humans with Extreme Obesity.

Objective: Up-regulated expression of transcription-factor E2F1 in human visceral adipose tissue (VAT) characterizes a dysmetabolic obesity sub-phenotype. An E2F1-miRNA network has been described in multiple cancers. Here we investigated whether elevated VAT-E2F1 in obesity is associated with VAT-miRNA alterations similar to, or distinct from, those described in cancer. Furthermore, we assessed if E2F1-associated miRNA changes may contribute to the link between high- VAT-E2F1 and a dysmetabolic obesity phenotype. Methods: We assembled a cohort of patients with obesity and high-VAT-E2F1, matched by age, sex, ±BMI to patients with low-VAT-E2F1, with and without obesity (8 patients/groupX3 groups). We performed Nanostring©-based miRNA profiling of VAT samples from all 24 patients. Candidate E2F1-related miRNAs were validated by qPCR in an independent cohort of patients with extreme obesity, with or without type-2-diabetes (T2DM) (n = 20). Bioinformatic tools and manipulation of E2F1 expression in cells were used to establish the plausibility of the functional VAT-E2F1-miRNA network in obesity. Results: Among n = 798 identified miRNAs, 17 were differentially expressed in relation to E2F1 and not to obesity itself. No evidence for the cancer-related E2F1-miRNA network was identified in human VAT in obesity. In HEK293-cells, overexpression/downregulation of E2F1 correspondingly altered the expression of miRNA-206 and miRNA-210-5p, two miRNAs with reported metabolic functions consistent with those of E2F1. In VAT from both cohorts, the expression of both miRNA-206 and 210-5p intercorrelated, and correlated with the expression of E2F1. In cohort 1 we did not detect significant associations with biochemical parameters. In cohort 2 of patients with extreme obesity, all those with high VAT-E2F1 showed a diabetes-complicated obesity phenotype and higher expression of miRNA-206 and miRNA-210-5p, which also correlated with fasting glucose levels (both miRNAs) and fasting insulin (miRNA-210-5p). Conclusions: Whilst the previously described cancer-related E2F1-miRNA network does not appear to operate in VAT in obesity, miRNAs-206 and 210-5p may link high-E2F1 expression in VAT with diabetes-complicated extreme obesity phenotype.

Hypothalamic orexin prevents non-alcoholic steatohepatitis and hepatocellular carcinoma in obesity.

Non-alcoholic steatohepatitis (NASH) occasionally occurs under obesity; however, factors modulating the natural history of fatty liver disease remain unknown. Since hypothalamic orexin that regulates physical activity and autonomic balance prevents obesity, we investigate its role in NASH development. Male orexin-deficient mice fed a high-fat diet (HFD) show severe obesity and progression of NASH with fibrosis in the liver. Hepatic fibrosis also develops in ovariectomized orexin-deficient females fed an HFD but not ovariectomized wild-type controls. Moreover, long-term HFD feeding causes hepatocellular carcinoma (HCC) in orexin-deficient mice. Intracerebroventricular injection of orexin A or pharmacogenetic activation of orexin neurons acutely activates hepatic mTOR-sXbp1 pathway to prevent endoplasmic reticulum (ER) stress, a NASH-causing factor. Daily supplementation of orexin A attenuates hepatic ER stress and inflammation in orexin-deficient mice fed an HFD, and autonomic ganglionic blocker suppresses the orexin actions. These results suggest that hypothalamic orexin is an essential factor for preventing NASH and associated HCC under obesity.

Assessing Obesity-Related Adipose Tissue Disease (OrAD) to Improve Precision Medicine for Patients Living With Obesity.

Obesity is a heterogenous condition that affects the life and health of patients to different degrees and in different ways. Yet, most approaches to treat obesity are not currently prescribed, at least in a systematic manner, based on individual obesity sub-phenotypes or specifically-predicted health risks. Adipose tissue is one of the most evidently affected tissues in obesity. The degree of adipose tissue changes - "adiposopathy", or as we propose to relate to herein as Obesity-related Adipose tissue Disease (OrAD), correspond, at least cross-sectionally, to the extent of obesity-related complications inflicted on an individual patient. This potentially provides an opportunity to better personalize anti-obesity management by utilizing the information that can be retrieved by assessing OrAD. This review article will summarize current knowledge on histopathological OrAD features which, beyond cross-sectional analyses, had been shown to predict future obesity-related endpoints and/or the response to specific anti-obesity interventions. In particular, the review explores adipocyte cell size, adipose tissue inflammation, and fibrosis. Rather than highly-specialized methods, we emphasize standard pathology laboratory approaches to assess OrAD, which are readily-available in most clinical settings. We then discuss how OrAD assessment can be streamlined in the obesity/weight-management clinic. We propose that current studies provide sufficient evidence to inspire concerted efforts to better explore the possibility of predicting obesity related clinical endpoints and response to interventions by histological OrAD assessment, in the quest to improve precision medicine in obesity.

CTRP6 rapidly responds to acute nutritional changes, regulating adipose tissue expansion and inflammation in mice.

In chronic obesity, activated adipose tissue proinflammatory cascades are tightly linked to metabolic dysfunction. Yet, close temporal analyses of the responses to obesogenic environment such as high-fat feeding (HFF) in susceptible mouse strains question the causal relationship between inflammation and metabolic dysfunction, and/or raises the possibility that certain inflammatory cascades play adaptive/homeostatic, rather than pathogenic roles. Here, we hypothesized that CTRP6, a C1QTNF family member, may constitute an early responder to acute nutritional changes in adipose tissue, with potential physiological roles. Both 3-days high-fat feeding (3dHFF) and acute obesity reversal [2-wk switch to low-fat diet after 8-wk HFF (8wHFF)] already induced marked changes in whole body fuel utilization. Although adipose tissue expression of classical proinflammatory cytokines (Tnf-α, Ccl2, and Il1b) exhibited no, or only minor, change, C1qtnf6 uniquely increased, and decreased, in response to 3dHFF and acute obesity reversal, respectively. CTRP6 knockout (KO) mouse embryonic fibroblasts (MEFs) exhibited increased adipogenic gene expression (Pparg, Fabp4, and Adipoq) and markedly reduced inflammatory genes (Tnf-α, Ccl2, and Il6) compared with wild-type MEFs, and recombinant CTRP6 induced the opposite gene expression signature, as assessed by RNA sequencing. Consistently, 3dHFF of CTRP6-KO mice induced a greater whole body and adipose tissue weight gain compared with wild-type littermates. Collectively, we propose CTRP6 as a gene that rapidly responds to acute changes in caloric intake, acting in acute overnutrition to induce a "physiological inflammatory response" that limits adipose tissue expansion.NEW & NOTEWORTHY CTRP6 (C1qTNF6), a member of adiponectin gene family, regulates inflammation and metabolism in established obesity. Here, short-term high-fat feeding in mice is shown to increase adipose tissue expression of CTRP6 before changes in the expression of classical inflammatory genes occur. Conversely, CTRP6 expression in adipose tissue decreases early in the course of obesity reversal. Gain- and loss-of-function models suggest CTRP6 as a positive regulator of inflammatory cascades, and a negative regulator of adipogenesis and adipose tissue expansion.

Adaptation of fuel selection to acute decrease in voluntary energy expenditure is governed by dietary macronutrient composition in mice.

In humans, exercise-induced thermogenesis is a markedly variable component of total energy expenditure, which had been acutely affected worldwide by COVID-19 pandemic-related lockdowns. We hypothesized that dietary macronutrient composition may affect metabolic adaptation/fuel selection in response to an acute decrease in voluntary activity. Using mice fed short-term high-fat diet (HFD) compared to low-fat diet (LFD)-fed mice, we evaluated whole-body fuel utilization by metabolic cages before and 3 days after omitting a voluntary running wheel in the cage. Short-term (24-48 h) HFD was sufficient to increase energy intake, fat oxidation, and decrease carbohydrate oxidation. Running wheel omission did not change energy intake, but resulted in a significant 50% decrease in total activity and a ~20% in energy expenditure in the active phase (night-time), compared to the period with wheel, irrespective of the dietary composition, resulting in significant weight gain. Yet, while in LFD wheel omission significantly decreased active phase fat oxidation, thereby trending to increase respiratory exchange ratio (RER), in HFD it diminished active phase carbohydrate oxidation. In conclusion, acute decrease in voluntary activity resulted in positive energy balance in mice on both diets, and decreased oxidation of the minor energy (macronutrient) fuel source, demonstrating that dietary macronutrient composition determines fuel utilization choices under conditions of acute changes in energetic demand.

Human adipose tissue is a putative direct target of daytime orexin with favorable metabolic effects: A cross-sectional study.

OBJECTIVE: Orexin/hypocretin (Ox) and its receptors (OxR), a neuroendocrine system centrally regulating sleep/wakefulness, were implicated in the regulation of peripheral metabolism. It was hypothesized that human adipose tissue constitutes a direct target of the OxA/OxR system that associates with distinct metabolic profile(s). METHODS: Serum Ox levels and abdominal subcutaneous and visceral adipose tissue expression of Ox/HCRT, OxR1/HCRTR1, and OxR2/HCRTR2 were measured in n = 81 patients. RESULTS: Higher morning circulating Ox levels were associated with improved lipid profile and insulin sensitivity, independently of BMI (ß = -0.363, p = 0.018 for BMI-adjusted homeostatic model of insulin resistance). Adipose HCRT mRNA was detectable in <20% of patients. Visceral HCRT expressers were mostly (80%) males and, compared with nonexpressers, had lower total and LDL cholesterol. HCRTR1 was readily detectable, and HCRTR2 was undetectable. HCRTR1 mRNA and OxR1 protein expression were higher in subcutaneous than visceral adipose tissue, and among nonobese patients, patients with obesity, and patients with obesity and T2DM were 3.4 (1.0), 0.7 (0.1), 0.6 (0.1) (AU) (p < 0.001) and 1.0 (0.2), 0.5 (0.1), 0.4 (0.1) (AU) (p = NS), respectively. Higher visceral HCRTR1 expression was associated with lower fasting insulin and homeostatic model of insulin resistance, also after adjusting for BMI. In human adipocytes, HCRTR1 expression did not exhibit significant oscillation. CONCLUSIONS: Human adipose tissue is a putative direct target of the OxA-OxR1 system, with higher morning input being associated with improved metabolic profile.

Effects of Diet-Modulated Autologous Fecal Microbiota Transplantation on Weight Regain.

BACKGROUND & AIMS: We evaluated the efficacy and safety of diet-modulated autologous fecal microbiota transplantation (aFMT) for treatment of weight regain after the weight-loss phase. METHODS: In the DIRECT PLUS (Dietary Intervention Randomized Controlled Trial Polyphenols-Unprocessed) weight-loss trial (May 2017 through July 2018), abdominally obese or dyslipidemic participants in Israel were randomly assigned to healthy dietary guidelines, Mediterranean diet, and green-Mediterranean diet weight-loss groups. All groups received free gym membership and physical activity guidelines. Both isocaloric Mediterranean groups consumed 28 g/d walnuts (+440 mg/d polyphenols provided). The green-Mediterranean dieters also consumed green tea (3-4 cups/d) and a Wolffia globosa (Mankai strain, 100 g/d) green shake (+800 mg/d polyphenols provided). After 6 months (weight-loss phase), 90 eligible participants (mean age, 52 years; mean weight loss, 8.3 kg) provided a fecal sample that was processed into aFMT by frozen, opaque, and odorless capsules. The participants were then randomly assigned to groups that received 100 capsules containing their own fecal microbiota or placebo until month 14. The primary outcome was regain of the lost weight over the expected weight-regain phase (months 6-14). Secondary outcomes were gastrointestinal symptoms, waist circumference, glycemic status, and changes in the gut microbiome, as measured by metagenomic sequencing and 16s ribosomal RNA. We validated the results in a parallel in vivo study of mice specifically fed with Mankai compared with control chow diet. RESULTS: Of the 90 participants in the aFMT trial, 96% ingested at least 80 of 100 oral aFMT or placebo frozen capsules during the transplantation period. No aFMT-related adverse events or symptoms were observed. For the primary outcome, although no significant differences in weight regain were observed among the participants in the different lifestyle interventions during months 6-14 (aFMT, 30.4% vs placebo, 40.6%; P = .28), aFMT significantly attenuated weight regain in the green-Mediterranean group (aFMT, 17.1%, vs placebo, 50%; P = .02), but not in the dietary guidelines (P = .57) or Mediterranean diet (P = .64) groups (P for the interaction = .03). Accordingly, aFMT attenuated waist circumference gain (aFMT, 1.89 cm vs placebo, 5.05 cm; P = .01) and insulin rebound (aFMT, -1.46 ± 3.6 µIU/mL vs placebo, 1.64 ± 4.7 µIU/mL; P = .04) in the green-Mediterranean group but not in the dietary guidelines or Mediterranean diet (P for the interaction = .04 and .03, respectively). The green-Mediterranean diet was the only intervention to induce a significant change in microbiome composition during the weight-loss phase, and to prompt preservation of weight-loss-associated specific bacteria and microbial metabolic pathways (mainly microbial sugar transport) after the aFMT. In mice, Mankai-modulated aFMT in the weight-loss phase compared with control diet aFMT, significantly prevented weight regain and resulted in better glucose tolerance during a high-fat diet-induced regain phase (all, P < .05). CONCLUSIONS: Autologous FMT, collected during the weight-loss phase and administrated in the regain phase, might preserve weight loss and glycemic control, and is associated with specific microbiome signatures. A high-polyphenols, green plant-based or Mankai diet better optimizes the microbiome for an aFMT procedure. ClinicalTrials.gov number, NCT03020186.

The effect of green Mediterranean diet on cardiometabolic risk; a randomised controlled trial.

BACKGROUND: A Mediterranean diet is favourable for cardiometabolic risk. OBJECTIVE: To examine the residual effect of a green Mediterranean diet, further enriched with green plant-based foods and lower meat intake, on cardiometabolic risk. METHODS: For the DIRECT-PLUS parallel, randomised clinical trial we assigned individuals with abdominal obesity/dyslipidaemia 1:1:1 into three diet groups: healthy dietary guidance (HDG), Mediterranean and green Mediterranean diet, all combined with physical activity. The Mediterranean diets were equally energy restricted and included 28 g/day walnuts. The green Mediterranean diet further included green tea (3-4 cups/day) and a Wolffia globosa (Mankai strain; 100 g/day frozen cubes) plant-based protein shake, which partially substituted animal protein. We examined the effect of the 6-month dietary induction weight loss phase on cardiometabolic state. RESULTS: Participants (n=294; age 51 years; body mass index 31.3 kg/m2; waist circumference 109.7 cm; 88% men; 10 year Framingham risk score 4.7%) had a 6-month retention rate of 98.3%. Both Mediterranean diets achieved similar weight loss ((green Mediterranean -6.2 kg; Mediterranean -5.4 kg) vs the HDG group -1.5 kg; p<0.001), but the green Mediterranean group had a greater reduction in waist circumference (-8.6 cm) than the Mediterranean (-6.8 cm; p=0.033) and HDG (-4.3 cm; p<0.001) groups. Stratification by gender showed that these differences were significant only among men. Within 6 months the green Mediterranean group achieved greater decrease in low-density lipoprotein cholesterol (LDL-C; green Mediterranean -6.1 mg/dL (-3.7%), -2.3 (-0.8%), HDG -0.2 mg/dL (+1.8%); p=0.012 between extreme groups), diastolic blood pressure (green Mediterranean -7.2 mm Hg, Mediterranean -5.2 mm Hg, HDG -3.4 mm Hg; p=0.005 between extreme groups), and homeostatic model assessment for insulin resistance (green Mediterranean -0.77, Mediterranean -0.46, HDG -0.27; p=0.020 between extreme groups). The LDL-C/high-density lipoprotein cholesterol (HDL-C) ratio decline was greater in the green Mediterranean group (-0.38) than in the Mediterranean (-0.21; p=0.021) and HDG (-0.14; p<0.001) groups. High-sensitivity C-reactive protein reduction was greater in the green Mediterranean group (-0.52 mg/L) than in the Mediterranean (-0.24 mg/L; p=0.023) and HDG (-0.15 mg/L; p=0.044) groups. The green Mediterranean group achieved a better improvement (-3.7% absolute risk reduction) in the 10-year Framingham Risk Score (Mediterranean-2.3%; p=0.073, HDG-1.4%; p<0.001). CONCLUSIONS: The green MED diet, supplemented with walnuts, green tea and Mankai and lower in meat/poultry, may amplify the beneficial cardiometabolic effects of Mediterranean diet. TRIAL REGISTRATION NUMBER: This study is registered under ClinicalTrials.gov Identifier no NCT03020186.

The Obesity-Susceptibility Gene TMEM18 Promotes Adipogenesis through Activation of PPARG.

TMEM18 is the strongest candidate for childhood obesity identified from GWASs, yet as for most GWAS-derived obesity-susceptibility genes, the functional mechanism remains elusive. We here investigate the relevance of TMEM18 for adipose tissue development and obesity. We demonstrate that adipocyte TMEM18 expression is downregulated in children with obesity. Functionally, downregulation of TMEM18 impairs adipocyte formation in zebrafish and in human preadipocytes, indicating that TMEM18 is important for adipocyte differentiation in vivo and in vitro. On the molecular level, TMEM18 activates PPARG, particularly upregulating PPARG1 promoter activity, and this activation is repressed by inflammatory stimuli. The relationship between TMEM18 and PPARG1 is also evident in adipocytes of children and is clinically associated with obesity and adipocyte hypertrophy, inflammation, and insulin resistance. Our findings indicate a role of TMEM18 as an upstream regulator of PPARG signaling driving healthy adipogenesis, which is dysregulated with adipose tissue dysfunction and obesity.

Calcium-Sensing Receptor in Adipose Tissue: Possible Association with Obesity-Related Elevated Autophagy.

Autophagy is upregulated in adipose tissue (AT) from people with obesity. We showed that activation of the calcium-sensing receptor (CaSR) elevates proinflammatory cytokines through autophagy in preadipocytes. Our aim is to understand the role of CaSR on autophagy in AT from humans with obesity. We determined mRNA and protein levels of CaSR and markers of autophagy by qPCR and western blot in human visceral AT explants or isolated primary preadipocytes (60 donors: 72% female, 23-56% body fat). We also investigated their association with donors' anthropometric variables. Donors' % body fat and CaSR mRNA expression in AT were correlated (r = 0.44, p < 0.01). CaSR expression was associated with mRNA levels of the autophagy markers atg5 (r = 0.37, p < 0.01), atg7 (r = 0.29, p < 0.05) and lc3b (r = 0.40, p < 0.01). CaSR activation increased becn and atg7 mRNA expression in AT. CaSR activation also upregulated LC3II by ~50%, an effect abolished by the CaSR inhibitor. Spermine (CaSR agonist) regulates LC3II through the ERK1/2 pathway. Structural equation model analysis suggests a link between donors' AT CaSR expression, AT autophagy and expression of Tumor Necrosis Factor alpha TNF-α. CaSR expression in visceral AT is directly associated with % body fat, and CaSR activation may contribute to obesity-related disruption in AT autophagy.

Depletion of ASK1 blunts stress-induced senescence in adipocytes.

Increasing energy expenditure via induction of browning in white adipose tissue has emerged as a potential strategy to treat obesity and associated metabolic complications. We previously reported that ASK1 inhibition in adipocytes protected from high-fat diet (HFD) or lipopolysaccharide (LPS)-mediated downregulation of UCP1 both in vitro and in vivo. Conversely, adipocyte-specific ASK1 overexpression attenuated cold-induction of UCP-1 in inguinal fat. Herein, we provide evidence that both TNFα-mediated and HFD-induced activation of p38 MAPK in white adipocytes are ASK1-dependent. Moreover, expression of senescence markers was reduced in HFD-fed adipocyte-specific ASK1 knockout mice. Similarly, LPS-induced upregulation of senescence markers was blunted in ASK1-depleted adipocytes. Thus, our study identifies a previously unknown role for ASK1 in the induction of stress-induced senescence.

Distinct infrastructure of lipid networks in visceral and subcutaneous adipose tissues in overweight humans.

BACKGROUND: Adipose tissue plays important roles in health and disease. Given the unique association of visceral adipose tissue with obesity-related metabolic diseases, the distribution of lipids between the major fat depots located in subcutaneous and visceral regions may shed new light on adipose tissue-specific roles in systemic metabolic perturbations. OBJECTIVE: We sought to characterize the lipid networks and unveil differences in the metabolic infrastructure of the 2 adipose tissues that may have functional and nutritional implications. METHODS: Paired visceral and subcutaneous adipose tissue samples were obtained from 17 overweight patients undergoing elective abdominal surgery. Ultra-performance LC-MS was used to measure 18,640 adipose-derived features; 520 were putatively identified. A stem cell model for adipogenesis was used to study the functional implications of the differences found. RESULTS: Our analyses resulted in detailed lipid metabolic maps of the 2 major adipose tissues. They point to a higher accumulation of phosphatidylcholines, triacylglycerols, and diacylglycerols, although lower ceramide concentrations, in subcutaneous tissue. The degree of unsaturation was lower in visceral adipose tissue (VAT) phospholipids, indicating lower unsaturated fatty acid incorporation into adipose tissue. The differential abundance of phosphatidylcholines we found can be attributed at least partially to higher expression of phosphatidylethanolamine methyl transferase (PEMT). PEMT-deficient embryonic stem cells showed a dramatic decrease in adipogenesis, and the resulting adipocytes exhibited lower accumulation of lipid droplets, in line with the lower concentrations of glycerolipids in VAT. Ceramides may inhibit the expression of PEMT by increased insulin resistance, thus potentially suggesting a functional pathway that integrates ceramide, PEMT, and glycerolipid biosynthetic pathways. CONCLUSIONS: Our work unveils differential infrastructure of the lipid networks in visceral and subcutaneous adipose tissues and suggests an integrative pathway, with a discriminative flux between adipose tissues.

A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue Dysfunction Downstream of E2F1 in Human Obesity.

Elevated expression of E2F1 in adipocyte fraction of human visceral adipose tissue (hVAT) associates with a poor cardiometabolic profile. We hypothesized that beyond directly activating autophagy and MAP3K5 (ASK)-MAP kinase signaling, E2F1 governs a distinct transcriptome that contributes to adipose tissue and metabolic dysfunction in obesity. We performed RNA sequencing of hVAT samples from age-, sex-, and BMI-matched patients, all obese, whose visceral E2F1 protein expression was either high (E2F1high) or low (E2F1low). Tumor necrosis factor superfamily (TNFSF) members, including TRAIL (TNFSF10), TL1A (TNFSF15), and their receptors, were enriched in E2F1high While TRAIL was equally expressed in adipocytes and stromal vascular fraction (SVF), TL1A was mainly expressed in SVF, and TRAIL-induced TL1A was attributed to CD4+ and CD8+ subclasses of hVAT T cells. In human adipocytes, TL1A enhanced basal and impaired insulin-inhibitable lipolysis and altered adipokine secretion, and in human macrophages it induced foam cell biogenesis and M1 polarization. Two independent human cohorts confirmed associations between TL1A and TRAIL expression in hVAT and higher leptin and IL6 serum concentrations, diabetes status, and hVAT-macrophage lipid content. Jointly, we propose an intra-adipose tissue E2F1-associated TNFSF paracrine loop engaging lymphocytes, macrophages, and adipocytes, ultimately contributing to adipose tissue dysfunction in obesity.

Higher Mast Cell Accumulation in Human Adipose Tissues Defines Clinically Favorable Obesity Sub-Phenotypes.

The identification of human obesity sub-types may improve the clinical management of patients with obesity and uncover previously unrecognized obesity mechanisms. Here, we hypothesized that adipose tissue (AT) mast cells (MC) estimation could be a mark for human obesity sub-phenotyping beyond current clinical-based stratifications, both cross-sectionally and prospectively. We estimated MC accumulation using immunohistochemistry and gene expression in abdominal visceral AT (VAT) and subcutaneous (SAT) in a human cohort of 65 persons with obesity who underwent elective abdominal (mainly bariatric) surgery, and we validated key results in two clinically similar, independent cohorts (n = 33, n = 56). AT-MC were readily detectable by immunostaining for either c-kit or tryptase and by assessing the gene expression of KIT (KIT Proto-Oncogene, Receptor Tyrosine Kinase), TPSB2 (tryptase beta 2), and CMA1 (chymase 1). Participants were characterized as VAT-MClow if the expression of both CMA1 and TPSB2 was below the median. Higher expressers of MC genes (MChigh) were metabolically healthier (lower fasting glucose and glycated hemoglobin, with higher pancreatic beta cell reserve (HOMA-ß), and lower triglycerides and alkaline-phosphatase) than people with low expression (MClow). Prospectively, higher MC accumulation in VAT or SAT obtained during surgery predicted greater postoperative weight-loss response to bariatric surgery. Jointly, high AT-MC accumulation may be used to clinically define obesity sub-phenotypes, which are associated with a "healthier" cardiometabolic risk profile and a better weight-loss response to bariatric surgery.