Isthmin-1 (ISM1), a novel adipokine that reflects abdominal adipose tissue distribution in individuals with obesity.

BACKGROUND: The assessment of obesity-related health risks has traditionally relied on the Body Mass Index and waist circumference, but their limitations have propelled the need for a more comprehensive approach. The differentiation between visceral (VIS) and subcutaneous (SC) fat provides a finer-grained understanding of these risks, yet practical assessment methods are lacking. We hypothesized that combining the SC-VIS fat ratio with non-invasive biomarkers could create a valuable tool for obesity-related risk assessment. METHODS AND RESULTS: A clinical study of 125 individuals with obesity revealed significant differences in abdominal fat distribution measured by CT-scan among genders and distinct models of obesity, including visceral, subcutaneous, and the SC/VIS ratio. Stratification based on these models highlighted various metabolic changes. The SC/VIS ratio emerged as an excellent metric to differentiate metabolic status. Gene expression analysis identified candidate biomarkers, with ISM1 showing promise. Subsequent validation demonstrated a correlation between ISM1 levels in SC and plasma, reinforcing its potential as a non-invasive biomarker for fat distribution. Serum adipokine levels also correlated with the SC/VIS ratio. The Receiver Operating Characteristic analysis revealed ISM1's efficacy in discriminating individuals with favorable metabolic profiles based on adipose tissue distribution. Correlation analysis also suggested that ISM1 was involved in glucose regulation pathways. CONCLUSION: The study's results support the hypothesis that the SC-VIS fat ratio and its derived non-invasive biomarkers can comprehensively assess obesity-related health risks. ISM1 could predict abdominal fat partitioning and be a potential biomarker for evaluating obesity-related health risks.

CRISPR/Cas9-mediated deletion of adipocyte genes associated with NAFLD alters adipocyte lipid handling and reduces steatosis in hepatocytes in vitro.

Obesity is a major risk factor for the development of nonalcoholic fatty liver disease (NAFLD), and the subcutaneous white adipose tissue (scWAT) is the primary lipid storage depot and regulates lipid fluxes to other organs. Our previous work identified genes upregulated in scWAT of patients with NAFLD: SOCS3, DUSP1, and SIK1. Herein, we knocked down (KD) their expression in human adipose-derived mesenchymal stem cells (hADMSCs) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterized their phenotype. We found that SOCS3, DUSP1, and SIK1 expression in hADMSC-derived adipocytes was not critical for adipogenesis. However, the metabolic characterization of the cells suggested that the genes played important roles in lipid metabolism. Reduction of SIK1 expression significantly increased both de novo lipogenesis (DNL) and palmitate-induced lipogenesis (PIL). Editing out SOCS3 reduced DNL while increasing isoproterenol-induced lipolysis and insulin-induced palmitate accumulation. Conversely, DUSP1 reduced PIL and DNL. Moreover, RNA-sequencing analysis of edited cells showed that these genes not only altered lipid metabolism but also other biological pathways related to inflammatory processes, in the case of DUSP1, extracellular matrix remodeling for SOCS3, or cellular transport for SIK1. Finally, to evaluate a possible adipocyte-hepatocyte axis, human hepatoma HepG2 cells were cocultured with edited hADMSCs-derived adipocytes in the presence of [3H]-palmitate. All HepG2 cells cultured with DUSP1-, SIK1-, or SOCS3-KD adipocytes decreased [3H]-palmitate accumulation compared with control adipocytes. These results support our hypotheses that SOCS3, DUSP1, and SIK1 regulate multiple aspects of adipocyte function, which may play a role in the progression of obesity-associated comorbidities, such as NAFLD.NEW & NOTEWORTHY Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology successfully edited genomic DNA of human adipose-derived mesenchymal stem cells (hADMSC). SOCS3, SIK1, and DUSP1 regulate adipocyte lipid handling. Silencing SOCS3, SIK1, and DUSP1 expression in hADMSC-derived adipocytes reduces hepatocyte lipid storage in vitro.

Triglyceride-rich lipoproteins and insulin resistance in patients with chronic hepatitis C receiving direct-acting antivirals.

BACKGROUND & AIMS: Hepatitis C virus (HCV) interferes with carbohydrate and lipid metabolism causing cardiovascular disease and insulin resistance (IR). Direct-acting antivirals (DAAs) are highly effective for the eradication of HCV, with positive effects on metabolic health although paradoxically associated with increased total and LDL-cholesterol. The aims of this study were 1) to characterize dyslipidemia (lipoprotein content, number, and size) in naive HCV-infected individuals and 2) to evaluate the longitudinal association of metabolic changes and lipoparticle characteristics after DAA therapy. METHODS: We conducted a prospective study with one-year follow-up. 83 naive outpatients treated with DAAs were included. Those co-infected with HBV or HIV were excluded. IR was analyzed using the HOMA index. Lipoproteins were studied by fast-protein liquid chromatography (FPLC) and Nuclear Magnetic Resonance Spectroscopy (NMR). RESULTS: FPLC analysis showed that lipoprotein-borne HCV was only present in the VLDL region most enriched in APOE. There was a lack of association between HOMA and total cholesterol or cholesterol carried by LDL or HDL at baseline. Alternatively, a positive association was found between HOMA and total circulating triglycerides (TG), as well as with TG transported in VLDL, LDL, and HDL. HCV eradication with DAAs resulted in a strong and significant decrease in HOMA (-22%) and HDL-TG (-18%) after one-year follow-up. CONCLUSIONS: HCV-dependent lipid abnormalities are associated with IR and DAA therapy can reverse this association. These findings may have potential clinical implications as the HDL-TG trajectory may inform the evolution of glucose tolerance and IR after HCV eradication.

Gene Therapy Based on Mesenchymal Stem Cells Derived from Adipose Tissue for the Treatment of Obesity and Its Metabolic Complications.

Obesity is a highly prevalent condition often associated with dysfunctional adipose tissue. Stem cell-based therapies have become a promising tool for therapeutic intervention in the context of regenerative medicine. Among all stem cells, adipose-derived mesenchymal stem cells (ADMSCs) are the most easily obtained, have immunomodulatory properties, show great ex vivo expansion capacity and differentiation to other cell types, and release a wide variety of angiogenic factors and bioactive molecules, such as growth factors and adipokines. However, despite the positive results obtained in some pre-clinical studies, the actual clinical efficacy of ADMSCs still remains controversial. Transplanted ADMSCs present a meager rate of survival and proliferation, possibly because of the damaged microenvironment of the affected tissues. Therefore, there is a need for novel approaches to generate more functional ADMSCs with enhanced therapeutic potential. In this context, genetic manipulation has emerged as a promising strategy. In the current review, we aim to summarize several adipose-focused treatments of obesity, including cell therapy and gene therapy. Particular emphasis will be given to the continuum from obesity to metabolic syndrome, diabetes, and underlying non-alcoholic fatty liver disease (NAFLD). Furthermore, we will provide insights into the potential shared adipocentric mechanisms involved in these pathophysiological processes and their remediation using ADMSCs.

Identification of novel targets in adipose tissue involved in non-alcoholic fatty liver disease progression.

Obesity is a major risk factor for the development of Nonalcoholic fatty liver disease (NAFLD). We hypothesize that a dysfunctional subcutaneous white adipose tissue (scWAT) may lead to an accumulation of ectopic fat in the liver. Our aim was to investigate the molecular mechanisms involved in the causative role of scWAT in NALFD progression. We performed a RNA-sequencing analysis in a discovery cohort (n = 45) to identify genes in scWAT correlated with fatty liver index, a qualitative marker of liver steatosis. We then validated those targets in a second cohort (n = 47) of obese patients who had liver biopsies available. Finally, we obtained scWAT mesenchymal stem cells (MSCs) from 13 obese patients at different stages of NAFLD and established in vitro models of human MSC (hMSC)-derived adipocytes. We observed impaired adipogenesis in hMSC-derived adipocytes as liver steatosis increased, suggesting that an impaired adipogenic capacity is a critical event in the development of NAFLD. Four genes showed a differential expression pattern in both scWAT and hMSC-derived adipocytes, where their expression paralleled steatosis degree: SOCS3, DUSP1, SIK1, and GADD45B. We propose these genes as key players in NAFLD progression. They could eventually constitute potential new targets for future therapies against liver steatosis.

Adiponectin overexpression in C2C12 myocytes increases lipid oxidation and myofiber transition

Metabolic syndrome and obesity have detrimental effects on the metabolic function of the skeletal muscle. Mounting evidence indicates that patients with those conditions may present an increased ratio of glycolytic to oxidative fibers associated with a decrease in oxidative capacity. In this regard, adiponectin, a hormone mainly secreted by adipocytes that regulates glucose and lipid metabolism, has emerged as a myokine that could play an important role in this process. We aimed to investigate whether adiponectin overexpression in skeletal muscle might be a local protective mechanism, favoring fatty acid utilization. To that end, we generated an in vitro model of myocytes with upregulated endogenous adiponectin using a lentiviral carrier. We demonstrated that the adiponectin-transduced myocytes were able to produce and secrete fully functional adiponectin complexes. Adiponectin overexpression remarkably upregulated the mRNA level of myogenic regulatory factors as well as genes implicated in lipolysis (HSL, ATGL) and cellular and mitochondrial fatty acid transport (LPL, CD36, CPT1B). This was accompanied by increased isoproterenol-induced lipolysis and β-oxidation and reduced lipogenesis, whereas insulin-stimulated glucose uptake was unaltered in transduced myocytes. Lastly, the relative expression of the more glycolytic myofibers (MyHC IIb) compared to the more oxidative ones (MyHC I) was notably reduced. Our results showed that the released adiponectin acted in an autocrine/paracrine manner, increasing lipid oxidation in myocytes and leading to a transition of myofibers from the glycolytic to the oxidative type. In conclusion, muscle adiponectin overexpression might be a way to relieve muscle diseases caused by oxidative muscle fiber deficiency. (AU)

Adiponectin overexpression in C2C12 myocytes increases lipid oxidation and myofiber transition.

Metabolic syndrome and obesity have detrimental effects on the metabolic function of the skeletal muscle. Mounting evidence indicates that patients with those conditions may present an increased ratio of glycolytic to oxidative fibers associated with a decrease in oxidative capacity. In this regard, adiponectin, a hormone mainly secreted by adipocytes that regulates glucose and lipid metabolism, has emerged as a myokine that could play an important role in this process. We aimed to investigate whether adiponectin overexpression in skeletal muscle might be a local protective mechanism, favoring fatty acid utilization. To that end, we generated an in vitro model of myocytes with upregulated endogenous adiponectin using a lentiviral carrier. We demonstrated that the adiponectin-transduced myocytes were able to produce and secrete fully functional adiponectin complexes. Adiponectin overexpression remarkably upregulated the mRNA level of myogenic regulatory factors as well as genes implicated in lipolysis (HSL, ATGL) and cellular and mitochondrial fatty acid transport (LPL, CD36, CPT1B). This was accompanied by increased isoproterenol-induced lipolysis and ß-oxidation and reduced lipogenesis, whereas insulin-stimulated glucose uptake was unaltered in transduced myocytes. Lastly, the relative expression of the more glycolytic myofibers (MyHC IIb) compared to the more oxidative ones (MyHC I) was notably reduced. Our results showed that the released adiponectin acted in an autocrine/paracrine manner, increasing lipid oxidation in myocytes and leading to a transition of myofibers from the glycolytic to the oxidative type. In conclusion, muscle adiponectin overexpression might be a way to relieve muscle diseases caused by oxidative muscle fiber deficiency.

Amino Acid Profile in Malnourished Patients with Liver Cirrhosis and Its Modification with Oral Nutritional Supplements: Implications on Minimal Hepatic Encephalopathy.

Low plasma levels of branched chain amino acids (BCAA) in liver cirrhosis are associated with hepatic encephalopathy (HE). We aimed to identify a metabolic signature of minimal hepatic encephalopathy (MHE) in malnourished cirrhotic patients and evaluate its modification with oral nutritional supplements (ONS) enriched with ß-Hydroxy-ß-methylbutyrate (HMB), a derivative of the BCAA leucine. Post hoc analysis was conducted on a double-blind placebo-controlled trial of 43 individuals with cirrhosis and malnutrition, who were randomized to receive, for 12 weeks, oral supplementation twice a day with either 220 mL of Ensure® Plus Advance (HMB group, n = 22) or with 220 mL of Ensure® Plus High Protein (HP group, n = 21). MHE evaluation was by psychometric hepatic encephalopathy score (PHES). Compared to the HP group, an HMB-specific treatment effect led to a larger increase in Val, Leu, Phe, Trp and BCAA fasting plasma levels. Both treatments increased Fischer's ratio and urea without an increase in Gln or ammonia fasting plasma levels. MHE was associated with a reduced total plasma amino acid concentration, a reduced BCAA and Fischer´s ratio, and an increased Gln/Glu ratio. HMB-enriched ONS increased Fischer´s ratio without varying Gln or ammonia plasma levels in liver cirrhosis and malnutrition, a protective amino acid profile that can help prevent MHE.

Phenolic compounds apigenin, hesperidin and kaempferol reduce in vitro lipid accumulation in human adipocytes.

BACKGROUND: Adipocytes derived from human mesenchymal stem cells (MSCs) are widely used to investigate adipogenesis. Taking into account both the novelty of these MSCs and the scarcity of studies focused on the effects of phenolic compounds, the aim of the present study was to analyze the effect of apigenin, hesperidin and kaempferol on pre-adipocyte and mature adipocytes derived from this type of cells. In addition, the expression of genes involved in TG accumulation was also measured. METHODS: Pre-adipocytes were cultured from day 0 to day 8 and mature adipocytes for 48 h with the polyphenols at doses of 1, 10 and 25 µM. RESULTS: Apigenin did not show an anti-adipogenic action. Pre-adipocytes treated with hesperidin and kaempferol showed reduced TG content at the three experimental doses. Apigenin did not modify the expression of the main adipogenic genes (c/ebpß, c/ebpα, pparγ and srebp1c), hesperidin inhibited genes involved in the three phases of adipogenesis (c/ebpß, srebp1c and perilipin) and kaempferol reduced c/ebpß. In mature adipocytes, the three polyphenols reduced TG accumulation at the dose of 25 µM, but not at lower doses. All compounds increased mRNA levels of atgl. Apigenin and hesperidin decreased fasn expression. The present study shows the anti-adipogenic effect and delipidating effects of apigenin, hesperidin and kaempferol in human adipocytes derived from hMSCs. While hesperidin blocks all the stages of adipogenesis, kaempferol only inhibits the early stage. Regarding mature adipocytes, the three compounds reduce TG accumulation by activating, at least in part, lipolysis, and in the case of hesperidin and apigenin, also by reducing lipogenesis. CONCLUSIONS: The present study shows for the first time the anti-adipogenic effect and delipidating effect of apigenin, hesperidin and kaempferol in human adipocytes derived from MSCs for the first time.

Apolipoprotein E4 association with metabolic syndrome depends on body fatness.

BACKGROUND AND AIMS: The human Apolipoprotein E (APOE) gene is polymorphic. The APOE*4 allele is a risk factor for cardiovascular disease and could contribute to the development of the metabolic syndrome (MetS) as it may affect all MetS components. We hypothesize that the common APOE4 polymorphism differentially regulates MetS risk and that this association might be modulated by body fatness. METHODS & RESULTS: We used body mass index (BMI) as surrogate of fatness and cross-sectionally studied the prevalence of MetS in 4408 middle-aged men of the Aragon Workers Health Study (AWHS). Our analysis revealed i) a gene dose-dependent association between APOE*4 allele and increased risk for MetS, ii) this association primarily derived from the overweight subjects. For these individuals, the MetS risk was higher in APOE*4 carriers than in non-carriers (Odds Ratio = 1.31; 95% CI, 1.03-1.67). Additionally, we examined 3908 healthy young individuals from the Coronary Artery Risk Development in Young Adults (CARDIA) cohort, followed-up for 25 years. Compared with APOE*4 non-carriers, APOE*4 presence significantly increased the risk of developing MetS (Hazard Ratio, 1.12; 95% CI, 1.00-1.26). Again, an interplay between APOE*4 and the longitudinal development of fatness towards the onset of MetS occurred throughout the study. For individuals with BMI gain below the median, the cumulative onset rate of MetS was significantly higher in APOE*4 carriers than in the non-carriers (HR, 1.29; 95% CI, 1.07-1.55). CONCLUSIONS: Carrying APOE*4 alleles increases MetS in a dose-dependent manner, characterizing individual's APOE genotype might help identify at-risk subjects for preventive intervention.

The FAT expandability (FATe) Project: Biomarkers to determine the limit of expansion and the complications of obesity.

BACKGROUND: Obesity is an excessive accumulation of fat frequently, but not always, associated with health problems, mainly type 2 diabetes and cardiovascular disease. During a positive energy balance, as caused by excessive intake or sedentary lifestyle, subcutaneous adipose tissue expands and accumulates lipids as triglycerides. However, the amount of adipose tissue per se is unlikely to be the factor linking obesity and metabolic complications. The expandability hypothesis states that, if this positive energy balance is prolonged, a point is eventually reached where subcutaneous adipose tissue can not further expand and energy surplus no longer can be safely stored. Once the limit on storage capacity has been exceeded, the dietary lipids start spilling and accumulate ectopically in other organs (omentum, liver, muscle, pancreas) forming lipid byproducts toxic to cells. METHODS/DESIGN: FATe is a multidisciplinary clinical project aimed to fill gaps that still exist in the expandability hypothesis. Imaging techniques (CT-scan), metabolomics, and transcriptomics will be used to identify the factors that set the limit expansion of subcutaneous adipose tissue in a cohort of caucasian individuals with varying degrees of adiposity. Subsequently, a set of biomarkers that inform the individual limits of expandability will be developed using computational and mathematical modeling. A different validation cohort will be used to minimize the risk of false positive rates and increase biomarkers' predictive performance. DISCUSSION: The work proposed here will render a clinically useful screening method to predict which obese individuals will develop metabolic derangements, specially diabetes and cardiovascular disease. This study will also provide mechanistic evidence that promoting subcutaneous fat expansion might be a suitable therapy to reduce metabolic complications associated with positive energy balance characteristic of Westernized societies.

The apolipoprotein E polymorphism rs7412 associates with body fatness independently of plasma lipids in middle aged men.

BACKGROUND: The apolipoprotein E (APOE) gene is polymorphic, encoding one of 3 common alleles (ε2, ε3, ε4) produced from combinations of 2 non-synonymous SNPs (rs429358 and rs7412). APOE plays an important role controlling plasma lipids but its association with adipocyte functionality and body fatness remains to be determined. METHODS: We analyzed fasting plasma lipids and genotyped the two main APOE-SNPs (rs429358 and rs7412), both located in the fourth exon of the APOE, in 4660 Caucasian middle-aged men free of cardiovascular disease. RESULTS: The rs7412 SNP, which determines the APOE2 isoform, was significantly associated with Body Mass Index (BMI) and Waist Girth (WG) in a multivariate model accounting for age, smoking status and plasma lipids. BMI and WG were highest in TT homozygotes and lowest in CC homozygotes. This effect was independent of the rs429358 SNP, which failed to show any association with the BMI and WG variables. The odds ratio of being obese (BMI>30) for individuals carrying the APOε2 allele, present in 14% of the cohort and defined by the rs7412 SNP, was also significant in this multivariate model, with an OR of 1.27 (95% CI: 1.01-1.59). CONCLUSIONS: This study provides an evidence of a lipid-independent association between the APOE SNP rs7412 and body fatness surrogates, BMI and WG, in a large cohort of middle-aged males.