Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Gut Microbiota Composition and Functionality Are Associated With REM Sleep Duration and Continuous Glucose Levels.

CONTEXT: Sleep disruption is associated with worse glucose metabolic control and altered gut microbiota in animal models. OBJECTIVE: We aimed to evaluate the possible links among rapid eye movement (REM) sleep duration, continuous glucose levels, and gut microbiota composition. METHODS: This observational, prospective, real-life, cross-sectional case-control study included 118 (60 with obesity), middle-aged (39.1-54.8 years) healthy volunteers recruited at a tertiary hospital. Glucose variability and REM sleep duration were assessed by 10-day continuous glucose monitoring (CGM) (Dexcom G6) and wrist actigraphy (Fitbit Charge 3), respectively. The coefficient of variation (CV), interquartile range (IQR), and SD of glucose variability was assessed and the percentage of time in range (% TIR), at 126-139 mg/dL (TIR2), and 140-199 mg/dL (TIR3) were calculated. Shotgun metagenomics sequencing was applied to study gut microbiota taxonomy and functionality. RESULTS: Increased glycemic variability (SD, CV, and IQR) was observed among subjects with obesity in parallel to increased % TIR2 and % TIR3. REM sleep duration was independently associated with % TIR3 (ß = -.339; P < .001) and glucose variability (SD, ß = -.350; P < .001). Microbial taxa from the Christensenellaceae family (Firmicutes phylum) were positively associated with REM sleep and negatively with CGM levels, while bacteria from Enterobacteriacea family and bacterial functions involved in iron metabolism showed opposite associations. CONCLUSION: Decreased REM sleep duration was independently associated with a worse glucose profile. The associations of species from Christensenellaceae and Enterobacteriaceae families with REM sleep duration and continuous glucose values suggest an integrated picture of metabolic health.

The relevance of EGFR, ErbB receptors and neuregulins in human adipocytes and adipose tissue in obesity.

OBJECTIVE: To investigate the potential role of EGFR, ErbBs receptors and neuregulins in human adipose tissue physiology in obesity. METHODS: Gene expression analysis in human subcutaneous (SAT) and visceral (VAT) adipose tissue in three independent cohorts [two cross-sectional (N = 150, N = 87) and one longitudinal (n = 25)], and in vitro gene knockdown and overexpression experiments were performed. RESULTS: While both SAT and VAT ERBB2 and ERBB4 mRNA increased in obesity, SAT EGFR mRNA was negatively correlated with insulin resistance, but did not change in obesity. Of note, both SAT and VAT EGFR mRNA were significantly associated with adipogenesis and increased during human adipocyte differentiation. In vitro experiments revealed that EGFR, but not ERBB2 and ERBB4, gene knockdown in preadipocytes and in fully differentiated human adipocytes resulted in decreased expression of adipogenic-related genes. ERBB2 gene knockdown also reduced gene expression of fatty acid synthase in fully differentiated adipocytes. In addition, neuregulin 2 (NRG2) mRNA was associated with expression of adipogenic genes in human adipose tissue and adipocytes, and its overexpression increased expression of EGFR and relevant adipogenic genes. CONCLUSIONS: This study demonstrates the association between adipose tissue ERBB2 and obesity, confirms the relevance of EGFR on human adipogenesis, and suggests a possible adipogenic role of NRG2.

Serum neuregulin 4 is negatively correlated with insulin sensitivity in humans and impairs mitochondrial respiration in HepG2 cells.

Neuregulin 4 (NRG4) has been described to improve metabolic disturbances linked to obesity status in rodent models. The findings in humans are controversial. We aimed to investigate circulating NRG4 in association with insulin action in humans and the possible mechanisms involved. Insulin sensitivity (euglycemic hyperinsulinemic clamp) and serum NRG4 concentration (ELISA) were analysed in subjects with a wide range of adiposity (n = 89). In vitro experiments with human HepG2 cell line were also performed. Serum NRG4 was negatively correlated with insulin sensitivity (r = -0.25, p = 0.02) and positively with the inflammatory marker high-sensitivity C reative protein (hsCRP). In fact, multivariant linear regression analyses showed that insulin sensitivity contributed to BMI-, age-, sex-, and hsCRP-adjusted 7.2% of the variance in serum NRG4 (p = 0.01). No significant associations were found with adiposity measures (BMI, waist circumference or fat mass), plasma lipids (HDL-, LDL-cholesterol, or fasting triglycerides) or markers of liver injury. Cultured hepatocyte HepG2 treated with human recombinant NRG4 had an impact on hepatocyte metabolism, leading to decreased gluconeogenic- and mitochondrial biogenesis-related gene expression, and reduced mitochondrial respiration, without effects on expression of lipid metabolism-related genes. Similar but more pronounced effects were found after neuregulin 1 administration. In conclusion, sustained higher serum levels of neuregulin-4, observed in insulin resistant patients may have deleterious effects on metabolic and mitochondrial function in hepatocytes. However, findings from in vitro experiments should be confirmed in human primary hepatocytes.

Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation.

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP-Lbp UNA-siRNA) on the development of fatty liver. Weekly LNP-Lbp UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD). In mice fed a high-fat and high-sucrose diet, which displayed induced liver lipogenesis, LBP downregulation led to reduced liver lipid accumulation, lipogenesis (mainly stearoyl-coenzyme A desaturase 1 [Scd1]) and lipid peroxidation-associated oxidative stress markers. LNP-Lbp UNA-siRNA also resulted in significantly decreased blood glucose levels during an insulin tolerance test. In mice fed a standard chow diet or an MCD, in which liver lipogenesis was not induced or was inhibited (especially Scd1 mRNA), liver LBP downregulation did not impact on liver steatosis. The link between hepatocyte LBP and lipogenesis was further confirmed in palmitate-treated Hepa1-6 cells, in primary human hepatocytes, and in subjects with morbid obesity. Altogether, these data indicate that siRNA against liver Lbp mRNA constitutes a potential target therapy for obesity-associated fatty liver through the modulation of hepatic Scd1.

Presence of Blastocystis in gut microbiota is associated with cognitive traits and decreased executive function.

Growing evidence implicates the gut microbiome in cognition. Blastocystis is a common gut single-cell eukaryote parasite frequently detected in humans but its potential involvement in human pathophysiology has been poorly characterized. Here we describe how the presence of Blastocystis in the gut microbiome was associated with deficits in executive function and altered gut bacterial composition in a discovery (n = 114) and replication cohorts (n = 942). We also found that Blastocystis was linked to bacterial functions related to aromatic amino acids metabolism and folate-mediated pyrimidine and one-carbon metabolism. Blastocystis-associated shifts in bacterial functionality translated into the circulating metabolome. Finally, we evaluated the effects of microbiota transplantation. Donor's Blastocystis subtypes led to altered recipient's mice cognitive function and prefrontal cortex gene expression. In summary, Blastocystis warrant further consideration as a novel actor in the gut microbiome-brain axis.

Downregulation of peripheral lipopolysaccharide binding protein impacts on perigonadal adipose tissue only in female mice.

BACKGROUND AND AIMS: The sexual dimorphism in fat-mass distribution and circulating leptin and insulin levels is well known, influencing the progression of obesity-associated metabolic disease. Here, we aimed to investigate the possible role of lipopolysaccharide-binding protein (LBP) in this sexual dimorphism. METHODS: The relationship between plasma LBP and fat mass was evaluated in 145 subjects. The effects of Lbp downregulation, using lipid encapsulated unlocked nucleomonomer agent containing chemically modified-siRNA delivery system, were evaluated in mice. RESULTS: Plasma LBP levels were associated with fat mass and leptin levels in women with obesity, but not in men with obesity. In mice, plasma LBP downregulation led to reduced weight, fat mass and leptin gain after a high-fat and high-sucrose diet (HFHS) in females, in parallel to increased expression of adipogenic and thermogenic genes in visceral adipose tissue. This was not observed in males. Plasma LBP downregulation avoided the increase in serum LPS levels in HFHS-fed male and female mice. Serum LPS levels were positively correlated with body weight and fat mass gain, and negatively with markers of adipose tissue function only in female mice. The sexually dimorphic effects were replicated in mice with established obesity. Of note, LBP downregulation led to recovery of estrogen receptor alpha (Esr1) mRNA levels in females but not in males. CONCLUSION: LBP seems to exert a negative feedback on ERα-mediated estrogen action, impacting on genes involved in thermogenesis. The known decreased estrogen action and negative effects of metabolic endotoxemia may be targeted through LBP downregulation.

Microbiota alterations in proline metabolism impact depression.

The microbiota-gut-brain axis has emerged as a novel target in depression, a disorder with low treatment efficacy. However, the field is dominated by underpowered studies focusing on major depression not addressing microbiome functionality, compositional nature, or confounding factors. We applied a multi-omics approach combining pre-clinical models with three human cohorts including patients with mild depression. Microbial functions and metabolites converging onto glutamate/GABA metabolism, particularly proline, were linked to depression. High proline consumption was the dietary factor with the strongest impact on depression. Whole-brain dynamics revealed rich club network disruptions associated with depression and circulating proline. Proline supplementation in mice exacerbated depression along with microbial translocation. Human microbiota transplantation induced an emotionally impaired phenotype in mice and alterations in GABA-, proline-, and extracellular matrix-related prefrontal cortex genes. RNAi-mediated knockdown of proline and GABA transporters in Drosophila and mono-association with L. plantarum, a high GABA producer, conferred protection against depression-like states. Targeting the microbiome and dietary proline may open new windows for efficient depression treatment.

Caudovirales bacteriophages are associated with improved executive function and memory in flies, mice, and humans.

Growing evidence implicates the gut microbiome in cognition. Viruses, the most abundant life entities on the planet, are a commonly overlooked component of the gut virome, dominated by the Caudovirales and Microviridae bacteriophages. Here, we show in a discovery (n = 114) and a validation cohort (n = 942) that subjects with increased Caudovirales and Siphoviridae levels in the gut microbiome had better performance in executive processes and verbal memory. Conversely, increased Microviridae levels were linked to a greater impairment in executive abilities. Microbiota transplantation from human donors with increased specific Caudovirales (>90% from the Siphoviridae family) levels led to increased scores in the novel object recognition test in mice and up-regulated memory-promoting immediate early genes in the prefrontal cortex. Supplementation of the Drosophila diet with the 936 group of lactococcal Siphoviridae bacteriophages resulted in increased memory scores and upregulation of memory-involved brain genes. Thus, bacteriophages warrant consideration as novel actors in the microbiome-brain axis.

Specific adipose tissue Lbp gene knockdown prevents diet-induced body weight gain, impacting fat accretion-related gene and protein expression.

Lipopolysaccharide binding protein (Lbp) has been recently identified as a relevant component of innate immunity response associated to adiposity. Here, we aimed to investigate the impact of adipose tissue Lbp on weight gain and white adipose tissue (WAT) in male and female mice fed an obesogenic diet. Specific adipose tissue Lbp gene knockdown was achieved through lentiviral particles containing shRNA-Lbp injected through surgery intervention. In males, WAT Lbp mRNA levels increased in parallel to fat accretion, and specific WAT Lbp gene knockdown led to reduced body weight gain, decreased fat accretion-related gene and protein expression, and increased inguinal WAT basal lipase activity, in parallel to lowered plasma free fatty acids, leptin, triglycerides but higher glycerol levels, resulting in slightly improved insulin action in the insulin tolerance test. In both males and females, inguinal WAT Lbp gene knockdown resulted in increased Ucp1 and Ppargc1a mRNA and Ucp1 protein levels, confirming adipose Lbp as a WAT browning repressor. In perigonadal WAT, Lbp gene knockdown also resulted in increased Ucp1 mRNA levels, but only in female mice, in which it was 500-fold increased. These data suggest specific adipose tissue Lbp gene knockdown as a possible therapeutic approach in the prevention of obesity-associated fat accretion.

ITCH E3 ubiquitin ligase downregulation compromises hepatic degradation of branched-chain amino acids.

OBJECTIVE: Metabolic syndrome, obesity, and steatosis are characterized by a range of dysregulations including defects in ubiquitin ligase tagging proteins for degradation. The identification of novel hepatic genes associated with fatty liver disease and metabolic dysregulation may be relevant to unravelling new mechanisms involved in liver disease progression METHODS: Through integrative analysis of liver transcriptomic and metabolomic obtained from obese subjects with steatosis, we identified itchy E ubiquitin protein ligase (ITCH) as a gene downregulated in human hepatic tissue in relation to steatosis grade. Wild-type or ITCH knockout mouse models of non-alcoholic fatty liver disease (NAFLD) and obesity-related hepatocellular carcinoma were analyzed to dissect the causal role of ITCH in steatosis RESULTS: We show that ITCH regulation of branched-chain amino acids (BCAAs) degradation enzymes is impaired in obese women with grade 3 compared with grade 0 steatosis, and that ITCH acts as a gatekeeper whose loss results in elevation of circulating BCAAs associated with hepatic steatosis. When ITCH expression was specifically restored in the liver of ITCH knockout mice, ACADSB mRNA and protein are restored, and BCAA levels are normalized both in liver and plasma CONCLUSIONS: Our data support a novel functional role for ITCH in the hepatic regulation of BCAA metabolism and suggest that targeting ITCH in a liver-specific manner might help delay the progression of metabolic hepatic diseases and insulin resistance.

A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG.

The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets γ-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism of fat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.

Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Iron status influences non-alcoholic fatty liver disease in obesity through the gut microbiome.

BACKGROUND: The gut microbiome and iron status are known to play a role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), although their complex interaction remains unclear. RESULTS: Here, we applied an integrative systems medicine approach (faecal metagenomics, plasma and urine metabolomics, hepatic transcriptomics) in 2 well-characterised human cohorts of subjects with obesity (discovery n = 49 and validation n = 628) and an independent cohort formed by both individuals with and without obesity (n = 130), combined with in vitro and animal models. Serum ferritin levels, as a markers of liver iron stores, were positively associated with liver fat accumulation in parallel with lower gut microbial gene richness, composition and functionality. Specifically, ferritin had strong negative associations with the Pasteurellaceae, Leuconostocaceae and Micrococcaea families. It also had consistent negative associations with several Veillonella, Bifidobacterium and Lactobacillus species, but positive associations with Bacteroides and Prevotella spp. Notably, the ferritin-associated bacterial families had a strong correlation with iron-related liver genes. In addition, several bacterial functions related to iron metabolism (transport, chelation, heme and siderophore biosynthesis) and NAFLD (fatty acid and glutathione biosynthesis) were also associated with the host serum ferritin levels. This iron-related microbiome signature was linked to a transcriptomic and metabolomic signature associated to the degree of liver fat accumulation through hepatic glucose metabolism. In particular, we found a consistent association among serum ferritin, Pasteurellaceae and Micrococcacea families, bacterial functions involved in histidine transport, the host circulating histidine levels and the liver expression of GYS2 and SEC24B. Serum ferritin was also related to bacterial glycine transporters, the host glycine serum levels and the liver expression of glycine transporters. The transcriptomic findings were replicated in human primary hepatocytes, where iron supplementation also led to triglycerides accumulation and induced the expression of lipid and iron metabolism genes in synergy with palmitic acid. We further explored the direct impact of the microbiome on iron metabolism and liver fact accumulation through transplantation of faecal microbiota into recipient's mice. In line with the results in humans, transplantation from 'high ferritin donors' resulted in alterations in several genes related to iron metabolism and fatty acid accumulation in recipient's mice. CONCLUSIONS: Altogether, a significant interplay among the gut microbiome, iron status and liver fat accumulation is revealed, with potential significance for target therapies. Video abstract.

Activation of Endogenous H2S Biosynthesis or Supplementation with Exogenous H2S Enhances Adipose Tissue Adipogenesis and Preserves Adipocyte Physiology in Humans.

Aims: To investigate the impact of exogenous hydrogen sulfide (H2S) and its endogenous biosynthesis on human adipocytes and adipose tissue in the context of obesity and insulin resistance. Results: Experiments in human adipose tissue explants and in isolated preadipocytes demonstrated that exogenous H2S or the activation of endogenous H2S biosynthesis resulted in increased adipogenesis, insulin action, sirtuin deacetylase, and PPARγ transcriptional activity, whereas chemical inhibition and gene knockdown of each enzyme generating H2S (CTH, CBS, MPST) led to altered adipocyte differentiation, cellular senescence, and increased inflammation. In agreement with these experimental data, visceral and subcutaneous adipose tissue expression of H2S-synthesising enzymes was significantly reduced in morbidly obese subjects in association with attenuated adipogenesis and increased markers of adipose tissue inflammation and senescence. Interestingly, weight-loss interventions (including bariatric surgery or diet/exercise) improved the expression of H2S biosynthesis-related genes. In human preadipocytes, the expression of CTH, CBS, and MPST genes and H2S production were dramatically increased during adipocyte differentiation. More importantly, the adipocyte proteome exhibiting persulfidation was characterized, disclosing that different proteins involved in fatty acid and lipid metabolism, the citrate cycle, insulin signaling, several adipokines, and PPAR, experienced the most dramatic persulfidation (85-98%). Innovation: No previous studies investigated the impact of H2S on human adipose tissue. This study suggests that the potentiation of adipose tissue H2S biosynthesis is a possible therapeutic approach to improve adipose tissue dysfunction in patients with obesity and insulin resistance. Conclusion: Altogether, these data supported the relevance of H2S biosynthesis in the modulation of human adipocyte physiology. Antioxid. Redox Signal. 35, 319-340.

Adipose tissue knockdown of lysozyme reduces local inflammation and improves adipogenesis in high-fat diet-fed mice.

Chronic systemic low-level inflammation in metabolic disease is known to affect adipose tissue biology. Lysozyme (LYZ) is a major innate immune protein but its role in adipose tissue has not been investigated. Here, we aimed to investigate LYZ in human and rodents fat depots, and its possible role in obesity-associated adipose tissue dysfunction. LYZ mRNA and protein were identified to be highly expressed in adipose tissue from subjects with obesity and linked to systemic chronic-low grade inflammation, adipose tissue inflammation and metabolic disturbances, including hyperglycemia, dyslipidemia and decreased markers of adipose tissue adipogenesis. These findings were confirmed in experimental models after a high-fat diet in mice and rats and also in ob/ob mice. Importantly, specific inguinal and perigonadal white adipose tissue lysozyme (Lyz2) gene knockdown in high-fat diet-fed mice resulted in improved adipose tissue inflammation in parallel to reduced lysozyme activity. Of note, Lyz2 gene knockdown restored adipogenesis and reduced weight gain in this model. In conclusion, altogether these observations point to lysozyme as a new actor in obesity-associated adipose tissue dysfunction. The therapeutic targeting of lysozyme production might contribute to improve adipose tissue metabolic homeostasis.

Obesity-associated deficits in inhibitory control are phenocopied to mice through gut microbiota changes in one-carbon and aromatic amino acids metabolic pathways.

BACKGROUND: Inhibitory control (IC) is critical to keep long-term goals in everyday life. Bidirectional relationships between IC deficits and obesity are behind unhealthy eating and physical exercise habits. METHODS: We studied gut microbiome composition and functionality, and plasma and faecal metabolomics in association with cognitive tests evaluating inhibitory control (Stroop test) and brain structure in a discovery (n=156), both cross-sectionally and longitudinally, and in an independent replication cohort (n=970). Faecal microbiota transplantation (FMT) in mice evaluated the impact on reversal learning and medial prefrontal cortex (mPFC) transcriptomics. RESULTS: An interplay among IC, brain structure (in humans) and mPFC transcriptomics (in mice), plasma/faecal metabolomics and the gut metagenome was found. Obesity-dependent alterations in one-carbon metabolism, tryptophan and histidine pathways were associated with IC in the two independent cohorts. Bacterial functions linked to one-carbon metabolism (thyX,dut, exodeoxyribonuclease V), and the anterior cingulate cortex volume were associated with IC, cross-sectionally and longitudinally. FMT from individuals with obesity led to alterations in mice reversal learning. In an independent FMT experiment, human donor's bacterial functions related to IC deficits were associated with mPFC expression of one-carbon metabolism-related genes of recipient's mice. CONCLUSION: These results highlight the importance of targeting obesity-related impulsive behaviour through the induction of gut microbiota shifts.

Permanent cystathionine-ß-Synthase gene knockdown promotes inflammation and oxidative stress in immortalized human adipose-derived mesenchymal stem cells, enhancing their adipogenic capacity.

In the present study, we aimed to investigate the impact of permanent cystathionine-ß-Synthase (CBS) gene knockdown in human telomerase reverse transcriptase (hTERT) immortalized human adipose-derived mesenchymal stem cells (ASC52telo) and in their capacity to differentiate into adipocytes. CBS gene KD in ASC52telo cells led to increased cellular inflammation (IL6, CXCL8, TNF) and oxidative stress markers (increased intracellular reactive oxygen species and decreased reduced glutathione levels) in parallel to decreased H2S production and rejuvenation (LC3 and SIRT1)-related gene expression. In addition, CBS gene KD in ASC52telo cells resulted in altered mitochondrial respiratory function, characterised by decreased basal respiration (specifically proton leak) and spare respiratory capacity, without significant effects on cell viability and proliferation. In this context, shCBS-ASC52telo cells displayed enhanced adipogenic (FABP4, ADIPOQ, SLC2A4, CEBPA, PPARG)-, lipogenic (FASN, DGAT1)- and adipocyte (LEP, LBP)-related gene expression markers, decreased expression of proinflammatory cytokines, and increased intracellular lipid accumulation during adipocyte differentiation compared to control ASC52telo cells. Otherwise, the increased adipogenic potential of shCBS-ASC52telo cells was detrimental to the ability to differentiate into osteogenic linage. In conclusion, this study demonstrated that permanent CBS gene KD in ASC52telo cells promotes a cellular senescence phenotype with a very increased adipogenic potential, promoting a non-physiological enhanced adipocyte differentiation with excessive lipid storage.

Morbidly obese subjects show increased serum sulfide in proportion to fat mass.

BACKGROUND AND OBJECTIVES: The importance of hydrogen sulfide is increasingly recognized in the pathophysiology of obesity and type 2 diabetes in animal models. Very few studies have evaluated circulating sulfides in humans, with discrepant results. Here, we aimed to investigate serum sulfide levels according to obesity. SUBJECTS AND METHODS: Serum sulfide levels were analyzed, using a selective fluorescent probe, in two independent cohorts [cross-sectionally in discovery (n = 139) and validation (n = 71) cohorts, and longitudinally in 82 participants from discovery cohort]. In the validation cohort, blood gene expression of enzymes contributing to H2S generation and consumption were also measured. RESULTS: In the discovery cohort, serum sulfide concentration was significantly increased in subjects with morbid obesity at baseline and follow-up, and positively correlated with BMI and fat mass, but negatively with total cholesterol, haemoglobin, serum ferritin, iron and bilirubin after adjusting by age, gender and fat mass. Fat mass (ß = 0.51, t = 3.67, p < 0.0001) contributed independently to age-, gender-, insulin sensitivity- and BMI-adjusted serum sulfide concentration variance. Importantly, receiver operating characteristic analysis demonstrated the relevance of fat mass predicting serum sulfide levels, which was replicated in the validation cohort. In addition, serum sulfide concentration was decreased in morbidly obese subjects with impaired compared to those with normal fasting glucose. Longitudinally, weight gain resulted in increased serum sulfide concentration, whereas weight loss had opposite effects, being the percent change in serum sulfide positively correlated with the percent change in BMI and waist circumference, but negatively with bilirubin. Whole blood CBS, CTH, MPST, SQOR, TST and MPO gene expression was not associated to obesity or serum sulfide concentration. CONCLUSIONS: Altogether these data indicated that serum sulfide concentrations were increased in subjects with morbid obesity in proportion to fat mass and inversely associated with circulating markers of haem degradation.

Lysozyme is a component of the innate immune system linked to obesity associated-chronic low-grade inflammation and altered glucose tolerance.

BACKGROUND & AIMS: Several proteins of the innate immune system are known to be deregulated with insulin resistance. We here aimed to investigate the relationship among circulating lysozyme (both plasma concentration and activity) and obesity-associated metabolic disturbances. METHODS: Plasma lysozyme concentration was determined cross-sectionally in a discovery (Cohort 1, n = 137) and in a replication cohort (Cohort 2, n = 181), in which plasma lysozyme activity was also analyzed. Plasma lysozyme was also evaluated longitudinally in participants from the replication cohort (n = 93). Leukocyte lysozyme expression (LYZ mRNA) were also investigated in an independent cohort (Cohort 3, n = 76), and adipose tissue (AT) LYZ mRNA (n = 25) and plasma peptidoglycan levels (n = 61) in subcohorts from discovery cohort. RESULTS: Translocation of peptidoglycan (as inferred from its increased circulating levels) was linked to plasma lysozyme, hyperinsulinemia and dyslipidemia in obese subjects. In both discovery and replication cohorts, plasma lysozyme levels and activity were significantly increased in obesity in direct association with obesity-associated metabolic disturbances and inflammatory parameters, being circulating lysozyme negatively correlated with fasting glucose, HbA1c and insulin resistance (HOMA-IR) in obese subjects. Of note, total cholesterol (p < 0.0001) and LDL cholesterol (p = 0.003) contributed independently to age-, gender- and BMI adjusted plasma lysozyme activity. Longitudinally, changes in HbA1c levels and serum LDL cholesterol were negatively associated with circulating lysozyme antimicrobial activity. On the contrary, the change in glucose infusion rate during the clamp (insulin sensitivity) was positively associated with lysozyme concentration. CONCLUSIONS: Increased plasma lysozyme levels and activity are found in obese subjects. The longitudinal findings suggest that plasma lysozyme might be protective on the development of obesity-associated metabolic disturbances.