Gut microbiota signatures of vulnerability to food addiction in mice and humans.

OBJECTIVE: Food addiction is a multifactorial disorder characterised by a loss of control over food intake that may promote obesity and alter gut microbiota composition. We have investigated the potential involvement of the gut microbiota in the mechanisms underlying food addiction. DESIGN: We used the Yale Food Addiction Scale (YFAS) 2.0 criteria to classify extreme food addiction in mouse and human subpopulations to identify gut microbiota signatures associated with vulnerability to this disorder. RESULTS: Both animal and human cohorts showed important similarities in the gut microbiota signatures linked to food addiction. The signatures suggested possible non-beneficial effects of bacteria belonging to the Proteobacteria phylum and potential protective effects of Actinobacteria against the development of food addiction in both cohorts of humans and mice. A decreased relative abundance of the species Blautia wexlerae was observed in addicted humans and of Blautia genus in addicted mice. Administration of the non-digestible carbohydrates, lactulose and rhamnose, known to favour Blautia growth, led to increased relative abundance of Blautia in mice faeces in parallel with dramatic improvements in food addiction. A similar improvement was revealed after oral administration of Blautia wexlerae as a beneficial microbe. CONCLUSION: By understanding the crosstalk between this behavioural alteration and gut microbiota, these findings constitute a step forward to future treatments for food addiction and related eating disorders.

Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins.

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress.

Liver lipopolysaccharide binding protein prevents hepatic inflammation in physiological and pathological non-obesogenic conditions.

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels. As expected, the MCD increased hepatocyte vacuolation, liver inflammation and fibrosis markers, also increasing liver Lbp mRNA. In this model, liver Lbp gene knockdown resulted in a pronounced worsening of the markers of liver inflammation (also including CD68 and MPO activity), fibrosis, ER stress and protein carbonyl levels, all indicative of non-alcoholic steatohepatitis (NASH) progression. At cellular level, Lbp gene knockdown also increased expression of the proinflammatory mediators (Il6, Ccl2), and markers of fibrosis (Col1a1, Tgfb1) and protein carbonyl levels. In agreement with these findings, liver LBP mRNA in humans positively correlated with markers of liver damage (circulating hsCRP, ALT activity, liver CRP and KRT18 gene expression), and with a network of genes involved in liver inflammation, innate and adaptive immune system, endoplasmic reticulum stress and neutrophil degranulation (all with q-value<0.05). In conclusion, current findings suggest that a significant downregulation in liver LBP levels promotes liver oxidative stress and inflammation, aggravating NASH progression, in physiological and pathological non-obesogenic conditions.

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.

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.

Comparison of Outcomes between Obese and Nonobese Patients in Laparoscopic Adrenalectomy: A Cohort Study.

INTRODUCTION: Obesity is usually considered a risk factor for surgical complications. Laparoscopic adrenalectomy has replaced open adrenalectomy as the standard operation for adrenal tumors. OBJECTIVE: To compare the safety of laparoscopic adrenalectomy to treat adrenal tumors in obese versus nonobese patients. METHODS: This observational cohort study analyzed consecutive patients who underwent laparoscopic adrenalectomy with a lateral transperitoneal approach at a single center (2003-2020). Data and outcomes of obese (body mass index ≥30 kg/m2) and nonobese patients were compared. To analyze the association between operative time and other variables, we used simple and multivariate linear regression. RESULTS: N = 160 (90 obese/70 nonobese) patients underwent laparoscopic adrenalectomy. Cushing syndrome and pheochromocytoma were the most frequent indications. Obese patients were older (58 vs. 52 years, p < 0.001). A greater proportion of obese patients were ASA grade III + IV (71.1 vs. 48.6%, p = 0.004). Obesity was associated with a longer operative time (72.5 vs. 60 min, p < 0.001) and greater blood loss (40 vs. 20 mL, p = 0.022). There were no differences in conversion, morbidity, or hospital stay. After adjustment for confounding factors, operative time was positively correlated with BMI ≥30 kg/m2, learning curve, estimated blood loss, 2D laparoscopy, and specimen size. CONCLUSION: Lateral transperitoneal laparoscopic adrenalectomy is safe in patients with a BMI 30-35 kg/m2, so these patients also benefit from this minimally invasive surgery.

Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

During adipogenesis, preadipocytes' cytoskeleton reorganizes in parallel with lipid accumulation. Failure to do so may impact the ability of adipose tissue (AT) to shift between lipid storage and mobilization. Here, we identify cytoskeletal transgelin 2 (TAGLN2) as a protein expressed in AT and associated with obesity and inflammation, being normalized upon weight loss. TAGLN2 was primarily found in the adipose stromovascular cell fraction, but inflammation, TGF-ß, and estradiol also prompted increased expression in human adipocytes. Tagln2 knockdown revealed a key functional role, being required for proliferation and differentiation of fat cells, whereas transgenic mice overexpressing Tagln2 using the adipocyte protein 2 promoter disclosed remarkable sex-dependent variations, in which females displayed "healthy" obesity and hypertrophied adipocytes but preserved insulin sensitivity, and males exhibited physiologic changes suggestive of defective AT expandability, including increased number of small adipocytes, activation of immune cells, mitochondrial dysfunction, and impaired metabolism together with decreased insulin sensitivity. The metabolic relevance and sexual dimorphism of TAGLN2 was also outlined by genetic variants that may modulate its expression and are associated with obesity and the risk of ischemic heart disease in men. Collectively, current findings highlight the contribution of cytoskeletal TAGLN2 to the obese phenotype in a gender-dependent manner.-Ortega, F. J., Moreno-Navarrete, J. M., Mercader, J. M., Gómez-Serrano, M., García-Santos, E., Latorre, J., Lluch, A., Sabater, M., Caballano-Infantes, E., Guzmán, R., Macías-González, M., Buxo, M., Gironés, J., Vilallonga, R., Naon, D., Botas, P., Delgado, E., Corella, D., Burcelin, R., Frühbeck, G., Ricart, W., Simó, R., Castrillon-Rodríguez, I., Tinahones, F. J., Bosch, F., Vidal-Puig, A., Malagón, M. M., Peral, B., Zorzano, A., Fernández-Real, J. M. Cytoskeletal transgelin 2 contributes to gender-dependent adipose tissue expandability and immune function.

Adipose tissue TSH as a new modulator of human adipocyte mitochondrial function.

BACKGROUND/OBJECTIVES: Recent studies indicate a possible role of TSH/TSHR signalling axis on adipogenesis and adipose tissue physiology. Here, we aimed to investigate the relationship between adipose tissue TSHB and adipose tissue physiology-related gene expression. SUBJECTS/METHODS: Subcutaneous and visceral adipose tissue TSHB gene expression was analysed in two independent cohorts [Cohort1 (N = 96) and Cohort2 (N = 45)] and after bariatric surgery-induced weight loss [Cohort3 (N = 22)]. Adipose tissue TSH protein expression was also analysed in a subgroup of participants from Cohort 1 (N = 16). The effects of recombinant TSH on human subcutaneous preadipocytes and adipocytes were investigated. RESULTS: In cohort 1, both visceral and subcutaneous adipose tissue TSHB gene expression was positively correlated with the expression of mitochondrial function (PPARGC1A, ISCA2, CISD1, SIRT1, NFE2L2, NRF1) and fatty acid mobilization (CAV1, ENGL1), but not with adipogenic-related genes. Of note, adipose tissue TSH protein levels were also associated with some of these markers of mitochondrial function and fatty acid mobilization. These associations were replicated in cohort 2. Bariatric surgery-induced weight loss resulted in increased subcutaneous adipose tissue TSHB in parallel to increased PPARGC1A. In human subcutaneous adipocytes, rh-TSH administration led to increased mitochondrial respiratory capacity in parallel to increased mitochondrial function- and adipogenic-related gene expression, but no significant effects were observed during differentiation of human preadipocytes. CONCLUSION: These data point to a possible role of adipose tissue TSH in the maintenance of adipocyte mitochondrial function.

Decreased TLR3 in Hyperplastic Adipose Tissue, Blood and Inflamed Adipocytes is Related to Metabolic Inflammation.

BACKGROUND/AIMS: Obesity is characterized by the immune activation that eventually dampens insulin sensitivity and changes metabolism. This study explores the impact of different inflammatory/ anti-inflammatory paradigms on the expression of toll-like receptors (TLR) found in adipocyte cultures, adipose tissue, and blood. METHODS: We evaluated by real time PCR the impact of acute surgery stress in vivo (adipose tissue) and macrophages (MCM) in vitro (adipocytes). Weight loss was chosen as an anti-inflammatory model, so TLR were analyzed in fat samples collected before and after bariatric surgery-induced weight loss. Associations with inflammatory and metabolic parameters were analyzed in non-obese and obese subjects, in parallel with gene expression measures taken in blood and isolated adipocytes/ stromal-vascular cells (SVC). Treatments with an agonist of TLR3 were conducted in human adipocyte cultures under normal conditions and upon conditions that simulated the chronic low-grade inflammatory state of obesity. RESULTS: Surgery stress raised TLR1 and TLR8 in subcutaneous (SAT), and TLR2 in SAT and visceral (VAT) adipose tissue, while decreasing VAT TLR3 and TLR4. MCM led to increased TLR2 and diminished TLR3, TLR4, and TLR5 expressions in human adipocytes. The anti-inflammatory impact of weight loss was concomitant with decreased TLR1, TLR3, and TLR8 in SAT. Cross-sectional associations confirmed increased V/ SAT TLR1 and TLR8, and decreased TLR3 in obese patients, as compared with non-obese subjects. As expected, TLR were predominant in SVC and adipocyte precursor cells, even though expression of all of them but TLR8 (very low levels) was also found in ex vivo isolated and in vitro differentiated adipocytes. Among SVC, CD14+ macrophages showed increased TLR1, TLR2, and TLR7, but decreased TLR3 mRNA. The opposite patterns shown for TLR2 and TLR3 in V/ SAT, SVC, and inflamed adipocytes were observed in blood as well, being TLR3 more likely linked to lymphocyte instead of neutrophil counts. On the other hand, decreased TLR3 in adipocytes challenged with MCM dampened lipogenesis and the inflammatory response to Poly(I:C). CONCLUSION: Functional variations in the expression of TLR found in blood and hypertrophied fat depots, namely decreased TLR3 in lymphocytes and inflamed adipocytes, are linked to metabolic inflammation.

Adipose TSHB in Humans and Serum TSH in Hypothyroid Rats Inform About Cellular Senescence.

BACKGROUND/AIMS: Thyroid hormones have been recently linked to senescence and longevity. Given the recent description of TSHB mRNA in human adipose tissue (AT), we aimed to investigate the relationship between local AT TSH and adipose tissue senescence. METHODS: TSHB mRNA (measured by real-time PCR) and markers of adipose tissue senescence [BAX, DBC1, TP53, TNF (real-time PCR), telomere length (Telo TAGGG Telomere Length Assay) and lipidomics (liquid chromatography mass spectrometry)] were analysed in subcutaneous (SAT) and visceral (VAT) AT from euthyroid subjects. The chronic effects of TSH were also investigated in AT from hypothyroid rats and after recombinant human TSH (rhTSH) administration in human adipocytes. RESULTS: Both VAT and SAT TSHB gene expression negatively correlated with markers of AT cellular senescence (BAX, DBC1, TP53, TNF gene expression and specific glucosylceramides) and positively associated with telomere length. Supporting these observations, both rhTSH administration in human adipocytes and increased TSH in hypothyroid rats resulted in decreased markers of cellular senescence (Bax and Tp53 mRNA) in both gonadal and subcutaneous white adipose tissue. CONCLUSION: These data point to a possible role of TSH in AT cellular senescence.

HMOX1 as a marker of iron excess-induced adipose tissue dysfunction, affecting glucose uptake and respiratory capacity in human adipocytes.

AIMS/HYPOTHESIS: Iron excess in adipose tissue is known to promote adipose tissue dysfunction. Here, we aimed to investigate the possible role of haem oxygenase 1 (HMOX1) in iron excess-induced adipose tissue dysfunction. METHODS: Cross-sectionally, HMOX1 gene expression in subcutaneous and visceral adipose tissue was analysed in two independent cohorts (n = 234 and 40) in relation to obesity. We also evaluated the impact of weight loss (n = 21), weight gain (in rats, n = 20) on HMOX1 mRNA; HMOX1 mRNA levels during human adipocyte differentiation; the effects of inflammation and iron on adipocyte HMOX1; and the effects of HMOX1-induced activity on adipocyte mitochondrial respiratory function, glucose uptake and adipogenesis. RESULTS: Adipose tissue HMOX1 was increased in obese participants (p = 0.01) and positively associated with obesity-related metabolic disturbances, and markers of iron accumulation, inflammation and oxidative stress (p < 0.01). HMOX1 was negatively correlated with mRNAs related to mitochondrial biogenesis, the insulin signalling pathway and adipogenesis (p < 0.01). These associations were replicated in an independent cohort. Bariatric surgery-induced weight loss led to reduced HMOX1 (0.024 ± 0.010 vs 0.010 ± 0.004 RU, p < 0.0001), whereas in rats, high-fat diet-induced weight gain resulted in increased Hmox1 mRNA levels (0.22 ± 0.15 vs 0.54 ± 0.22 RU, p = 0.005). These changes were in parallel with changes in BMI and adipose tissue markers of iron excess, adipogenesis and inflammation. In human adipocytes, iron excess and inflammation led to increased HMOX1 mRNA levels. HMOX1 induction (by haem arginate [hemin] administration), resulted in a significant reduction of mitochondrial respiratory capacity (including basal respiration and spare respiratory capacity), glucose uptake and adipogenesis in parallel with increased expression of inflammatory- and iron excess-related genes. CONCLUSIONS/INTERPRETATION: HMOX1 is an important marker of iron excess-induced adipose tissue dysfunction and metabolic disturbances in human obesity.

Inflammatory response to bacterial lipopolysaccharide drives iron accumulation in human adipocytes.

The association among increased inflammation, disrupted iron homeostasis, and adipose tissue dysfunction in obesity has been widely recognized. However, the specific impact of inflammation on iron homeostasis during human adipogenesis and in adipocytes remains poorly understood. In this study, we investigated the effects of bacterial lipopolysaccharide (LPS) on iron homeostasis during human adipocyte differentiation, in fully differentiated adipocytes, and in human adipose tissue. We found that LPS-induced inflammation hindered adipogenesis and led to a gene expression profile indicative of intracellular iron accumulation. This was accompanied by increased expression of iron importers (TFRC and SLC11A2), markers of intracellular iron accumulation (FTH, CYBA, FTL, and LCN2), and decreased expression of iron exporter-related genes (SLC40A1), concomitant with elevated intracellular iron levels. Mechanistically, RNA-seq analysis and gene knockdown experiments revealed the significant involvement of iron importers SLC39A14, SLC39A8, and STEAP4 in LPS-induced intracellular iron accumulation in human adipocytes. Notably, markers of LPS signaling pathway-related inflammation were also associated with a gene expression pattern indicative of intracellular iron accumulation in human adipose tissue, corroborating the link between LPS-induced inflammation and iron accumulation at the tissue level. In conclusion, our findings demonstrate that induction of adipocyte inflammation disrupts iron homeostasis, resulting in adipocyte iron overload.

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.

Downregulated Adipose Tissue Expression of Browning Genes With Increased Environmental Temperatures.

CONTEXT: Climate change and global warming have been hypothesized to influence the increased prevalence of obesity worldwide. However, the evidence is scarce. OBJECTIVE: We aimed to investigate how outside temperature might affect adipose tissue physiology and metabolic traits. METHODS: The expression of genes involved in thermogenesis/browning and adipogenesis were evaluated (through quantitative polymerase chain reaction) in the subcutaneous adipose tissue (SAT) from 1083 individuals recruited in 5 different regions of Spain (3 in the North and 2 in the South). Plasma biochemical variables and adiponectin (enzyme-linked immunosorbent assay) were collected through standardized protocols. Mean environmental outdoor temperatures were obtained from the National Agency of Meteorology. Univariate, multivariate, and artificial intelligence analyses (Boruta algorithm) were performed. RESULTS: The SAT expression of genes associated with browning (UCP1, PRDM16, and CIDEA) and ADIPOQ were significantly and negatively associated with minimum, average, and maximum temperatures. The latter temperatures were also negatively associated with the expression of genes involved in adipogenesis (FASN, SLC2A4, and PLIN1). Decreased SAT expression of UCP1 and ADIPOQ messenger RNA and circulating adiponectin were observed with increasing temperatures in all individuals as a whole and within participants with obesity in univariate, multivariate, and artificial intelligence analyses. The differences remained statistically significant in individuals without type 2 diabetes and in samples collected during winter. CONCLUSION: Decreased adipose tissue expression of genes involved in browning and adiponectin with increased environmental temperatures were observed. Given the North-South gradient of obesity prevalence in these same regions, the present observations could have implications for the relationship of the obesity pandemic with global warming.

Potential therapeutic implications of histidine catabolism by the gut microbiota in NAFLD patients with morbid obesity.

The gut microbiota contributes to the pathophysiology of non-alcoholic fatty liver disease (NAFLD). Histidine is a key energy source for the microbiota, scavenging it from the host. Its role in NAFLD is poorly known. Plasma metabolomics, liver transcriptomics, and fecal metagenomics were performed in three human cohorts coupled with hepatocyte, rodent, and Drosophila models. Machine learning analyses identified plasma histidine as being strongly inversely associated with steatosis and linked to a hepatic transcriptomic signature involved in insulin signaling, inflammation, and trace amine-associated receptor 1. Circulating histidine was inversely associated with Proteobacteria and positively with bacteria lacking the histidine utilization (Hut) system. Histidine supplementation improved NAFLD in different animal models (diet-induced NAFLD in mouse and flies, ob/ob mouse, and ovariectomized rats) and reduced de novo lipogenesis. Fecal microbiota transplantation (FMT) from low-histidine donors and mono-colonization of germ-free flies with Enterobacter cloacae increased triglyceride accumulation and reduced histidine content. The interplay among microbiota, histidine catabolism, and NAFLD opens therapeutic opportunities.

Lysozyme Gene Expression in 3T3-L1 Cells Sustains Expression of Adipogenic Genes and Adipocyte Differentiation.

Substantial levels of lysozyme in adipose tissue in association to obesity have been recently demonstrated in mice and humans. In addition, experiments in mice suggest that lysozyme might impact on adipose tissue adipogenesis. To further investigate the relationship between lysozyme and adipogenesis, in the present study, we aimed to study lysozyme (Lyz2) during 3T3-L1 adipocyte differentiation and its possible role in adipogenesis. Time course experiment during 3T3-L1 adipocyte differentiation indicated that Lyz2 gene expression decreased at day 4, which was caused by isobutylmethylxanthine administration, and recovered at the end of the process (day 8). Importantly, the impact of isobutylmethylxanthine-induced downregulation of Lyz2 gene expression on adipogenesis was not comparable to that observed in the full cocktail, questioning whether the reduction in lysozyme at early stage of adipocyte differentiation is relevant to this process. In fact, the depletion in Lyz2 expression had a negative impact on adipogenesis, and rosiglitazone administration failed to compensate for the anti-adipogenic effect observed in Lyz2 gene knockdown cells. Otherwise, when Lyz2 gene knockdown cells were co-cultured with control cells, these cells had higher expression of adipogenic genes than those co-cultured with themselves at the end of adipocyte differentiation. In conclusion, this study suggests that lysozyme expression in 3T3-L1 cells sustains expression of adipogenic genes and adipocyte differentiation.

The Combined Partial Knockdown of CBS and MPST Genes Induces Inflammation, Impairs Adipocyte Function-Related Gene Expression and Disrupts Protein Persulfidation in Human Adipocytes.

Recent studies in mice and humans demonstrated the relevance of H2S synthesising enzymes, such as CTH, CBS, and MPST, in the physiology of adipose tissue and the differentiation of preadipocyte into adipocytes. Here, our objective was to investigate the combined role of CTH, CBS, and MPST in the preservation of adipocyte protein persulfidation and adipogenesis. Combined partial CTH, CBS, and MPST gene knockdown was achieved treating fully human adipocytes with siRNAs against these transcripts (siRNA_MIX). Adipocyte protein persulfidation was analyzed using label-free quantitative mass spectrometry coupled with a dimedone-switch method for protein labeling and purification. Proteomic analysis quantified 216 proteins with statistically different levels of persulfidation in KD cells compared to control adipocytes. In fully differentiated adipocytes, CBS and MPST mRNA and protein levels were abundant, while CTH expression was very low. It is noteworthy that siRNA_MIX administration resulted in a significant decrease in CBS and MPST expression, without impacting on CTH. The combined partial knockdown of the CBS and MPST genes resulted in reduced cellular sulfide levels in parallel to decreased expression of relevant genes for adipocyte biology, including adipogenesis, mitochondrial biogenesis, and lipogenesis, but increased proinflammatory- and senescence-related genes. It should be noted that the combined partial knockdown of CBS and MPST genes also led to a significant disruption in the persulfidation pattern of the adipocyte proteins. Although among the less persulfidated proteins, we identified several relevant proteins for adipocyte adipogenesis and function, among the most persulfidated, key mediators of adipocyte inflammation and dysfunction as well as some proteins that might play a positive role in adipogenesis were found. In conclusion, the current study indicates that the combined partial elimination of CBS and MPST (but not CTH) in adipocytes affects the expression of genes related to the maintenance of adipocyte function and promotes inflammation, possibly by altering the pattern of protein persulfidation in these cells, suggesting that these enzymes were required for the functional maintenance of adipocytes.

Adipose tissue cysteine dioxygenase type 1 is associated with an anti-inflammatory profile, impacting on systemic metabolic traits.

BACKGROUND: Adipose tissue is a source of multiple factors that modulate systemic insulin sensitivity and cardiovascular risk. Taurine is obtained from the diet but it is less known that it is endogenously synthesized by cysteine dioxygenase type 1 (CDO1). CDO1 exerts a role in adipose tissue from rodent models, but the potential translational value in humans is not available in the literature. METHODS: CDO1 gene expression was analysed in visceral and subcutaneous adipose tissue samples in association with metabolic traits in participants with different degrees of obesity in four independent cohorts. CDO1 was also evaluated in isolated human adipocytes in vitro. Mechanistically, CDO1gene knockdown (KD) of human preadipocytes and adipose-derived mesenchymal stem cells (ASC52telo) (using lentiviral particles) was also evaluated. Mitochondrial respiratory function of adipocytes was evaluated using Seahorse. FINDINGS: Both visceral (VAT) and subcutaneous adipose tissue (SAT) CDO1 mRNA was associated with gene expression markers of adipose tissue function in the four cohorts. Higher CDO1 expression was linked to decreased fasting triglycerides and blood HbA1c even after adjusting by age, BMI and sex. In addition, CDO1 mRNA positively correlated with the expression of genes involved in adipogenesis and negatively with different inflammatory markers. Both VAT and SAT CDO1 mRNA was mainly expressed in adipocytes and significantly increased during adipocyte differentiation, but attenuated under inflammatory conditions. Mechanistically, CDO1 gene KD reduced taurine biosynthesis, evidencing lower CDO1 activity. In both human preadipocytes and ASC52telo cells, CDO1 gene KD resulted in decreased gene expression markers of adipogenesis (ADIPOQ, FABP4, FASN, SLC2A4, CEBPA) and increased inflammatory genes (TNF and IL6) during adipocyte differentiation. Of note, CDO1 gene KD led to decreased mitochondrial respiratory function in parallel to decreased expression of mitochondrial function-, but not biogenesis-related genes. INTERPRETATION: Current findings show the relevance of CDO1 in adipose tissue physiology, suggesting its contribution to an improved systemic metabolic profile. FUNDING: This work was partially supported by research grants PI16/01173, PI19/01712, PI20/01090 and PI21/01361 from the Instituto de Salud Carlos III from Spain, Fondo Europeo de Desarrollo Regional (FEDER) funds, and VII Spanish Diabetes Association grants to Basic Diabetes Research Projects led by young researchers.

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.

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.