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.

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.

Adipose tissue coregulates cognitive function.

Obesity is associated with cognitive decline. Recent observations in mice propose an adipose tissue (AT)-brain axis. We identified 188 genes from RNA sequencing of AT in three cohorts that were associated with performance in different cognitive domains. These genes were mostly involved in synaptic function, phosphatidylinositol metabolism, the complement cascade, anti-inflammatory signaling, and vitamin metabolism. These findings were translated into the plasma metabolome. The circulating blood expression levels of most of these genes were also associated with several cognitive domains in a cohort of 816 participants. Targeted misexpression of candidate gene ortholog in the Drosophila fat body significantly altered flies memory and learning. Among them, down-regulation of the neurotransmitter release cycle-associated gene SLC18A2 improved cognitive abilities in Drosophila and in mice. Up-regulation of RIMS1 in Drosophila fat body enhanced cognitive abilities. Current results show previously unidentified connections between AT transcriptome and brain function in humans, providing unprecedented diagnostic/therapeutic targets in AT.

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.

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.

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.

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.

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.

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.

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.

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.

Compounds that modulate AMPK activity and hepatic steatosis impact the biosynthesis of microRNAs required to maintain lipid homeostasis in hepatocytes.

BACKGROUND: While the impact of metformin in hepatocytes leads to fatty acid (FA) oxidation and decreased lipogenesis, hepatic microRNAs (miRNAs) have been associated with fat overload and impaired metabolism, contributing to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). METHODS: We investigated the expression of hundreds of miRNAs in primary hepatocytes challenged by compounds modulating steatosis, palmitic acid and compound C (as inducers), and metformin (as an inhibitor). Then, additional hepatocyte and rodent models were evaluated, together with transient mimic miRNAs transfection, lipid droplet staining, thin-layer chromatography, quantitative lipidomes, and mitochondrial activity, while human samples outlined the translational significance of this work. FINDINGS: Our results show that treatments triggering fat accumulation and AMPK disruption may compromise the biosynthesis of hepatic miRNAs, while the knockdown of the miRNA-processing enzyme DICER in human hepatocytes exhibited increased lipid deposition. In this context, the ectopic recovery of miR-30b and miR-30c led to significant changes in genes related to FA metabolism, consistent reduction of ceramides, higher mitochondrial activity, and enabled ß-oxidation, redirecting FA metabolism from energy storage to expenditure. INTERPRETATION: Current findings unravel the biosynthesis of hepatic miR-30b and miR-30c in tackling inadequate FA accumulation, offering a potential avenue for the treatment of NAFLD. FUNDING: Instituto de Salud Carlos III (ISCIII), Govern de la Generalitat (PERIS2016), Associació Catalana de Diabetis (ACD), Sociedad Española de Diabetes (SED), Fondo Europeo de Desarrollo Regional (FEDER), Xunta de Galicia, Ministerio de Economía y Competitividad (MINECO), "La Caixa" Foundation, and CIBER de la Fisiopatología de la Obesidad y Nutrición (CIBEROBN).