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.
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•LPS-induced inflammation promotes intracellular iron accumulation during adipocyte differentiation and in human adipocytes.•SLC39A14, SLC39A8 and STEAP4 are involved in LPS-induced intracellular iron accumulation.•The link between LPS-induced inflammation and iron accumulation is confirmed in human adipose tissue.
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.
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 H
S generation and consumption were also measured.
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 (β = 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.
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.
Abstract
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.
In the present study, we aimed to investigate the impact of permanent cystathionine-β-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.
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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.
Most of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function ...of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified.
Here, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis.
We have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release.
These findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.
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.
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The current study provides further evidence about the relevance of adipose tissue LBP in obesity, suggesting that specific adipose tissue Lbp gene knockdown might constitute a novel therapeutic approach in the prevention of obesity-associated fat accretion, dyslipidemia, and insulin resistance.
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.
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).
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.
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 β-oxidation, redirecting FA metabolism from energy storage to expenditure.
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.
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).
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.
Absence of H19X‐encoded miR‐424(322)/503 in mice results in altered progenitor/adipose stem cell homeostasis, adipose tissue expansion, and its expression is reduced in obese subjects. Mechanistically, epigenetically‐controlled miR‐424(322)/503 modulates adipocyte differentiation by regulating γ‐Synuclein (SNCG). Expression levels of miR‐424(322)/503 and tissular/soluble SNCG co‐segregate with obesity in humans and can be modulated by weight loss surgically induced by gastric bypass.
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