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The role of hepatic cell senescence in human non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is not well understood. To examine this, we performed liver biopsies and extensive characterization of 58 individuals with or without NAFLD/NASH. Here, we show that hepatic cell senescence is strongly related to NAFLD/NASH severity, and machine learning analysis identified senescence markers, the BMP4 inhibitor Gremlin 1 in liver and visceral fat, and the amount of visceral adipose tissue as strong predictors. Studies in liver cell spheroids made from human stellate and hepatocyte cells show BMP4 to be anti-senescent, anti-steatotic, anti-inflammatory and anti-fibrotic, whereas Gremlin 1, which is particularly highly expressed in visceral fat in humans, is pro-senescent and antagonistic to BMP4. Both senescence and anti-senescence factors target the YAP/TAZ pathway, making this a likely regulator of senescence and its effects. We conclude that senescence is an important driver of human NAFLD/NASH and that BMP4 and Gremlin 1 are novel therapeutic targets.

Objective Gremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects. Methods Adult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time. Results Liver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation. Conclusion GREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered.

Objective Peroxisome proliferator-activated receptors (PPARs) are key transcription factors that regulate adipose development and function, and the conversion of white into brown-like adipocytes. Here we investigated whether PPARα and PPARγ activation synergize to induce the browning of white fat. Methods A selection of PPAR activators was tested for their ability to induce the browning of both mouse and human white adipocytes in vitro, and in vivo in lean and obese mice. Results All dual PPARα/γ activators tested robustly increased uncoupling protein 1 (Ucp1) expression in both mouse and human adipocytes in vitro, with tesaglitazar leading to the largest Ucp1 induction. Importantly, dual PPARα/γ activator tesaglitazar strongly induced browning of white fat in vivo in both lean and obese male mice at thermoneutrality, greatly exceeding the increase in Ucp1 observed with the selective PPARγ activator rosiglitazone. While selective PPARγ activation was sufficient for the conversion of white into brown-like adipocytes in vitro, dual PPARα/γ activation was superior to selective PPARγ activation at inducing white fat browning in vivo. Mechanistically, the superiority of dual PPARα/γ activators is mediated at least in part via a PPARα-driven increase in fibroblast growth factor 21 (FGF21). Combined treatment with rosiglitazone and FGF21 resulted in a synergistic increase in Ucp1 mRNA levels both in vitro and in vivo. Tesaglitazar-induced browning was associated with increased energy expenditure, enhanced insulin sensitivity, reduced liver steatosis, and an overall improved metabolic profile compared to rosiglitazone and vehicle control groups. Conclusions PPARγ and PPARα synergize to induce robust browning of white fat in vivo, via PPARγ activation in adipose, and PPARα-mediated increase in FGF21., Graphical abstract Dual PPARα/γ activation is superior to selective PPARγ activation at inducing browning of white fat in vivo: via PPARγ action in the adipose and a PPARα-mediated action in the liver resulting in increased circulating fibroblast growth factor 21 (FGF21).Image 1, Highlights • Dual PPARα/γ activators robustly induce browning of white fat in vitro and in vivo. • PPARγ action alone is sufficient to convert white into brown-like adipocytes in vitro. • Dual PPARα/γ activators are superior to PPARγ activators for browning of white fat in vivo. • PPARγ action in adipose and PPARα-mediated increase in FGF21 synergize to induce browning of white fat in vivo.

Vitamin D metabolism is actively modulated in adipose tissue during obesity. To better investigate this process, we develop a specific LC-HRMS/MS method that can simultaneously quantify three vitamin D metabolites, i.e., cholecalciferol, 25-hydroxyvitamin D3 (25(OH)D3), and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a complex matrix, such as mouse adipose tissue and plasma. The method uses pretreatment with liquid&ndash, liquid or solid&ndash, phase extraction followed by derivatization using Amplifex&reg, reagents to improve metabolite stability and ionization efficiency. Here, the method is optimized by co-eluting stable isotope-labelled internal standards to calibrate each analogue and to spike biological samples. Intra-day and inter-day relative standard deviations were 0.8&ndash, 6.0% and 2.0&ndash, 14.4%, respectively for the three derivatized metabolites. The limits of quantification (LoQ) achieved with Amplifex&reg, derivatization were 0.02 ng/mL, 0.19 ng/mL, and 0.78 ng/mL for 1,25(OH)2D3, 25(OH)D3 and cholecalciferol, respectively. Now, for the first time, 1,25(OH)2D3 can be co-quantified with cholecalciferol and 25(OH)D3 in mouse adipose tissue. This validated method is successfully applied to study the impact of obesity on vitamin D status in mice.

The BMP2/4 antagonist and novel adipokine Gremlin 1 is highly expressed in human adipose cells and increased in hypertrophic obesity. As a secreted antagonist, it inhibits the effect of BMP2/4 on adipose precursor cell commitment/differentiation. We examined mRNA levels of Gremlin 1 in key target tissues for insulin and also measured tissue and serum levels in several carefully phenotyped human cohorts. Gremlin 1 expression was high in adipose tissue, higher in visceral than in subcutaneous tissue, increased in obesity, and further increased in type 2 diabetes (T2D). A similar high expression was seen in liver biopsies, but expression was considerably lower in skeletal muscles. Serum levels were increased in obesity but most prominently in T2D. Transcriptional activation in both adipose tissue and liver as well as serum levels were strongly associated with markers of insulin resistance in vivo (euglycemic clamps and HOMA of insulin resistance), and the presence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). We also found Gremlin 1 to antagonize insulin signaling and action in human primary adipocytes, skeletal muscle, and liver cells. Thus, Gremlin 1 is a novel secreted insulin antagonist and biomarker as well as a potential therapeutic target in obesity and its complications T2D and NAFLD/NASH.