Obese individuals differ in their risk of developing metabolic and cardiovascular complications depending on fat distribution (subcutaneous versus visceral) and adipose tissue (AT) phenotype (hyperplasic versus hypertrophic). However, the exact mechanisms which determine whether an obese individual is metabolically healthy or unhealthy are not clear, and analyses of the underlying pathomechanisms are limited by the lack of suitable in vivo models in which metabolically healthy versus metabolically unhealthy AT accumulation can be specifically induced. In the current study, we aimed to establish a protocol for the use of zebrafish as a model for obesity-related metabolically healthy versus metabolically unhealthy AT accumulation. We overfed adult male zebrafish of the AB strain with normal fat diet (NFD) or high fat diet (HFD) for 8 weeks and compared parameters related to obesity, i.e. body weight, body mass index, condition index and body fat percentage, to control zebrafish fed under physiological conditions. In addition, we investigated the presence of early obesity-related metabolic alterations by quantifying blood glucose levels, plasma triglyceride and cholesterol levels, and by assessing ectopic lipid accumulation in the liver of zebrafish. Finally, we determined gene expression levels of marker genes related to lipid metabolism, inflammation and fibrosis in visceral AT and liver. We show that 8-weeks overfeeding with either NFD or HFD leads to a significant increase in body weight and AT mass compared to controls. In contrast to NFD-overfed zebrafish, HFD-overfed zebrafish additionally present metabolic alterations, e.g. hyperglycemia and ectopic lipid accumulation in the liver, and a metabolically unhealthy AT phenotype with adipocyte hypertrophy especially in the visceral AT depot, which is accompanied by changes in the expression of marker genes for lipid metabolism, inflammation and fibrosis. In summary, we have established a method for the specific induction of metabolically distinct obesity phenotypes in zebrafish. Our results indicate that zebrafish represents an attractive model to study regulatory mechanisms involved in the determination of AT phenotype during development of metabolically healthy versus metabolically unhealthy obesity.

Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal–regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 ( Nlrp3 ) mutant mice. Tamoxifen-inducible Nlrp3 knock-in ( Nlrp3 ^A350V/+ CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3 -KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 ( Col1a1 ) in hepatic stellate cells isolated from Nlrp3 -KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.

Obesity is associated with a dysregulation of beta-cell and adipocyte function. The molecular interactions between adipose tissue and beta-cells are not yet fully elucidated. We investigated, whether or not the adipocytokine Nicotinamide phosphoribosyltransferase (Nampt) and its enzymatic product Nicotinamide mononucleotide (NMN), which has been associated with obesity and type 2 diabetes mellitus (T2DM) directly influence beta-cell survival and function. The effect of Nampt and NMN on viability of INS-1E cells was assessed by WST-1 assay. Apoptosis was measured by Annexin V/PI and TUNEL assay. Activation of apoptosis signaling pathways was evaluated. Adenylate kinase release was determined to assess cytotoxicity. Chronic and acute effects of the adipocytokine Nampt and its enzymatic product NMN on insulin secretion were assessed by glucose stimulated insulin secretion in human islets. While stimulation of beta-cells with the cytokines IL-1β, TNFα and IFN-γ or palmitate significantly decreased viability, Nampt and NMN showed no direct effect on viability in INS-1E cells or in human islets, neither alone nor in the presence of pro-diabetic conditions (elevated glucose concentrations and palmitate or cytokines). At chronic conditions over 3 days of culture, Nampt and its product NMN had no effects on insulin secretion. In contrast, both Nampt and NMN potentiated glucose stimulated insulin secretion acutely during 1 h incubation of human islets. Nampt and NMN neither influenced beta-cell viability nor apoptosis but acutely potentiated glucose stimulated insulin secretion.