Identification of Mfn2-S249 as a Phosphoregulatory Switch of Mitochondrial Fusion Dynamics

Mitochondrial remodeling is a fundamental process underlying cellular respiration and metabolism. Here we report TAK1 as a direct regulator of mitochondrial fusion. TAK1 is activated by a variety of mitogenic factors, cytokines and environmental stimuli, which we find induces rapid fragmentation through Mfn2 inactivation. TAK1 phosphorylates Mfn2 at Ser249, which inhibits the binding of GTP required for Mfn trans-dimerization and mitochondrial membrane fusion. Accordingly, expression of Mfn2-S249 phosphomimetics (Mfn2-E/D) constitutively promote fission whereas alanine mutant (Mfn2-A) yields hyperfused mitochondria and increased bioenergetics in cells. In mice, Mfn2-E knock-in yields embryonic lethality in homozygotes whereas heterozygotes are viable but exhibit increased visceral fat accumulation despite normal body weight and cognitive/motor functions compared to wildtype and Mfn2-A mice. Mature white adipocytes isolated from mutant mice reveal cell-autonomous TAK1-related effects on mitochondrial remodeling and lipid metabolism. These results identify Mfn2-S249 as a dynamic phosphoregulatory switch of mitochondrial fusion during development and energy homeostasis.

Adipokines: inflammation and the pleiotropic role of white adipose tissue

I had been working on the endocrine and signalling role of white adipose tissue (WAT) since 1994 following the identification of the ob (Lep) gene(1), this after some 15 years investigating the physiological role of brown adipose tissue. The ob gene, a mutation in which it is responsible for the profound obesity of ob/ob (Lepob/Lepob) mice, is expressed primarily in white adipocytes and encodes the pleiotropic hormone leptin. The discovery of this adipocyte hormone had wide-ranging implications, including that white fat has multiple functions that far transcend the traditional picture of a simple lipid storage organ.

HIF3A Inhibition Triggers Browning of White Adipocytes via Metabolic Rewiring

Maintenance of energy balance between intake and expenditure is a prerequisite of human health, disrupted in severe metabolic diseases, such as obesity and type 2 diabetes (T2D), mainly due to accumulation of white adipose tissue (WAT). WAT undergoes a morphological and energetic remodelling toward brown adipose tissue (BAT) and the BAT activation has anti-obesity potential. The mechanisms or the regulatory factors able to activate BAT thermogenesis have been only partially deciphered. Identifying novel regulators of BAT induction is a question of great importance for fighting obesity and T2D. Here, we evaluated the role of Hif3α in murine pre-adipocyte 3T3-L1 cell line, a versatile and well characterized biological model of adipogenesis, by gain- and loss-of function approaches and in thermogenesis-induced model in vivo. HIF3A is regulated by inflammation, it modulates lypolysis in adipose tissue of obese adults, but its role in energy metabolism has not previously been investigated. We characterized gene and protein expression patterns of adipogenesis and metabolic activity in vitro and mechanistically in vivo. Overexpression of Hif3α in differentiating adipocytes increases white fat cells, whereas silencing of Hif3α promotes “browning” of white cells, activating thermogenesis through upregulation of Ucp1, Elovl3, Prdm16, Dio2 and Ppargc1a genes. Investigating cell metabolism, Seahorse Real-Time Cell Metabolism Analysis showed that silencing of Hif3α resulted in a significant increase of mitochondrial uncoupling with a concomitant increase in acetyl-CoA metabolism and Sirt1 and Sirt3 expression. The causal Hif3α/Ucp1 inverse relation has been validated in Cannabinoid receptor 1 (CB1) knockout, a thermogenesis-induced model in vivo. Our data indicate that Hif3α inhibition triggers “browning” of white adipocytes activating the beneficial thermogenesis rewiring energy metabolism in vitro and in vivo. HIF3A is a novel player that controls the energy metabolism with potential applications in developing therapy to fight metabolic disorders, as obesity, T2D and ultimately cancer.

Apigenin Induces Browning in White Adipocytes Mediated by VEGF-PRDM16 Signaling.

The white adipose tissues are metabolically inert which results in deranged biological signalling disorders resulting in obesity. Lack of vascularisation in these tissues is mainly responsible to make them metabolically inert. Not much work has been done in this direction to understand the role of angiogenesis in white adipocytes metabolism. In the present study, we evaluated the effect of angiogenic modulator in modulating the metabolism in white adipocyte. Nutraceuticals apigenin (Apg) was employed as angiogenic modulator. The results indicated that promoting angiogenesis by Apg enhanced the de novo differentiation and trans-differentiation of white adipocyte into brown like phenotype by triggering vascular endothelial growth factor A. Cross talk between endothelial and adipocytes were observed in co-culture studies. The metabolic shift in white adipocytes was observed to be due to the upregulation of PRDM16 cascade. The study provides new insights for inducing metabolic shift in white adipocytes by modulation of angiogenesis in white adipocyte to trigger browning for the treatment of obesity. Further the study opens scopes for development of medical devices for obese subjects, an area that needs to be addressed specifically with reference to soft tissue engineering as commercial soft tissue engineering scaffolds does not suit the obese patients.

Regulatory modules of human thermogenic adipocytes: functional genomics of large cohort and Meta-analysis derived marker-genes

Background Recently, ProFAT and BATLAS studies identified brown and white adipocytes marker genes based on analysis of large databases. They offered scores to determine the thermogenic status of adipocytes using the gene-expression data of these markers. In this work, we investigated the functional context of these genes. Results Gene Set Enrichment Analyses (KEGG, Reactome) of the BATLAS and ProFAT marker-genes identified pathways deterministic in the formation of brown and white adipocytes. The collection of the annotated proteins of the defined pathways resulted in expanded white and brown characteristic protein-sets, which theoretically contain all functional proteins that could be involved in the formation of adipocytes. Based on our previously obtained RNA-seq data, we visualized the expression profile of these proteins coding genes and found patterns consistent with the two adipocyte phenotypes. The trajectory of the regulatory processes could be outlined by the transcriptional profile of progenitor and differentiated adipocytes, highlighting the importance of suppression processes in browning. Protein interaction network-based functional genomics by STRING, Cytoscape and R-Igraph platforms revealed that different biological processes shape the brown and white adipocytes and highlighted key regulatory elements and modules including GAPDH-CS, DECR1, SOD2, IL6, HRAS, MTOR, INS-AKT, ERBB2 and 4-NFKB, and SLIT-ROBO-MAPK. To assess the potential role of a particular protein in shaping adipocytes, we assigned interaction network location-based scores (betweenness centrality, number of bridges) to them and created a freely accessible platform, the AdipoNET (https//adiponet.com), to conveniently use these data. The Eukaryote Promoter Database predicted the response elements in the UCP1 promoter for the identified, potentially important transcription factors (HIF1A, MYC, REL, PPARG, TP53, AR, RUNX, and FoxO1). Conclusion Our integrative approach-based results allowed us to investigate potential regulatory elements of thermogenesis in adipose tissue. The analyses revealed that some unique biological processes form the brown and white adipocyte phenotypes, which presumes the existence of the transitional states. The data also suggests that the two phenotypes are not mutually exclusive, and differentiation of thermogenic adipocyte requires induction of browning as well as repressions of whitening. The recognition of these simultaneous actions and the identified regulatory modules can open new direction in obesity research.

Liraglutide suppresses obesity and promotes browning of white fat via miR-27b in vivo and in vitro

Objective To investigate the effect of liraglutide on the browning of white fat and the suppression of obesity via regulating microRNA (miR)-27b in vivo and in vitro. Methods Sprague-Dawley rats were fed a high-fat (HF) diet and 3T3-L1 pre-adipocytes were differentiated into mature white adipocytes. Rats and mature adipocytes were then treated with different doses of liraglutide. The mRNA and protein levels of browning-associated proteins, including uncoupling protein 1 (UCP1), PR domain containing 16 (PRDM16), CCAAT enhancer binding protein β (CEBPβ), cell death-inducing DFFA-like effector A (CIDEA) and peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α), were detected using quantitative real-time polymerase chain reaction and Western blotting. Results Liraglutide decreased body weight and reduced the levels of blood glucose, triglyceride and low-density lipoprotein cholesterol in HF diet-fed rats. Liraglutide increased the levels of UCP1, PRDM16, CEBPβ, CIDEA and PGC-1α in vivo and vitro. The levels of miR-27b were upregulated in HF diet-fed rats, whereas liraglutide reduced the levels of miR-27b. In vitro, overexpression of miR-27b decreased the mRNA and protein levels of UCP1, PRDM16, CEBPβ, CIDEA and PGC-1α. Transfection with the miR-27b mimics attenuated the effect of liraglutide on the browning of white adipocytes. Conclusion Liraglutide induced browning of white adipose through regulation of miR-27b.