Biology subjects

Carobbio, S.

Publications and source records attributed to Carobbio, S..

5 recordsLinked to original sources

bioRxiv2025

miR-10b-5p regulates adipocyte lineage commitment and adipogenesis via targeting of Gata6 and Tubby.

22.1 BackgroundAdipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. 2.2 MethodsSmall RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. 2.3 ResultsBoth cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. 2.4 ConclusionsThese results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.

molecular biology↗

bioRxiv2025

12/15-Lipoxygenase orchestrates murine wound healing via PPARg-activating oxylipins acting holistically to dampen inflammation.

12/15-lipoxygenase (12/15-LOX, Alox15) generates bioactive oxygenated lipids during inflammation, however its homeostatic role(s) in normal healing are unclear. Here, the role of 12/15-LOX in resolving skin wounds was elucidated, focusing on how its lipids act together in physiologically relevant amounts. In mice, wounding caused acute appearance of 12/15-LOX-expressing macrophages and stem cells, coupled to early generation of [~]12 monohydroxy-oxylipins and enzymatically oxygenated phospholipids (eoxPL). Alox15 deletion increased -smooth muscle actin, collagen deposition, stem cell/fibroblast proliferation, IL6/pSTAT3, pSMAD3, and IFN-{gamma} levels. Conversely, CD206 expression, F480+ cells, MMP9 and MMP2 activities were reduced. Alox15-/- skin was deficient in PPAR{gamma}/adiponectin activity. Furthermore, while pro-inflammatory genes were upregulated as normal during wounding, many including Il6, Il1b, ccl4, Cd14, Cd274, Clec4d, Clec4e, Csf3, and Cxcl2 failed to revert to baseline during healing, indicating disruption of an anti-inflammatory brake. Reconstituting Alox15-/- wounds with a physiological mixture of Alox15-derived primary oxylipins generated by healing wounds restored MMP and dampened collagen deposition. The oxylipin mixture activated PPAR{gamma} in vitro, while in vivo, the PPAR{gamma} co-activator, Helz2, was significantly upregulated. Additional inflammatory and proliferative gene networks impacted by Alox15-/-included Elf4, Cebpb and Tcf3, with many of their associated genes significantly dysregulated. In summary, the impact of 12/15-LOX is ascribed to the deficiency of abundantly generated monohydroxy oxylipins acting together via PPAR{gamma}/adiponectin. The identification of multiple gene alterations reveals several new targets for treatment of non-healing wounds. Our studies demonstrate that abundant 12/15-LOX oxylipins act together, dampening inflammation in vivo, revealing a need to consider lipid signaling holistically. Significance statementDefective wound healing is a significant global clinical problem. Macrophage 12/15-lipoxygenase (12/15-LOX, Alox15) generates abundant lipid mediators termed oxylipins during inflammation. However, its physiological role during resolving wound healing is unclear, with studies so far assessing the bioactivity of individual lipids pharmacologically, rather than holistically in physiological amounts. Here, we report that Alox15 deficiency in mice caused a fibrotic response with failure to dampen inflammation, due to a dysregulated PPAR{gamma}/adiponectin axis. Treatment of Alox15-/- wounds with physiological mixtures of PPAR{gamma}-activating 12/15-LOX primary monohydroxy products restored the phenotype. Several transcriptional networks (Elf4, Cebpb and Tcf3) controlled by Alox15 were uncovered, identifying new targets for promoting physiological wound healing.

pharmacology and toxicology↗

bioRxiv2022

Oxygen is a critical regulator of cellular metabolism and function in cell culture

Cell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.

cell biology↗

bioRxiv2022

Semaphorin 4B is an ADAM17-cleaved inhibitor of adipocyte thermogenesis

ObjectiveThe metalloprotease ADAM17 (also called TACE) plays fundamental roles in homeostasis by shedding key signaling molecules from the cell surface. Although its importance for the immune system and epithelial tissues is well-documented, little is known about the role of ADAM17 in metabolic homeostasis. The purpose of this study was to determine the impact of ADAM17 expression, specifically in adipose tissues, on metabolic homeostasis. MethodsWe used histopathology, molecular, proteomic, transcriptomic, in vivo integrative physiological and ex vivo biochemical approaches to determine the impact of adipose tissue-specific deletion of ADAM17 upon adipocyte and whole organism metabolic physiology. ResultsADAM17adipoq-cre{Delta}/{Delta} mice exhibited a hypermetabolic phenotype characterized by elevated energy consumption and increased levels of adipocyte thermogenic gene expression. On a high fat diet, these mice were more thermogenic, while exhibiting elevated expression levels of genes associated with lipid oxidation and lipolysis. This hypermetabolic phenotype protected mutant mice from obesogenic challenge, limiting weight gain, hepatosteatosis and insulin resistance. Activation of beta-adrenoceptors by the neurotransmitter norepinephrine, a key regulator of adipocyte physiology, triggered the shedding of ADAM17 substrates, and regulated ADAM17 expression at the mRNA and protein levels, hence identifying a functional connection between thermogenic licensing and the regulation of ADAM17. Proteomic studies identified Semaphorin 4B (SEMA4B), as a novel ADAM17-shed adipokine, whose expression is regulated by physiological thermogenic cues that acts to dampen thermogenic responses in adipocytes. Transcriptomic data showed that cleaved SEMA4B acts in an autocrine manner in brown adipocytes to dampen the expression of genes involved in thermogenesis, adipogenesis, lipid uptake, storage and catabolism. ConclusionOur findings identify a novel ADAM17-dependent axis, regulated by beta-adrenoceptors and mediated by the ADAM17-cleaved form of SEMA4B, that may act to limit uncontrolled energy depletion during thermogenesis.

physiology↗

bioRxiv2018

Inactivation of Ppp1r15a minimises weight gain and insulin resistance during caloric excess in mice

Phosphorylation of the translation initiation factor eIF2 within the mediobasal hypothalamus is known to suppress food intake, but the role of the eIF2 phosphatases in regulating body weight is poorly understood. Mice deficient in active PPP1R15A, a stress-inducible eIF2 phosphatase, are healthy and more resistant to endoplasmic reticulum stress than wild type controls. We report that when Ppp1r15a mutant mice are fed a high fat diet they gain less weight than wild type littermates owing to reduced food intake. This results in healthy leaner Ppp1r15a mutant animals with reduced hepatic steatosis and improved insulin sensitivity, albeit with a modest defect in insulin secretion. By contrast, no weight differences are observed between wild type and Ppp1r15a deficient mice fed a standard diet. We conclude that mice lacking the C-terminal PP1-binding domain of PPP1R15A show reduced dietary intake and preserved glucose tolerance. Our data indicate that this results in reduced weight gain and protection from diet-induced obesity.

physiology↗