Obesity and metabolic syndrome are associated with mitochondrial dysfunction and deranged regulation of metabolic genes. Peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) is a transcriptional coactivator that regulates metabolism and mitochondrial biogenesis through stimulation of nuclear hormone receptors and other transcription factors. We report that the PGC-1β gene encodes two microRNAs (miRNAs), miR-378 and miR-378*, which counterbalance the metabolic actions of PGC-1β. Mice genetically lacking miR-378 and miR-378* are resistant to high-fat diet-induced obesity and exhibit enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. Among the many targets of these miRNAs, carnitine O-acetyltransferase, a mitochondrial enzyme involved in fatty acid metabolism, and MED13, a component of the Mediator complex that controls nuclear hormone receptor activity, are repressed by miR-378 and miR-378*, respectively, and are elevated in the livers of miR-378/378* KO mice. Consistent with these targets as contributors to the metabolic actions of miR-378 and miR-378*, previous studies have implicated carnitine O-acetyltransferase and MED13 in metabolic syndrome and obesity. Our findings identify miR-378 and miR-378* as integral components of a regulatory circuit that functions under conditions of metabolic stress to control systemic energy homeostasis and the overall oxidative capacity of insulin target tissues. Thus, these miRNAs provide potential targets for pharmacologic intervention in obesity and metabolic syndrome.fatty acid oxidation | adipocytes | mitochondrial CO 2 production

Adipocyte differentiation is a well defined process that is under the control of transcriptional activators and repressors. We show that histone deacetylase (HDAC) inhibitors efficiently block adipocyte differentiation in vitro. This effect is specific to adipogenesis, as another mesenchymal differentiation process, osteoblastogenesis, is enhanced upon HDAC inhibition. Through the systematic genetic deletion of HDAC genes in cultured mesenchymal precursor cells, we show that deletion of HDAC1 and HDAC2 leads to reduced lipid accumulation, revealing redundant and requisite roles of these class I HDACs in adipogenesis. These findings unveil a previously unrecognized role for HDACs in the control of adipogenesis.In humans, unused caloric energy resulting from an excessive net caloric intake is converted to triglycerides and stored in fat depots for further usage. In principle, the fat mass of these depots can increase either by hypertrophy (an increase of adipocyte size) or by hyperplasia (and increase in adipocyte number). It has been recently demonstrated that fat cell number is primarily determined by early adulthood and that subsequent changes in fat mass occur mainly through increases in adipocyte volume (1). However, ϳ10% of fat cells are renewed annually in adults. The molecular mechanisms driving the turnover of adipocyte tissue in adults are incompletely understood, but it has been speculated that a combination of cell death and neoadipogenesis from mesenchymal precursor cells is responsible for maintaining the fat cell number pre-set in early adulthood (1).Adipogenesis is a tightly orchestrated process in which mesenchymal precursor cells differentiate into mature fat cells and express batteries of genes encoding enzymes involved in lipid biosynthesis, transport, and storage. This process is under the control of a cascade of well characterized transcription factors, including C/EBP␤, 3 SREBPs, and PPAR␥ (2). Studies in cultured cells have shown that these adipogenic core transcription factors interact with histone acetyltransferases, which stimulate transcription by acetylating nucleosomal histones, thereby relaxing chromatin structure (3). Histone deacetylases (HDACs), a conserved family of chromatin-modifying enzymes that repress transcription by deacetylating nucleosomal histones, also associate with these adipogenic transcription factors (3), counteracting the functions of histone acetyltransferases. Thus, in the classic model of adipocyte differentiation, HDACs are thought to inhibit the adipogenic program by directly repressing the transcriptional activity of pro-adipogenic transcription factors (4). There are five classes of HDACs that display distinct patterns of expression, regulation, and substrate preference. Class I HDACs (HDAC1, -2, -3, and -8) are expressed in a wide range of tissues and efficiently deacetylate histones (5). In contrast, class IIa HDACs (HDAC4, -5, -7, and -9) display preferential expression in muscle and neural tissues and contain a divergent catalytic domain that has minim...

Maintenance of skeletal muscle structure and function requires efficient and precise metabolic control. Autophagy plays a key role in metabolic homeostasis of diverse tissues by recycling cellular constituents, particularly under conditions of caloric restriction, thereby normalizing cellular metabolism. Here we show that histone deacetylases (HDACs) 1 and 2 control skeletal muscle homeostasis and autophagy flux in mice. Skeletal muscle-specific deletion of both HDAC1 and HDAC2 results in perinatal lethality of a subset of mice, accompanied by mitochondrial abnormalities and sarcomere degeneration. Mutant mice that survive the first day of life develop a progressive myopathy characterized by muscle degeneration and regeneration, and abnormal metabolism resulting from a blockade to autophagy. HDAC1 and HDAC2 regulate skeletal muscle autophagy by mediating the induction of autophagic gene expression and the formation of autophagosomes, such that myofibers of mice lacking these HDACs accumulate toxic autophagic intermediates. Strikingly, feeding HDAC1/2 mutant mice a high-fat diet from the weaning age releases the block in autophagy and prevents myopathy in adult mice. These findings reveal an unprecedented and essential role for HDAC1 and HDAC2 in maintenance of skeletal muscle structure and function and show that, at least in some pathological conditions, myopathy may be mitigated by dietary modifications.autophagosome formation | muscle disease | muscle metabolism | epigenetic regulation C ellular homeostasis is maintained by a balance between protein biosynthesis and degradation. Macroautophagy (herein referred to as autophagy) is a catabolic pathway responsible for the degradation of various cellular constituents or deleterious cellular components. The process involves formation of vesicles, called autophagosomes, that capture and deliver proteins to lysosomes for degradation (1). The resulting breakdown products are recycled and used to support cellular metabolism (2). Genetic studies with mice mutant for genes involved in autophagy substantiated the importance of basal autophagy for organelle turnover and cellular homeostasis (3-11). Various stress conditions, such as fasting, exercise, hypoxia, oxidative stress, and pathogen infection, trigger autophagy as an adaptive response to normalize cellular metabolism (12, 13).Deregulation of autophagy has been implicated in cancer (14, 15), neurodegenerative disorders (4, 5, 10), and muscular diseases (16,17). Epigenetic factors have been implicated in regulating autophagy during various pathological conditions (12). Alteration of the acetylation status of histones or other proteins through histone deacetylases (HDACs) is a key mechanism that controls gene transcription and protein function. Removal of acetyl groups from histone tails by HDACs promotes transcriptional repression by allowing chromatin compaction (18). HDACs also modulate the activity of a variety of transcription factors and large macromolecular complexes involved in diverse cellular processes (19)....

Aberrant hepatocyte Notch activity is critical to the development of nonalcoholic steatohepatitis (NASH)–induced liver fibrosis, but mechanisms underlying Notch reactivation in developed liver are unclear. Here, we identified that increased expression of the Notch ligand Jagged1 (JAG1) tracked with Notch activation and nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in human liver biopsy specimens and mouse NASH models. The increase in Jag1 was mediated by hepatocyte Toll-like receptor 4 (TLR4)–nuclear factor κB (NF-κB) signaling in pericentral hepatocytes. Hepatocyte-specific Jag1 overexpression exacerbated fibrosis in mice fed a high-fat diet or a NASH-provoking diet rich in palmitate, cholesterol, and sucrose and reversed the protection afforded by hepatocyte-specific TLR4 deletion, whereas hepatocyte-specific Jag1 knockout mice were protected from NASH-induced liver fibrosis. To test therapeutic potential of this biology, we designed a Jag1-directed antisense oligonucleotide (ASO) and a hepatocyte-specific N-acetylgalactosamine (GalNAc)–modified siRNA, both of which reduced NASH diet–induced liver fibrosis in mice. Overall, these data demonstrate that increased hepatocyte Jagged1 is the proximal hit for Notch-induced liver fibrosis in mice and suggest translational potential of Jagged1 inhibitors in patients with NASH.

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Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain–of–function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan. Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix–loop–helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.

Notch receptors are determinants of cell fate and function, and play an important role in the regulation of bone development and skeletal remodeling. Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. LMS presents with neurological developmental abnormalities and bone loss. We created a mouse model (Notch3em1Ecan) harboring a 6691TAATGA mutation in the Notch3 locus, and heterozygous Notch3em1Ecan mice exhibit cancellous and cortical bone osteopenia. In the present work, we explored whether Notch3 antisense oligonucleotides (ASO) downregulate Notch3 and have the potential to ameliorate the osteopenia of Notch3em1Ecan mice. Notch3 ASOs decreased the expression of Notch3 wild type and Notch36691-TAATGA mutant mRNA expressed by Notch3em1Ecan mice in osteoblast cultures without evidence of cellular toxicity. The effect was specific since ASOs did not downregulate Notch1, Notch2 or Notch4. The expression of Notch3 wild type and Notch36691-TAATGA mutant transcripts also was decreased in bone marrow stromal cells and osteocytes following exposure to Notch3 ASOs. In vivo, the subcutaneous administration of Notch3 ASOs at 25 to 50 mg/Kg decreased Notch3 mRNA in the liver, heart and bone. Microcomputed tomography demonstrated that the administration of Notch3 ASOs ameliorates the cortical osteopenia of Notch3em1Ecan mice, and ASOs decreased femoral cortical porosity and increased cortical thickness and bone volume. However, the administration of Notch3 ASOs did not ameliorate the cancellous bone osteopenia of Notchem1Ecan mice. In conclusion, Notch3 ASOs downregulate Notch3 expression in skeletal cells and their systemic administration ameliorates cortical osteopenia in Notch3em1Ecan mice; as such ASOs may become useful strategies in the management of skeletal diseases affected by Notch gain-of-function.

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