SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.     

Mycardial histone acetylation

Myocardial histone acetylation was investigated in an isolated perfused heart preparation. Radioactive acetate rapidly accumulated in the intracellular compartment which preceded the covalent modification of histones. The acetylation of nucleohistones was rapid and reached a maximum during the first 20 min of perfusion. After 60 min of continuous perfusion there was a three-fold decrease in the amount of acetate bound to histone proteins. Histone H3 appeared to be preferred substrate for acetylation and was most responsive to a change in the concentration of radioactive acetate as well as the perfusion time.     

Demethylase activity is directed by histone acetylation

Mammalian genomes are compartmentalized into dense inactive chromatin that is hypermethylated and active open chromatin that is hypomethylated. It is generally accepted that this bimodal pattern of methylation is established during development and is then faithfully inherited through subsequent cell divisions by a maintenance DNA methyltransferase (DNMT1). The pattern of methylation is believed to direct local histone acetylation states. In contrast to this well accepted consensus, we show here using a transient transfection model that an active demethylase is involved in shaping patterns of methylation in somatic cells. Demethylase activity is directed by the state of histone acetylation, and therefore, the resulting methylation pattern is determined by local histone acetylation states contrary to the accepted model. Our data support a new model suggesting that the pattern of methylation is maintained by a dynamic balance of methylation and demethylation activities and the local state of histone acetylation. This provides a simple mechanism for explaining why active genes are not methylated.     

Schizosaccharomyces pombe Hst4 functions in DNA damage response by regulating histone H3 K56 acetylation

The packaging of eukaryotic DNA into chromatin is likely to be crucial for the maintenance of genomic integrity. Histone acetylation and deacetylation, which alter chromatin accessibility, have been implicated in DNA damage tolerance. Here we show that Schizosaccharomyces pombe Hst4, a homolog of histone deacetylase Sir2, participates in S-phase-specific DNA damage tolerance. Hst4 was essential for the survival of cells exposed to the genotoxic agent methyl methanesulfonate (MMS) as well as for cells lacking components of the DNA damage checkpoint pathway. It was required for the deacetylation of histone H3 core domain residue lysine 56, since a strain with a point mutation of its catalytic domain was unable to deacetylate this residue in vivo. Hst4 regulated the acetylation of H3 K56 and was itself cell cycle regulated. We also show that MMS treatment resulted in increased acetylation of histone H3 lysine 56 in wild-type cells and hst4Delta mutants had constitutively elevated levels of histone H3 K56 acetylation. Interestingly, the level of expression of Hst4 decreased upon MMS treatment, suggesting that the cell regulates access to the site of DNA damage by changing the level of this protein. Furthermore, we find that the phenotypes of both K56Q and K56R mutants of histone H3 were similar to those of hst4Delta mutants, suggesting that proper regulation of histone acetylation is important for DNA integrity. We propose that Hst4 is a deacetylase involved in the restoration of chromatin structure following the S phase of cell cycle and DNA damage response.     

Core histone acetylation is regulated by linker histone stoichiometry in vivo

We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.