Enhancer of zeste homolog 2 (EZH2) regulates adipocyte lipid metabolism independent of adipogenic differentiation: Role of apolipoprotein E.

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.

Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics.

Mutations within the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are the most common genetic cause of autosomal and sporadic Parkinson's disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation-all of which could lead to neurodegeneration associated with PD.

Transgenic Overexpression of HDAC9 Promotes Adipocyte Hypertrophy, Insulin Resistance and Hepatic Steatosis in Aging Mice.

Histone deacetylase (HDAC) 9 is a negative regulator of adipogenic differentiation, which is required for maintenance of healthy adipose tissues. We reported that HDAC9 expression is upregulated in adipose tissues during obesity, in conjunction with impaired adipogenic differentiation, adipocyte hypertrophy, insulin resistance, and hepatic steatosis, all of which were alleviated by global genetic deletion of Hdac9. Here, we developed a novel transgenic (TG) mouse model to test whether overexpression of Hdac9 is sufficient to induce adipocyte hypertrophy, insulin resistance, and hepatic steatosis in the absence of obesity. HDAC9 TG mice gained less body weight than wild-type (WT) mice when fed a standard laboratory diet for up to 40 weeks, which was attributed to reduced fat mass (primarily inguinal adipose tissue). There was no difference in insulin sensitivity or glucose tolerance in 18-week-old WT and HDAC9 TG mice; however, at 40 weeks of age, HDAC9 TG mice exhibited impaired insulin sensitivity and glucose intolerance. Tissue histology demonstrated adipocyte hypertrophy, along with reduced numbers of mature adipocytes and stromovascular cells, in the HDAC9 TG mouse adipose tissue. Moreover, increased lipids were detected in the livers of aging HDAC9 TG mice, as evaluated by oil red O staining. In conclusion, the experimental aging HDAC9 TG mice developed adipocyte hypertrophy, insulin resistance, and hepatic steatosis, independent of obesity. This novel mouse model may be useful in the investigation of the impact of Hdac9 overexpression associated with metabolic and aging-related diseases.

Impact of housing temperature on adipose tissue HDAC9 expression and adipogenic differentiation in high fat-fed mice.

OBJECTIVE: Impaired adipogenic differentiation exacerbates metabolic disease in obesity. This study reported that high-fat diet (HFD)-fed mice housed at thermoneutrality exhibited impaired adipogenic differentiation, attributed to increased expression of histone deacetylase 9 (HDAC9). However, the impact of HFD on adipogenic differentiation is reportedly variable, possibly reflecting divergent environmental conditions such as housing temperature. METHODS: C57BL/6J (wild-type [WT]) mice were housed at either thermoneutral (28-30°C) or ambient (20-22°C) temperature and fed HFD or chow diet (CD) for 12 weeks. For acute exposure experiments, WT or transient receptor potential cation channel subfamily M member 8 (TRPM8) knockout mice housed under thermoneutrality were acutely exposed to ambient temperature for 6 to 24 h. RESULTS: WT mice fed HFD and housed at thermoneutrality, compared with ambient temperature, gained more weight despite reduced food intake. They likewise exhibited increased inguinal adipose tissue HDAC9 expression and reduced adipogenic differentiation in vitro and in vivo compared with CD-fed mice. Conversely, HFD-fed mice housed at ambient temperature exhibited minimal change in adipose HDAC9 expression or adipogenic differentiation. Acute exposure of WT mice to ambient temperature reduced adipose HDAC9 expression independent of sympathetic ß-adrenergic signaling via a TRPM8-dependent mechanism. CONCLUSIONS: Adipose HDAC9 expression is temperature sensitive, regulating adipogenic differentiation in HFD-fed mice housed under thermoneutrality.

Perivascular adipose tissue promotes vascular dysfunction in murine lupus.

Introduction: Patients with systemic lupus erythematosus (SLE) are at elevated risk for Q10 cardiovascular disease (CVD) due to accelerated atherosclerosis. Compared to heathy control subjects, lupus patients have higher volumes and densities of thoracic aortic perivascular adipose tissue (PVAT), which independently associates with vascular calcification, a marker of subclinical atherosclerosis. However, the biological and functional role of PVAT in SLE has not been directly investigated. Methods: Using mouse models of lupus, we studied the phenotype and function of PVAT, and the mechanisms linking PVAT and vascular dysfunction in lupus disease. Results and discussion: Lupus mice were hypermetabolic and exhibited partial lipodystrophy, with sparing of thoracic aortic PVAT. Using wire myography, we found that mice with active lupus exhibited impaired endothelium-dependent relaxation of thoracic aorta, which was further exacerbated in the presence of thoracic aortic PVAT. Interestingly, PVAT from lupus mice exhibited phenotypic switching, as evidenced by "whitening" and hypertrophy of perivascular adipocytes along with immune cell infiltration, in association with adventitial hyperplasia. In addition, expression of UCP1, a brown/beige adipose marker, was dramatically decreased, while CD45-positive leukocyte infiltration was increased, in PVAT from lupus mice. Furthermore, PVAT from lupus mice exhibited a marked decrease in adipogenic gene expression, concomitant with increased pro-inflammatory adipocytokine and leukocyte marker expression. Taken together, these results suggest that dysfunctional, inflamed PVAT may contribute to vascular disease in lupus.

Hepatocyte-specific disruption of soluble epoxide hydrolase attenuates abdominal aortic aneurysm formation: novel role of the liver in aneurysm pathogenesis.

Introduction: Inflammation is a key pathogenic feature of abdominal aortic aneurysm (AAA). Soluble epoxide hydrolase (sEH) is a pro-inflammatory enzyme that converts cytochrome P450-derived epoxides of fatty acids to the corresponding diols, and pharmacological inhibition of sEH prevented AAA formation. Both cytochrome P450 enzymes and sEH are highly expressed in the liver. Here, we investigated the role of hepatic sEH in AAA using a selective pharmacological inhibitor of sEH and hepatocyte-specific Ephx2 (which encodes sEH gene) knockout (KO) mice in two models of AAA [angiotensin II (AngII) infusion and calcium chloride (CaCl 2 ) application]. Methods and results: sEH expression and activity were strikingly higher in mouse liver compared with aorta and further increased the context of AAA, in conjunction with elevated expression of the transcription factor Sp1 and the epigenetic regulator Jarid1b, which have been reported to positively regulate sEH expression. Pharmacological sEH inhibition, or liver-specific sEH disruption, achieved by crossing sEH floxed mice with albumin-cre mice, prevented AAA formation in both models, concomitant with reduced expression of hepatic sEH as well as complement factor 3 (C3) and serum amyloid A (SAA), liver-derived factors linked to AAA formation. Moreover, sEH antagonism markedly reduced C3 and SAA protein accumulation in the aortic wall. Co-incubation of liver ex vivo with aneurysm-prone aorta resulted in induction of sEH in the liver, concomitant with upregulation of Sp1, Jarid1b, C3 and SAA gene expression, suggesting that the aneurysm-prone aorta secretes factors that activate sEH and downstream inflammatory signaling in the liver. Using an unbiased proteomic approach, we identified a number of dysregulated proteins [ e.g., plastin-2, galectin-3 (gal-3), cathepsin S] released by aneurysm-prone aorta as potential candidate mediators of hepatic sEH induction. Conclusion: We provide the first direct evidence of the liver's role in orchestrating AAA via the enzyme sEH. These findings not only provide novel insight into AAA pathogenesis, but they have potentially important implications with regard to developing effective medical therapies for AAA.

Integrative multiomics analysis of neointima proliferation in human saphenous vein: implications for bypass graft disease.

Introduction: Human saphenous veins (SV) are widely used as grafts in coronary artery bypass (CABG) surgery but often fail due to neointima proliferation (NP). NP involves complex interplay between vascular smooth muscle cells (VSMC) and fibroblasts. Little is known, however, regarding the transcriptomic and proteomic dynamics of NP. Here, we performed multi-omics analysis in an ex vivo tissue culture model of NP in human SV procured for CABG surgery. Methods and results: Histological examination demonstrated significant elastin degradation and NP (indicated by increased neointima area and neointima/media ratio) in SV subjected to tissue culture. Analysis of data from 73 patients suggest that the process of SV adaptation and NP may differ according to sex and body mass index. RNA sequencing confirmed upregulation of pro-inflammatory and proliferation-related genes during NP and identified novel processes, including increased cellular stress and DNA damage responses, which may reflect tissue trauma associated with SV harvesting. Proteomic analysis identified upregulated extracellular matrix-related and coagulation/thrombosis proteins and downregulated metabolic proteins. Spatial transcriptomics detected transdifferentiating VSMC in the intima on the day of harvesting and highlighted dynamic alterations in fibroblast and VSMC phenotype and behavior during NP. Specifically, we identified new cell subpopulations contributing to NP, including SPP1 + , LGALS3 + VSMC and MMP2 + , MMP14 + fibroblasts. Conclusion: Dynamic alterations of gene and protein expression occur during NP in human SV. Identification of the human-specific molecular and cellular mechanisms may provide novel insight into SV bypass graft disease.

Sex-Dependent Role of Adipose Tissue HDAC9 in Diet-Induced Obesity and Metabolic Dysfunction.

Obesity is a major risk factor for both metabolic and cardiovascular disease. We reported that, in obese male mice, histone deacetylase 9 (HDAC9) is upregulated in adipose tissues, and global deletion of HDAC9 protected against high fat diet (HFD)-induced obesity and metabolic disease. Here, we investigated the impact of adipocyte-specific HDAC9 gene deletion on diet-induced obesity in male and female mice. The HDAC9 gene expression was increased in adipose tissues of obese male and female mice and HDAC9 expression correlated positively with body mass index in humans. Interestingly, female, but not male, adipocyte-specific HDAC9 KO mice on HFD exhibited reduced body weight and visceral adipose tissue mass, adipocyte hypertrophy, and improved insulin sensitivity, glucose tolerance and adipogenic differentiation gene expression. Furthermore, adipocyte-specific HDAC9 gene deletion in female mice improved metabolic health as assessed by whole body energy expenditure, oxygen consumption, and adaptive thermogenesis. Mechanistically, compared to female mice, HFD-fed male mice exhibited preferential HDAC9 expression in the stromovascular fraction, which may have offset the impact of adipocyte-specific HDAC9 gene deletion in male mice. These results suggest that HDAC9 expressed in adipocytes is detrimental to obesity in female mice and provides novel evidence of sex-related differences in HDAC9 cellular expression and contribution to obesity-related metabolic disease.

Arc/Arg3.1 has an activity-regulated interaction with PICK1 that results in altered spatial dynamics.

Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be the first 126 amino acids of the PICK1 BAR domain. Overall, our data support a novel interaction and model where PICK1 mediates Arc regulation of AMPARs particularly under depolarizing conditions.