No abstract

Increased oxidative stress and abundance of reactive oxygen species (ROS) are positively correlated with a variety of pathophysiologies, including cardiovascular disease, type 2 diabetes, Alzheimer's disease, and neuroinflammation. In adipose biology, diabetic obesity is correlated with increased ROS in an ageand depot-specific manner and is mechanistically linked to mitochondrial dysfunction, endoplasmic reticulum (ER) stress, potentiated lipolysis, and insulin resistance. The cellular quality control systems that homeostatically regulate oxidative stress in the lean state are down-regulated in obesity as a consequence of inflammatory cytokine pressure leading to the accumulation of oxidized biomolecules. New findings have linked protein, DNA, and lipid oxidation at the biochemical level, and the structures and potential functions of protein adducts such as carbonylation that accumulate in stressed cells have been characterized. The sum total of such regulation and biochemical changes results in alteration of cellular metabolism and function in the obese state relative to the lean state and underlies metabolic disease progression. In this review, we discuss the molecular mechanisms and events underlying these processes and their implications for human health and disease. Adipose oxidative stress is a major contributor to insulin resistance and cellular dysfunction (1) and is regulated in an ageand depot-specific manner. Adipose oxidative stress increases with age in both humans and experimental mice and varies between depots. For example, in contrast to younger mice (6 months), aged C57BL/6 mice (23 months) exhibit increased ROS 2 in the visceral adipose depot (epididymal) as compared with the subcutaneous depot (inguinal) (2). Similar experiments on human tissue have revealed that subcutaneous adipose tissue from obese diabetic subjects exhibit increased H 2 O 2 production compared with that from age-matched obese, nondiabetic subjects or to lean controls (3). Moreover, ROS levels are higher in epicardial fat as compared with subcutaneous adipose tissue (4). Furthermore, oxidized lipids and proteins accumulate to a greater extent in visceral depots compared with subcutaneous depots (5, 6). These observations point out the complexity of linking oxidative stress to disease and the molecular mechanisms that drive dysfunction and dysregulation. Major challenges exist in defining the specifics of reactive oxygen species-driven pathology and its connectivity to human health and disease.

Histone deacetylase 3 (HDAC3) is unique among the HDAC superfamily of chromatin modifiers that silence transcription through enzymatic modification of histones because interaction with nuclear receptor corepressors (NCoR1/2) is required for engagement of its catalytic activity 1 – 3 . However, loss of HDAC3 also represses transcription 4 – 8 . Here we report that, during lipopolysaccharide (LPS) activation of macrophages, recruitment of HDAC3 to ATF2-bound sites without NCoR1/2 non-canonically activates inflammatory gene expression. By contrast, HDAC3 deacetylase activity is selectively engaged at ATF3-bound sites that suppress toll-like receptor (TLR) signaling. Deletion of HDAC3 in macrophages safeguards mice from lethal exposure to LPS, but this protection is not conferred by genetic or pharmacological abolition of HDAC3 catalytic activity. Thus, HDAC3 is a dichotomous transcriptional activator and repressor whose non-canonical deacetylase-independent functions are vital for the innate immune system.

The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding-fasting cycles result in substrate availability fluctuations which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBα and β are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, here we generated mice with cardiomyocyte (CM)-specific deletion of both Rev-erb s, which died prematurely due to dilated cardiomyopathy. Loss of Rev-erbs markedly downregulated fatty acid oxidation genes prior to overt pathology, which was mediated by induction of the transcriptional repressor E4BP4, a direct target of cardiac REV-ERBs. E4BP4 directly controls circadian expression of Nampt and its biosynthetic product NAD + via distal cis -regulatory elements. Thus, REV-ERB-mediated E4BP4 repression is required for Nampt expression and NAD + production by the salvage pathway. Together, these results highlight the indispensable role of circadian REV-ERBs in cardiac gene expression, metabolic homeostasis and function.

In obesity-linked insulin resistance, oxidative stress in adipocytes leads to lipid peroxidation and subsequent carbonylation of proteins by diffusible lipid electrophiles. Reduction in oxidative stress attenuates protein carbonylation and insulin resistance, suggesting that lipid modification of proteins may play a role in metabolic disease, but the mechanisms remain incompletely understood. Herein, we show that , diet-induced obesity in mice surprisingly results in preferential carbonylation of nuclear proteins by 4-hydroxy--2,3-nonenal (4-HNE) or 4-hydroxy--2,3-hexenal (4-HHE). Proteomic and structural analyses revealed that residues in or around the sites of zinc coordination of zinc finger proteins, such as those containing the C2H2 or MATRIN, RING, C3H1, or N4-type DNA-binding domains, are particularly susceptible to carbonylation by lipid aldehydes. These observations strongly suggest that carbonylation functionally disrupts protein secondary structure supported by metal coordination. Analysis of one such target, the nuclear protein estrogen-related receptor γ (ERR-γ), showed that ERR-γ is modified by 4-HHE in the obese state. carbonylation decreased the DNA-binding capacity of ERR-γ and correlated with the obesity-linked down-regulation of many key genes promoting mitochondrial bioenergetics. Taken together, these findings reveal a novel mechanistic connection between oxidative stress and metabolic dysfunction arising from carbonylation of nuclear zinc finger proteins, such as the transcriptional regulator ERR-γ.

Oxidative stress is a hallmark of metabolic disease, though the mechanisms that define this link are not fully understood. Irreversible modification of proteins by reactive lipid aldehydes (protein carbonylation) is a major consequence of oxidative stress in adipose tissue and the substrates and specificity of this modification are largely unexplored. Here we show that histones are avidly modified by 4-hydroxynonenal (4-HNE) in vitro and in vivo. Carbonylation of histones by 4-HNE increased with age in male flies and visceral fat depots of mice and was potentiated in genetic (ob/ob) and high-fat feeding models of obesity. Proteomic evaluation of in vitro 4-HNE- modified histones led to the identification of both Michael and Schiff base adducts. In contrast, mapping of sites in vivo from obese mice exclusively revealed Michael adducts. In total, we identified 11 sites of 4-hydroxy hexenal (4-HHE) and 10 sites of 4-HNE histone modification in visceral adipose tissue. In summary, these results characterize adipose histone carbonylation as a redox-linked epigenomic mark associated with metabolic disease and aging.

Brown adipose tissue (BAT) is a key thermogenic organ whose expression of uncoupling protein 1 (UCP1) and ability to maintain body temperature in response to acute cold exposure require histone deacetylase 3 (HDAC3). HDAC3 exists in tight association with nuclear receptor corepressors (NCoRs) NCoR1 and NCoR2 (also known as silencing mediator of retinoid and thyroid receptors [SMRT]), but the functions of NCoR1/2 in BAT have not been established. Here we report that as expected, genetic loss of NCoR1/2 in BAT (NCoR1/2 BAT-dKO) leads to loss of HDAC3 activity. In addition, HDAC3 is no longer bound at its physiological genomic sites in the absence of NCoR1/2, leading to a shared deregulation of BAT lipid metabolism between NCoR1/2 BAT-dKO and HDAC3 BAT-KO mice. Despite these commonalities, loss of NCoR1/2 in BAT does not phenocopy the cold sensitivity observed in HDAC3 BAT-KO, nor does loss of either corepressor alone. Instead, BAT lacking NCoR1/2 is inflamed, particularly with respect to the interleukin-17 axis that increases thermogenic capacity by enhancing innervation. Integration of BAT RNA sequencing and chromatin immunoprecipitation sequencing data revealed that NCoR1/2 directly regulate Mmp9 , which integrates extracellular matrix remodeling and inflammation. These findings reveal pleiotropic functions of the NCoR/HDAC3 corepressor complex in BAT, such that HDAC3-independent suppression of BAT inflammation counterbalances stimulation of HDAC3 activity in the control of thermogenesis.

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