Summary Nicotinamide riboside supplements (NRS) have been touted as a nutraceutical that promotes cardiometabolic and musculoskeletal health by enhancing nicotinamide adenine dinucleotide (NAD + ) biosynthesis, mitochondrial function, and/or the activities of NAD-dependent sirtuin deacetylase enzymes. This investigation examined the impact of NRS on whole body energy homeostasis, skeletal muscle mitochondrial function, and corresponding shifts in the acetyl-lysine proteome, in the context of diet-induced obesity using C57BL/6NJ mice. The study also included a genetically modified mouse model that imposes greater demand on sirtuin flux and associated NAD + consumption, specifically within muscle tissues. In general, whole body glucose control was marginally improved by NRS when administered at the midpoint of a chronic high-fat diet, but not when given as a preventative therapy upon initiation of the diet. Contrary to anticipated outcomes, the study produced little evidence that NRS increases tissue NAD + levels, augments mitochondrial function, and/or mitigates diet-induced hyperacetylation of the skeletal muscle proteome.

SUMMARYA hallmark of type 2 diabetes (T2D), a major cause of world-wide morbidity and mortality, is dysfunction of insulin-producing pancreatic islet β cells1–3. T2D genome-wide association studies (GWAS) have identified hundreds of signals, mostly in the non-coding genome and overlapping β cell regulatory elements, but translating these into biological mechanisms has been challenging4–6. To identify early disease-driving events, we performed single cell spatial proteomics, sorted cell transcriptomics, and assessed islet physiology on pancreatic tissue from short-duration T2D and control donors. Here, through integrative analyses of these diverse modalities, we show that multiple gene regulatory modules are associated with early-stage T2D β cell-intrinsic defects. One notable example is the transcription factor RFX6, which we show is a highly connected β cell hub gene that is reduced in T2D and governs a gene regulatory network associated with insulin secretion defects and T2D GWAS variants. We validated the critical role of RFX6 in β cells through direct perturbation in primary human islets followed by physiological and single nucleus multiome profiling, which showed reduced dynamic insulin secretion and large-scale changes in the β cell transcriptome and chromatin accessibility landscape. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs, and individuals and thus we anticipate this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits with GWAS data.

Mounting evidence has shown that CETP has important physiological roles in adapting to chronic nutrient excess, specifically, to protect against diet-induced insulin resistance. However, the underlying mechanisms for the protective roles of CETP in metabolism are not yet clear. Mice naturally lack CETP expression. We used transgenic mice with a human CETP minigene (huCETP) controlled by its natural flanking region to further understand CETP-related physiology in response to obesity. Female huCETP mice and their wild-type littermates were fed a high-fat diet for 6 months. Blood lipid profile and liver lipid metabolism were studied. Insulin sensitivity was analyzed with euglycemic-hyperinsulinemic clamp studies combined with 3H-glucose tracer techniques. While high-fat diet feeding induced obesity for huCETP mice and their wild-type littermates lacking CETP expression, insulin sensitivity was higher for female huCETP mice than for their wild-type littermates. There was no difference in insulin sensitivity for male huCETP mice vs. littermates. The increased insulin sensitivity in females was largely caused by the better insulin-mediated suppression of hepatic glucose production. In huCETP females, CETP in the circulation decreased HDL-cholesterol content and increased liver cholesterol uptake and liver cholesterol and oxysterol contents, which was associated with the upregulation of LXR target genes in long-chain polyunsaturated fatty acid biosynthesis and PPARα target genes in fatty acid β-oxidation in the liver. The upregulated fatty acid β-oxidation may account for the improved fatty liver and liver insulin action in female huCETP mice. This study provides further evidence that CETP has beneficial physiological roles in the metabolic adaptation to nutrient excess by promoting liver fatty acid oxidation and hepatic insulin sensitivity, particularly for females.

Early life stress (ELS) is associated with cardiovascular disease (CVD) risk in adulthood, but the underlying vascular mechanisms are poorly understood. Increased hemoglobin and heme have recently been implicated to mediate endothelial dysfunction in several vascular diseases. Chronic physiological stress is associated with alterations in the heme pathway that have been well-described in the literature. However, very little is known about the heme pathway with exposure to ELS or chronic psychosocial stress. Utilizing a mouse model of ELS, maternal separation with early weaning (MSEW), we previously reported that MSEW induces endothelial dysfunction via increased superoxide production. We reasoned that heme dysregulation may be one of the culprits induced by MSEW and sustained throughout adulthood; thus, we hypothesized that MSEW induces heme dysfunction. We investigated whether circulating levels of heme, a circulating pro-oxidant mediator, are increased by MSEW and examined the role of the heme metabolic pathway and heme homeostasis in this process.We found that circulating levels of heme are increased in mice exposed to MSEW and that plasma from MSEW mice stimulated higher superoxide production in cultured mouse aortic endothelial cells (MAECs) compared to plasma from normally reared mice. The heme scavenger hemopexin blunted this enhanced superoxide production. Splenic haptoglobin abundance was significantly lower and hemoglobin levels per red blood cell were significantly higher in MSEW versus control mice. These findings lead us to propose that ELS induces increased circulating heme through dysregulation of the haptoglobin-hemoglobin system representing a mechanistic link between ELS

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Early life stress (ELS) has been correlated with an increased risk of developing cardiovascular disease (CVD) in adulthood; however, mechanisms linking ELS and CVD risk are unknown. Toll‐like receptor (TLR) activation, specifically TLR4, has been implicated as a key link between inflammation and CVD. TLR4 signaling can activated NADPH oxidases (NOXs) that produce superoxide to increase CVD risk. Further, multiple studies have found that NF‐kB activation, an important downstream step in the TLR4 signaling pathway, requires the activation of certain NOX isoforms, including NOX2 and NOX4. We utilized a mouse model of maternal separation with early weaning (MSEW) to investigate the effect of ELS on the expression and activation of genes encoding these pro‐oxidant, proinflammatory proteins. MSEW mice were separated 4hr/day during postnatal day (PD)2–5, 8 hr/day during PD6‐16, and weaned at PD17. Normally‐reared control mice were left undisturbed and weaned at PD21. Experiments were conducted on 3–6 month old male mice. We have previously shown that MSEW induces NOX‐dependent superoxide production and vascular endothelial dysfunction. We also found increased gene expression of NOX2, NOX4, and TLR4 in the aorta of MSEW mice. Recent immunohistochemical studies have also revealed increased aortic TLR4 protein expression in MSEW mice (n=5/group). Due to similar patterns of expression of genes involved in the TLR4 signaling pathway, we further hypothesized that a common upstream transcription factor could be a driver of these ELS‐induced gene changes. We focused this study on histone deacetylases (HDACs), as numerous studies have implicated HDACs in the modulation of proinflammatory responses and the progression of a variety of CVDs. Interestingly, we found that MSEW induced aortic HDAC9 protein expression, exclusive of other HDAC isoforms. A 3‐hr ex vivo pretreatment with trichostatin A (TSA), a pan‐inhibitor of HDACs, improved vascular endothelial function in MSEW mice (n=5/group, p=0.047). Further, ex vivo TSA treatment led to a trend of decreased expression of NOX2, NOX4, and TLR4 genes (n=4–8/group). These results suggest that ELS leads to HDAC9‐dependent pro‐oxidative and proinflammatory gene expression that increase CVD risk in adulthood. They also further emphasize the role of TLR4 signaling on superoxide production and vascular endothelial function. Future studies aim to clarify the role of TLR4 signaling in these phenotypes, as well as the effect of increased HDAC9 expression on the transcriptional regulation of TLR4 and other genes that could confer CVD risk in adulthood.Support or Funding InformationThis research was funded by PO1HL69999 and the UAB PREP Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure to adverse childhood experiences, also known as early life stress (ELS), is an independent risk factor for the development of cardiovascular disease (CVD) in adulthood. Endothelial dysfunction is a major contributor to the development of CVD and has been linked to childhood adversity induced CVD risk. We previously reported circulating ET‐1 levels are significantly elevated in young adults exposed to childhood adversity in humans. Endothelin‐1 (ET‐1) via the ETA receptor promotes endothelial dysfunction and vascular inflammation. However, the regulation of ET‐1 by ELS and the mechanistic link between elevated ET‐1 levels and ELS‐induced increase in CVD risk remains unknown. In a mouse model of ELS involving maternal separation and early weaning (MSEW), we previously reported that ELS induced elevated plasma ET‐1 as well as increased vascular histone deacetylase 9 (HDAC9) protein expression with no changes in expression of other HDAC isoforms. Moreover, MSEW mice develop endothelial dysfunction in adulthood that is attenuated by a pan‐inhibitor of Class I and class II HDAC proteins, trichostatin A (TSA). HDACs are a family of epigenetic enzymes that regulate transcription through deacetylation of histone and non‐histone proteins. In humans, previous studies have shown SNPs in the HDAC9 gene are prominent in large artery stroke and coronary artery disease and that upregulated HDAC9 expression is localized in endothelial cells of carotid atherosclerotic human tissue suggesting HDAC9 may play an important role in atherogenesis. Therefore, we hypothesized that vascular HDAC9 upregulation promotes endothelial dysfunction in MSEW mice via increased ET‐1. MSEW involves maternal separation 4 h/day (postnatal (PD) 2 to 5) and 8 h/day (PD6 to 16) and weaned at PD17. Normally reared litters weaned at PD21 were used as controls (CON). In adult male CON and MSEW mice, we determined aortic localization of HDAC9 and ET‐1. Immunohistolocalization showed aortic HDAC9 is upregulated specifically in the endothelial cells of MSEW mice (n=5/group). Confocal immunofluorescence imaging revealed aortic ET‐1 expression is also localized in the endothelium of both CON and MSEW mice. To examine the effects of endothelial HDAC9 on ET‐1 expression, we used mouse aortic endothelial cells (MAECs) transfected with control vector or HDAC9 plasmid incubated in the presence or absence of TSA. Cultured MAECs treated with TSA blunted ET‐1 expression (70% decrease from vehicle, p<0.05, n=3/group). Furthermore, HDAC9 over‐expression in cultured MAECs significantly increased ET‐1 expression (relative to control vector group, 1.0±0.08 vs 1.73±0.44 arbitrary units, p<0.05, n=3/group) and TSA reduced the HDAC9‐dependent ET‐1 expression (1.68 fold decrease from control vector vehicle group, p<0.05, n=3/group). Our findings indicate endothelial dysfunction previously observed in our MSEW mouse model may be mediated by HDAC9‐dependent elevated ET‐1 production. In conclusion, this study highlights the importance of ELS in the development of CVD and the implications of an epigenetic pathway as a potential mechanistic link.Support or Funding InformationNIH P01 HLP0169999; NIH K01 DK105038This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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