Human genetic studies have repeatedly associated ADAMTS7 with atherosclerotic cardiovascular disease. Subsequent investigations in mice demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury. However, the cell-specific mechanisms governing ADAMTS7 proatherogenicity remain unclear. To determine which vascular cell types express ADAMTS7, we interrogated single-cell RNA-seq of human carotid atherosclerosis and found ADAMTS7 expression in smooth muscle cells (SMCs), endothelial cells (ECs), and fibroblasts. We subsequently created SMC- and EC-specific Adamts7 conditional KO and transgenic mice. Conditional KO of Adamts7 in either cell type did not reduce atherosclerosis, whereas transgenic induction in either cell type increased atherosclerosis. In SMC transgenic mice, this increase coincides with an expansion of lipid-laden SMC foam cells and a decrease in fibrous cap formation. RNA-seq of Adamts7-overexpressing SMCs revealed an upregulation of lipid genes typically assigned to macrophages. Mechanistically, ADAMTS7 increases SMC oxidized LDL uptake through CD36, whose expression is upregulated by PU.1. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) and motif analysis revealed increased chromatin accessibility at AP-1-enriched regions, consistent with AP-1-dependent remodeling of PU.1-regulated lipid-handling loci. In summary, ADAMTS7 promotes atherosclerosis by driving SMC foam cell formation through an AP-1/PU.1/CD36 regulatory axis.

OBJECTIVE:The distribution of excess white adipose tissue (WAT) in obesity correlates with risk for comorbidities. Thus, understanding depot-specific WAT developmental mechanisms is translationally relevant. SNPs near the gene CEBPA associate with waist to hip ratio, and while C/EBPα is a recognized regulator of adipogenesis, there is no previously known role for C/EBPα in regulating adipose distribution. METHODS:We crossed Cebpa floxed mice to the AdipoQ-Cre transgenic mouse strain, generating mice with adipocyte-specific knockout of Cebpa (Cebpa_ASKO). Mice were phenotyped on a chow diet and after prolonged high-fat diet (HFD) feeding. RESULTS:Cebpa_ASKO mice almost entirely lack gonadal WAT (gWAT), while inguinal WAT (iWAT) is present in near normal amounts. Despite developing, Cebpa_ASKO iWAT contains fewer and larger adipocytes, fails to expand under HFD challenge, and is dysfunctional as evidenced by transcriptomics and functional studies. Finally, Cebpa_ASKO mice have lipid-laden brown adipose tissue (BAT), increased hepatic triglycerides, and increased plasma cholesterol, all of which worsen with prolonged HFD feeding. CONCLUSIONS:These results highlight a previously unrecognized difference in the essentiality of C/EBPα for gWAT and iWAT development and highlight novel interorgan relationships between WAT and other metabolic tissues. Further studies of these specific mechanisms could have clinical relevance for targeting visceral adiposity in humans.

Rates of obesity and its associated metabolic comorbidities continue to rise in the developed world. It is well established that in obesity, the distribution and not just amount of excess white adipose tissue (WAT) correlates with a person's risk for comorbidities such as coronary artery disease and type 2 diabetes. Thus, understanding the specific mechanisms that drive WAT development in specific adipose depots could elucidate novel mechanisms of metabolic disease. SNPs near the gene CEBPA have been associated with multiple cardiometabolic traits by human genome-wide association studies, including waist-to-hip ratio, suggesting that CEBPA regulates WAT distribution. CEBPA encodes a well characterized transcription factor (C/EBPα) that is long recognized as a master regulator of adipocyte differentiation, yet depot-specific roles for C/EBPα have not been previously described. To further investigate this genetic link, we generated mice with adipocyte-specific Cebpa knockout (Cebpa_ASKO) and found that these mice are almost entirely lacking gonadal WAT (gWAT) despite the inguinal WAT (iWAT) being present in near normal amounts. Despite developing, Cebpa_ASKO iWAT contains fewer and larger adipocytes, and fails to expand when challenged with high fat diet. RNA-seq and functional studies demonstrate evidence of altered lipid metabolism and adipocyte function in Cebpa_ASKO iWAT. Finally, Cebpa_ASKO mice have multiple other metabolic phenotypes, including lipid-laden BAT, increased hepatic triglycerides, and increased plasma cholesterol, all of which worsen with prolonged high fat diet feeding. Taken together, these data highlight depot-specific roles for C/EBPα in adipose tissue development, as well as the importance of adipocyte C/EBPα in maintaining metabolic homeostasis.

Obesity, defined as excess white adipose tissue (WAT), associates with a higher risk of metabolic disease and comorbidities, and continues to be on the rise in the developed world. However, different WAT depots associate with differing levels of cardiometabolic risk. Expansion of visceral WAT (vWAT) correlates with increased risk for cardiovascular disease and type 2 diabetes, while expansion of subcutaneous WAT (sWAT) does not. These observations suggest mechanistic differences between the two depots, and elucidating these mechanisms may provide insights into promoting cardiometabolic health in humans. Human GWAS have shown that SNPs near the gene CEBPA associate with waist-to-hip ratio, a metric of vWAT mass in humans, suggesting CEBPA plays a specific role in vWAT. CEBPA encodes the transcription factor C/EBPα, a known regulator of adipogenesis. We established adipocyte-specific Cebpa knockout (Cebpa_ASKO) mice and found they lack gonadal WAT (gWAT), the largest vWAT depot in mice. Conversely, sWAT is still present in Cebpa_ASKO mice, although with reduced mass (-23%, p<0.05) and larger adipocytes. Additionally, Cebpa_ASKO mice have reduced lipolysis (-26% NEFAs, p<0.05), plasma adiponectin (-89%, p<0.0001) and leptin (-82%, p<0.05), suggesting the remaining sWAT is dysfunctional. Together, these data suggest that C/EBPα has WAT depot-specific roles, being required for vWAT development, but only for function in mature sWAT. Given the myriad roles for vWAT in metabolism and the absence of vWAT in Cebpa_ASKO mice, we investigated downstream metabolic consequences of this phenotype. Cebpa_ASKO mice have increased brown adipose tissue (BAT) mass (+118%, p<0.001) due to increased ectopic lipid deposition. Cebpa_ASKO mice also have increases in plasma cholesterol (+20%, p<0.05), liver mass (+24%, p<0.01), and hepatic fat content (+329%, p<0.01). Overall, these results demonstrate that adipocyte C/EBPα significantly influences whole body metabolism. Ongoing studies center on delineating the different mechanisms through which C/EBPα functions in vWAT and sWAT. Understanding the depot-specific mechanisms of C/EBPα could have practical implications for new clinical targets of obesity and metabolic disease.

Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity -related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non -coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN (adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAIN knockdown (KD) increases KLF5 and interleukin-8 (IL -8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL -8, via linc-ADAIN KD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitro and in vivo . KD of linc-ADAIN in human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity -induced adipose tissue remodeling. linc-ADAIN is an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

The gene tribbles pseudokinase 1 ( TRIB1 ) has been repeatedly linked to multiple human cardiometabolic traits through genome-wide association studies, including coronary artery disease, plasma cholesterol and triglycerides, and circulating liver transaminases, signaling that TRIB1 is a key regulator of liver metabolism and health. Studies in liver specific Trib1 KO mice have shown that hepatic TRIB1 regulates de novo lipogenesis and steatosis through the regulation of protein levels of the transcription factor C/EBPα, yet the mechanism governing this relationship in hepatocytes has not been investigated. We demonstrate here that human TRIB1 promotes the degradation of C/EBPα in both a COP1- and proteasome-dependent manner in human hepatoma cells. We also observe rapid degradation of TRIB1 protein in hepatoma cells, and find that this is also COP1- and proteasome-dependent. To identify hepatocyte-specific interacting partners of TRIB1 that regulate these processes, we performed tandem-affinity purification of TRIB1 in Huh7 cells and subsequent mass-spec analysis, and identified multiple novel TRIB1 binding partners including the pseudokinases serine/threonine kinase 38 (STK38) and 40 (STK40). We confirmed these interactions in vitro and found that STK40, but not STK38, is required for TRIB1-mediated proteasomal degradation of C/EBPα. Together, our results reveal that TRIB1 induces proteasomal degradation of CEBP/α in a COP1- and proteasome-dependent manner in human hepatocytes. Further, we identify the pseudokinase STK40 as a novel regulator of hepatic TRIB1 function, and ongoing work aims to investigate the role of STK40 in hepatic lipid metabolism. Finally, we show that TRIB1 is itself degraded by the proteosome in a COP1-dependent manner. As increased hepatic Trib1 confers a beneficial metabolic profile in mice, these findings could provide a novel avenue for therapeutic targeting of TRIB1 in the treatment of cardiometabolic disease. Overall, our findings add greater detail to the molecular mechanisms governing the regulation of metabolism by hepatic TRIB1, a gene which human genetics highlights as a crucial regulator of cardiometabolic traits in humans.

Objective: Multiple genome-wide association studies (GWAS) have identified SNPs in the 8q24 locus near TRIB1 that are significantly associated with plasma lipids and other markers of cardiometabolic health, and prior studies have revealed the roles of hepatic and myeloid Trib1 in plasma lipid regulation and atherosclerosis. The same 8q24 SNPs are additionally associated with plasma adiponectin levels in humans, implicating TRIB1 in adipocyte biology. Here, we hypothesize that TRIB1 in adipose tissue regulates plasma adiponectin, lipids, and metabolic health. Methods: We investigate the metabolic phenotype of adipocyte-specific Trib1 knockout mice (Trib1_ASKO) fed on chow and high-fat diet (HFD). Through secretomics of adipose tissue explants and RNA-seq of adipocytes and livers from these mice, we further investigate the mechanism of TRIB1 in adipose tissue. Results: Trib1_ASKO mice have an improved metabolic phenotype with increased plasma adiponectin levels, improved glucose tolerance, and decreased plasma lipids. Trib1_ASKO adipocytes have increased adiponectin production and secretion independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice indicates that alterations in adipocyte function underlie the observed plasma lipid changes. Adipose tissue explant secretomics further reveals that Trib1_ASKO adipose tissue has decreased ANGPTL4 production, and we demonstrate an accompanying increase in the lipoprotein lipase (LPL) activity that likely underlies the triglyceride phenotype. Conclusions: This study shows that adipocyte Trib1 regulates multiple aspects of metabolic health, confirming previously observed genetic associations in humans and shedding light on the further mechanisms by which TRIB1 regulates plasma lipids and metabolic health.