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While increased levels of high-density lipoprotein (HDL)-cholesterol correlate with protection against cardiovascular disease, recent findings demonstrate that HDL function, rather than HDL-cholesterol levels, may be a better indicator of cardiovascular risk. One mechanism by which HDL function can be compromised is through modification by reactive aldehydes such as acrolein (Acro), 4-hydroxynonenal, and malondialdehyde (MDA). In this study, we tested the hypothesis that modification of HDL with reactive aldehydes would impair HDL's athero-protective functions in macrophages. Compared to native HDL, Acro- and MDA-modified HDL have impaired abilities to promote migration of primary peritoneal macrophages isolated from C57BL6/J mice. Incubation of macrophages with MDA-HDL also led to an increased ability to generate reactive oxygen species. Our studies revealed that the changes in HDL function following aldehyde modification are likely not through activation of canonical nuclear factor-kappa B signaling pathways. Consistent with this finding, treatment of either noncholesterol-loaded macrophages or foam cells with modified forms of HDL does not lead to significant changes in expression levels of inflammatory markers. Importantly, our data also demonstrate that changes in HDL function are dependent on the type of modification present on the HDL particle. Our findings suggest that modification of HDL with reactive aldehydes can impair some, but not all, of HDL's athero-protective functions in macrophages.

Bariatric surgeries are integral to the management of obesity and its metabolic complications. However, these surgeries cause bone loss and increase fracture risk through poorly understood mechanisms. In a mouse model, vertical sleeve gastrectomy (VSG) caused trabecular and cortical bone loss that was independent of sex, body weight, and diet, and this loss was characterized by impaired osteoid mineralization and bone formation. VSG had a profound effect on the bone marrow niche, with rapid loss of marrow adipose tissue, and expansion of myeloid cellularity, leading to increased circulating neutrophils. Following VSG, circulating granulocyte-colony stimulating factor (G-CSF) was increased in mice, and was transiently elevated in a longitudinal study of humans. Elevation of G-CSF was found to recapitulate many effects of VSG on bone and the marrow niche. In addition to stimulatory effects of G-CSF on myelopoiesis, endogenous G-CSF suppressed development of marrow adipocytes and hindered accrual of peak cortical and trabecular bone. Effects of VSG on induction of neutrophils and depletion of marrow adiposity were reduced in mice deficient for G-CSF; however, bone mass was not influenced. Although not a primary mechanism for bone loss with VSG, G-CSF plays an intermediary role for effects of VSG on the bone marrow niche.

Mechanisms by which autosomal recessive mutations in Lmna cause familial partial lipodystrophy type 2 (FPLD2) are poorly understood. To investigate function of lamin A/C in adipose tissues, we created mice with an adipocyte-specific loss of Lmna (LmnaADKO). Although LmnaADKO mice develop and maintain adipose tissues in early postnatal life, they show a striking and progressive loss of white and brown adipose tissues as they approach sexual maturity. LmnaADKO mice exhibit a surprisingly mild metabolic dysfunction on a chow diet, but on a high fat diet they share many characteristics of FPLD2 including hyperglycemia, hepatic steatosis, hyperinsulinemia, and almost undetectable circulating adiponectin and leptin. Whereas LmnaADKO mice have reduced regulated and constitutive bone marrow adipose tissue with a concomitant increase in cortical bone, FPLD2 patients have reduced bone mass and bone mineral density compared to controls. In cell culture models of Lmna deficiency, mesenchymal precursors undergo adipogenesis without impairment, whereas fully-differentiated adipocytes have increased lipolytic responses to adrenergic stimuli. LmnaADKO mice faithfully reproduce many characteristics of FPLD2 and thus provide a unique animal model to investigate mechanisms underlying Lmna-dependent loss of adipose tissues.

Objective Obesity is a key risk factor for many secondary chronic illnesses, including type 2 diabetes and cardiovascular disease. Canonical Wnt/β-catenin signaling is established as an important endogenous inhibitor of adipogenesis. This pathway is operative in mature adipocytes; however, its roles in this context remain unclear due to complexities of Wnt signaling and differences in experimental models. In this study, we used novel cultured cell and mouse models to investigate functional roles of Wnts secreted from adipocytes. Methods We generated adipocyte-specific Wntless (Wls) knockout mice and cultured cell models to investigate molecular and metabolic consequences of disrupting Wnt secretion from mature adipocytes. To characterize Wls-deficient cultured adipocytes, we evaluated the expression of Wnt target and lipogenic genes and the downstream functional effects on carbohydrate and lipid metabolism. We also investigated the impact of adipocyte-specific Wls deletion on adipose tissues and global glucose metabolism in mice fed normal chow or high-fat diets. Results Many aspects of the Wnt signaling apparatus are expressed and operative in mature adipocytes, including the Wnt chaperone Wntless. Deletion of Wntless in cultured adipocytes results in the inhibition of de novo lipogenesis and lipid monounsaturation, likely through repression of Srebf1 (SREBP1c) and Mlxipl (ChREBP) and impaired cleavage of immature SREBP1c into its active form. Adipocyte-specific Wls knockout mice (Wls-/-) have lipogenic gene expression in adipose tissues and isolated adipocytes similar to that of controls when fed a normal chow diet. However, closer investigation reveals that a subset of Wnts and downstream signaling targets are upregulated within stromal-vascular cells of Wls-/- mice, suggesting that adipose tissues defend loss of Wnt secretion from adipocytes. Interestingly, this compensation is lost with long-term high-fat diet challenges. Thus, after six months of a high-fat diet, Wls-/- mice are characterized by decreased adipocyte lipogenic gene expression, reduced visceral adiposity, and improved glucose homeostasis. Conclusions Taken together, these studies demonstrate that adipocyte-derived Wnts regulate de novo lipogenesis and lipid desaturation and coordinate the expression of lipogenic genes in adipose tissues. In addition, we report that Wnt signaling within adipose tissues is defended, such that a loss of Wnt secretion from adipocytes is sensed and compensated for by neighboring stromal-vascular cells. With chronic overnutrition, this compensatory mechanism is lost, revealing that Wls-/- mice are resistant to diet-induced obesity, adipocyte hypertrophy, and metabolic dysfunction., Graphical abstract Image 1, Highlights • Wntless in adipocytes is required for the expression of a network of lipogenic genes. • Regulation of lipogenic genes by Wntless is likely mediated by Srebf1 and Mlxipl. • Adipose tissues defend loss of adipocyte-derived Wnt secretion. • Compensation for Wntless deficiency is lost with diet-induced obesity. • Wntless deficiency causes reduced adipocyte size and improved metabolic function.

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here we developed brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin (Adipoq), adipose triglyceride lipase (Atgl), fatty acid synthase (Fasn), perilipin 1 (Plin1), or stearoyl-CoA desaturase 1 (Scd1) by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 (FGF21), and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies, with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

Although adipogenesis is mainly controlled by a small number of master transcription factors, including CCAAT/enhancer-binding protein family members and peroxisome proliferator-activated receptor γ (PPARγ), other transcription factors also are involved in this process. Thyroid cancer cells expressing a paired box 8 (PAX8)–PPARγ fusion oncogene trans-differentiate into adipocyte-like cells in the presence of the PPARγ ligand pioglitazone, but this trans-differentiation is inhibited by the transcription factor NK2 homeobox 1 (NKX2-1). Here, we tested whether NKX family members may play a role also in normal adipogenesis. Using quantitative RT-PCR (RT-qPCR), we examined the expression of all 14 NKX family members during 3T3-L1 adipocyte differentiation. We found that most NKX members, including NKX2-1, are expressed at very low levels throughout differentiation. However, mRNA and protein expression of a related family member, NKX1-2, was induced during adipocyte differentiation. NKX1-2 also was up-regulated in cultured murine ear mesenchymal stem cells (EMSCs) during adipogenesis. Importantly, shRNA-mediated NKX1-2 knockdown in 3T3-L1 preadipocytes or EMSCs almost completely blocked adipocyte differentiation. Furthermore, NKX1-2 overexpression promoted differentiation of the ST2 bone marrow–derived mesenchymal precursor cell line into adipocytes. Additional findings suggested that NKX1-2 promotes adipogenesis by inhibiting expression of the antiadipogenic protein COUP transcription factor II. Bone marrow mesenchymal precursor cells can differentiate into adipocytes or osteoblasts, and we found that NKX1-2 both promotes ST2 cell adipogenesis and inhibits their osteoblastogenic differentiation. These results support a role for NKX1-2 in promoting adipogenesis and possibly in regulating the balance between adipocyte and osteoblast differentiation of bone marrow mesenchymal precursor cells.

Adipose tissue undergoes distinct structural remodeling in response to increases in fat mass, as in obesity. During this process, the extracellular matrix (ECM) plays a pivotal role with respect to both adipocyte receptor signaling and structural remodeling. One ECM protein, procollagen endopeptidase enhancer 2 (PCPE2), has been shown by our lab to work in partnership with the scavenger receptor class B type I (SR-BI) to regulate adipocyte cholesterol homeostasis. To investigate the functional impact of the partnership between SR-BI and PCPE2, we studied mature adipocytes differentiated from murine embryonic fibroblasts (MEF) and compared to our adipose specific PCPE2 knockout mice. Our studies showed the presence of an SR-BI-PCPE2 protein complex based on the co-immunoprecipitation of PCPE2 with SR-BI in both differentiated MEFs and mouse adipose tissue. Furthermore, stimulated emission depletion (STED) microscopy showed SR-BI and PCPE2 localized on the lipid droplet surface separated by distances ranging from 24 – 90 nm, suggesting that the SR-BI-PCPE2 complex may contain other proteins. Most significantly, a loss of SR-BI function was shown in PCPE2-deficient adipocytes by the SR-BI mediated HDL binding and cholesteryl ester (CE) uptake assays. Compared to differentiated MEFs from Ldlr -/- mice, binding of HDL to differentiated MEFs from Ldlr -/- Pcpe2 -/- mice was reduced by 75%, and HDL cholesteryl oleyl ether uptake was reduced by 50%. Disruption of adipocyte SR-BI function in the absence of PCPE2 also reduced free cholesterol efflux indicating substantial disruption to intracellular cholesterol homeostasis. Moreover, analysis of RNAseq from several human clinical studies show that PCPE2 mRNA expression is positively correlated with fat mass in specific adipose depots, while negatively correlated with plasma insulin, glucose and triglyceride levels, with the latter two more robust in type 2 diabetic populations (FDR < 0.004). Overall, these results suggest that the partnership between SR-BI and PCPE2 in adipocytes plays an important role in cholesterol trafficking and homeostasis which influences fat mass expansion, glucose tolerance and insulin resistance.