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SummaryTo investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

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.

Although visceral adipocytes located within the body’s central core are maintained at ~37°C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell, and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well-studied; however, comparatively little is known about mechanisms by white adipocytes adapt to temperatures below 37°C. Here we report that adaptation of cultured adipocytes to 31°C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, induces extensive changes in gene expression, increased anabolic and catabolic lipid metabolism, and elevated oxygen consumption with reduced reliance on glucose and preferential use of pyruvate, glutamine and fatty acids as energy sources. Cool temperatures up-regulate stearoyl-CoA desaturase-1 expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues, and stearoyl-CoA desaturase-1 activity is required for acquisition of maximal oxygen consumption at 31°C.

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.

Gene transfer using viral or nonviral vectors enables the ability to manipulate specific cells and tissues for gene silencing, protein overexpression, or genome modification. Despite the widespread application of viral- and non-viral-mediated gene transfer to liver, heart, skeletal muscle, and the central nervous system, its use in adipose tissue has been limited. This is largely because adipose tissue is distributed throughout the body in distinct depots and adipocytes make up a minority of the cells within the tissue, making transduction difficult. Currently, there is no consensus methodology for efficient gene transfer to adipose tissue and many studies report conflicting information with regard to transduction efficiency and vector biodistribution. In this review, we summarize the challenges associated with gene transfer to adipose tissue and report on innovations that improve efficacy. We describe how vector and route of administration are the two key factors that influence transduction efficiency and outline a “gold standard” approach and experimental workflow for validating gene transfer to adipose tissue. Lastly, we speculate on how CRISPR/Cas9 can be integrated to improve adipose tissue research.

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 visceral adipocytes located within the body’s central core are maintained at approximately 37°C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well studied; however, comparatively little is known about mechanisms by which white adipocytes adapt to temperatures below 37°C. Here, we report that adaptation of cultured adipocytes to 31°C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, increases anabolic and catabolic lipid metabolism, and elevates oxygen consumption. Cool adipocytes rely less on glucose and more on pyruvate, glutamine, and, especially, fatty acids as energy sources. Exposure of cultured adipocytes and gluteal white adipose tissue (WAT) to cool temperatures activates a shared program of gene expression. Cool temperatures induce stearoyl-CoA desaturase-1 (SCD1) expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues (BMATs), and SCD1 activity is required for acquisition of maximal oxygen consumption at 31°C., Adipocytes in bone marrow, subcutaneous and dermal sites generally exist at temperatures below 37°C. This study identifies the molecular and metabolic program that adapts white adipocytes to these cooler environments.