👤 Ludger Scheja

Brown adipose tissue (BAT) comprises a heterogeneous population of adipocytes and non-adipocyte cell types. To characterize these cellular subpopulations and their adaptation to cold, we performed single-nucleus mRNA-sequencing (snRNA-seq) on interscapular BAT from mice maintained at room temperature or exposed to acute (24h) or chronic (10 days) cold (6 °C). To investigate the role of the de novo lipogenesis (DNL)-regulating transcription factor carbohydrate response element-binding protein (ChREBP), we analyzed control and brown adipocyte-specific ChREBP knockout mice. We identified different cell populations, including seven brown adipocyte subtypes with distinct metabolic profiles. One of them highly expressed ChREBP and DNL enzymes. Notably, these lipogenic adipocytes were highly sensitive to acute cold exposure, showing a marked depletion in BAT of control mice that was compensated by other brown adipocyte subtypes maintaining DNL. Chronic cold exposure resulted in an expansion of basal brown adipocytes and adipocytes putatively derived from stromal and endothelial precursors. In ChREBP-deficient mice, lipogenic adipocytes were almost absent under all conditions, identifying the transcription factor as a key determinant of this adipocyte subtype. Detailed expression analyses revealed Ttc25 as a specific marker of lipogenic brown adipocytes and as a downstream target of ChREBP. Furthermore, pathway and cell-cell interaction analyses implicated a Wnt-ChREBP axis in the maintenance of lipogenic adipocytes, with Wnt ligands from stromal and muscle cells providing instructive cues. Our findings provide a comprehensive atlas of BAT cellular heterogeneity and reveal a critical role for ChREBP in lipogenic adipocyte identity, with implications for BAT plasticity and metabolic function. Show less

Recombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. To achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. We demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. Taken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes. Show less

Brown and beige adipocytes combust nutrients for thermogenesis and through their metabolic activity decrease pro-atherogenic remnant lipoproteins in hyperlipidemic mice. However, whether the activation of thermogenic adipocytes affects the metabolism and anti-atherogenic properties of high-density lipoproteins (HDL) is unknown. Here, we report a reduction in atherosclerosis in response to pharmacological stimulation of thermogenesis linked to increased HDL levels in APOE*3-Leiden.CETP mice. Both cold-induced and pharmacological thermogenic activation enhances HDL remodelling, which is associated with specific lipidomic changes in mouse and human HDL. Furthermore, thermogenic stimulation promotes HDL-cholesterol clearance and increases macrophage-to-faeces reverse cholesterol transport in mice. Mechanistically, we show that intravascular lipolysis by adipocyte lipoprotein lipase and hepatic uptake of HDL by scavenger receptor B-I are the driving forces of HDL-cholesterol disposal in liver. Our findings corroborate the notion that high metabolic activity of thermogenic adipocytes confers atheroprotective properties via increased systemic cholesterol flux through the HDL compartment. Show less

Clinical interest in de novo lipogenesis has been sparked by recent studies in rodents demonstrating that de novo lipogenesis specifically in white adipose tissue produces the insulin-sensitizing fatty acid palmitoleate. By contrast, hepatic lipogenesis is thought to contribute to metabolic disease. How de novo lipogenesis in white adipose tissue versus liver is altered in human obesity and insulin resistance is poorly understood. Here we show that lipogenic enzymes and the glucose transporter-4 are markedly decreased in white adipose tissue of insulin-resistant obese individuals compared with non-obese controls. By contrast, lipogenic enzymes are substantially upregulated in the liver of obese subjects. Bariatric weight loss restored de novo lipogenesis and glucose transporter-4 gene expression in white adipose tissue. Notably, lipogenic gene expression in both white adipose tissue and liver was strongly linked to the expression of carbohydrate-responsive element-binding protein-β and to metabolic risk markers. Thus, de novo lipogenesis predicts metabolic health in humans in a tissue-specific manner and is likely regulated by glucose-dependent carbohydrate-responsive element-binding protein activation. Show less

Changes in fatty acid metabolism associated with insulin resistance have been described in rats and humans but have not been well characterized in the frequently used mouse model of diet-induced obesity. To analyse the early phase as well as established insulin resistance, C57BL/6 mice were placed for 1 or 16 weeks on a high fat diet (1w-HFD, 16w-HFD). Endocrine and metabolic parameters indicated that 1w-HFD mice showed a moderate but significant induction of insulin resistance while 16w-HFD mice exhibited profound obesity-associated insulin resistance and dyslipidemias. Significant alterations in fatty acid composition were observed in plasma and liver in both groups. Liver phospholipid-associated arachidonate and docosahexaenoate were increased in both 1w-HFD and 16w-HFD mice, possibly due to increased expression of the desaturases Fads1 and Fads2. Unexpectedly, SCD1 activity and gene expression in liver were decreased in the 1w-HFD group accompanied by diminished total hepatic lipid levels, while they were increased in chronically fed mice. Our data indicate that the early phase of HFD-induced insulin resistance is not associated with elevated liver lipid concentration. Furthermore, the early and persistent rise of arachidonate and docosahexaenoate indicates that insulin resistance is not due to insufficient availability (or concentrations) of polyunsaturated fatty acids as postulated previously. Show less