Bexarotene (Targretin) is a clinically used antitumoral agent which exerts its action through binding to and activation of the retinoid-X-receptor (RXR). The most frequent side-effect of bexarotene administration is an increase in plasma triglycerides, an independent risk factor of cardiovascular disease. The molecular mechanism behind this hypertriglyceridemia remains poorly understood.Using wild-type and LXR alpha/beta-deficient mice, we show here that bexarotene induces hypertriglyceridemia and activates hepatic LXR-target genes of lipogenesis in an LXR-dependent manner, hence exerting a permissive effect on RXR/LXR heterodimers. Interestingly, RNA analysis and Chromatin Immunoprecipitation assays performed in the liver reveal that the in vivo permissive effect of bexarotene on the RXR/LXR heterodimer is restricted to lipogenic genes without modulation of genes controlling cholesterol homeostasis.These findings demonstrate that the hypertriglyceridemic action of bexarotene occurs via the RXR/LXR heterodimer and show that RXR heterodimers can act with a selective permissivity on target genes of specific metabolic pathways in the liver.
The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of adipocyte differentiation in vivo and ex vivo and has been shown to control the expression of several adipocyte-specific genes. In this study, we used chromatin immunoprecipitation combined with deep sequencing to generate genome-wide maps of PPARγ and retinoid X receptor (RXR)-binding sites, and RNA polymerase II (RNAPII) occupancy at very high resolution throughout adipocyte differentiation of 3T3-L1 cells. We identify >5000 high-confidence shared PPARγ:RXR-binding sites in adipocytes and show that during early stages of differentiation, many of these are preoccupied by non-PPARγ RXR-heterodimers. Different temporal and compositional patterns of occupancy are observed. In addition, we detect co-occupancy with members of the C/EBP family. Analysis of RNAPII occupancy uncovers distinct clusters of similarly regulated genes of different biological processes. PPARγ:RXR binding is associated with the majority of induced genes, and sites are particularly abundant in the vicinity of genes involved in lipid and glucose metabolism. Our analyses represent the first genome-wide map of PPARγ:RXR target sites and changes in RNAPII occupancy throughout adipocyte differentiation and indicate that a hitherto unrecognized high number of adipocyte genes of distinctly regulated pathways are directly activated by PPARγ:RXR.
Insulin treatment is associated with increased adipose mass in both humans and mice. However, the underlying dynamic basis of insulin induced lipid accumulation in adipose tissue remains elusive. To assess this, young female C57BL6/J mice were fed a low fat diet for 3 weeks, treated subsequently with 7 days of constant subcutaneous insulin infusion by osmotic minipumps and compared to mice with only buffer infused. To track changes in lipid deposition during insulin treatment, metabolic labeling was conducted with heavy water for the final 4 days. Blood glucose was significantly lowered within one hour after implantation of insulin loaded mini pumps and remained lower throughout the study. Insulin treated animals gained significantly more weight during treatment and the mean weight of the subcutaneous adipose depots was significantly higher with the highest dose of insulin. Surprisingly, de novo palmitate synthesis within the subcutaneous and the gonadal depots was not affected significantly by insulin treatment. In contrast insulin treatment caused accumulation of triglycerides in both depots due to either deposition of newly synthesised triglycerides (subcutaneous depot) or inhibition of lipolysis (gonadal depot).
The C/EBPalpha transcription factor regulates growth and differentiation of several tissues during embryonic development. Several hypotheses as to how C/EBPalpha inhibits cellular growth in vivo have been derived, mainly from studies of tissue culture cells. In fetal liver it has been proposed that a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPalpha is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(DeltaPHR/DeltaPHR) (DeltaPHR) mice, carrying a modified cebpa allele lacking amino acids 180 to 194, were born at the Mendelian ratio, reached adulthood, and displayed no apparent adverse phenotypes. When fetal livers from the DeltaPHR mice were analyzed for their expression of cell cycle markers, bromodeoxyuridine incorporation, cyclin-dependent kinase 2 kinase activity, and global gene expression, we failed to detect any cell cycle or developmental differences between the DeltaPHR mice and their control littermates. These in vivo data demonstrate that any C/EBPalpha-mediated growth repression via the PHR as well as the basic region is dispensable for proper embryonic development of, and cell cycle control in, the liver. Surprisingly, control experiments performed in C/EBPalpha null fetal livers yielded similar results.
Insulin resistance impairs the cellular insulin response, and often precedes metabolic disorders, like type 2 diabetes, impacting an increasing number of people globally. Understanding the molecular mechanisms in hepatic insulin resistance is essential for early preventive treatments. To elucidate changes in insulin signal transduction associated with hepatocellular resistance, we employed a multi-layered mass spectrometry-based proteomics approach focused on insulin receptor (IR) signaling at the interactome, phosphoproteome, and proteome levels in a long-term hyperinsulinemia-induced insulin-resistant HepG2 cell line with a knockout of the insulin-like growth factor 1 receptor (IGF1R KO). The analysis revealed insulin-stimulated recruitment of the PI3K complex in both insulin-sensitive and -resistant cells. Phosphoproteomics showed attenuated signaling via the metabolic PI3K-AKT pathway but sustained extracellular signal-regulated kinase (ERK) activity in insulin-resistant cells. At the proteome level, the ephrin type-A receptor 2 (EphA2) showed an insulin-induced increase in expression, which occurred through the ERK signaling pathway and was concordantly independent of insulin resistance. Induction of EphA2 by insulin was confirmed in additional cell lines and observed uniquely in cells with high IR-to-IGF1R ratio. The multi-layered proteomics dataset provided insights into insulin signaling, serving as a resource to generate and test hypotheses, leading to an improved understanding of insulin resistance.
Insulin icodec is a novel insulin analog in clinical development with a terminal elimination half-life of ~196 hours, designed to cover a full week’s basal insulin requirements with a single subcutaneous injection. The insulin molecule was modified to achieve an albumin-bound circulating depot of icodec which acts just as human insulin (HI) but is more slowly cleared.Addition of a C20 fatty diacid containing side chain at B29K via a hydrophilic linker imparts strong but reversible albumin binding. Three amino acid substitutions (A14E, B16H and B25H) ensure reduced enzymatic degradation and contribute to attenuating insulin receptor (IR) binding and clearance, further prolonging the half-life. In vitro studies demonstrated that icodec is a specific and full agonist of human IR, that displays the same dose-dependent mode of action as HI, exemplified by its ability to phosphorylate the IR and stimulate intracellular signaling pathways (phospho-AKT and -ERK). Functional assays have demonstrated that icodec elicits the same pattern of metabolic effects as HI. The affinity of icodec for the IGF-1 receptor was found to be proportionately lower than its binding to the IR. In vitro mitogenic effect of icodec in primary human mammary cells (HMEC), as well as in mammary and colon carcinoma cells (MCF-7 and COLO 205) was found to be low relative to that of HI. Conclusion: icodec is a new insulin analog designed to achieve a slowly cleared, albumin-bound circulating depot which results in a long half-life suited for once weekly injections, covering the basal insulin requirements for a full week.
Healthy adipose tissue has a remarkable ability to adapt to energy intake through contraction and expansion. A large body of evidence pin-points the ability expand adipose tissue as vital to maintain whole body lipid homeostasis and insulin sensitivity in humans as well as in mice. Increases in fat mass can be driven by expansion of existing adipocytes or by recruitment of new adipocytes from adipose tissue pre-courser cells or preadipocytes (Adipogenesis). Failure to expand adipose tissues as well as appearance of dysfunctional large adipocytes both result in local and systemic insulin resistance. Conversely, strategies to preserve adipocyte function during tissue expansion through enhanced adipogenesis could preserve insulin function in the face of obesity or high caloric intake. In order to study the hormonal control of adipose tissue expansion we have examined the ability of insulin and IGF-1 to induce adipogenesis in isolated rodent primary SVF cells. We find that both insulin and IGF-1 dose dependently induce lipid droplet accumulation and expression of adipocyte genes in primary cells isolated from mouse epididymal and subcutaneous adipose depots. Surprisingly, adipogenesis in mouse SVF cells is a very insulin sensitive process and more specifically we identify CD45neg/Ly6c low SVF cells isolated from epididymal fat pads as extremely sensitive to insulin induced adipogenesis. These results indicate that insulin signaling directly in preadipocytes could drive adipocyte differentiation in vivo e.g., during high insulin levels in serum in response to prolonged periods of high caloric intake or lipid induced insulin resistance. Disclosure T.Å. Pedersen: Employee; Spouse/Partner; Cook Medical. Employee; Self; Novo Nordisk A/S. J. Peics: Employee; Self; Novo Nordisk A/S. G.S. Olsen: Employee; Self; Novo Nordisk A/S.
The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFRβ+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, "FIPs") are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFRβ+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.
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