Browning of White Adipose Tissue Uncouples Glucose Uptake from Insulin Signaling

<div><p>Presence of thermogenically active adipose tissue in adult humans has been inversely associated with obesity and type 2 diabetes. While it had been shown that insulin is crucial for the development of classical brown fat, its role in development and function of inducible brown-in-white (brite) adipose tissue is less clear. Here we show that insulin deficiency impaired differentiation of brite adipocytes. However, adrenergic stimulation almost fully induced the thermogenic program under these settings. Although brite differentiation of adipocytes as well as browning of white adipose tissue entailed substantially elevated glucose uptake by adipose tissue, the capacity of insulin to stimulate glucose uptake surprisingly was not higher in the brite state. Notably, in line with the insulin-independent stimulation of glucose uptake, our data revealed that brite recruitment results in induction of solute carrier family 2 (GLUT-1) expression in adipocytes and inguinal WAT. These results for the first time demonstrate that insulin signaling is neither essential for brite recruitment, nor is it improved in cells or tissues upon browning.</p></div

Lack of insulin impairs differentiation but not browning capacity of primary pre-adipocytes.

<p>(<b>A</b>) Heatmap showing differential mRNA expression between confluent primary inguinal white adipose tissue (iWAT) precursor cells differentiated for 24 h with white (EtOH treated) or brite (cPGI₂ treated) differentiation cocktail and between absence or presence of insulin (Ins) in the medium. Higher and lower expression is displayed in red and blue, respectively. (n = 3). (<b>B</b>) mRNA expression of UCP-1 and CIDEA or (<b>C</b>) FABP4 and RETN in primary iWAT precursor cells differentiated into white (EtOH treated) or brite (cPGI₂ treated) adipocytes for 8 days with insulin present in the differentiation medium for the indicated timepoints (n = 3). All values in bar graphs are expressed as means ± SEM, #p<0.05, ##p<0.01, ###p<0.001 white (EtOH treated) vs. brite (cPGI₂ treated) cells, *p<0.05, **p<0.01, ***p<0.001 normal conditions vs. insulin deprived conditions.</p

Brite adipose cells and tissues exhibit elevated glucose uptake independent of insulin stimulation, thereby enhancing glucose clearance from blood.

<p>(<b>A, B</b>) ³H-2-deoxy-D-glucose (3H-2DOG) uptake by primary inguinal white adipose tissue (iWAT) precursor cells (<b>A</b>) absolute or (<b>B</b>) relative to unstimulated basal uptake. Cells were differentiated into white (EtOH treated) or brite (cPGI₂ treated) adipocytes for 8 days and stimulated with different doses of Insulin for 20 minutes. (<b>C, D</b>) Intraperitoneal insulin (Ins) tolerance test (0.5 U/kg body weight insulin) of mice treated with CL316,243 (CL, 1 µg/g/day) or NaCl via s.c. implanted osmotic pumps for 10 days. (<b>C</b>) Absolute blood glucose levels and (<b>D</b>) levels relative to non-insulin-stimulated are shown. (<b>E, F</b>) 3H-2DOG uptake rate into inguinal or abdominal white (iWAT, aWAT) or brown (BAT) adipose tissue and heart of the same mice as in B, C. (<b>E</b>) Absolute uptake and (<b>F</b>) uptake relative to non-insulin-stimulated conditions is shown. Uptake rates were measured 45 minutes after intraperitoneal injection of insulin or vehicle. All values are expressed as means ± SEM, n = 3–6, #p<0.05, ##p<0.01, ###p<0.001 white vs. brite, *p<0.05, **p<0.01, ***p<0.001 no insulin vs. insulin stimulated.</p

MYB deletion affects KSL cell number and differentiation.

<p>(A) Flow cytometric analysis of bone marrow from <i>Myb</i>^<i>F/F</i>:<i>MxCre</i> mutant mice and <i>Myb</i>^<i>+/+</i>:<i>MxCre</i> control animals, 24 or 48 hours following in vivo induction of the <i>Myb</i>^<i>F/F</i> allele deletion by p(I:C) injection. The representative two-dimensional plots show the gating on lineage negative cells and the typical KIT⁺/SCA-1⁺/LIN^- (KSL) staining. Histograms represent the percentage of KSL cells within the total bone marrow population, with numbers presented as mean ± SEM, determined from 3 independent experiments (***, p<0.0001). (B) Control and <i>Myb</i>-deleted KSL were sorted and seeded in methylcellulose. Colony numbers and size were assessed after 6 days. The right histograms represent total colony numbers and the relative proportions of colonies based on size respectively. Pictures of the counted colonies are presented in the left panels. (C) Sorted KSL from control and <i>Myb</i>-deleted animals 24 hours post injection of poly(I:C) were cultured into liquid medium containing SCF, FL and TPO. After culturing for 2 days, the cells were re-stained and analysed by flow cytometry for expression of CD11 and CD41 (left panel). Cells from the <i>Myb</i>^<i>F/F</i>:<i>MxCre</i> bone marrow were sorted on the basis of CD11 and CD41. The sorted cells were spun onto glass slides and stained with Diff-Quick (upper right panel). The images show representative cells demonstrating monocytic (CD11b⁺) and megakaryocytic (CD11b⁺CD41⁺ and CD11b^-CD41⁺) morphologies.</p

<i>Flt3</i> intronic element hypersensitivity coincides with FLT3 expression in primary haematopoietic stem/progenitor cells.

<p>Upper panel gives a schematic representation of the murine <i>Flt3</i> promoter and first intron (filled box indicates exon1) and shows the level of cross-species sequence conservation and the position of repeated sequences. The underlined regions A B and C indicate the general location of the regions of hypersensitivity to DNAseI digestion in HPC7 cells. Lower panel plots show the assessment of DNaseI digestion sensitivity in the HPC7 cell line and in primary KSL FIT3⁺, KSL FITt3^- cells, CMP (LIN^-/KIT⁺/SCA-1^-/ CD34^-/CD16/32⁺), GMP (LIN^-/KIT⁺/SCA-1^-/CD34⁺/CD16/32⁺) and MEP (LIN^-/KIT⁺/SCA-1^-/CD34^-/CD16/32^-). For primary cells, the analysis are restricted to the digestion sites observed in HCP7 at -1.45 kb (HS-A), 0.15kb (HS-B) and +7.5kb (HS-C) from the murine <i>Flt3</i> initiation codon.</p

<i>Flt3</i> regulatory regions enable the recruitment of a combination of master regulators of haematopoiesis.

<p>Detection of in vivo transcription factor binding at the sites of hypersensitivity to nuclease digestion was achieved by ChIP. Relative enrichments from X-ChIP material were determined against the IgG control material by Q-PCR at the location of the hypersensitive regions and normalised against two internal control regions (located at -3.5kb and -0.27kb from the ATG). Error bars represent the standard error of the mean. All plots are representative of a minimum of three independent experiments.</p

<i>Myb and Cebpa</i> knockdown in primary KIT⁺ enriched bone marrow cells.

<p>(A) Two-dimensional flow cytometry dot plot showing the analysis of the KSL compartment of a KIT⁺ enriched population transfected with siRNA control or undergoing siRNA-mediated silencing of <i>Myb</i> or <i>Cebpa</i> for 20 hours. (B) Grouping four independent experiments, boxplots depict the variations in percentage of FLT3⁺ cells within the KSL populations. (C) Quantitative PCR analysis of <i>Myb</i> and <i>Cebpa</i> RNA expression in sorted transfected KSL cells 20 hours post transfection. Expression is normalised to <i>β</i>_<i>2</i><i>-microglobulin</i> and standardised to the control samples. Error bars represent the standard error of the mean. Plots are representative of 4 independent experiments. Numbers are plotted as mean ± SEM (**, p<0.001 and *, p<0.05).</p

Induced <i>Myb</i> ablation results in FLT3 down-regulation in a population highly enriched in LMPP.

<p>(A) Representative histogram and two-dimensional flow cytometric plot analysis of cells in the KSL compartment of the bone marrow of the cKO and control mice 24 hours post poly(I:C) injection. (B) The cells were further analysed for surface expression of FIT3 and the proportion of different KSL sub-fractions were assessed based on the surface expression of VCAM-1 and CD62L. Regrouping 7 experiments, the right panel shows their relative contribution in percentage of the gated population, indicated as plot header. (C) Serial gating defined a population highly enriched in LMPP (KSL/CD62L⁺/VCAM^-) for which the percentage of FIT3^hi cells was measured. (D) Quantitative PCR analysis of <i>Myb</i>, <i>Flt3</i>, <i>Il7r</i>α, <i>Dntt and Notch1</i> RNA expression in KSL cells isolated from the bone marrow of poly(I:C)-induced <i>Myb</i> cKOs and litter-mate controls 24 hours post injection. Expression is normalised to <i>gapdh</i> and standardised the <i>Myb</i>^<i>+/+</i>:MxCre control samples, set as 1. Error bars represent the standard error of the mean. Plots are representative of 7 independent experiments. Numbers are plotted as mean ± SEM (***, p<0.0001 and **, p<0.01).</p