Human 'brite/beige' adipocytes develop from capillary networks, and their implantation improves metabolic homeostasis in mice - PubMed (original) (raw)

doi: 10.1038/nm.4031. Epub 2016 Jan 25.

Jamie Kady 1 3, Minwoo Nam 2 4, Raziel Rojas-Rodriguez 1 2, Aaron Berkenwald 5, Jong Hun Kim 1, Hye-Lim Noh 1, Jason K Kim 1, Marcus P Cooper 4, Timothy Fitzgibbons 4, Michael A Brehm 1 3, Silvia Corvera 1

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Human 'brite/beige' adipocytes develop from capillary networks, and their implantation improves metabolic homeostasis in mice

So Yun Min et al. Nat Med. 2016 Mar.

Abstract

Uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots, in adipocytes that are termed 'brite' (brown-in-white) or 'beige'. In humans, the presence of brite or beige (brite/beige) adipocytes is correlated with a lean, metabolically healthy phenotype, but whether a causal relationship exists is not clear. Here we report that human brite/beige adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform in response to adenylate cyclase activation from being UCP1 negative to being UCP1 positive, which is a defining feature of the beige/brite phenotype, while displaying uncoupled respiration. When implanted into normal chow-fed, or into high-fat diet (HFD)-fed, glucose-intolerant NOD-scid IL2rg(null) (NSG) mice, brite/beige adipocytes activated in vitro enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Pro-angiogenic conditions therefore drive the proliferation of human beige/brite adipocyte progenitors, and activated beige/brite adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism.

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Figures

Figure 1

Figure 1

Proliferation of human adipogenic precursors requires angiogenesis. All RT-PCR results are expressed as the fold over the minimum detectable value in the series, and represent the means and range of 2 technical replicates, of representative experiments that have been replicated a minimum of three times with cells from separate individuals. In cases where error bars are not apparent, replicates were too close to result in a visible range. (a) Explant growth in DMEM (top), DMEM + angiogenic growth factors (middle) or EGM-2 MV + angiogenic growth factors (bottom). Scale bars, 1 cm (left panels) and 200 µm (right panels). (b) Growth area (arbitrary units) from explants grown for 11 days in the absence (− GF) or presence (+ GF) of angiogenic growth factors. Plotted are means and s.e.m, from 6 explants per condition from 2 different individuals (n = 12). Statistical significance was calculated using the Mann Whitney test *P < 0.05, **P < 0.01. (c) High resolution representative images of explants (n = 27 images per time point) grown for 5 (left), 12 (middle) or 18 (right) days, exposed to MDI at day 12. Arrowheads indicate elongated cells forming the sprouts, and arrows indicate lipid droplets. Scale bars, 200 µm and 50 µm (inset). (d) RT-PCR for genes indicated in non-differentiated (− MDI) or differentiated (+ MDI) explants 7 days after induction of differentiation. (e) A representative field (n = 30 images from independent wells) of capillary network cells at days 0 (top left), 6 (top right), 12 (bottom left) and 18 (bottom right) after induction of differentiation; arrows indicate growth of lipid droplets within a single adipocyte. Scale bars, 50 µm. (f) RT-PCR for genes indicated in non-differentiated (− MDI) or differentiated (+ MDI) cells seven days after induction of differentiation. (g) Example images (n = 1, though 35 independent clones were examined in total) of three adipogenic and one non-adipogenic clones identified by lipid droplet content, Scale bars, 50 µm. (h) Human adiponectin concentration detected in culture medium from three non-adipogenic and three adipogenic clones.

Figure 2

Figure 2

Induction of human ‘brite/beige’ phenotype in adipocytes derived from capillary networks. (a) Experimental scheme (top) and RT-PCR of indicated genes (bottom), expressed as the fold over the minimum detectable value in the series. 0.25d refers to 6h. Shown are means and range of 2 technical replicates. In cases where error bars are not apparent, replicates were too close to result in a visible range. These results have been replicated with cells from 3 separate individuals. (b) Experimental scheme (top) and UCP1 mRNA expression (bottom left) in cells exposed to isoproterenol (Iso) or forskolin (Fsk) as indicated in the scheme, or in response to different concentrations of adrenergic agonists (bottom right). Plotted are means and s.e.m. of 3 biological replicates (n = 3); statistical difference relative to CL316,243 was determined at each concentration using Student t-tests corrected for multiple comparisons using the Holm-Sidak method *P < 0.05, **P < 0.01, ****P < 0.0001. (c) Representative images (n = 30 images (10 fields each of cells from 3 separate individuals)) of differentiated cells exposed to Fsk for 0 (left panel), 3 (middle panel) and 7 (right panel) days showing UCP1 (green), lipid droplets (red), and nuclei (blue). Scale bars, 200 µm. Plotted are means and s.e.m. of UCP1 staining intensity from 2 fields per coverslip, of cells from 3 different individuals (n = 6). Statistical significance was assessed using the Mann-Whitney test versus non-differentiated cells, **P < 0.01 ***P < 0.001. (d) Mitochondrial Hsp70 (left panel) and UCP1 (right panel) in adipocytes exposed to Fsk for 1 week. Arrowheads indicate linear mitochondrial structures in cell devoid of UCP1. Arrows indicate rounded mitochondrial structures containing both UCP1 and Hsp70. Scale bars, 20 µm. (e) Oxygen consumption by adipocytes exposed to vehicle or Fsk for 1 week. Plotted are the means and s.e.m. of 4 experiments assayed in triplicate (n = 4). (f) Summary data for oxygen consumption parameters, calculated as described in Online methods, derived from the means and s.e.m. of cells from 4 separate individuals assayed in triplicate (n = 4). Statistical significance was assessed using 2-tailed unpaired Student t-tests: *P < 0.05, **P < 0.005. (g) Oxygen consumption rate by digitonin-permeabilized adipocytes exposed to vehicle or Fsk for 1 week. Plotted are the means and s.e.m. of four experiments (n=4). Statistical significance assessed using 2-tailed unpaired Student t-tests *P < 0.05. ns, not significant. (h) Lipid droplets (green), mitochondria (red) and nuclei (blue) in adipocytes without (left) or with (right) exposure to Fsk for 14 days. Scale bars, 20 µm. (i) RT-PCR of indicated genes, expressed as the fold relative to t = 0. Shown are means and range of 2 technical replicates. These results have been replicated a minimum of 3 times with cells from separate individuals.

Figure 3

Figure 3

Characteristics and metabolic effects of human ‘brite/beige’ adipocytes derived from capillary networks. (a) RT-PCR for indicated genes in non-differentiated (C), differentiated (MDI), and forskolin-treated adipocytes (FSK), and perivascular adipose tissue (PV). Values represent fold difference over the lowest detectable value in the series for the respective probe set. Plotted are means and s.e.m. from 2 technical replicates of samples from 3 (cells) or 4 (PV) individuals. (b) Phase image of cells for implantation (top left),; suspension of cells (bottom left); dorsal area of mouse injected with Matrigel alone (top middle); collected remnants of the hydrogel (top right); dorsal area of mouse injected with cells (bottom middle); excised adipose structure (bottom right, scale bar) displaying vascularization (arrows). Scale bars, 100 µm (top left) and 1 cm. (c) Representative H&E staining of implant (n = 40 sections with 5 sections from each of 8 independent implants) ; dotted red line separating implant from surrounding mouse adipose tissue, blood vessels within the implant (arrow). Scale bars, 300 µm and 75 µm (inset). Similar results were seen in 8 additional implants. (d) Human adiponectin in sera from 6 mice implanted with either Matrigel alone or with cells in Matrigel, in serum from a control C57BL6 mouse, and in a 1:500 dilution of normal human serum. (e) RT-PCR for mouse Ucp1, human UCP1 and human PLIN1 in mouse adipose depots (mBAT, mIng and mEpi), human capillary-derived cells (C, MDI and FSK), and adipose structures from three mice, excised 7 weeks following implantation. Values represent the fold difference relative to the lowest detectable value in the series for each respective probe set, a value of 0 was given to non-detectable values. Error bars represent range of 2 technical replicates. (f) Fasting glucose levels, (g) Glucose tolerance curves (left) and areas under the curve (A.U.C) of the glucose excursion after 16h fast in male mice implanted with Matrigel (n = 8) or Matrigel+cells (n = 10). (h) Glucose tolerance curves after an 8 h fast in mice housed at 30°C for 2 weeks, fed a normal (n = 5) or a 60% HFD (n = 5). (i) Glucose tolerance curves after 11 weeks of 60% HFD feeding and an 8 h fast in mice housed at 30°C, implanted with either Matrigel (n = 5) or Matrigel+cells (n = 3) after 2 weeks of a HFD. Statistical analysis was performed using 2-tailed unpaired Student-t tests at each time point of glucose tolerance curves, and Mann-Whitney test between groups for fasting blood glucose and area under the curves. *P < 0.05, **P < 0.01, ***P < 0.0001. (j) Livers from mice used in i immediately following sacrifice at 11 weeks. Scale bars, 1 cm.

Figure 4

Figure 4

Mechanism for metabolic effects of human ‘brite/beige’ adipocytes. (a) Glucose turnover (µmol/Kg/min) normalized to serum human adiponectin (ng/ml) in male mice implanted with non-stimulated (MDI, n = 5) or forskolin-stimulated (FSK, n = 5) adipocytes, seven weeks prior to hyperinsulinemic-euglycemic glucose clamps. Statistical analysis was performed using the Mann-Whitney test, *P < 0.05. (b) Relationship between serum human adiponectin levels and glucose turnover in implanted mice (n = 10). Linear regression P value = 0.0027, R2 = 0.601. (c) RT-PCR for human UCP1 in implanted cell structures from mice studied in a. Statistical analysis was done using unpaired 2-tailed Student t-test, *P < 0.05. (d) Temperature recordings from subcutaneous iButtons in mice implanted with Matrigel or cells 7 weeks following implantation. Arrow represents time at which mice were placed at 5°C. (e) Glucose uptake into epididymal fat (WAT), interscapular brown fat (BAT) and implanted cell structures from mice studied in b, n = 10. Statistical analysis was performed using unpaired 2-tailed Student-t tests *P < 0.05, ***P < 0.0001. (f) Relationship between glucose uptake into implanted cell structures and glucose turnover (n = 10). (g) Volcano plot of differential gene expression in adipocytes without or with forskolin treatment for 7 days, indicating genes of interest. (h) RT-PCR of PCSK1, PENK and IL-33 expression in non-differentiated (C), differentiated (MDI), and forskolin-treated adipocytes (FSK), and perivascular adipose tissue (PV). Values represent fold difference over the lowest detectable value in the series for the respective probe set. Plotted are means and s.e.m. from 2 technical replicates of samples from 3 (cells) or 4 (PV) individuals.

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