Fibroblast growth factor 8b induces uncoupling protein 1 expression in epididymal white preadipocytes

Figures

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  Figure 1. Paracrine fibroblast growth factor 8b enhances Ucp1 mRNA und protein expression in white adipocytes. (A) Screening of all paracrine FGFs in immortalized epididymal white adipocytes. FGF8b and FGF17 led to increased expression of Ucp1 mRNA expression, n = 3. (B) Peptide products of four different splice isoforms of the Fgf8 transcript were compared. Fgf8b and Fgf8f were able to increase Ucp1 transcript abundance, Fgf8a and e were not, n = 3. (C) Fgf8b dose dependently increased Ucp1 transcript abundance, n = 5. (D) The Fgf8b induced increase in transcript abundance was accompanied by increased Ucp1 protein. Depicted is a representative Western Blot and quantified signal intensities, n = 3.

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  Figure 2. Metabolic alterations in Fgf8b treated adipocytes. (A) Adipocytes were treated with 125 ng/ml FGF8b or not (control). Depicted are lactate concentration and medium colour of treated versus untreated cells at the end of a six day differentiation course and 48 hours after a medium change. (B) Relative glucose uptake was measured in untreated (control) or Fgf8b treated (62.5 ng/ml and 125 ng/ml, respectively) adipocytes in response to insulin (ins, 20 nM, 10 min) or isoproterenol (iso, 500 nm, 10 min), n = 3. (C) Transcript abundance of genes associated with glycolysis. Lactate exporters monocarboxylate transporter 1 and 4 (Mct1, Mct4) as well as pyruvate dehydrogenase kinase 1 (Pdk1) and glucose transporter 1 (Glut1) were increased by Fgf8b treatment, while glucose transporter 4 (Glut4) was downregulated. Lactate dehydrogenase (Ldha) remained unchanged, n = 5, *p < 0.05, ***p < 0.001, Holm-Sidak multiple test. (D) Oxygen consumption of differentiated adipocytes treated for 24 hours with 125 ng/ml Fgf8b and measured in initially glucose free medium. Injections are indicated by arrows in this order: 10 mM glucose, 1 µM rotenone plus 5 µM antimycin A (anti), 400 µM monensin plus 2 µM…

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  Figure 3. Gene expression profile of Fgf8b and PPARy agonist treated white adipocytes. White adipocytes were treated with Fgf8b (125 ng/ml) and/or rosiglitazone (20 µM) during differentiation. (A,B) Transcript abundance of genes indicative for brown adipocytes, brite markers, adipogenesis, and FGF signalling (abbreviations below), n = 3–6. Fgf8b treatment leads to significantly different abundance of all transcripts analysed except Tbx1, Slc21a1 and Fgfrs 3 and 4 both in the absence and in the presence of rosiglitazone and except Fgf21 in the presence of rosiglitazone (Holm-Sidak multiple testing). (C) Representative Western Blot detecting terminal adipogenic differentiation markers HSL and perilipin and mitochondrial marker COX4. (D) Quantified, normalized band intensity of proteins detected in (C), n = 3, bars represent mean values ± SD. Stars indicate a significantly different abundance of this transcript as compared to the control. Ucp1, uncoupling protein 1; Cidea, cell death-inducing DNA fragmentation factor alpha-like effector A; Pgc1a, peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; Cs, citrate synthase; Prdm16, PR domain containing 16; Tmem26, Transmembrane…

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  Figure 4. Fgf8b interferes in adipocyte differentiation. (A) The number of mature, lipid-laden adipocytes is reduced at the end of differentiation in a medium containing Fgf8b (125 ng/ml) versus control treated cells (con). Orange colour indicates automatic detection of a lipid droplet by an image analysis algorithm. (B,C) Final lipid droplet number is decreased (B), while size distribution is similar (C) after Fgf8b treatment. (D) Unstimulated (left panel) and isoproterenol-stimulated (iso, right panel) lipolytic rate determined by glycerol release are both decrease after differentiation in the presence of Fgf8b. Bars depict mean values ± SD, n = 3–4. (E) Transcript abundance of brown/brite adipocyte markers UCP1, Cidea and Pgc1a was increased by Fgf8b when normalized to the marker of terminal adipocytes differentiation AP2/Fabp4. Ucp1, uncoupling protein 1; Cidea, cell death-inducing DNA fragmentation factor alpha-like effector A; Pgc1a, peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; Cs, citrate synthase; bars are mean values ± SD, n = 3. Stars indicate a significant difference (p < 0.05, Sidak’s multiple comparisons test).

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  Figure 5. Fgf8b reprograms proliferating preadipocytes. (A) White adipocytes were treated with Ffgf8b (125 ng/ml) for 48 hours either during proliferation, induction, early or late differentiation and compared to treatment during the entire differentiation. Black squares indicate days of Fgf8b treatment. Treatment during the entire differentiation period led to strongest Ucp1 induction. During proliferation and early differentiation cells were most susceptible for Fgf8b treatment, while inclusion of the induction phase into the treatment abrogated its effect. Bars are mean values ± SD, n = 5. (B) Time course of indicated mRNA expression following a 48 hours Fgf8b treatment pulse on proliferating preadipocytes. Points are mean values ± SD, n = 3.

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  Figure 6. In vivo application of Fgf8b into epididymal white adipose. Implantation of pellets releasing 100 ng FGF8b per day and placebo pellets into the epididymal white adipose tissue results in a brownish aureole around the implantation site of FGF8b, but not of placebo pellets after three weeks of treatment. (A) Photographs of epididymal depots from three independent experiments of 6 mice each. (B) Visual browning score on a scale of 1 (least) to 10 (most) browning around a pellet was assessed by six blinded observers. Each dot represents the mean value for one specific depot, n = 18, p = 0.0068 (t test). (C) Visual browning assessment by keywords describing multilocular adipocyte occurrence and UCP1 immunostaining on histological sections was assessed by six blinded observers. Data is provided as percentage of 30 sections per depots that fit the keyword description. Each dot represents the mean value for one specific depot; no significant differences (Holm-Sidak multiple test). (E) Indirect calorimetry and injection of norepinephrine did not reveal a difference in maximal thermogenic capacity in Fgf8b versus placebo treated mice. (D) Transcript abundance of brite adipocyte markers…