Exposure to cold alters glucose and lipid metabolism of white and brown adipose tissue via activation of β-adrenergic receptor (ADRB). Fibroblast growth factor 21 (FGF21) has been shown to be locally released from adipose tissue upon activation of ADRBs and FGF21 increases glucose uptake in adipocytes. Therefore, FGF21 may play an autocrine role in inducing glucose uptake after β-adrenergic stimulation. To determine the putative autocrine role of FGF21, we stimulated three different types of adipocytes in vitro with Isoprenaline (Iso), an ADRB agonist, in the presence or absence of the FGF receptor (FGFR) inhibitor PD 173074. The three cell lines represent white (3T3-L1), beige (ME3) and brown (WT-1) adipocyte phenotypes, respectively. All three cells systems expressed β-klotho (KLB) and FGFR1 after differentiation and treatment with recombinant FGF21 increased glucose uptake in 3T3-L1 and WT-1 adipocytes, while no significant effect was observed in ME3. Oppositely, all three cell lines responded to Iso treatment and an increase in glucose uptake and lipolysis were observed. Interestingly, in response to the Iso treatment only the WT-1 adipocytes showed an increase in FGF21 in the medium. This was consistent with the observation that PD 173074 decreased Iso-induced glucose uptake in the WT-1 adipocytes. This suggests that FGF21 plays an autocrine role and increases glucose uptake after β-adrenergic stimulation of cultured brown WT-1 adipocytes.

Introduction

Fibroblast growth factor 21 (FGF21) has shown a great therapeutic potential in regulating body weight, insulin sensitivity and plasma lipids in obese subjects with type 2 diabetes as reviewed by Sonoda et al. [1]. FGF21 belongs to the FGF19 subfamily of endocrine FGFs [2] and is highly expressed in and released from the liver in response to increases in free fatty acids (FFA), high glucose and low protein [3,4]. FGF21 binds to FGF receptor (FGFR) 1c and 3c, but only the in presence of the co-receptor β-klotho (KLB) [5]. FGFR1c and KLB are highly expressed in adipose tissue [6] and in specific regions of the central nervous system (CNS) [7]. In adipocytes FGF21 increases glucose uptake through an induction of glucose transporter 1 (GLUT1) encoded by the Slc2a1 gene [8–10] and FGF21 normalizes blood glucose in animal models of type 2 diabetes [8,11,12]. FGF21 has furthermore been shown to induce thermogenic gene expression in inguinal white adipose tissue (iWAT) and brown adipose tissue (BAT) [13,14] and the increase in glucose uptake has been shown to be dependent on uncoupling protein 1 (UCP1) [15]. UCP1 is highly expressed in BAT, and the abundance and activity of UCP1 contributes to the thermogenic and energy combusting capacity of BAT [16–19]. In contrast, the WAT is specialized in storing energy in the form of triacylglycerol [19,20]. However, in some WAT depots like the iWAT, cold exposure can promote the appearance of brown-like adipocytes. In these brown-like adipocytes thermogenic genes like Ucp1 are induced and these fat depots are called beige [21–23]. The increase in Ucp1 expression and the beiging of WAT have been suggested to be involved in both the anti-diabetic [15] as well as the anti-obesity effect of FGF21 [24].

Cold exposure stimulates β-adrenergic receptors (ADRB1-3) at the adipocyte cell surface via release of noradrenalin [25,26] from the nerve endings. In adipocytes the primarily subtype is ADRB3 [27,28], and in rodents stimulation with an ADRB3 agonist causes an increase in glucose uptake by increasing GLUT1 levels in WAT and BAT [28,29]. Furthermore, β-adrenergic stimulation induces the expression of thermogenic genes, like Fgf21 and Ucp1, in adipose tissue [14,30–32] and reduces expression of the gene Ob coding for the adipocyte-selective hormone, Leptin [33].

In response to β-adrenergic stimuli FGF21 is released from WAT and BAT depots [14,30]. Therefore, FGF21 has been suggested to play an autocrine role in the response to cold exposure as both FGF21 and β-adrenergic stimulation induce metabolic changes (e.g. glucose uptake) in the adipose tissue [14,30,34]. To investigate if FGF21 plays an autocrine role upon β-adrenergic stimulation in cultured adipocytes, the effect of Isoprenaline (Iso)-induced glucose uptake and lactate production was studied in the presence of an FGFR inhibitor (PD 173074) [35,36] in white, beige and brown adipocytes in vitro. The advance of the in vitro systems is that the potential role of CNS in FGF21-mediated glucose uptake [37] is excluded. Our studies showed that FGF21 release from brown mouse WT-1 adipocytes contributes to the increase in glucose uptake induced by β-adrenergic stimulation.

Materials and methods

Cell culture

3T3-L1 (American Type Culture Collection) and WT-1 adipocytes (generous gift from C. Ronald Kahn, Joslin Diabetes Center, Boston [38]) were propagated in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, 31966-021) with 10% fetal bovine serum (FBS) (Gibco, 16000-044) and 100 U/ml Penicillin-Streptomycin (Pen-Strep) (Gibco, 15140). ME3 adipocytes [39] were propagated in AmnioMax Basal Medium (Gibco, 17001-074) supplemented with 14% AmnioMax Supplement (Gibco, 12556-023). 3T3-L1, ME3, and WT-1 adipocytes were differentiated after the same procedure. The differentiation procedure was initiated with one-day post-confluent cells (noted as day 0) with DMEM containing 10% FBS, 100 U/ml Pen-Strep, 1 µM Troglitazone (Sigma–Aldrich, T2573), 0.5 mM 3-Isobutyl-1-methyl-xanthine (Sigma–Aldrich, I5879), 0.25 µM Dexamethasone (Sigma–Aldrich, D4902), and 100 nM Human Insulin (Novo Nordisk, Denmark). At day 3, the medium was changed, and DMEM with 10% FBS, 100 U/ml Pen-Strep, 1 µM Troglitazone, and 100 nM Human Insulin were added. The cells were refed at day 6 with DMEM containing 10% FBS, 100 U/ml Pen-Strep, and 100 nM Human Insulin. At day 8, the medium was changed to DMEM only supplemented with 10% FBS and 100 U/ml Pen-Strep. The cells were ready to be used for experiments at day 9.

Supernatant analysis

The cells were stimulated for ∼24 h with one or two of the following substances; 0.03–100 nM FGF21 (Novo Nordisk, Denmark), and 10-10,000 nM Iso (Acros Organics, 6700-39-6). The medium was collected at the end of the stimulation to determine glycerol, lactate and FGF21. The amount of free glycerol was determined calorimetrically with the Free Glycerol Assay Kit (Abcam, ab65337) (lower limit of quantification (LLOQ): 1 µM). Lactate in the supernatant was determined calorimetrically with the L-Lactate Assay Kit (Abcam, ab65331) (LLOQ:0.02 mM). FGF21 in the medium was measured with the use of the FGF21 Mouse/Rat ELISA (Biovendor, RD29118200R LLOQ: 18.4 pg/ml). For the three types of measurements, the absorbance was measured with the Spectramax Plus (Molecular Devices, California, U.S.A.).

qPCR

After stimulation the cells were lysed using a lysis buffer (Qiagen, 74106) supplemented with 1% 2-mercapto-ethanol (Gibco, 31350-010). The mRNA was purified using Qiacube (Qiagen, Hilden, Germany) and the RNeasy Mini Kit (Qiagen, 74106). The mRNA levels and quality were determined using a Nanodrop 8000 Spectrophotometer (Thermo Scientific, Massachusetts, U.S.A.). mRNA was reverse transcribed into cDNA at either a Surecycler 8800 (Agilent Technologies, California, U.S.A.) or PTC-200 DNA-Engine (MJ Research, Quebec, Canada) by using the iScript cDNA Synthesis Kit (Bio-Rad, 170-8891). Real time qPCR was performed at a ViaaA7 (Applied Biosystems, California, U.S.A.). In each well, primers (Table 1), cDNA (25 ng), RNase-free water (Qiagen, 74106), and TaqMan Fast Advanced Master Mix (Applied Biosystems, 4444 557) were added. For data processing the threshold cycle (CT) values were recalculated to 2−CT and normalized to the internal housekeeping genes, either Nono or Ppib, which were not regulated in response to treatment.

Table 1
Primers (Applied Biosystems) used for real time qPCR
GenBank accession numberGene symbolGene description
mm02601819_g1 Adrb3 β-adrenergic receptor 3 
mm00840165_g1 Fgf21 Fibroblast growth factor 21 
mm00438930_m1 Fgfr1 Fibroblast growth factor receptor 1 
mm00433294_m1 Fgfr3 Fibroblast growth factor receptor 3 
mm00434759_m1 Ob Leptin 
mm00473122_m1 Klotho beta Beta-klotho (co-receptor) 
mm00834875_g1 Nono Internal control 
mm00478295_m1 Ppib Internal control 
mm00441480_m1 Slc2a1 Glucose transporter 1 
mm01244861_m1 Ucp1 Uncoupling protein 1 
GenBank accession numberGene symbolGene description
mm02601819_g1 Adrb3 β-adrenergic receptor 3 
mm00840165_g1 Fgf21 Fibroblast growth factor 21 
mm00438930_m1 Fgfr1 Fibroblast growth factor receptor 1 
mm00433294_m1 Fgfr3 Fibroblast growth factor receptor 3 
mm00434759_m1 Ob Leptin 
mm00473122_m1 Klotho beta Beta-klotho (co-receptor) 
mm00834875_g1 Nono Internal control 
mm00478295_m1 Ppib Internal control 
mm00441480_m1 Slc2a1 Glucose transporter 1 
mm01244861_m1 Ucp1 Uncoupling protein 1 

Glucose uptake

Adipocytes were stimulated with 0.03–100 nM FGF21 and/or 1–3000 nM Iso for 24 h in the presence or absence of 10–1000 nM PD 173074 (Tocris Bioscience, 21580-11-7). Hereafter, the cells were washed twice with warm assay buffer (Dulbecco's phosphate buffered saline (DPBS) (Gibco, 14040-091) with 1 mM MgCl2 (Sigma–Aldrich, M2670), 2 mM KCl (Merch, K37722736), 15 mM HEPES (Gibco, 15630-056), and 0.1% human serum albumin (Sigma–Aldrich, A1887)). 250 µl/well warm assay buffer containing 1 µCi [3H]-2-Deoxy-d-glucose (PerkinElmer, NET328001MC), was added, and the cells incubated for one hour at 37°C and the assay was stopped by adding ice cold assay buffer and subsequently the cells were washed twice. To determine the glucose uptake the cells were lysed with 200 µl 1% Triton-X 100 (Sigma–Aldrich, T9284) and 100 µl lysate was transferred to an OptiPlate and 200 µl Microscint 40 (PerkinElmer, 6013641) were added and the plate was left for half an hour. The radioactivity in each well was thereafter measured using a TopCount NXT (PerkinElmer, Massachusetts, U.S.A.).

Statistics

GraphPad Prism 8 was used for statistical analysis, and it was assumed the data were normal distributed and had equal variance. The data are presented as mean ± standard error of mean (SEM) or mean ± standard error. Unpaired or paired two-tailed t-test or two-way ANOVA were used to test for significance. Statistical difference is denoted as *P < 0.05: **P < 0.01, and ***P < 0.001. The EC50 values are stated with the mean (95% confidence interval, asymmetrical), and extra sum-of-squares F-test was used to test for significant difference (*P < 0.05) between the values for EC50, top, and bottom. Replicates (n) is defined as an individual experiment with new plating and differentiation of cells.

Results

Characterization of the three cell lines

The cell lines 3T3-L1, ME3, and WT-1 were cultured and differentiated as described in Materials and methods. The three cell lines were, based on literature findings, expected to represent white adipocytes (3T3-L1), beige adipocytes (ME3), and brown adipocytes (WT-1). After differentiation, all cell lines expressed Fgfr1 and Klb mRNA (Figure 1A). The lowest expression of Klb mRNA was observed in ME3 adipocytes. The 3T3-L1 and ME3 adipocytes also expressed Fgfr3 mRNA while Fgfr3 mRNA was barely detectable in WT-1 (Figure 1A). Fgfr1 mRNA was significantly higher expressed compared with Fgfr3 mRNA in all three cell lines. All three cell lines expressed similar levels of Adrb3 mRNA (Supplementary Figure S1).

Basal gene expression in differentiated 3T3-L1, ME3 and WT-1 adipocytes.

Figure 1.
Basal gene expression in differentiated 3T3-L1, ME3 and WT-1 adipocytes.

(A) Basal gene expression of FGF21 receptor complex in differentiated 3T3-L1, ME3, and WT-1 adipocytes (B) Basal gene expression of selected genes involved in browning of 3T3-L1, ME3, and WT-1 adipocytes. Data are mean ± SEM. Average of three independent experiments. CT-value of each gene is stated above the bar. Statistical difference is denoted *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired, two tailed t-test).

Figure 1.
Basal gene expression in differentiated 3T3-L1, ME3 and WT-1 adipocytes.

(A) Basal gene expression of FGF21 receptor complex in differentiated 3T3-L1, ME3, and WT-1 adipocytes (B) Basal gene expression of selected genes involved in browning of 3T3-L1, ME3, and WT-1 adipocytes. Data are mean ± SEM. Average of three independent experiments. CT-value of each gene is stated above the bar. Statistical difference is denoted *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired, two tailed t-test).

The mRNA expression of four genes expected to be regulated by browning (e.g. induced by FGF21 or Iso stimulation), Fgf21, Ucp1, Ob, and Slc2a1, were also measured. All four genes were expressed in 3T3-L1, ME3, and WT-1 in the basal state (Figure 1B). The highest gene expression of Slc2a1 mRNA was seen in 3T3-L1 adipocytes, together with the lowest gene expression of Ucp1 mRNA. Oppositely, WT-1 adipocytes had the highest gene expression of Ucp1 mRNA. The mRNA expression of Fgf21 was not significantly different between the three cell lines in the basal state. ME3 adipocytes had a significantly lower expression of Ob mRNA compared with 3T3-L1 and WT-1 adipocytes. In summary, all three cell lines expressed the obligate co-receptor Klb after differentiation as well as the Fgfr1. Moreover, Ucp1 mRNA expression was highest in the WT-1 adipocytes and lowest in the 3T3-L1 adipocytes, in agreement with our hypothesis of WT-1 being a model system for brown adipocytes.

Effect of FGF21

Recombinant human FGF21 dose-dependently increased the glucose uptake in 3T3-L1 and WT-1 adipocytes while the FGF21 response in the ME3 cells was impaired and only a slight response was observed at 100 nM (Figure 2A–C). The EC50 values for the FGF21-mediated glucose uptake were determined to be in the low nM range for 3T3-L1 and WT-1 adipocytes. Significant increases in glucose uptake was observed from FGF21 concentrations of 0.3 nM and 0.1 nM FGF21 in the 3T3-L1 and WT-1 cells, respectively. The EC50 was not determined in ME3 adipocytes as no clear saturation level was obtained (Figure 2B). Moreover, a significant increase (P < 0.05) in lactate in the medium was observed in response to 3 nM FGF21 in the 3T3-L1, while the increase in lactate did not reach significance in the WT-1 adipocytes (P = 0.053) (Figure 2D–F). A non-significant increase in lactate was observed in ME3 adipocytes in agreement with the less pronounced effect of FGF21 on glucose uptake in these cells. FGF21-induced glucose uptake in adipose tissue has been linked to UCP1 up regulation in vivo [15] but FGF21 did not induce a significant increase in Ucp1 mRNA expression in any of the three cell lines however, clear tendencies (from 2–6-fold up-regulation) were observed (Supplementary Figure S2A–C). Oppositely, FGF21 significantly decreased the expression of Ob mRNA in 3T3-L1 adipocytes, while no significant regulation of Ob mRNA was observed in ME3 and WT-1 adipocytes (Supplementary Figure S2D–F).

FGF21-mediated glucose uptake and lactate release in 3T3-L1, ME3, and WT-1 adipocytes.

Figure 2.
FGF21-mediated glucose uptake and lactate release in 3T3-L1, ME3, and WT-1 adipocytes.

FGF21-mediated glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C). Data mean ± SEM. Average of 3–4 independent experiments. Mean EC50 values (95% confidence interval, asymmetrical). Lactate released after 24 h of stimulation with 3 nM of FGF21 in 3T3-L1 adipocytes (D), ME3 adipocytes (E) and in WT-1 adipocytes (F). Data are mean ± SEM. Average of 3–5 independent experiments. Statistical difference is denoted *P < 0.05 (paired, two tailed t-test).

Figure 2.
FGF21-mediated glucose uptake and lactate release in 3T3-L1, ME3, and WT-1 adipocytes.

FGF21-mediated glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C). Data mean ± SEM. Average of 3–4 independent experiments. Mean EC50 values (95% confidence interval, asymmetrical). Lactate released after 24 h of stimulation with 3 nM of FGF21 in 3T3-L1 adipocytes (D), ME3 adipocytes (E) and in WT-1 adipocytes (F). Data are mean ± SEM. Average of 3–5 independent experiments. Statistical difference is denoted *P < 0.05 (paired, two tailed t-test).

The increase in Ucp1 mRNA is however, not necessarily associated with an increase in UCP1 activity [40] and during cold exposure lipolysis is induced through ADRB3 agonism. The resulting increase in fatty acid concentrations stimulates UCP1 activity [41]. The direct effect of FGF21 on lipolysis has however, been quite controversial with contradicting data [42,43]. As free fatty acids (FFA) can be utilized as energy or re-esterified in the adipocytes, glycerol released into the medium was measured to determine the lipolytic activity of FGF21. FGF21 (3 nM) did not stimulate glycerol release in any of the three cell lines (Supplementary Figure S3A–C). The basal lipolytic activity was ∼6-fold higher in the white 3T3-L1 adipocytes (1200 µM glycerol) compared with the more beige and brown cell lines where only 100–200 µM glycerol was released, however, the numbers of differentiated adipocytes between the three cell lines may vary which may impact the result, and normalization to cell number or amount of protein is required to compare amount of glycerol release directly. In summary, FGF21 induced glucose uptake and lactate production in 3T3-L1 and WT-1 adipocytes, while no significant effect was observed in ME3 adipocytes. FGF21 did not induce lipolysis in any of the cell lines.

Effect of isoprenaline

To mimic cold exposure the adipocytes were exposed to Iso, a β-adrenergic agonist [28,44]. Iso dose-dependently increased glucose uptake in brown WT-1 adipocytes, while no significant effect was observed in 3T3-L1 and ME3 adipocytes (Figure 3A–C). The release of lactate in response to Iso was however, significantly increased in all three cell lines (Figure 3D–F). The increase in lactate in the medium represents an increase in glycolysis and interestingly, the effect of Iso on lactate production in the WT-1 cells is more pronounced than the effect on glucose uptake. This indicate that in the more brown-like cell line glucose is more likely to be converted to lactate whereas glucose may be converted to triglycerides in the white adipocytes. To determine if the FGF21- and Iso-induced glucose uptake was additive or synergistic a FGF21 dose response on top of 300 nM Iso was performed. The potency of FGF21 was not changed in response to Iso, but as Iso increased glucose uptake itself, both basal and maximal glucose uptake of the dose response curves of Iso are above the FGF21 dose response curves (Supplementary Figure S4). As previously observed for FGF21, the Iso-mediated glucose uptake was limited in the ME3 cells (Figure 2B and Supplementary Figure S4B). An increase in Slc2a1 mRNA expression was however, observed in ME3 adipocytes in response to 24 h of Iso treatment, while no effect was observed in 3T3-L1 and WT-1 (Supplementary Figure S5), indicating that other glucose transporters may mediate the Iso-induced glucose uptake in 3T3-L1 and WT-1 adipocytes or that the amount of Slc2a1 mRNA decline over time, which has been shown by others [45]. Interestingly, like FGF21 Iso aIso elicited a reciprocal up and down-regulation of Ucp1 (Supplementary Figure S6A–C) and Ob mRNA levels, respectively (Supplementary Figure S6D–F) in all three cellular systems.

Glucose uptake and lactate production in 3T3-L1, ME3, and WT-1 adipocytes after stimulation with Iso.

Figure 3.
Glucose uptake and lactate production in 3T3-L1, ME3, and WT-1 adipocytes after stimulation with Iso.

Glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) in response to increasing concentrations of Iso. Data mean ± SEM. Lactate released after 24 h of stimulation with increasing concentrations of Iso in 3T3-L1 adipocytes (D), ME3 adipocytes (E) and in WT-1 adipocytes (F). Data are mean ± SEM. Average of three independent experiments. Statistical difference in response to non-stimulated cells is denoted *P < 0.05 (paired, two tailed t-test).

Figure 3.
Glucose uptake and lactate production in 3T3-L1, ME3, and WT-1 adipocytes after stimulation with Iso.

Glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) in response to increasing concentrations of Iso. Data mean ± SEM. Lactate released after 24 h of stimulation with increasing concentrations of Iso in 3T3-L1 adipocytes (D), ME3 adipocytes (E) and in WT-1 adipocytes (F). Data are mean ± SEM. Average of three independent experiments. Statistical difference in response to non-stimulated cells is denoted *P < 0.05 (paired, two tailed t-test).

In contrast with FGF21, Iso dose-dependently increased lipolysis in 3T3-L1 and ME3 adipocytes measured as free glycerol in the medium (Figure 4). Due to large variations in the induction of glycerol (from 12- to 73-fold) in response to Iso the lipolytic effect of Iso did not reach statistical significance in the WT-1 adipocytes. The lipolytic effect of Iso was however, more pronounced in the more brown-like phenotype (ME3 and WT-1 adipocytes) where a 30–40-fold increase in glycerol was observed compared with a 2–3-fold increase in the white 3T3-L1 adipocytes.

Lipolysis in 3T3-L1, ME3, and WT-1 adipocytes after stimulation with Iso.

Figure 4.
Lipolysis in 3T3-L1, ME3, and WT-1 adipocytes after stimulation with Iso.

Iso-mediated glycerol release in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C). Free glycerol in the medium in the non-stimulated state is shown above the bars for each cell line. Data are mean ± SEM. Average of three independent experiments. Statistical difference in response to none-stimulated is denoted *P < 0.05, **P < 0.01, and ***P < 0.001. (paired, two tailed t-test).

Figure 4.
Lipolysis in 3T3-L1, ME3, and WT-1 adipocytes after stimulation with Iso.

Iso-mediated glycerol release in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C). Free glycerol in the medium in the non-stimulated state is shown above the bars for each cell line. Data are mean ± SEM. Average of three independent experiments. Statistical difference in response to none-stimulated is denoted *P < 0.05, **P < 0.01, and ***P < 0.001. (paired, two tailed t-test).

The autocrine effect of FGF21

To study if Iso induced FGF21, Fgf21 mRNA and FGF21 protein released into the medium were measured. A 6-fold increase in Fgf21 mRNA was observed in WT-1 adipocytes after 24 h stimulation with 300 nM Iso; however, the increase did not reach statistical significance (Supplementary Figure S7). No effect was observed in 3T3-L1 and ME3 adipocytes. The effect of higher doses of Iso and different incubations times on Fgf21 mRNA expression and release were not tested but a clear dose-response indicated that only the WT-1 adipocytes released FGF21 protein into the medium upon 24 h of Iso stimulation (Figure 5A–C).

Iso-mediated FGF21 release from 3T3-L1, ME3, and WT-1 adipocytes.

Figure 5.
Iso-mediated FGF21 release from 3T3-L1, ME3, and WT-1 adipocytes.

Iso-mediated release of FGF21 into the medium of 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) after 24 h of stimulation. Data are mean ± SEM. Average of 2–4 independent experiments.

Figure 5.
Iso-mediated FGF21 release from 3T3-L1, ME3, and WT-1 adipocytes.

Iso-mediated release of FGF21 into the medium of 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) after 24 h of stimulation. Data are mean ± SEM. Average of 2–4 independent experiments.

To investigate the putative autocrine role of FGF21 on glucose metabolism after β-adrenergic stimulation the three cell lines were stimulated for 24 h with Iso in the absence or presence of an FGFR inhibitor. Three different kinase inhibitors (SU 5402 (10 µM), PD 173074 (200 nM), or U0126(10 µM) were tested and PD 173074 was the only one which inhibited FGF21-mediated glucose uptake (Supplementary Figure S8). PD 173074 inhibits FGFR3 and FGFR1 with IC50 in the nM range and at a concentration of 200 nM PD 173074 inhibited FGF21 dependent glucose uptake in 3T3-L1 and WT-1 adipocytes (Figure 6). The FGFR inhibitor PD 173074 has in several studies been shown to inhibit FGFR kinase activity with high selectivity [46,47], and the IC50 for inhibition of FGFR1 has been determined to be ∼20 nM (MedChem Express). In the WT-1 adipocytes which released FGF21 in response to Iso the Iso-stimulated glucose uptake was decrease in the presence of PD 173074 while no effect was observed in 3T3-L1 and ME3 adipocytes (Figure 7). The limited effect of Iso on glucose uptake in the 3T3-L1 and ME3 is in agreement with the observations in Figure 3. A dose-dependent glucose uptake was still observed in response to Iso in the presence of PD 173074 and other FGFR1/FGF21-independent mechanisms must therefore be involved; likewise, the limited effect on Iso on glucose uptake in 3T3-L1 and ME3 adipocytes must be FGFR1/FGF21 independent. Surprisingly, a significant reduction in the basal glucose uptake was observed in the WT-1 adipocytes (Figure 7C) and tendencies for a similar patterns are observed in 3T3-L1 and ME3 adipocytes, indicating that during basal condition other FGFs (e.g. FGF1 [48]) which also signal through FGFR1 or FGFR3 [46] may be involved in basal glucose uptake in adipocytes.

PD 173074 inhibits FGF21-mediated glucose uptake in 3T3-L1 and WT-1 adipocytes.

Figure 6.
PD 173074 inhibits FGF21-mediated glucose uptake in 3T3-L1 and WT-1 adipocytes.

FGF21-mediated glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) in the absence (circles) or presence of the FGFR inhibitor PD 173074 (triangles) (200 nM) for 24 h. Data are mean ± SEM. Average of four independent experiments. Statistical difference of the PD effect (stimulation at 300 nM FGF21) is denoted *P < 0.05.

Figure 6.
PD 173074 inhibits FGF21-mediated glucose uptake in 3T3-L1 and WT-1 adipocytes.

FGF21-mediated glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) in the absence (circles) or presence of the FGFR inhibitor PD 173074 (triangles) (200 nM) for 24 h. Data are mean ± SEM. Average of four independent experiments. Statistical difference of the PD effect (stimulation at 300 nM FGF21) is denoted *P < 0.05.

Autocrine effect of FGF21 in WT-1 adipocytes in response to Iso stimulation.

Figure 7.
Autocrine effect of FGF21 in WT-1 adipocytes in response to Iso stimulation.

Iso-mediated glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) in the presence or absence of PD 173074 (PD) for 24 h. Closed bars: glucose uptake in response to increasing concentrations of Iso and open bars: glucose uptake in response to increasing conc. of Iso and 200 nM PD 173074. Data are mean ± SEM. Average of three independent experiments. Statistical effect determined by two-way ANOVA of Iso and PD 173074 is denoted as &P < 0.05, &&P < 0.01 and *P < 0.05, **P < 0.01, respectively.

Figure 7.
Autocrine effect of FGF21 in WT-1 adipocytes in response to Iso stimulation.

Iso-mediated glucose uptake in 3T3-L1 adipocytes (A), ME3 adipocytes (B) and in WT-1 adipocytes (C) in the presence or absence of PD 173074 (PD) for 24 h. Closed bars: glucose uptake in response to increasing concentrations of Iso and open bars: glucose uptake in response to increasing conc. of Iso and 200 nM PD 173074. Data are mean ± SEM. Average of three independent experiments. Statistical effect determined by two-way ANOVA of Iso and PD 173074 is denoted as &P < 0.05, &&P < 0.01 and *P < 0.05, **P < 0.01, respectively.

In summary, in response to Iso, an increase in FGF21 protein was observed in the medium from WT-1 brown adipocytes, and in the presence of the FGFR inhibitor PD 173074, the effect of Iso on glucose uptake was attenuated. This indicates an autocrine role of FGF21 on glucose uptake in these brown adipocytes.

Discussion

The three cell lines all expressed Klb and Fgfr1 mRNA after differentiation. The lowest expression level of Klb mRNA was observed in ME3 cells. The highest basal gene expression of Ucp1 mRNA was found in WT-1 brown adipocytes while 3T3-L1 adipocytes had the lowest basal gene expression of Ucp1 as well as the lowest induction of Ucp1 after Iso stimulation. This agrees with Murholm et al. [17] and substantiates that 3T3-L1 cells resemble white adipocytes and WT-1 cells, brown adipocytes. The third cell line, ME3, showed the greatest induction of Ucp1 in response to Iso, indicating that ME3 cells are likely beige adipocytes, in agreement with the literature [18]. The increase in glucose uptake in response to FGF21 was less pronounced in ME3 adipocytes compared with 3T3-L1 and WT-1 adipocytes, potentially linked to the lower Klb mRNA expression. In adipocytes the increase in glucose uptake via GLUT1 and GLUT4 [49] has been shown to correlate with an increased expression of Ucp1 [31] indicating that UCP1 activity increases glucose uptake. However, in our hands, 10 nM FGF21 did not significantly increase Ucp1 mRNA despite EC50 for glucose uptake in the low nM range in the 3T3-L1 and WT-1 adipocytes. The FGF21-induced glucose uptake may therefore not be directly mediated by changes in UCP1 activity in these cellular systems opposite to what Challa et al. [50] find in vivo. However, other authors have shown that selective FGFR1 activation of BAT does not stimulate glucose utilization in mice [51] and more studies are required to correlate FGF21 induced glucose uptake to UCP1 activity in adipocytes.

FGF21 has also been shown to induce lipogenesis in 3T3-L1 adipocytes [43] and glucose may therefore be converted to triglycerides. This may however, be cell dependent and is more likely to take place in the more white adipocytes [43]. In response to Iso, a dose-dependent increase in Ucp1 mRNA expression was observed in all three cells lines however, a significant increase in glucose uptake was only observed in the WT-1 adipocytes. Interestingly, the increase in lactate production in response to Iso was also larger in WT-1 cells compared with 3T3-L1 and ME3 cells. The large conversion of glucose to lactate has previously been described in adipocytes [31,52] and may be a surrogate for glucose uptake in adipocytes. The conversion rate may, however, depend of the amount of activated UCP1, and more data are required to understand why lactate production is increased in WT-1 adipocytes stimulated with Iso. FGF21, Iso, and insulin all increase glucose uptake in adipocytes [8,28] but the fate of the glucose differs in response to the different stimuli as well as on the adipocyte phenotype (brown, beige or white). In response to insulin, glucose is converted to triglycerides (lipogenesis) as well as lactate [53] while Iso in our hands, also induced lactate production. It is however, controversial how ADRB3 affects lipogenesis as a futile cycle of both a decrease and an increase in lipogenesis have been described [54–56]. However, in general ADRB3 agonism increases lipolysis in adipocytes which fuels UCP1-dependent thermogenesis [40].

The effect of FGF21 on lipid metabolism in adipocytes is controversial as both an induction of lipogenesis [43] and lipolysis [42] have been observed in vitro. Other studies have clearly showed that FGF21 inhibits lipolysis in vivo [57,58], similarly to the action of insulin [59,60]. The inhibition of lipolysis by insulin has moreover, been shown to be regulated by inhibition of the Gi-coupled receptor GPR81 [53]. GPR81 is activated by lactate and mediates an anti-lipolytic effect [53] with an EC50 estimated to be between 1.5–7 mM lactate [53]. In our study, FGF21 (Figure 2) and Iso (Figure 3) stimulated lactate production in the medium reached concentrations of ∼9–18 mM, depending on the type of adipocytes, which is above the EC50 for GPR81. Therefore, it would be interesting to understand if GPR81 also affects FGF21 and Iso-induced lipolysis. Interestingly, GPR81 stimulation has also been shown to induce browning [61], assigning lactate as an important metabolite in adipocyte metabolism.

In this study we determined to autocrine effect of FGF21 by using the PD 173074 inhibitor. However, other FGF21 inhibitor peptides [62] and antibodies [63] could have been used. Importantly, PD 173074 is a potent inhibitor of FGFR3 [64] but as our cellular systems expressed very little FGFR3 we expect that the inhibitory effect of PD 173074 is obtained by FGFR1 inhibition. Surprisingly, PD 173074 reduced basal glucose uptake in WT-1 adipocytes, indicating that other FGFs (e.g. FGF1) [48] may be involved in the basal glucose uptake in ME3 and WT-1 adipocytes or an unspecific effect of PD 173074 independent of FGFR. To confirm our findings in vivo adipose specific FGF21 knockout mice are required but interestingly hepatic FGF21 is released in response to cold [65] indicating an important cross-talk between the adipose tissue and the liver in the adaptive response to cold exposure.

Finally, the Iso-induced FGF21 release from the WT-1 adipocytes was surprisingly low and below the estimated EC50 value for glucose uptake of 1 nM. However, the amount of FGF21 released into the medium agrees with previous reports in immortalized and primary brown adipocytes [30,66]. Moreover, the amount of FGF21 might have been higher in close proximity to the surface of the adipocytes [67], and FGF21 could furthermore have been internalized [68] or degraded [69].

In conclusion, an autocrine loop exists in mouse brown WT-1 adipocytes, showing that FGF21 contributes to increased glucose uptake secondary to Iso stimulation in cultured cells.

Conflict of interest

K.V.H. and B.A. are full time employees and minor stockholders of Novo Nordisk A/S.

Funding

The studies were performed at Novo Nordisk A/S and no specific funding was obtained for this study.

Author Contribution

S.J., K.H.V., J.B.H., H.S.H. and B.A. designed the study. S.J. and B.A. wrote the manuscript.

Abbreviations

     
  • ADRB

    activation of β-adrenergic receptor

  •  
  • BAT

    brown adipose tissue

  •  
  • CNS

    central nervous system

  •  
  • DMEM

    Dulbecco's Modified Eagle's Medium

  •  
  • FBS

    fetal bovine serum

  •  
  • FFA

    free fatty acids

  •  
  • FGF21

    fibroblast growth factor 21

  •  
  • GLUT1

    glucose transporter 1

  •  
  • UCP1

    uncoupling protein 1

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Supplementary data