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. 2025 Dec 3;53(1):157. doi: 10.1007/s11033-025-11307-3

Irisin-induced cardiac fibroblast proliferation is dependent on glucose concentration and is inhibited by WZB117.

Marta Drobnik 1, Małgorzata Gałdyszyńska 2, Aleksander Kucner 2, Jacek Szymański 3, Marlena Juszczak 4, Jacek Drobnik 2,✉
PMCID: PMC12675697  PMID: 41335262

Abstract

Backgrounds

Fibroblast proliferation is influenced by inter alia the cellular transport of glucose and its metabolism. The aim of the study is to confirm whether irisin is involved in the regulation of human cardiac fibroblast proliferation, and whether its effect is dependent on glucose concentration or glucose transporter activity (GLUT1). The study also examines the expression of glucose transporters on the cardiac fibroblast cell membrane.

Methods and results

Human cardiac fibroblasts were cultured under hypoglycemia (1 mmol/l), normoglycemia (5 mmol/l) or hyperglycemia (25 mmol/l), and treated with different concentrations of irisin (10− 7 M, 10− 8 M and 10− 9 M). The results indicate that glucose concentration had no influence on cardiac fibroblast proliferation measured by BrdU. Increased proliferation was noted at 10− 8 M and 10− 9 M irisin in hyperglycemia, and 10− 9 M in normoglycemia; no such change was observed in hypoglycemia. Treatment with 10− 8 M irisin increased glucose uptake by cardiac fibroblasts and decreased its concentration in the medium. Flow cytometry confirmed GLUT 1, 3 and 4 expression on the fibroblast surface. The GLUT 1 inhibitor WZB117 decreased fibroblast proliferation (10− 6 M) in normoglycemic and hyperglycemic conditions, and negated the effect of irisin at 10− 9 M. WZB117 treatment increased the glucose concentration in the culture medium.

Conclusion

Irisin accelerates cardiac fibroblast proliferation and glucose uptake; however, this effect is dependent on glucose concentration and GLUT1 activity. GLUT1, 3 and 4 protein expression was identified on human cardiac fibroblasts.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11033-025-11307-3.

Keywords: Cardiac fibroblasts, Glucose transporters, GLUT1, GLUT3, GLUT4, Fibrosis, Proliferation

Introduction

In patients with heart failure, systolic dysfunction or preserved ejection fraction, the presence of diabetes mellitus represents a key independent predictor of morbidity and mortality [1]. Diabetes itself is associated with diffuse cardiac fibrosis within the heart [1, 2]. While fibrosis is known to impair heart function and potentially lead to heart failure when combined with other factors, its mechanism remains unclear. Also, while inhibiting myocardial fibrosis may attenuated the course of diabetic cardiomyopathy [3], no effective method has yet been found for treating or preventing heart fibrosis in diabetics.

Cardiac fibroblasts play an important role in the metabolism of the cardiac extracellular matrix. The degree of collagen accumulation within the organ is regulated to varying degrees by both physical [4] and chemical [5] factors, such as the synthesis and cleavage of collagen. It is also determined to an extent by the level of fibroblast proliferation, which is dependent on extracellular glucose concentration, and has been found to be inhibited [6] or stimulated [7, 8] under hyperglycemic conditions.

The energetic status of a cell may influence its function, e.g. extracellular matrix synthesis or fibroblast proliferation. As glucose is not able to diffuse across the cell membrane, its influx is dependent on glucose transporters. Two main types of glucose transporters have been described: sodium-glucose-linked transporters (SGLT) and facilitated diffusion glucose transporters (GLUTs) [9].

These GLUTs facilitate the diffusion of glucose across the plasma membrane; while these proteins include 12 membrane-spanning regions, both their carboxy and amino terminals are localized intracellularly. GLUT 1, GLUT 3 and GLUT 4 are all Class 1 glucose transporters [10].

GLUT 1, responsible for the basal uptake of glucose, is present on various cells, including mouse L929 fibroblast [11]. Its overexpression has been reported on cancer cells, where its presence supports cell proliferation by facilitating increased glucose uptake and aerobic fermentation [11]. GLUT 3 is mainly present in cells with higher glucose requirements, such as those in the brain, testis and placenta, as well as cancer cells [12]. GLUT 4 is an insulin-responsive glucose transporter found in the heart, skeletal muscle, adipose tissue, brain and 3T3-L1 fibroblasts [13].

Studies have found glucose homeostasis and insulin resistance to be improved in the presence of higher concentrations of irisin (myokine). The structure of irisin comprises 112 amino acids; it was first described in 2012 as a hormone released from muscle cells, whose precursor is the fibronectin type III domain [14]. The protein is released during exercise and is associated with elevated expenditure of energy [14, 15]. It has been identified in skeletal muscle and heart tissue, as well as in the brain, liver, pancreas, stomach and testis of rats [16]. It levels tend to be lower in patients with type 2 diabetes mellitus [17].

The aim of the present study is to confirm whether glucose concentration may influence the effect of irisin on cardiac fibroblast proliferation, and whether its activity is dependent on GLUT 1 activity. The study hypothesis is that irisin regulates the activity of glucose transporters in fibroblasts, and by doing so, it influences glucose transport by the cells, and thus their metabolism and energetic status. This in turn may affect fibroblast proliferation, which requires increased glucose uptake and lactate excretion, and is characterized by elevated basal respiration and ATP synthesis [18]. It is believed that irisin exerts its beneficial effects by regulating glucose transporter activity.

Materials and methods

Cell culture

The experiments were carried out on an immortalized human cardiac fibroblast cell line (ABM, Richmond, BC, Canada). The cells were cultured according to the supplier’s recommendations, i.e. in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific Waltham, MA, USA), supplemented with 3% Fetal Bovine Serum (Biowest Nuaille, France), 25 µg/ml gentamycin (Thermo Fisher Scientific Waltham, MA, USA), 2.5 µg/ml amphotericin B (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 5 µg/ml insulin (Fisher Scientific Waltham, MA, USA), 50 µg/ml sodium pyruvate (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), and 50 µg/ml vitamin C (Sigma Aldrich, St. Louis, MO, USA).

The cultures were divided into three experimental variants based on exposure to glucose: one grown under hypoglycemic conditions (Group G1: 1 mmol/l glucose), another under normoglycemic conditions (Group G2: 5 mmol/l) and another under hyperglycemic conditions (Group G3: 25 mmol/l). The glucose solution was supplied by Thermo Fisher Scientific Waltham (MA, USA). The cells were seeded at an initial density of 4 × 103 /well (96-well plates) or 4 × 104/cm2 (12-well plates) on plates coated with 10 µg/cm2 type I collagen (Sigma Aldrich, St. Louis, MO, USA). The cultures were then maintained at 37 °C (5% CO2) and allowed to settle.

To determine the effect of irisin (Biorbyt Ltd, Cambridge, UK) on fibroblast proliferation, the culture groups were divided into four subgroups treated with different levels of irisin: 0 M (control), 10-7M, 10-8M and 10-9M. Briefly, irisin was dissolved in the medium, the cultures were incubated for four days, and cell proliferation was analyzed using bromodeoxyuridine (BrdU) according to the manufacturer’s instructions. Briefly, BrdU is incorporated to the newly-synthesized DNA of proliferating cells, where it is detected by anti-BrdU antibodies.

The normoglycemic cultures were also used to determine the optimal concentration of the GLUT 1 inhibitor WZB 117 (Tocris, Bristol, UK) on fibroblast proliferation. The cells were divided into the following five groups: Group 1, an untreated control; Group 2, consisting of cells treated with 0.01% DMSO (the solvent for WZB117); Group 3, treated with 10-8M WZB117; Group 4, treated with 10-7M WZB117; Group 5, treated with 10-6M WZB117. Fibroblast proliferation was then evaluated.

In addition, it was examined whether WZB117 may reduce the effect of irisin under hyperglycemic conditions. The hyperglycemic cultures divided into the following subgroups for testing: Group 1, untreated control; Group 2, DMSO; Group 3, 10-6M WZB117; Group 4, 10-8M irisin with DMSO; Group 5, 10-8M irisin with 10-6M WZB117. Fibroblast proliferation was then evaluated.

Expression of GLUT 1, 3 and 4

The expression of GLUT 1, 3 and 4 on the surface of cardiac fibroblasts was confirmed by flow cytometry. Initially the cells were fixed with BD Cytofix (BD Biosciences, Franklin Lakes NY, USA) for 20 min at 4 °C. After washing, the cells were suspended in BD Pharmingen Stain Buffer (BD Biosciences, Franklin Lakes NY, USA) and stained at 4 °C for 25 min. The following antibodies were applied consecutively: rabbit GLUT1 antibody orb128739 (Biorbyt Ltd, Cambridge, UK), followed by rabbit GLUT3 antibody orb126368 (Biorbyt Ltd, Cambridge, UK) and then rabbit GLUT4 antibody orb10728 (Biorbyt Ltd, Cambridge, UK). The cells were then washed again and incubated at 4 °C for 25 min with FITC Goat anti-rabbit IgG labelled with fluorescein isothiocyanate as the secondary antibody (BD Pharmingen TM, San Diego, CA, USA). The cells were then counted using a FACS CytoFLEX Cytometer (Beckman Couler, Inc), and the resulting data analyzed by CytExpert 2.1 software (Beckman Couler, Inc).

Glucose uptake test

The test was performed according the supplier’s recommendations (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were grown under normoglycemic conditions on 96-well plates; these were divided into three groups: one set of untreated controls, one set treated with 10− 8M irisin and another with 10− 9M irisin. All groups were administered with serum-free medium (100 µl/well) and incubated overnight before assay. They were then washed three times with PBS and glucose-starved by plating with 100µL of KRPH buffer with 2% bovine BSA, and then incubated for 40 min. Each well received 10 µL of 10 mM 2-deoxyglucose (2-DG), and the cultures were incubated for 20 min before being washed with PBS. Extraction buffer was then applied, and the samples were frozen at -80 °C and then warmed to 85 °C over 40 min. The resulting lysates were cooled and neutralization buffer was added (10 µL /well).

For the assay itself, the samples were first centrifuged (13 000 g, 1 min, room temperature), and then diluted tenfold in assay buffer. All wells were then treated with reaction mix A (10 µL /well) to generate NADPH; the samples were then shaken and incubated in darkness (37 °C) for 60 min. Following this, 90 µL /well of Extraction Buffer was added to degrade the NADPH. The samples were then heated to 90 °C for 40 min, cooled to 4 °C (5 min), and neutralization buffer was added (12 µL /well). Each received 38 µL of Reaction Mix B and the samples were shaken. Absorbance was measured at 412 nm every five minutes using an Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA).

The next stage examined whether irisin influences glucose uptake. The cells were divided into the following three subgroups: untreated controls, cultures treated with 10− 8 M irisin, and cultures treated with 10− 9 M irisin. The level of glucose in the medium was determined by colorimetry (oxidase/peroxidase; GOT-POD). Briefly, 1 ml of GOT-POD reagent was added to the tube containing 10 µL of sample; they were then mixed and the mixture incubated for 10 min, following which the optical density was measured at 505 nm. The inhibitory effect of WZB117 on glucose uptake was also determined in medium taken from controls, 0.01% DMSO cultures, and WZB117 (10− 6 M) cultures. All experiments were performed in medium with 5mM glucose concentration.

Statistical analysis

The obtained data were analyzed using Statistica 13.1 software (StatSoft, Tulsa, OK, USA). The normality of the results was evaluated with the Shapiro-Wilk test. Homogenity of variance was assessed by Levene’s test. If the data had a normal distribution and homogenous variance, the ANOVA test was applied with the post hoc Shaffer’s test. When the distribution was not normal, or the variance was not homogenous, the results were tested with the Kruskal-Wallis test, and the differences between groups evaluated with the Mann-Whitney U-test. The results were considered significant at p < 0.05.

Results

The BrdU analysis identified similar proliferation rates between hypoglycemic (G1–1mmol/l), normoglycemic (G5–5mmol/l) and hyperglycemic (G25–25mmol/l) conditions. These data indicate that cardiac fibroblast proliferation does not seem to be influenced by glucose concentration (Fig. 1A).

Fig. 1.

Fig. 1

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Fig. 1A. The effect of various glucose concentrations on the proliferation of human cardiac fibroblasts cultured in three glucose media: hypoglycemic (G1, 1mmol/L) normoglycemic (G5, 5mmol/L) and hyperglycemic (G25, 25mmol/L); Fig. 1B. Influence of different concentrations of irisin on human cardiac fibroblast proliferation in hypoglycemic medium: G1 (hypoglycemic control), G1 I-7 irisin at concentration of 10− 7 M, G1 I-8 irisin at concentration of 10− 8 M and G1 I-9 irisin at concentration of 10− 9 M; Fig. 1C. Effects of various concentrations of irisin on human cardiac fibroblast proliferation in normoglycemic medium: G5 (normoglycemic control), G5 I-7 irisin at concentration of 10− 7 M, G5 I-8 irisin at concentration of 10− 8 M and G5 I-9 irisin at concentration of 10− 9 M; Fig. 1D. The effect of irisin concentration on human cardiac fibroblast proliferation in hyperglycemic medium: G25 (hyperglycemic control), G25 I-7 10− 7 M irisin at concentration of 10− 7 M, G25 I-8 irisin at concentration of 10− 8 M and G25 I-9 irisin at concentration of 10− 9 M. Each value represents mean ± SD.

Under hypoglycemic conditions (G1–1mmol/l), no differences in cell proliferation were found between the untreated controls (G1) and the treated cells, i.e. those receiving 10-7M (G1 I-7), 10-8M (G1 I-8) or 10-9M (G1 I-9) irisin. This suggests that irisin did not modify cardiac fibroblast proliferation at any applied concentration (Fig. 1B).

Under normoglycemic conditions (G5–5 mmol/l), the cells treated with 10-9M irisin (G1 I-9) demonstrated significantly greater proliferation than the normoglycemic controls (G5, p = 0.05). However, no significant differences were observed between controls and either 10-7M (G1 I-7) or 10-8M (G1 I-8) (Fig. 1C).

Under hyperglycemic conditions, treatment with 10-8M (G25 I-8) and 10-9M irisin (G25 I-9) increased fibroblast proliferation compared to controls (G25) (p = 0.006 and p = 0.035 respectively). However, no significant difference was observed between controls and 10-7M irisin (G25 I-7; Fig. 1D).

Under normoglycemic conditions, 10− 8 M irisin significantly increased glucose uptake compared to controls (p = 0.03). However, no significant increase was observed at 10-9M irisin (Fig. 2A).

Fig. 2.

Fig. 2

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The effect of irisin: I-8 (10-8 M) and I-9 (10-9 M) on glucose uptake by human cardiac fibroblasts (Fig.2A) as well as on glucose concentration in extracellular medium (Fig.2B). Influence of WZB117 (10-6 M), (Fig.2C) on glucose concentration in extracellular medium comparing with DMSO treated cultures (DMSO) and untreated control (CTR). (Each value represents mean ± SD.

Treatment with 10− 8 M or 10− 9 M irisin resulted in significantly greater glucose uptake by fibroblasts compared with controls, accompanied by lower glucose concentration in the medium (p < 0.001; Fig. 2B). This uptake determines the rate of glucose utilization within the cell.

Administration of the GLUT1 inhibitor WZB117 at 10− 6 M (WZB-6) increased glucose concentration within the extracellular medium compared with DMSO alone (p < 0.001) and controls (CTR, p < 0.001; Fig. 2C).

GLUT 1, GLUT3 and GLUT 4 expression was found to correlate with their protein levels on the cardiac fibroblast surface. The GLUT distribution across the cell surface is compared with the autofluorescence data and isotypic controls in Fig. 3.

Fig. 3.

Fig. 3

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Expression of GLUT 1 (Fig. 3A), GLUT 3 (Fig. 3B) and GLUT 4 (Fig. 3C) on cell membrane of human cardiac fibroblasts. The curves in each figure represents: left – autofluorescence of the cells, middle – isotypic control, right – appropriate glucose transporter fluorescence.

Under normoglycemic conditions, no significant difference in cell proliferation was observed between 0.01% DMSO and intact control cultures, nor between DMSO and 10-8M WZB117 (WZB-8). However, WZB117 at 10-7M (WZB-7) and 10-6M (WZB-6) decreased proliferation compared to the DMSO groups. Only 10-6M WZB117 (WZB-6) significantly reduced proliferation compared to the DMSO group (p = 0.00008) and controls (CTR, p = 0.0017). No significant difference was observed between 10 and 7 M WZB117 (WZB-7) and DMSO-treated cultures (Fig. 4A).

Fig. 4.

Fig. 4

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Human cardiac fibroblast proliferation in controls (CTR), DMSO-treated cultures, cells treated with; WZB 117 (GLUT 1, 3 or 4 inhibitor) at concentrations of 10− 9 M (WZB-9), 10− 8 M (WZB-8), 10− 7 M (WZB-7) or 10− 6 M (WZB-6); cultures showed on Fig. 3A were performed in normoglycemic medium or in hyperglycemic medium (Fig. 3B). Proliferation of human cardiac fibroblasts in controls (CTR) or DMSO-treated cultures; other cultres received WZB 117 (10− 6 M) alone, irisine (10− 8 M) with DMSO, or both irisine (10− 8 M) and WZB 117 (10− 6 M), (Fig. 3C). The experiment was performed in hyperglycemic conditions. Each value represents mean ± SD.

In the hyperglycemic medium, similar proliferation was noted in both the control (CTR) and DMSO groups. Compared to the DMSO group, treatment with WZB117 (WZB-8) insignificantly increased proliferation at 10-8M, but lowered proliferation at 10-7M (WZB-7) and 10-6M (WZB-6). The WZB-6 group demonstrated significantly lower proliferation compared to DMSO (p = 0.007) and controls (CTR, p = 0.003), while no significant differences were found between WZB-7 and DMSO (p = 0.47; Fig. 4B).

In all variants except the untreated controls (CTR), DMSO was present at a concentration of 0.01% (Fig. 4C). No significant differences in proliferation were observed between controls and the DMSO group for either normoglycemic or hyperglycemic conditions. The WZB-6 group demonstrated significantly slower proliferation compared with the DMSO group (p = 0.024). Irisin (10-8M) insignificantly increased proliferation compared to DMSO; however, this effect was significantly reduced (p = 0.02) by the combined application of WZB117 (10-6M) and irisin (10-8M) in the Ir/WZB cultures (Fig. 4C).

Interestingly, the results of irisin treatment varied significantly depending on the concentration and the glycemic environment viz. normoglycemia or hyperglycemia (Fig. 1C, D). However irisin treatment only achieved insignificant augmentation of fibroblast proliferation under hyperglycemic conditions (Fig. 4C); this was most likely due to the addition of DMSO (i.e. the WZB117 solvent) to the medium, which seems to attenuate the effect of irisin.

Discussion

Our findings indicate that treatment with glucose at medium concentrations ranging from 1 mol/L to 25 mol/L did not alter the proliferation rate of the tested human cardiac fibroblasts. Similarly, Gorski et al. report no significant difference in fibroblast cell activation or glucose influx between hyperglycemic conditions (25mM) and normoglycemic controls (5.5mM). Also, while the cells responded to insulin, indicated by a rise in protein kinase B phosphorylation following acute insulin application, increasing the insulin concentration in the hyperglycemic medium did not support intracellular glucose transport: no changes in glucose uptake or hyaluronic acid synthesis or secretion were noted [19].

However, previous studies also report that high glucose concentrations impaired the proliferation and migration of dermal fibroblasts and inhibited their transformation into myofibroblasts [20]; however, these conditions increased the proliferation of fibroblasts from obese diabetic Zucker adult male rats compared with controls [8]. Hyperglycemia also induced cardiac fibroblast proliferation and collagen synthesis in diabetic mice [7]. Tarnowski-Babey et al. report that beta cells demonstrated accelerated proliferation following acute hyperglycemia, but exhibited toxic effects during chronic hyperglycemia [21].

Initially, the influence of irisin on fibroblast proliferation was examined in mediums with different glucose concentrations, as these conditions may determine the level of glucose transporter expression or glucose uptake. Indeed, lower uptake by lymphocyte T and B was noted at abnormal glucose levels, which was attributed to decreased expression of glucose transporters [22]. Similarly, both increases and decreases in glucose concentration in the extracellular fluid were associated with underexpression of GLUT1 and SGLT1 in trophoblast cells [23]; such changes also influenced transporter expression in rat skeletal muscle [24]. In the present study, while extracellular glucose concentration did not modify cardiac fibroblast proliferation, it modulated the effect of irisin: increasing the proliferation of human cardiac fibroblasts in normoglycemic or hyperglycemic media, but not under hypoglycemic conditions. In addition, irisin increased proliferation when administered at 10-9M and 10-8M at high glucose concentrations, but only at 10-9M in normoglycemia. It is possible that glucose concentration influences irisin activity by glycosylation; indeed, glycosylated irisin has demonstrated better bioactivity than the unconjugated form [25], and irisin was found to reduce glucose concentration in diabetic mice but not in normoglycemic controls [20].

Our findings also indicate that human cardiac fibroblasts act as targets for irisin. This is not surprising, as irisin is known to influence the activity of a wide number of cell types. It was found to increase proliferation among rat primary osteoblasts and a mouse osteoblastic cell line [26] as well as mouse hemopoietic stem cells [27]. In mice, low doses of irisin inhibited endothelial-to-mesenchymal transition induced by high glucose concentrations, while at high doses, it accelerated the proliferation and migration of cardiac fibroblasts; this was linked with excessive collagen deposition within the heart [28]. Irisin has also been found to increase the proliferation of inter alia tendon-derived stem/progenitor cells [29], osteoclast precursor cells [30], and human hepatocellular carcinoma cells [31], C2C12 myoblasts [32].

However, our present findings indicate, for the first time, that irisin (10− 8M) also augments the uptake of glucose in cardiac fibroblasts under normoglycemic conditions (Fig. 2); furthermore, the lower concentration (10− 9M) was also found to increase glucose uptake. Irisin has previously been reported to increase glucose uptake into human myoblasts and skeletal muscle cells [33]; it was also found to affect glucose uptake in mice with diabetes mellitus, but not in normoglycemic animals [31].

Our present findings confirm that irisin modulated glucose uptake in normoglycemic conditions (5mM of glucose concentration in medium); however, this effect was not tested in hyperglycemic conditions, which can be regarded as a limitation. The rate of glucose uptake typically determines its rate of utilization inside the cells; as such, the increase in glucose influx into the fibroblasts observed following irisin treatment (Fig. 2A and B) may result in increased intracellular metabolism.

The present findings also indicate a medium glucose level fell significantly following treatment with 10− 8 M and 10− 9 M irisin; these results are in line with the observed increased glucose uptake by the cells (Fig. 2A). Similarly, pronounced acceleration of glucose metabolism was also noted previously in an adipocyte model treated with irisin, which was also attributed to intracellular transport of glucose [34].

The flow cytometry analysis confirmed the expression of GLUT 1, 3 and 4 on the membranes of the human cardiac fibroblasts. GLUT 1 has previously been reported on mouse L929 fibroblast membranes [11], and GLUT4 on 3T3-L1 murine embryonic fibroblasts [13]. Also, GLUT1, GLUT3 and GLUT 4 mRNA were detected in insulin-responsive human dermal fibroblasts by Northern blot, although no GLUT 2 mRNA was found [35]; GLUT1 and GLUT3 proteins have been identified in fibroblasts [36].

Both insulin and protein kinase C activators (phorbol esters) were found to increase 3-O-methyl-D-glucose levels in human skin fibroblasts; this increase has been found to depend on protein synthesis and correlates with the level of GLUT1 and GLUT3 mRNA synthesis. However, phorbol esters act through a different mechanisms than insulin, in that they do not modify the levels of GLUT1 and GLUT3 mRNA [12]. Our present results confirm the expression of the selected glucose transporters on the tested human cardiac fibroblasts.

Our findings indicate that treatment with the GLUT 1 inhibitor WZB117 could inhibit the unstimulated proliferation of fibroblasts within the culture; this compound also cancels the effect of irisin on proliferation. Indeed, WZB117 was found to inhibit glucose metabolism within the tested fibroblasts, with the WZB117-treated cells exhibiting elevated glucose concentrations in extracellular medium than the DMSO cultures and the untreated controls (Fig. 3C). Hence, WZB117 appears to inhibit glucose uptake.

Similarly, WZB117 also decreases GLUT1 expression in breast cancer cells [37], and partially reduces enhanced glycolysis in colorectal cancer cells caused by SAP30 gene overexpression [38]; it also decreases intracellular glucose levels in tumor cells [39]. These results suggest that inhibition of glucose transporters, and the resulting decrease of intracellular glucose influx, may reduce the proliferation of human cardiac fibroblasts. Hence, the effect of irisin on fibroblast proliferation is dependent on glucose transporter activity.

These results also suggest that glucose metabolism may be an important determinant of cardiac fibroblast proliferation. Irisin is known to increase the intracellular transport of glucose [33], to induce intracellular uptake of glucose in various skeletal muscle cells and myoblasts [17], and to reduce insulin resistance [40]. Likewise, our present findings also indicate that irisin treatment increased glucose uptake by the cardiac fibroblasts, while also decreasing the glucose concentration in the extracellular medium (Fig. 3A, B).

Cellular energy storage (ATP) and biomass accumulation play important parts in cell metabolism, which in turn determines cell proliferation. Proliferation requires the biosynthesis of various macromolecules, which is driven by various pathways: one of these is glycolysis, which initiates both ATP storage and biosynthesis. The rate of glycolysis is dependent on glucose influx into the cell, and predictably, this influx is greater in proliferating cells and those undergoing fibrosis. Intracellular glucose influx is preceded by an increase in GLUT 1 in the cell membrane, which is associated with the activation of hexokinase, and other enzymes participating in glycolysis [41, 42]. Hence, it is possible that treatment with WZB117, a GLUT1 inhibitor, blocked irisin-stimulated cell proliferation by inhibiting glucose uptake, and that the effect of irisin on proliferation may be dependent on the glucose metabolism within the cells.

Conclusion: Irisin induces human cardiac fibroblast proliferation, and the level of this induction is dependent on the glucose concentration within the medium. Treatment with irisin supports glucose uptake by cardiac fibroblasts. Interestingly, irisin appears to increase fibroblast proliferation under normoglycemic and hyperglycemic conditions, but not hypoglycemia. GLUT1, GLUT 3 and GLUT 4 protein expression was noted on human cardiac fibroblast cell membranes. It was found that GLUT1 blockade inhibited irisin activity, and thus, human cardiac fibroblast proliferation. Hence, both irisin secretion and the GLUT1 glucose transporter may represent potential targets for pharmacotherapy preventing heart fibrosis and the development of diabetic cardiomyopathy.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (231.5KB, doc)
Supplementary Material 2 (29.5KB, doc)
Supplementary Material 3 (229.6KB, pdf)

Acknowledgements

The study was supported by grant from Medical University of Lodz 503/6-103-04/503-01.

Author contributions

Marta Drobnik and Jacek Drobnik contributed to whole study conception and design. Material preparation, investigation, data analysis and interpretation, writing the main manuscript were performed by Marta Drobnik, Małgorzata Gałdyszyńska, Jacek Szymański, Marlena Juszczak, Jacek Drobnik. Małgorzata Gałdyszyńska and Jacek Szymański were responsible for conceptualization and design of experiments in Fig. 2 and Fig. 3 respectively. Marlena Juszczak participated in collection of the data, their analysis as well as commented and reviewed the manuscript. Aleksander Kucner performed experiments on Fig. 2B and 2C. All authors read and approved the final manuscript

Funding

The study was supported by grant from Medical University of Lodz 503/6-103-04/503-01.

Data availability

The data will be submitted after request.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

Conflict of interest is not declared by the authors.

Ethics, consent to Participate, and consent to publish declarations

Not applicable.

Footnotes

Publisher’s note

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References

  • 1.Nghiem TP, Tran HM, Nguyen DNQ, Dao LT, Tran CC, Vo TM (2025) Subclinical myocardial dysfunction in type 2 diabetes mellitus: insights from left ventricular diastolic function and global longitudinal strain assessment. Med Sci (Basel) 13(4):237 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kelly DJ, Zhang Y, Connelly K, Cox AJ, Martin J, Krum H et al (2007) Tranilast attenuates diastolic dysfunction and structural injury in experimental diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 293(5):H2860–H2869 [DOI] [PubMed] [Google Scholar]
  • 3.Zhang T, Hu J, Li Z, Tian T, Huang R, Li N, Wang D (2025) 1,25-dihydroxyvitamin D3 attenuates type 1 diabetes-related myocardial apoptosis and fibrosis by influencing the interaction of FoxO1 and p-Smad3. Int Immunopharmacol 162:115128 [DOI] [PubMed] [Google Scholar]
  • 4.Gałdyszyńska M, Zwoliński R, Piera L, Szymański J, Jaszewski R, Drobnik J (2023) Stiff substrates inhibit collagen accumulation via integrin α2β1, FAK and Src kinases in human atrial fibroblast and myofibroblast cultures derived from patients with aortal stenosis. Biomed Pharmacother 159:114289 [DOI] [PubMed] [Google Scholar]
  • 5.Radwańska P, Gałdyszyńska M, Piera L, Drobnik J (2023) Kisspeptin-10 increases collagen content in the myocardium by focal adhesion kinase activity. Sci Rep 13(1):19977 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Liu YY, Lu RJ, Guo B, Gao LL, Zhang N, Chang WX (2021) The effects of hyperglycemia on the biological behavior of human gingival fibroblasts on a titanium surface. Ann Palliat Med 10(9):9685–9691 [DOI] [PubMed] [Google Scholar]
  • 7.Liu Z, Zhang S, Wang T, Shao H, Gao J, Wang Y et al (2019) Neferine inhibits MDA-MB-231 cells growth and metastasis by regulating miR-374a/FGFR-2. Chem Biol Interact 309:108716 [DOI] [PubMed] [Google Scholar]
  • 8.Fowlkes V, Clark J, Fix C, Law BA, Morales MO, Qiao X, Ako-Asare K, Goldsmith JG, Carver W, Murray DB, Goldsmith EC (2023) Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts. Life Sci 92(11):669–676 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wang J, Ye C, Chen C, Xiong H, Xie B, Zhou J et al (2017) Glucose transporter GLUT1 expression and clinical outcome in solid tumors: a systematic review and meta-analysis. Oncotarget 8(10):16875–16886 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Singh G, Ansari S, Aran KR (2025) Targeting glucose transporters in parkinson’s disease: a novel metabolic approach for disease modification. Metab Brain Dis 40(7):253 [DOI] [PubMed] [Google Scholar]
  • 11.Rylaarsdam LE, Johnecheck GN, Looyenga BD, Louters LL et al (2019) GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells. Biochimie 162:88–96 [DOI] [PubMed] [Google Scholar]
  • 12.Longo N, Griffin LD, Langley SD, Elsas LJ (1992) Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin. Biochim Biophys Acta 1104(1):24–30 [DOI] [PubMed] [Google Scholar]
  • 13.Lee HS, Lee HJ, Suh HJ (2011) Silk protein hydrolysate increases glucose uptake through up-regulation of GLUT 4 and reduces the expression of leptin in 3T3-L1 fibroblast. Nutr Res 31(12):937–943 [DOI] [PubMed] [Google Scholar]
  • 14.Boström P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, Rasbach KA et al (2012) A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481(7382):463–468 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Roca-Rivada A, Castelao C, Senin LL, Landrove MO, Baltar J, Belén Crujeiras A et al (2013) FNDC5/irisin is not only a myokine but also an adipokine. PLoS ONE 8(4):e60563 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Aydin S, Kuloglu T, Aydin S, Kalayci M, Yilmaz M, Cakmak T et al (2014) A comprehensive immunohistochemical examination of the distribution of the fat-burning protein Irisin in biological tissues. Peptides 61:130–136 [DOI] [PubMed] [Google Scholar]
  • 17.Rongjing S, Xuecheng Z, Da-qi Z, Rong W, Yufei F (2021) Lower levels of Irisin in patients with type 2 diabetes mellitus: a meta-analysis. Diabetes Res Clin Pract 175:108788 [DOI] [PubMed] [Google Scholar]
  • 18.Yao CH, Wang R, Wang Y, Kung CP, Weber JD, Patti GJ (2019) Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation. Elife 29:8:e41351 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Gorski DJ, Petz A, Reichert C, Twarock S, Grandoch M, Fischer JW (2019) Cardiac fibroblast activation and hyaluronan synthesis in response to hyperglycemia and diet-induced insulin resistance. Sci Rep 9(1):1827 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Li B, Bian X, Hu W, Wang X, Li Q, Wang F, Sun M, Ma K, Zhang C, Chang J, Fu X (2020) Regenerative and protective effects of calcium silicate on senescent fibroblasts induced by high glucose. Wound Repair Regen 28(3):315–325 [DOI] [PubMed] [Google Scholar]
  • 21.Tornovsky-Babeay S, Weinberg-Corem N, Ben-Haroush Schyr R, Avrahami D, Lavi J, Feleke E, Kaestner KH, Dor Y, Glaser B (2021) Biphasic dynamics of beta cell mass in a mouse model of congenital hyperinsulinism: implications for type 2 diabetes. Diabetologia 64(5):1133–1143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Oleszczak B, Szablewski L, Pliszka M (2012) The effect of hyperglycemia and hypoglycemia on glucose transport and expression of glucose transporters in human lymphocytes B and T: an in vitro study. Diabetes Res Clin Pract 96(2):170–178 [DOI] [PubMed] [Google Scholar]
  • 23.Li H, Gu Y, Zhang Y, Lucas MJ, Wang Y (2004) High glucose levels down-regulate glucose transporter expression that correlates with increased oxidative stress in placental trophoblast cells in vitro. J Soc Gynecol Investig 11(2):75–81 [DOI] [PubMed] [Google Scholar]
  • 24.Mathoo JM, Shi ZQ, Klip A, Vranic M (1999) Opposite effects of acute hypoglycemia and acute hyperglycemia on glucose transport and glucose transporters in perfused rat skeletal muscle. Diabetes 48(6):1281–1288 [DOI] [PubMed] [Google Scholar]
  • 25.Zhang Y, Li R, Meng Y, Li S, Donelan W, Zhao Y, Qi L, Zhang M, Wang X, Cui T, Yang LJ, Tang D (2014) Irisin stimulates Browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling. Diabetes 63(2):514–525 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Qiao Q, Nie X, Ma Y, Chen Y, Cheng R, Yin Y, Hu W, Xu W, Xu L (2016) Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways. Sci Rep 7(6):18732 Erratum in: Sci Rep. 2016; 6:21053. Yong Qiao, Xiao [corrected to Qiao, Xiaoyong]; Xian Ma, Ya [corrected to Ma, Yaxian]; Yao Yinrg, Wei [corrected to Yin, Weiyao]; Ming Xu, Wen [corrected to Xu, Wenming]; Zhi Xu, Liang [corrected to Xu, Liangzhi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Li H, Kong D, Zhao Y, Liu X, Xiao F, Li X, Hu J et al (2023) Irisin protected Hemopoietic stem cells and improved outcome of severe bone marrow failure. Biomed Pharmacother 31:169:115863 [DOI] [PubMed] [Google Scholar]
  • 28.Liu X, Mujahid H, Rong B, Lu QH, Zhang W, Li P, Li N, Liang ES, Wang Q, Tang DQ, Li NL, Ji XP, Chen YG, Zhao YX, Zhang MX (2018) Irisin inhibits high glucose-induced endothelial-to-mesenchymal transition and exerts a dose-dependent bidirectional effect on diabetic cardiomyopathy. J Cell Mol Med 22(2):808–822 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Xu L, Chen Z, Geng T, Ru B, Wan Q, Zhang J, Li S, Cai W (2022) Irisin promotes the proliferation and tenogenic differentiation of rat tendon-derived stem/progenitor cells via activating YAP/TAZ. Vitro Cell Dev Biol Anim 58(8):658–668 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ma Y, Qiao X, Zeng R, Cheng R, Zhang J, Luo Y, Nie Y, Hu Y, Yang Z, Zhang J, Liu L, Xu W, Xu CC, Xu L (2018) Irisin promotes proliferation but inhibits differentiation in osteoclast precursor cells. FASEB J 17:fj201700983RR [DOI] [PubMed] [Google Scholar]
  • 31.Shi G, Tang N, Qiu J, Zhang D, Huang F, Cheng Y, Ding K, Li W, Zhang P, Tan X (2017) Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma. Biochem Biophys Res Commun 493(1):585–591 [DOI] [PubMed] [Google Scholar]
  • 32.Ye X, Shen Y, Ni C, Ye J, Xin Y, Zhang W, Ren Y (2019) Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1α pathway. Peptides 119:170120 [DOI] [PubMed] [Google Scholar]
  • 33.Lee HJ, Lee JO, Kim N, Kim JK, Kim HI, Lee YW, Kim SJ, Choi JI, Oh Y, Kim JH, Suyeon-Hwang, Park SH, Kim HS (2015) Irisin, a novel Myokine, regulates glucose uptake in skeletal muscle cells via AMPK. Mol Endocrinol 29(6):873–881 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.May JM, Mikulecky DC (1983) Glucose utilization in rat adipocytes. The interaction of transport and metabolism as affected by insulin. J Biol Chem 25(8):4771–4777 [PubMed] [Google Scholar]
  • 35.Pang Y, Zhu H, Xu J, Yang L, Liu L, Li J (2017) β-arrestin-2 is involved in Irisin induced glucose metabolism in type 2 diabetes via p38 MAPK signaling. Exp Cell Res 15(2):199–204 [DOI] [PubMed] [Google Scholar]
  • 36.Longo N, Bell GI, Shuster RC, Griffin LD, Langley SD, Elsas LJ (1990) Human fibroblasts express the insulin-responsive glucose transporter (GLUT4). Trans Assoc Am Physicians 103:202–213 [PubMed] [Google Scholar]
  • 37.Zhao F, Ming J, Zhou Y et al (2016) Inhibition of Glut1 by WZB117 sensitizes radioresistant breast cancer cells to irradiation. Cancer Chemother Pharmacol 77:963–972 [DOI] [PubMed] [Google Scholar]
  • 38.Zhang H, Wu X, Nie J, Li X, Li C, Li H (2025) The METTL14-YTHDF1-SAP30 axis promotes Glycolysis and oxaliplatin resistance in colorectal adenocarcinoma via m6A modification. J Gastroenterol Hepatol 40(7):1786–1796 [DOI] [PubMed] [Google Scholar]
  • 39.Liang Y, An R, Du P, Wei Y, Liu S, Zheng J, Lei P, Zhang H (2025) NIR-Activated Hollow upconversion nanocomposites for tumor therapy via GLUT1 Inhibition and mitochondrial function disruption. ACS Appl Mater Interfaces 17(14):20849–20858 [DOI] [PubMed] [Google Scholar]
  • 40.Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, Zoccola M, Pagnotta S, Carrozzo R, de Bot ST, Perluigi M, van Roon-Mom WMC, Squitieri F (2023) GLUT-1 changes in paediatric huntington disease brain cortex and fibroblasts: an observational case-control study. EBioMedicine 97:104849 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Boström P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC (2012) A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481(7382):463–468 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Rajesh R, Atallah R, Bärnthaler T (2023) Dysregulation of metabolic pathways in pulmonary fibrosis. Pharmacol Ther 246:108436 [DOI] [PubMed] [Google Scholar]

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