This study explored the role of irisin as a new pancreatic β-cell secretagogue and survival factor and its potential role in the communication between skeletal muscle and pancreatic β-cells under lipotoxic conditions. Recombinant irisin stimulated insulin biosynthesis and glucose-stimulated insulin secretion (GSIS) in a PKA-dependent manner and prevented saturated fatty acid–induced apoptosis in human and rat pancreatic β-cells, as well as in human and murine pancreatic islets, via AKT/BCL2 signaling. Treatment of myotubes with 0.5 mmol/L palmitate for 4 h, but not with oleate, promoted an increase in irisin release in the culture medium. Moreover, increased serum levels of irisin were observed in mice fed with a high-fat diet. Mouse serum rich in irisin and the conditioned medium from myotubes exposed to palmitate for 4 h significantly reduced apoptosis of murine pancreatic islets and insulin-secreting INS-1E cells, respectively, and this was abrogated in the presence of an irisin-neutralizing antibody. Finally, in vivo administration of irisin improved GSIS and increased β-cell proliferation. In conclusion, irisin can promote β-cell survival and enhance GSIS and may thus participate in the communication between skeletal muscle and β-cells under conditions of excess saturated fatty acids.
Irisin is a newly discovered muscle-derived hormone, produced by cleavage of the membrane protein fibronectin type III domain–containing protein 5 (FNDC5) and proposed to bridge exercise with metabolic homeostasis (1). Irisin levels correlate with markers of insulin resistance in subjects without diabetes, while they are reduced in overt type 2 diabetes (2,3). The biological functions of irisin include effects on multiple tissues (1,4–7). Irisin also improves glucose tolerance and insulin sensitivity and increases energy expenditure in both obese and diabetic mice (8). Irisin increases proliferation of insulin-secreting INS-1E cells and protects them from high glucose–induced apoptosis (9).
If chronically in excess, saturated free fatty acids (FFAs) can reduce insulin biosynthesis (10) and secretion (11) and promote β-cell apoptosis (12,13). Excessively high plasma levels of FFAs, particularly long-chain saturated FFAs, also foster insulin resistance (14). Palmitate can directly impair insulin signaling in skeletal muscle cells (15) and alter the expression of some myokines (16).
Skeletal muscle, the major site of insulin-stimulated glucose disposal (17), has recently been identified as a secretory organ able to release myokines. It is thus plausible that this organ might interact with the endocrine pancreas by releasing myokines that adjust insulin secretion to the actual insulin need for appropriate peripheral glucose utilization (18,19). In this study, we have investigated the effects of irisin on pancreatic β-cells and the release of this myokine by skeletal muscle cells.
Research Design and Methods
Animal experimentations were conducted after approval by the Ethics Committee (CESA) of Istituto di Ricerche Genetiche “Gaetano Salvatore” (IRGS), Biogem, Italy (internal ID 0907) in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1985) and regulations of Italy and the EU. CD-1 and C57BL/6 mice were purchased from Charles River Laboratories (Calco, Lecco, Italy). The high-fat diet (HFD) (60% of energy from fat) was from Mucedola (Settimo Milanese, Milan, Italy) (formulation given in Supplementary Table 1).
In Vivo Irisin Administration
Eighteen male 6-week-old C57BL/6 mice were randomized into three groups: 6 mice were immediately sacrificed; the remaining mice were intraperitoneally injected daily with irisin (0.5 μg/g body wt) or vehicle (PBS) for 14 days. Intraperitoneal glucose tolerance tests were carried out, and then animals were sacrificed. Pancreas, serum, and plasma were collected.
Pancreatic Islets and Cell Culture
Mouse and human islets were isolated and cultured as previously described (20,21). More details on cell lines and culture conditions are given in Supplementary Data.
Palmitate and oleate were obtained from Sigma-Aldrich (St. Louis, MO) and prepared as previously reported (13). All controls were treated with the same volume of 10% fatty acid–free BSA solution without palmitate or oleate.
Cells were preincubated with recombinant irisin (AdipoGen SA, Liestal, Switzerland) for the indicated doses and times. The chemical inhibitors AKT Inhibitor VIII (2.5 μmol/L) or H-89 (5 μmol/L) (Calbiochem; Merck Millipore, Darmstadt, Germany) were added 30 min or 60 min before irisin.
Cells lysates were obtained and analyzed by immunoblotting as previously described (13). A list of the antibodies used is shown in Supplementary Table 2.
Gene Expression Analysis by Quantitative RT-PCR
RNA isolation, cDNA synthesis, and mRNA quantitation were carried out as previously described (13). A list of the primer sequences used is shown in Supplementary Table 3.
Immunofluorescence and Immunohistochemistry
Immunofluorescence and immunohistochemistry were carried out as previously described (13,22). More details are given in Supplementary Data.
Apoptosis and BrdU Assays
Apoptosis was measured by using the Cell Death Detection ELISAPLUS Kit and In Situ Cell Death Detection Kit and TMR red (Roche Biochemicals, Indianapolis, IN) and by assessing caspase-3 cleavage. Proliferation was assessed with the BrdU ELISA kit (colorimetric) (Abcam, Cambridge, U.K.).
Irisin and Insulin Assays
Irisin concentrations were measured using an irisin-competitive ELISA kit (AdipoGen SA, Liestal, Switzerland). Insulin levels were measured by specific ELISA (Mercodia AB, Sylveniusgatan, Uppsala, Sweden). Glucose-stimulated insulin secretion (GSIS) was performed as previously described (13). More details are given in Supplementary Data.
Data were analyzed by the Student t test or ANOVA, as appropriate, and are presented as mean ± SD. Statistical significance was set at P value <0.05.
Irisin Prevents Palmitate-Induced β-Cell Apoptosis and Induces Insulin Secretion
INS-1E cells treated with 0.5 mmol/L palmitate for 24 h showed increased caspase-3 cleavage, as expected. However, pretreatment with recombinant irisin for 24 h reduced this response in a dose-dependent manner. A similar effect was observed in human pancreatic 1.1B4 cells and in murine and human pancreatic islets (Fig. 1A and B). Immunofluorescence of murine islets confirmed that β-cells showed increased apoptosis in response to palmitate and that this was counteracted by irisin (Fig. 1C). Since treatment of INS-1E cells with irisin increased AKT phosphorylation (Fig. 1D), which is known to upregulate B-cell lymphoma 2 (BCL2) expression (23), we investigated whether irisin prevented palmitate-induced apoptosis through a mechanism involving BCL2 and BCL2-associated X protein (BAX)—key apoptosis regulatory proteins. While palmitate increased BAX expression and, consequently, reduced the BCL2-to-BAX ratio, pretreatment with irisin increased BCL2 expression and restored the BCL2-to-BAX ratio (Fig. 1E). When INS-1E cells were pretreated with the AKT Inhibitor VIII, irisin-mediated AKT phosphorylation was abrogated, and augmentation of BCL2 protein expression and inhibition of palmitate-induced apoptosis were prevented (Fig. 1F and G).
Exposure of human and murine islets and rat INS-1E cells to irisin also increased insulin (Ins) mRNA levels and insulin content and augmented GSIS (Fig. 2A–K). These responses were dependent on PKA activation, since they were abrogated by the PKA inhibitor H-89, as was the irisin-induced CREB phosphorylation at 25 mmol/L glucose (Fig. 2H–L). Moreover, irisin increased proliferation of INS-1E cells and human pancreatic 1.1B4 cells (Supplementary Fig. 1).
Irisin Is Secreted in Response to Saturated FFAs and Promotes β-Cell Survival
Treatment of rat myotubes with palmitate for 4 h resulted in increased FNDC5 mRNA levels and protein content and caused a threefold higher release of irisin in the culture medium compared with untreated myotubes. By contrast, in myotubes treated with palmitate for 24 h, FNDC5 mRNA levels and protein content were significantly reduced and irisin release in the culture medium was unchanged (Fig. 3A–C). Validation of the ELISA kit and irisin antibody is shown in Supplementary Fig. 2. Oleate, a monounsaturated FFA, did not stimulate irisin release (Supplementary Fig. 3). Human myotubes showed a similar regulation of irisin release by palmitate (Fig. 3E), even though FNDC5 mRNA levels were unchanged (Fig. 3D). Furthermore, treatment with a small interfering RNA of FNDC5 prevented the palmitate-induced increase of FNDC5 protein expression, while irisin release was unaffected (Supplementary Fig. 4).
Plasma irisin levels were also measured in vivo in CD-1 mice fed with a standard diet (SD) or HFD for 63 days. The HFD caused a rapid increase of blood irisin concentrations, which persisted at later times (Fig. 3F). Interestingly, murine pancreatic islets cultured in the presence of serum from mice fed with an HFD containing high levels of irisin were protected from palmitate-induced apoptosis; the protective effect was attenuated in the presence of a neutralizing antibody against irisin (Fig. 3G), which inhibited the irisin-induced increase of UCP-1 expression in 3T3-L1 adipocytes (Supplementary Fig. 5). Moreover, culture medium was collected from L6 myotubes exposed to 0.5 mmol/L palmitate for 4 h (PM4h) or not (PM0h), and the effects on β-cell apoptosis were evaluated. Samples of L6 myotube-free culture medium with (CTR2) or without (CTR1) palmitate were used as additional controls. CTR2 increased INS-1E apoptosis, as expected, while apoptosis was significantly reduced when INS-1E cells were exposed to PM4h, and this effect was abrogated by the neutralizing antibody directed against irisin (Fig. 3H).
In Vivo Irisin Administration Improves Insulin Secretion and Glucose Tolerance and Increases the Pancreatic β-Cell Mass
Intraperitoneal injection of irisin for 14 days in C57BL/6 mice resulted in a 1.5-fold increase in irisin plasma levels compared with vehicle (Fig. 4A). Furthermore, in irisin- compared with vehicle-treated mice, insulin secretion was enhanced 90 min after intraperitoneal glucose injection, and this resulted in a faster decrease of glucose levels (Fig. 4B). In vivo dosing of irisin significantly augmented the β-cell area and slightly reduced the α-cell area within pancreatic islets, thus increasing the β-cell–to–α-cell ratio (Fig. 4C). Moreover, insulin content and β-cell proliferation in the islets were markedly increased after irisin administration (Fig. 4C and D).
Here, we demonstrate for the first time that recombinant irisin protects human and rodent β-cells and islets from palmitate-induced apoptosis through a mechanism involving AKT/BCL2 signaling (Fig. 1). Similarly, in the human umbilical vein endothelial cells and INS-1E cells irisin partially suppressed high glucose–induced apoptosis by affecting BCL2, BAX, and caspase expression (7,9). In vivo administration of irisin also improves GSIS and increases insulin content and β-cell mass and proliferation (Fig. 4). Furthermore, irisin promotes insulin biosynthesis and secretion in a PKA-dependent manner (Fig. 2). The beneficial effects of irisin on β-cells appear to be glucose dependent and comparable with those of glucagon-like peptide 1 and its analogs (24). Like glucagon-like peptide 1, irisin induces cyclic AMP generation (data not shown) and activates PKA and AKT, suggesting the existence of a specific receptor that has not been yet identified.
Short-term treatment of rat myotubes with palmitate results in increased FNDC5 mRNA levels and protein content and raises irisin levels in the culture medium threefold compared with control (Fig. 3A–C). The mechanism by which saturated FFAs induce irisin release from skeletal muscle cells is still unclear and may involve the activation of FNDC5 cleavage by specific protease(s). In human myotubes, higher irisin release in the culture medium in response to palmitate occurred in the absence of changes in FNDC5 mRNA levels (Fig. 3D and E). Furthermore, in rat myotubes, FNDC5 knockdown prevented the palmitate-induced increase of FNDC5 protein expression without affecting irisin release (Supplementary Fig. 4). Altogether, these results suggest independent regulation of FNDC5 expression and cleavage by FFAs. Irisin release from myotubes is also influenced by the type of FFA, since it is not stimulated by oleate, a monounsaturated FFA (Supplementary Fig. 3).
Mice receiving an HFD and gaining weight display an early increase in plasma irisin levels that persists thereafter (Fig. 3F). This is in line with the observed positive correlation between serum irisin levels and BMI, adiposity markers, and HOMA of insulin resistance index in humans (3). Adipose tissue also secretes irisin (25); however, ∼72% of circulating irisin is estimated to be released by skeletal muscle (1). The availability of tissue-specific FNDC5 knockout mice will help to address this issue.
We could not target the actions of irisin on pancreatic β-cells in vivo because of lack of identification of the irisin receptor and difficulty in neutralizing circulating irisin in mice. However, the serum from HFD-fed mice, containing high levels of irisin, protected murine pancreatic islets against palmitate-induced apoptosis, and this was largely abrogated in the presence of a neutralizing antibody against irisin (Fig. 3G). This is in line with the observation that myotube-derived irisin in the conditioned medium also prevented apoptosis of β-cells (PM4h) (Fig. 3H). However, in contrast to recombinant irisin, PM4h did not enhance GSIS (Supplementary Fig. 6). Since irisin elicits distinct biological effects at different doses (1,4–7), it is possible that higher irisin concentrations than those in PM4h are required to enhance GSIS. Alternatively, other myokines in PM4h could counteract the action of irisin on insulin secretion.
In conclusion, the myokine irisin has antiapoptotic actions on pancreatic β-cells and stimulates β-cell proliferation, insulin biosynthesis, and secretion. Under conditions of excess saturated FFAs, irisin release from skeletal muscle may be promoted as an adaptive response, signaling to the endocrine pancreas to compensate for the increased insulin resistance and initial deterioration of glucose tolerance.
Acknowledgments. The authors thank Dr. Paola Pontrelli, Dr. Chiara Divella, and Dr. Michele Lauriero (Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari Aldo Moro, Bari, Italy) for technical assistance in immunohistochemistry experiments and confocal images acquisition.
Funding. This work is supported by a grant from Ager - Agroalimentare e ricerca (Claims of Olive oil to iMProvE The market ValuE of the product [COMPETITIVE]) to F.G.
Duality of Interest. A.N., N.M., and F.G. are named inventors of a pending patent application related to the work described. F.G. has received grant support from Takeda, Eli Lilly, and LifeScan. L.L. is a consultant for and has received lecture fees from AstraZeneca, Eli Lilly, Boehringer Ingelheim, Takeda, Sanofi, Novo Nordisk, Roche, Medtronic, and Jannsen. F.G. is a consultant for and has received lecture fees from AstraZeneca, Eli Lilly, Novo Nordisk, Roche, Sanofi, and Takeda. No other potential conflicts of interest relevant to this article were reported.
Author Contributions. A.N., N.M., and F.G. designed the study. G.B. and R.S. acquired data. N.M., G.B., R.L., and I.P. performed experiments and data analysis. A.N., N.M., and G.B. performed data analysis and interpretation. G.B., R.S., R.L., A.C., and M.B. participated in interpretation and discussion of data. A.N., N.M., and F.G. wrote the manuscript. P.M., S.P., and L.L. critically reviewed the manuscript for intellectual content. All the authors approved the final version of the manuscript. F.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Prior Presentation. Parts of this study were presented in abstract form at the 76th Scientific Sessions of the American Diabetes Association, New Orleans, LA, 10–14 June 2016, and at the 52nd Annual Meeting of the European Association for the Study of Diabetes, Munich, Germany, 12–16 September 2016.