Impacts of rat hindlimb Fndc5/irisin overexpression on muscle and adipose tissue metabolism

Abstract

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D₂O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.

Keywords: FNDC5; glucose metabolism; irisin; muscle; overexpression.

Fig. 1.

Study schematic. 2DG, 2-deoxy-glucose (6 mg/kg); Ctrl, control group; D₂O, deuterium (7.2 mL/kg); Fndc5, fibronectin type III domain-containing 5; IPGTT, intraperitoneal glucose tolerance test (2 g/kg); IVE, in vitro electroporation; OE, overexpressed group.

Fig. 2.

Fibronectin type III domain-containing 5 (Fndc5) expression and irisin levels in control (Ctrl; n = 8) and Fndc5 overexpression (OE; n = 8) animals. A: Fndc5 mRNA in tibialis cranialis (TC) muscle (P = 0.014). B: Fndc5 protein expression in TC (P = 0.036). C: plasma irisin concentration (P = 0.049). Values are means ± SD. Statistical analysis was via unpaired t test. *P < 0.05 vs. Ctrl. Molecular mass is in kilodaltons.

Fig. 3.

Glycogen content and glucose uptake in control (Ctrl; n = 8) and fibronectin type III domain-containing 5 overexpression (OE; n = 8) animals. A: tibialis cranialis (TC) muscle glycogen content (P = 0.04). B: glycogen synthase 1 (GYS1) gene expression (P = 0.03) and phosphorylated glycogen synthase kinase-3α/β (P-GSK3 α/β) Ser21/9 protein (P = 0.007). C: glycogen synthase (GS) activation (i.e., P-GS divided by GS protein expression; P = 0.048). D: intraperitoneal glucose tolerance test (IPGTT). E: phosphorylated 5-AMP-activated protein kinase (P-AMPK) Thr172, P-P38 MAPK Thr180, and P-acetyl-CoA carboxylase (P-ACC) Ser79. F: P-AS160 Thr642 and glucose transporter 4 (GLUT4) protein expression. G: TC muscle 2-deoxy-d-glucose-6-phosphate (2DG6P). H: 2-deoxy-d-glucose (2DG) fractional uptake (i.e., 2DG6P divided by area under the curve of 2DG plasma). Values are means ± SD. Statistical analysis was via unpaired t test. *P < 0.05, **P < 0.01 vs. Ctrl. Molecular mass is in kilodaltons. AU, arbitrary units; T, total.

Fig. 4.

Markers of mitochondrial biogenesis and density in tibialis cranialis muscle. A: mitochondrial biogenesis regulatory genes: peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (TFAM), and nuclear respiratory factors (NRF). B: gene expression of uncoupling proteins 2 and 3 (UCP2/3). C: ATP synthase genes ATP5J2 and ATP2A3. D: mitochondrial density represented by citrate synthase (CS) activity. E: protein levels of mitochondrial biogenesis markers: PGC-1α, cytochrome c, succinate dehydrogenase enzyme A (SDHA), heat shock protein 60 (HSP60), and voltage-dependent anion channel (VDAC). F: electron (e-) transporter complexes (C) of mitochondria: CI NADH dehydrogenase [ubiquinone] 1β subunit 8 (NDUFB8), CII succinate dehydrogenase subunit B (SDHB), CIII ubiquinone cytochrome c reductase complex (UQCRC2), CIV mitochondria encoded cytochrome c oxidase (MTCO1), and CV mitochondria membrane ATP synthase 5A (ATP5A). Values are means ± SD. Statistical analysis was via unpaired t test. No statistically significant differences were observed. Molecular mass is in kilodaltons. Ctrl, control group; OE, overexpressed group.

Fig. 5.

A: gene expression of muscle regulatory growth factors and insulin-like growth factor-1 (IGF-1; P = 0.0002) in tibialis cranialis muscle. B: fractional synthesis rate (FSR) in myofibrillar and sarcoplasmic muscle fractions. C and D: protein expression of proteins involved in anabolic signaling: phosphorylated (P-) protein kinase B (Akt) ^Ser473, P-ERK ^{Thr202/Tyr204}, P-proline-rich Akt substrate of 40 kDa (P-PRAS40 ^Thr246), P-mechanistic target of rapamycin (P-mTOR ^Ser2448), P-ribosomal protein S6 kinase 1 (P-P70 S6K1 ^Thr389), P-4E-binding protein 1 (P-4EBP1 ^Ser65/Thr70), and P-eukaryotic initiation factor 4E (P-eIF4E ^Ser209). Values are means ± SD. Statistical analysis was via unpaired t test. ***P < 0.001 vs. control group (Ctrl). Molecular mass is in kilodaltons. MyoG, myogenin; OE, overexpressed group.

Fig. 6.

Gene expression levels in tibialis cranialis muscle of the following. A: negative growth regulatory factors: myostatin (MSTN), follistatin, and regulated in development and DNA damage response 1 (REDD1). B: apoptotic factors: B-cell lymphoma 2 (BCL-2) apoptosis suppressor and BCL-2-associated X (BAX) proapoptotic factor. C: autophagy-related genes: Atg7, Atg5, and cathepsin L. D: calpain factors: cysteine-aspartic acid protease (caspase-3) and m-calpain. E and F: protein expression of autophagy signaling pathway components: phosphorylated tuberin or tumor suppressor 2 (P-TSC2), phosphatidylinositol 3-kinase class III (PI3K III), beclin-1, autophagy marker light chain 3 (LC3B), atrogin-1, and muscle RING-finger protein-1 (Murf-1). Values are means ± SD. Statistical analysis was via unpaired t test. No statistically significant differences were observed. Molecular mass is in kilodaltons. Ctrl, control group; OE, overexpressed group.

Fig. 7.

Markers of mitochondrial biogenesis in subcutaneous fat. A: peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factors (NRF1), and mitochondrial transcription factor A (TFAM). B: uncoupling protein 1 (UCP1) and cell death activator A (CIDEA). Values are means ± SD. Statistical analysis was via unpaired t test. No statistically significant differences were observed. Ctrl, control group; OE, overexpressed group.