Growth Hormone Receptor Gene Is Essential for Chicken Mitochondrial Function in Vivo and in Vitro

International Journal of Molecular Sciences

Article Growth Hormone Receptor Gene is Essential for Chicken Mitochondrial Function In Vivo and In Vitro

Bowen Hu 1,2 , Shuang Hu 1,2, Minmin Yang 1,2, Zhiying Liao 1,2, Dexiang Zhang 1,2, Qingbin Luo 1,2, Xiquan Zhang 1,2,* and Hongmei Li 1,2,*

1 Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; [email protected] (B.H.); [email protected] (S.H.); [email protected] (M.Y.); [email protected] (Z.L.); [email protected] (D.Z.); [email protected] (Q.L.) 2 Guangdong Provincial Key Lab of AgroAnimal Genomics and Molecular Breeding and Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, Guangzhou 510642, China * Correspondence: [email protected] (X.Z.); [email protected] (H.L.)

Received: 7 March 2019; Accepted: 28 March 2019; Published: 31 March 2019

Abstract: The growth hormone receptor (GHR) gene is correlated with many phenotypic and physiological alternations in chicken, such as shorter shanks, lower body weight and muscle mass loss. However, the role of the GHR gene in mitochondrial function remains unknown in poultry. In this study, we assessed the function of mitochondria in sex-linked dwarf (SLD) chicken skeletal muscle and interfered with the expression of GHR in DF-1 cells to investigate the role of the GHR gene in chicken mitochondrial function both in vivo and in vitro. We found that the expression of key regulators of mitochondrial biogenesis and mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (OXPHOS) genes were downregulated and accompanied by reduced enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle and GHR knockdown cells. Then, we assessed mitochondrial function by measuring mitochondrial membrane potential (∆Ψm), mitochondrial swelling, reactive oxygen species (ROS) production, malondialdehyde (MDA) levels, ATP levels and the mitochondrial respiratory control ratio (RCR), and found that mitochondrial function was impaired in SLD chicken skeletal muscle and GHR knockdown cells. In addition, we also studied the morphology and structure of mitochondria in GHR knockdown cells by transmission electron microscopy (TEM) and MitoTracker staining. We found that knockdown of GHR could reduce mitochondrial number and alter mitochondrial structure in DF-1 cells. Above all, we demonstrated for the ﬁrst time that the GHR gene is essential for chicken mitochondrial function in vivo and in vitro.

Keywords: growth hormone receptor; mitochondrial function; sex-linked dwarf chicken; skeletal muscle; DF-1 cells

1. Introduction Mitochondria are dynamic organelles with a crucial role in cellular energy homeostasis and metabolism, with the generation of adenosine triphosphate (ATP) through the respiratory chain (RC) being one of their main functions [1]. The RC is composed of four complexes (I–IV) embedded in the inner mitochondrial membrane which are uniquely controlled by mitochondrial DNA (mtDNA) and the nuclear genome [2]. Similar to mammalian mtDNA, chicken mtDNA determines 37 gene products, of which 13 are oxidative phosphorylation (OXPHOS) subunits (complex I, III, IV and V) [3]. In addition, mitochondrial biogenesis is essential for its function, which is mainly regulated by nuclear genes through the PGC1α–NRF1–TFAM signaling pathway [4]. Peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), the master regulator of mitochondrial biogenesis, can activate the

Int. J. Mol. Sci. 2019, 20, 1608; doi:10.3390/ijms20071608 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 1608 2 of 13 expression of nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (TFAM) to regulate mtDNA replication and transcription in mammals [5]. Growth hormone (GH) and growth hormone receptor (GHR) come together in the GH–GHR–IGF1 signaling pathway to influence mitochondrial function in mammals. Growth hormone can regulate mitochondrial function through the Box 1 region of the GHR gene in CHO cells [6], and several alterations in OXPHOS complexes have been observed in GH-deficient Ames mice [7]. Knockout of GHR (GHRKO) in mice could also alter the expression of key regulators of mitochondrial biogenesis [8,9]. Furthermore, previous studies have reported that insulin-like growth factor 1 (IGF1) signaling can regulate mitochondrial biogenesis markers in the steroidogenic cells of prepubertal testis [10], and is essential for mitochondrial biogenesis in cancer cells [11]. These findings suggest that the GHR gene plays a pivotal role in mammalian mitochondrial function both in vivo and in vitro. Sex-linked dwarf (SLD) chickens, which are characterized by diverse mutations in GHR [12], are feasible models for understanding the role of GHR in poultry [13]. Previous studies have reported that these mutations in GHR can result in many phenotypic and physiological alterations in chicken, such as shorter shanks, lower body weight and muscle mass loss [14–16]. However, the role of GHR in mitochondrial function in poultry remains unknown. Based on the abovementioned findings, we assessed the function of mitochondria in SLD chicken skeletal muscle and interfered with the expression of GHR in DF-1 cells to investigate the role of GHR in chicken mitochondrial function both in vivo and in vitro. Here, we demonstrate for the first time that GHR is essential for chicken mitochondrial function both in vivo and in vitro.

2. Results

2.1. Low Expression of Key Regulators of Mitochondrial Biogenesis and mtDNA-Encoded OXPHOS Genes in SLD Chicken Skeletal Muscle In this study, we first assessed the relative mRNA expression of genes involved in the GH–GHR–IGF1 signaling pathway by qRT-PCR. The relative mRNA levels of the GH gene were not significantly different, however, for GHR and IGF1, were significantly downregulated in SLD chicken skeletal muscle compared with normal chicken skeletal muscle, indicating the low level of GH binding activity in SLD chicken skeletal muscle (Figure1A). In order to investigate the role of GHR in mitochondrial biogenesis in vivo, we assessed the relative mRNA expression of the genes involved in the PGC1α–NRF1–TFAM signaling pathway using qRT-PCR. We found the relative mRNA expression of PGC1α, NRF1, and TFAM to all be significantly downregulated in SLD chicken skeletal muscle compared with normal chicken skeletal muscle (Figure1B), indicating an inhibition of mitochondrial biogenesis in SLD chicken skeletal muscle. Since TFAM can regulate the transcription of mtDNA, we then determined the relative mRNA expression of mtDNA-encoded OXPHOS genes. We found that the expression of mtDNA-encoded OXPHOS genes was also downregulated in SLD chicken skeletal muscle compared with normal chicken skeletal muscle, indicating that mtDNA transcription was inhibited in SLD chicken skeletal muscle; however, we did not observe a significant difference in CYTB expression (Figure1C). Taken together, we found low expression of key regulators of mitochondrial biogenesis and mtDNA-encoded OXPHOS genes in SLD chicken skeletal muscle. Int. J. Mol. Sci. 2019, 20, 1608 3 of 13 Int. J. Mol. Sci. 2019, 20, x 3 of 13 Int. J. Mol. Sci. 2019, 20, x 3 of 13

Figure 1. Low expression of key regulators of mitochondrial biogenesis and mtDNA-encoded oxidative Figure 1. Low expression of key regulators of mitochondrial biogenesis and mtDNA-encoded phosphorylationFigure 1. Low (OXPHOS) expression genes of key in sex-linked regulators dwarf of mitochondrial (SLD) chicken biogenesis skeletal and muscle. mtDNA-encoded (A) The relative oxidative phosphorylation (OXPHOS) genes in sex-linked dwarf (SLD) chicken skeletal muscle. (A) mRNAoxidative expression phosphorylation of genes involved(OXPHOS) in genes the GH–GHR–IGF1in sex-linked dwarf signaling (SLD) chicken pathway skeletal was muscle. measured (A) by The relative mRNA expression of genes involved in the GH–GHR–IGF1 signaling pathway was qRT-PCRThe relativein SLD mRNAchicken expression skeletal muscle of genes as involved compared in with the GH–GHR–IGF1 normal chicken signaling skeletal pathway muscle. was (B) The measured by qRT-PCR in SLD chicken skeletal muscle as compared with normal chicken skeletal relativemeasured mRNA by expression qRT-PCR involvedin SLD chicken in the skeletal PGC1α –NRF1–TFAMmuscle as compared signaling with normal pathway chicken was measured skeletal by muscle. (B) The relative mRNA expression involved in the PGC1α–NRF1–TFAM signaling pathway qRT-PCRmuscle. in SLD(B) The chicken relative skeletal mRNA muscleexpression as comparedinvolved in withthe PGC1α–NRF1–TFAM normal chicken skeletal signaling muscle. pathway (C) The was measured by qRT-PCR in SLD chicken skeletal muscle as compared with normal chicken skeletal was measured by qRT-PCR in SLD chicken skeletal muscle as compared with normal chicken skeletal relativemuscle. mRNA (C) expressionThe relative ofmRNA mtDNA-encoded expression of mtDNA-encoded OXPHOS genes OXPHOS was measured genes was by qRT-PCRmeasured inby SLD muscle. (C) The relative mRNA expression of mtDNA-encoded OXPHOS genes was measured by chickenqRT-PCR skeletal in muscleSLD chicken as compared skeletal muscle with normal as compared chicken with skeletal normal muscle. chicken Data skeletal are muscle. expressed Data as are means qRT-PCR in SLD chicken skeletal muscle as compared with normal chicken skeletal muscle. Data are ± SEM,expressed * p < 0.05; as means ** p < ± 0.01; SEM, *** * pp < <0.05; 0.001. ** p < 0.01; *** p < 0.001. expressed as means ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. 2.2. Reduced2.2. Reduced Enzymatic Enzymatic Activity Activity of OXPHOSof OXPHOS Complexes Complexes in SLD Chicken Chicken Skeletal Skeletal Muscle Muscle 2.2. Reduced Enzymatic Activity of OXPHOS Complexes in SLD Chicken Skeletal Muscle To investigateTo investigate the role the of roleGHR of GHRon the on enzymatic the enzymatic activity activity of OXPHOS of OXPHOS proteins proteinsin vivo in , vivo, we assessed we To investigate the role of GHR on the enzymatic activity of OXPHOS proteins in vivo, we the enzymaticassessed the activity enzymatic of OXPHOS activity of complexesOXPHOS complexes in SLD chickenin SLD chicken skeletal skeletal muscle. muscle. We found We found that the assessed the enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle. We found that the enzymatic activities of OXPHOS complex I, II, III and IV were significantly reduced by 26%, enzymaticthat the activities enzymatic of activities OXPHOS of complex OXPHOS I, complex II, III and I, II, IV III were and IV signiﬁcantly were significantly reduced reduced by 26%, by 45%,26%, 53% 45%, 53% and 72% in SLD chicken skeletal muscle compared with normal chicken skeletal muscle, and 72%45%, in 53% SLD and chicken 72% in skeletalSLD chicken muscle skeletal compared muscle with compared normal with chicken normal skeletal chicken muscle, skeletal respectivelymuscle, respectively (Figure 2A–D), indicating the reduced enzymatic activity of OXPHOS complexes in SLD (Figurerespectively2A–D), indicating (Figure 2A–D), the reducedindicating enzymaticthe reduced activityenzymatic of activity OXPHOS of OXPHOS complexes complexes in SLD in SLD chicken chicken skeletal muscle. skeletalchicken muscle. skeletal muscle.

Figure 2. Reduced enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle. Figure 2. Reduced enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle. (A) Figure 2. Reduced enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle. (A) (A) EnzymaticEnzymatic activityactivity of complex I Iwas was measured measured by by the the change change in inabsorbance absorbance of NADH of NADH in SLD in SLDand and Enzymatic activity of complex I was measured by the change in absorbance of NADH in SLD and normalnormal chicken chicken skeletal skeletal muscle. muscle. (B ()B Enzymatic) Enzymatic activityactivity of of complex complex II IIwas was measured measured by the by change the change in in normal chicken skeletal muscle. (B) Enzymatic activity of complex II was measured by the change in absorbanceabsorbance of DCIP of DCIP in SLDin SLD and and normal normal chicken skeletal skeletal muscle. muscle. (C) (EnzymaticC) Enzymatic activity activity of complex of complex III absorbance of DCIP in SLD and normal chicken skeletal muscle. (C) Enzymatic activity of complex III III waswas measured measured by by the the change change inin absorbanceabsorbance of of reduced reduced cytochrome cytochrome c in c SLD in SLD and and normal normal chicken chicken was measured by the change in absorbance of reduced cytochrome c in SLD and normal chicken skeletalskeletal muscle. muscle. (D) ( EnzymaticD) Enzymatic activity activity of of complex complex IV was was measured measured by by the the change change in absorbance in absorbance of of skeletal muscle. (D) Enzymatic activity of complex IV was measured by the change in absorbance of reduced cytochrome c in SLD and normal chicken skeletal muscle. Data are expressed as means ± ± reducedreduced cytochrome cytochrome c in c SLDin SLD and and normal normal chicken chicken skeletalskeletal muscle. muscle. Data Data are are expressed expressed as means as means ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. SEM,SEM, * p < * 0.05; p < 0.05; ** p **< p 0.01; < 0.01; *** ***p

2.3. Impaired Mitochondrial Function in SLD Chicken Skeletal Muscle To further investigate the role of GHR in chicken mitochondrial function in vivo, we assessed mitochondrial function in SLD and normal chicken skeletal muscle by measuring mitochondrial Int. J. Mol. Sci. 2019, 20, x 4 of 13 Int. J. Mol. Sci. 2019, 20, 1608 4 of 13 2.3. Impaired Mitochondrial Function in SLD Chicken Skeletal Muscle To further investigate the role of GHR in chicken mitochondrial function in vivo, we assessed membranemitochondrial potential function (∆Ψm), in mitochondrial SLD and normal swelling, chicken skeletal reactive muscle oxygen by measuring species mitochondrial (ROS) production, malondialdehydemembrane potential (MDA) (ΔΨm),levels, ATPmitochondrial levels, and swelling, the mitochondrial reactive oxygen respiratory species (ROS) control production, ratio (RCR). ∆Ψm wasmalondialdehyde significantly reduced(MDA) levels, (Figure ATP3A) levels, and and accompanied the mitochondrial by a significant respiratory increase control ratio of mitochondrial (RCR). ΔΨm was significantly reduced (Figure 3A) and accompanied by a significant increase of swellingmitochondrial (Figure3B) inswelling SLD chicken(Figure 3B) skeletal in SLD musclechicken comparedskeletal muscle with compared normal with chicken normal skeletal chicken muscle. Reactiveskeletal oxygen muscle. species Reactive production oxygen was species significantly production was increased significantly (Figure increased3C) and (Figure accompanied 3C) and by a significantaccompanied increase by of a MDA significant level increase (Figure of3 MDAD) and level a (Figure significant 3D) and reduction a significant of ATPreduction level of(Figure ATP 3E) in SLD chickenlevel (Figure skeletal 3E) in muscle SLD chicken compared skeletal with muscle normal compared chicken with skeletalnormal chicken muscle. skeletal Consistently, muscle. RCR was significantlyConsistently, reduced RCR was in significantly SLD chicken reduced skeletal in SLD muscle chicken compared skeletal muscle with normalcompared chicken with normal (Figure 3F). chicken (Figure 3F). Results showed that mitochondrial function in SLD chicken skeletal muscle was Results showed that mitochondrial function in SLD chicken skeletal muscle was impaired. impaired.

Figure 3. Impaired mitochondrial function in SLD chicken skeletal muscle. (A) Mitochondrial ∆Ψm was measuredFigure 3. by Impaired the fluorescence mitochondrial of JC-1 function in SLD in SLD and chicken normal skeletal chicken muscle. skeletal (A) Mitochondrial muscle. Red fluorescenceΔΨm was measured by the fluorescence of JC-1 in SLD and normal chicken skeletal muscle. Red represents aggregation of JC-1, green fluorescence represents monomeric JC-1, and ∆Ψm was fluorescence represents aggregation of JC-1, green fluorescence represents monomeric JC-1, and ΔΨm representedwas represented as the ratio as of the aggregated ratio of aggregated and monomeric and monomeric JC-1. (B JC-1.) Mitochondrial (B) Mitochondrial swelling swelling was was measured by the absorbancemeasured by at the 540 absorbance nm of mitochondria at 540 nm of isolated mitochondria from isolated SLD and from normal SLD and chicken normal skeletal chicken muscle. (C) Reactiveskeletal oxygen muscle. species (C) Reactive (ROS) oxygen production species was (ROS) measured production by was the measured fluorescence by the of fluorescence dichlorofluorescein of (DCF) indichlorofluorescein SLD and normal (DCF) chicken in SLDskeletal and muscle. normal Thechicken level skeletal of (D ) muscle. malondialdehyde The level of (MDA) (D) and (E) ATPmalondialdehyde were measured (MDA) in SLD and and (E) ATP normal were chicken measured skeletal in SLD muscle.and normal (F chicken) Mitochondrial skeletal muscle. respiratory (F) Mitochondrial respiratory control ratio (RCR) was calculated as the ratio of state III to state IV control ratio (RCR) was calculated as the ratio of state III to state IV respiration rate in SLD and normal respiration rate in SLD and normal chicken skeletal muscle. Data are expressed as means ± SEM, *p < chicken0.05; skeletal ** p < muscle. 0.01; *** p Data < 0.001. are expressed as means ± SEM, *p < 0.05; ** p < 0.01; *** p < 0.001.

2.4. Knockdown2.4. Knockdown of GHR of GHR Downregulated Downregulated the the Expression Expression of Key Regulators Regulators of Mitochondrial of Mitochondrial Biogenesis Biogenesis and and mtDNA-EncodedmtDNA-Encoded OXPHOS OXPHOS Genes Genes in DF-1in DF-1 Cells Cells In orderIn to order determine to determine the role the ofroleGHR of GHRin chicken in chicken mitochondrial mitochondrial function function inin vitro, vitro ,we we interfered interfered with the expressionwith the of expressionGHR in DF-1of GHR cells. in DF-1 Transfection cells. Transfection efficiency efficiency was measured was measured by the by the fluorescence fluorescence intensity of FAMintensity siRNAand of FAMqRT-PCR. siRNA The and expression qRT-PCR. of The the expressionGHR gene ofwas the significantly GHR gene downregulated was significantly in GHR knockdowndownregulated cells, indicating in GHR that knockdown we had cells, successfully indicating interfered that we had with successfully the expression interfered of the withGHR the gene in expression of the GHR gene in DF-1 cells (Figure 4A,B). Then, we detected the relative mRNA DF-1 cellsexpression (Figure 4 ofA,B). the Then,genes involved we detected in the the GH–GHR–IGF1 relative mRNA signaling expression pathway. of the We genes found involved that the in the GH–GHR–IGF1expression signaling of GH was pathway. not significantly We found different that in the GHR expression knockdown of GHcells,was however, not significantly the expression different in GHRofknockdown IGF1 was significantly cells, however, downregulated the expression in GHR knockdown of IGF1 was cells, significantly indicating the downregulated low level of GH in GHR knockdown cells, indicating the low level of GH binding activity in GHR knockdown cells (Figure4B). In order to investigate the role of GHR in mitochondrial biogenesis in vitro, we then detected the expression of key regulators of mitochondrial biogenesis and mtDNA-encoded OXPHOS genes in DF-1 cells. PGC1α, NRF1, TFAM, and mtDNA-encoded OXPHOS genes were all downregulated in GHR knockdown cells, but we did not observe a significant difference in ND6 and ATP8 expression (Figure4C,D). Taken together, we demonstrated that knockdown of GHR downregulated the expression of key regulators of mitochondrial biogenesis and mtDNA-encoded OXPHOS genes in DF-1 cells. Int. J. Mol. Sci. 2019, 20, x 5 of 13

binding activity in GHR knockdown cells (Figure 4B). In order to investigate the role of GHR in mitochondrialInt. J. Mol. Sci. biogenesis 2019, 20, x in vitro, we then detected the expression of key regulators of mitochondrial 5 of 13 biogenesis and mtDNA-encoded OXPHOS genes in DF-1 cells. PGC1α, NRF1, TFAM, and mtDNA- binding activity in GHR knockdown cells (Figure 4B). In order to investigate the role of GHR in encoded OXPHOS genes were all downregulated in GHR knockdown cells, but we did not observe mitochondrial biogenesis in vitro, we then detected the expression of key regulators of mitochondrial a significantbiogenesis difference and mtDNA-encoded in ND6 and ATP8 OXPHOS expression genes in (FigureDF-1 cells. 4C,D). PGC1α Taken, NRF1 together,, TFAM, weand demonstrated mtDNA- that knockdownencoded OXPHOS of GHR genes downregulated were all downregulated the expression in GHR of knockdownkey regulators cells, of but mitochondrial we did not observe biogenesis and mtDNA-encodeda significant difference OXPHOS in ND6 genesand ATP8 in DF-1 expression cells. (Figure 4C,D). Taken together, we demonstrated Int. J. Mol. Sci. 2019, 20, 1608 5 of 13 that knockdown of GHR downregulated the expression of key regulators of mitochondrial biogenesis and mtDNA-encoded OXPHOS genes in DF-1 cells.

Figure 4. Knockdown of GHR reduced the GH binding activity and downregulated the expression of Figure 4. Knockdown of GHR reduced the GH binding activity and downregulated the expression of keyFigure regulators 4. Knockdown of mitochondrial of GHR reduced biogenesis the andGH mtDNA-encodedbinding activity and genes downregulated in DF-1 cells. the ( Aexpression) Transfection of key regulators of mitochondrial biogenesis and mtDNA-encoded genes in DF-1 cells. (A) Transfection key regulators of mitochondrial biogenesis and mtDNA-encoded genes in DF-1 cells. (A) Transfection efficiencyefficiency was measured was measured by theby the fluorescence fluorescence intensityintensity of of FAM FAM siRNA siRNA and andqRT-PCR qRT-PCR at 48 h at after 48 h after efficiency was measured by the fluorescence intensity of FAM siRNA and qRT-PCR at 48 h after transfectiontransfection with si-GHR with si-GHR and and si-NC si-NC fragments fragments in in DF-1 DF-1 cells. cells. Bar, Bar, 100 100 μm.µ Them. relative The relative mRNA mRNA transfection with si-GHR and si-NC fragments in DF-1 cells. Bar, 100 μm. The relative mRNA expressionexpression of genes of genes involved involved in the in the GH–GHR–IGF1 GH–GHR–IGF1 signaling pathway pathway (B) (andB) andPGC1α–NRF1–TFAM PGC1α–NRF1–TFAM signalingexpressionsignaling pathway of genespathway (C involved) were (C) were measured in measured the GH–GHR–IGF1 by by qRT-PCR qRT-PCR signaling at 48 48 h h after after pathway transfection transfection (B) withandwith PGC1α–NRF1–TFAMsi-GHR si-GHR and si-NC and si-NC signaling pathway (C) were measured by qRT-PCR at 48 h after transfection with si-GHR and si-NC fragmentsfragments in DF-1 in cells.DF-1 cells. (D) mtDNA(D) mtDNA transcription transcription waswas measured measured by by qRT-PCR qRT-PCR at 48 at h after 48 h transfection after transfection fragmentswith si-GHR in DF-1 and cells. si-NC (D) fragments mtDNA transcriptionin DF-1 cells. Data was aremeasured expressed by as qRT-PCR means ± SEM, at 48 *h p after < 0.05; transfection ** p < with si-GHR and si-NC fragments in DF-1 cells. Data are expressed as means ± SEM, * p < 0.05; with si-GHR0.01; *** pand < 0.001. si-NC fragments in DF-1 cells. Data are expressed as means ± SEM, * p < 0.05; ** p < ** p < 0.01; *** p < 0.001. 0.01; *** p < 0.001. 2.5. Knockdown of GHR Reduced the Enzymatic Activity of OXPHOS Complexes in DF-1 Cells 2.5. Knockdown of GHR Reduced the Enzymatic Activity of OXPHOS Complexes in DF-1 Cells 2.5. KnockdownIn order of GHRto investigate Reduced whether the Enzymatic the knockdown Activity of OXPHOSGHR could Complexes alter the enzymatic in DF-1 Cells activities of In orderOXPHOS to investigateproteins in DF-1 whether cells, we the assessed knockdown the enzymatic of GHR activitycould of alterOXPHOS the enzymaticcomplexes at activities48 h of In order to investigate whether the knockdown of GHR could alter the enzymatic activities of OXPHOSafter proteins transfection in DF-1 with cells, si-GHR we and assessed si-NC thefragments enzymatic in DF-1 activity cells. The of OXPHOS enzymatic complexes activities of at 48 h OXPHOS proteins in DF-1 cells, we assessed the enzymatic activity of OXPHOS complexes at 48 h after transfectionOXPHOS complex with si-GHR I, II, III and and si-NC IV were fragments significantly in DF-1reduced cells. by The53%, enzymatic34%, 36% and activities 25% in ofGHR OXPHOS after transfection with si-GHR and si-NC fragments in DF-1 cells. The enzymatic activities of complexknockdown I, II, III and cells, IV respectively were significantly (Figure 5A–D), reduced indicating by 53%, that 34%,the knockdown 36% and of 25% GHR in couldGHR reduceknockdown OXPHOSthe enzymatic complex activity I, II, III of andOXPHOS IV were complexes significantly in DF-1 cells.reduced by 53%, 34%, 36% and 25% in GHR cells, respectively (Figure5A–D), indicating that the knockdown of GHR could reduce the enzymatic knockdown cells, respectively (Figure 5A–D), indicating that the knockdown of GHR could reduce activity of OXPHOS complexes in DF-1 cells. the enzymatic activity of OXPHOS complexes in DF-1 cells.

Figure 5. Knockdown of GHR reduced the enzymatic activity of OXPHOS complexes in DF-1 cells. (A) Enzymatic activity of complex I was measured by the change in absorbance of NADH at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. (B) Enzymatic activity of complex II was measured by the change in absorbance of DCIP at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. (C) Enzymatic activity of complex III was measured by the change in absorbance of reduced cytochrome c at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. (D) Enzymatic activity of complex IV was measured by the change in absorbance of Int. J. Mol. Sci. 2019, 20, 1608 6 of 13 reduced cytochrome c at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. Data are expressed as means ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. 2.6. Knockdown of GHR Impaired Mitochondrial Function in DF-1 Cells 2.6. Knockdown of GHR Impaired Mitochondrial Function in DF-1 Cells In order to further investigate whether the knockdown of GHR could cause mitochondrial In order to further investigate whether the knockdown of GHR could cause mitochondrial dysfunction in DF-1 cells, we assessed mitochondrial function in GHR knockdown cells. ∆Ψm was dysfunction in DF-1 cells, we assessed mitochondrial function in GHR knockdown cells. ΔΨm was significantly reduced (Figure6A) and accompanied by a significant increase of mitochondrial swelling significantly reduced (Figure 6A) and accompanied by a significant increase of mitochondrial (Figure6C) in GHR knockdown cells. Reactive oxygen species production was significantly increased swelling (Figure 6C) in GHR knockdown cells. Reactive oxygen species production was significantly (Figureincreased6B)and (Figure accompanied 6B) and accompanied by a significant by a increasesignificant of increase MDA level of MDA (Figure level6D) (Figure and a 6D) significant and a reductionsignificant of reductionATP level of (Figure ATP 6 levelE) in (FigureGHR knockdown 6E) in GHR knockdown cells. Consistently, cells. Consistently, RCR was significantly RCR was reducedsignificantly in GHR reducedknockdown in GHR cells knockdown (Figure6 F).cells As (Figure a result, 6F). we As demonstrated a result, we demonstrated that the knockdown that the of GHRknockdownimpaired of mitochondrial GHR impaired function mitochondrial in DF-1 function cells. in DF-1 cells.

Figure 6. Knockdown of GHR caused mitochondrial dysfunction in DF-1 cells. (A) Mitochondrial ∆Ψm wasFigure measured 6. Knockdown by the fluorescence of GHR caused of JC-1 mitochondrial at 48 h after transfection dysfunction with in DF-1 si-GHR cells. and (A si-NC) Mitochondrial fragments in DF-1ΔΨm cells. was Red measured fluorescence by the represents fluorescence aggregation of JC-1 at of 48 JC-1, h after green transfection fluorescence with represents si-GHR and monomeric si-NC JC-1,fragments∆Ψm was in represented DF-1 cells. as Red the ratiofluorescence of aggregated represents and monomeric aggregation JC-1. of JC-1,(B) Reactive green oxygen fluorescence species productionrepresents was monomeric measured JC-1, by theΔΨm fluorescence was represented of DCF as at the 48 hratio after of transfectionaggregated withand monomeric si-GHR and JC-1. si-NC fragments(B) Reactive in DF-1 oxygen cells. species Bar, 100 productionµm. (C) Mitochondrialwas measured byswelling the fluorescence was measured of DCF by the at 48 absorbance h after attransfection 540 nm after with transfection si-GHR and with si-NC si-GHR fragments and si-NCin DF-1 fragments cells. Bar, 100 in DF-1 μm. ( cells.C) Mitochondrial The level of swelling (D) MDA andwas (E )measured ATP were by measured the absorbance at 48 h at after 540 transfectionnm after transfection with si-GHR with andsi-GHR si-NC and fragments si-NC fragments in DF-1 cells.in DF-1 cells. The level of (D) MDA and (E) ATP were measured at 48 h after transfection with si-GHR (F) Respiratory control ratio was calculated as the ratio of state III to state IV respiration rate at 48 h and si-NC fragments in DF-1 cells. (F) Respiratory control ratio was calculated as the ratio of state III after transfection with si-GHR and si-NC fragments in DF-1 cells. Data are expressed as means ± SEM, to state IV respiration rate at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. * p < 0.05; ** p < 0.01; *** p < 0.001. Data are expressed as means ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. 2.7. Knockdown of GHR Altered Mitochondrial Structure and Reduced Mitochondrial Number in DF-1 Cells Considering that the morphology and structure of mitochondria lays the foundation for mitochondrial function, we assessed them by TEM at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. We found some alterations in structure of mitochondria in GHR knockdown cells (see arrows), including increased mitochondrial vacuolation, absence of dense matrix granules and disappearance of mitochondria cristae (Figure7A). We also found that knockdown of GHR could significantly reduce mitochondrial number and the ratio of mitochondria to cytosol volume in DF-1 cells by TEM (Figure7B,C). In order to verify the consequences of TEM, we then labeled the mitochondria by MitoTracker staining, in which fluorescence intensity represented the mitochondrial mass [17]. The mitochondrial mass was significantly reduced in GHR knockdown cells, which was Int. J. Mol. Sci. 2019, 20, x 7 of 13

2.7. Knockdown of GHR Altered Mitochondrial Structure and Reduced Mitochondrial Number in DF-1 Cells Considering that the morphology and structure of mitochondria lays the foundation for mitochondrial function, we assessed them by TEM at 48 h after transfection with si-GHR and si-NC fragments in DF-1 cells. We found some alterations in structure of mitochondria in GHR knockdown cells (see arrows), including increased mitochondrial vacuolation, absence of dense matrix granules and disappearance of mitochondria cristae (Figure 7A). We also found that knockdown of GHR could significantly reduce mitochondrial number and the ratio of mitochondria to cytosol volume in DF-1 Int.cells J. Mol. by Sci. TEM2019 (Figure, 20, 1608 7B,C). In order to verify the consequences of TEM, we then labeled7 of the 13 mitochondria by MitoTracker staining, in which fluorescence intensity represented the mitochondrial mass [17]. The mitochondrial mass was significantly reduced in GHR knockdown cells, which was consistent with the result ofof TEMTEM (Figure(Figure7 7D–F).D–F). InInsummary, summary, we we demonstrated demonstrated that thatknockdown knockdown ofof GHRGHR couldcould reducereduce mitochondrialmitochondrial numbernumber andand alteralter mitochondrialmitochondrial structurestructure ininDF-1 DF-1cells. cells.

Figure 7. Knockdown of GHR altered mitochondrial structure and reduced mitochondrial number in DF-1Figure cells. 7. Knockdown (A) Mitochondrial of GHR ultrastructure altered mitochondrial was imaged structure by TEM and at 48reduced h after mitochondrial transfection with number si-GHR in andDF-1 si-NC cells. fragments(A) Mitochondrial in DF-1 cells, ultrastructure and abnormal was mitochondria imaged by TEM are indicatedat 48 h after by black transfection arrows. with (B) The si- numberGHR and of si-NC mitochondria fragments and in ( CDF-1) the cells, ratio and of mitochondria abnormal mitochondria to cytosol volume are indicated as determined by black from arrows. TEM images(B) The werenumber assessed of mitochondria in DF-1 cells and (n (=C) 5). the (D ratio) MitoTracker of mitochondria staining to ofcytosol DF-1 volume cells was as measured determined at 48from h after TEM transfection images were with assessed si-GHR in and DF-1 si-NC cells fragments (n = 5). ( inD)DF-1 MitoTracker cells. Bar, staining 100 µm. of (E DF-1) Fluorescence cells was intensitymeasured of at MitoTracker 48 h after transfection staining and with (F) si-GHR average and cell si-NC fluorescence fragments intensity in DF-1 of cells. MitoTracker Bar, 100 stainingμm. (E) wereFluorescence assessed inintensity DF-1 cells. of DataMitoTracker are expressed staining as means and (±F) SEM, average * p < cell 0.05; fluorescence ** p < 0.01; *** intensityp < 0.001. of MitoTracker staining were assessed in DF-1 cells. Data are expressed as means ± SEM, * p < 0.05; ** p 3. Discussion< 0.01; *** p < 0.001. Growth hormone can bind to GHR to regulate IGF-1 production through the JAK–STAT 3. Discussion pathway [18]. The mutation in GHR in SLD chickens interferes with the binding between GH and GHR, and furtherGrowth disrupts hormone the can GH–GHR–IGF1 bind to GHR signaling to regulate pathway, IGF-1 accompanied production by through low levels the of JAK–STAT IGF-1 [19]. Apathway previous [18] study. The hasmutation reported in GHR that comparedin SLD chickens with aginginterferes rats with that hadthe binding been administrated between GH with and exogenousGHR, and further IGF-1, untreateddisrupts the aging GH–GHR–IGF1 rats showed signaling significant pathway, mitochondrial accompanied dysfunction by low [20 levels], indicating of IGF- that1 [19] the. A level previous of IGF1 study is essential has reported for mammalian that compared mitochondrial with aging function. rats that In thishad study, been weadministrated found that thewith relative exogenous mRNA IGF-1, expression untreated of IGF1 agingwas significantly rats showed downregulated significant mitochondrial in both SLD chickens dysfunction and GHR[20], knockdownindicating that cells, the indicating level of IGF1 the lowis essential level of for GH mammalian binding activity mitochondrial and IGF-1 function. production. In this These study, results we furtherfound that prompted the relative us to investigate mRNA expression mitochondrial of IGF1 function was in significantly SLD chicken downregulated skeletal muscle in and both in GHR SLD knockdownchickens and cells. GHR knockdown cells, indicating the low level of GH binding activity and IGF-1 production.Mitochondrial These results biogenesis, further a dynamically prompted us regulated to investigate process, mitochondrial is mainly regulated function by in nuclear SLD chicken genes throughskeletal muscle the PGC1 andα in–NRF1–TFAM GHR knockdown signaling cells. pathway [4]. The activity of PGC1α is related to its expressionMitochondrial level [21 ],biogenesis, and the low a dynamically expression ofregulatedPGC1α is process, associated is mainly with mitochondrialregulated by nuclear dysfunction genes inthrough human the skeletal PGC1α–NRF1–TFAM muscle [22]. PGC1 signalingα can regulate pathway expression [4]. The activity of TFAM, of and PGC1α the isoverexpression related to its of TFAM can stimulate mitochondrial biogenesis in mice fibroblast cells [23]. The role of TFAM has been highlighted by regulating the transcription of mtDNA-encoded genes in human skeletal muscle [24]. Our results are consistent with these findings in mammals because we also found that the relative mRNA expression of PGC1α, TFAM and mtDNA-encoded OXPHOS genes were all downregulated in SLD chicken skeletal muscle and GHR knockdown cells. Furthermore, there is evidence of low expression of PGC1α and reduced enzymatic activity of OXPHOS complexes in the skeletal muscle of GH-deficient Ames mice [7], and downregulated relative mRNA expression of NRF1 and TFAM in the skeletal muscle of GHRKO mice [8]. These findings are also consistent with our results, in which we observed the low expression of genes involved in the PGC1α–NRF1–TFAM Int. J. Mol. Sci. 2019, 20, 1608 8 of 13 signaling pathway and reduced enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle. However, our results show that the enzymatic activity of complexes I, II, III and IV was reduced by 26%, 45%, 53% and 72% in SLD chicken skeletal muscle, respectively, while in GHR knockdown cells the enzymatic activity was reduced by 53%, 34%, 36% and 25%, respectively, that means an exactly opposite trend. This is probably due to the mutation in GHR in vivo that is different from the interference with GHR in vitro. The mutation in GHR completely alters the function of the GHR in vivo, which might lead to a larger impact on the activity of complex IV. As the interference with GHR cannot completely inhibit the function of the GHR in vitro, which might primarily affect the activity of complex I, we argue that the regulatory mechanism of the enzymatic activities of OXPHOS complexes might be different between SLD chicken skeletal muscle and GHR knockdown cells. Impaired mitochondrial biogenesis and inhibited mtDNA transcription will further lead to mitochondrial dysfunction in mammals [25]. ∆Ψm is essential for mitochondrial function; loss of ∆Ψm indicates mitochondrial dysfunction [26] and is normally accompanied by increased mitochondrial swelling [27]. Reduced ∆Ψm may lead to uncoupling of OXPHOS, and the impairment of OXPHOS complex activities may increase ROS production accompanied by elevated MDA levels and reduced ATP levels [28]. Meanwhile, RCR is a sensitive indicator of mitochondrial respiratory function [29]. In cases of hepatic mitochondrial dysfunction in a murine model of peanut allergy, decreased RCR can be observed with a simultaneous increase in ROS production [30]. Here, we found that reduced ∆Ψm, increased mitochondrial swelling, excessive ROS production, elevated MDA levels, reduced ATP levels and decreased RCR were observed in SLD chicken skeletal muscle and GHR knockdown cells. Our results are in line with previous studies in mammals showing that the function of mitochondria is compromised in GHRKO osteocytes [31] and inhibition of IGF1 signaling leads to mitochondrial dysfunction in cancer cells [11]. Therefore, we argue that the low expression of IGF1 might cause mitochondrial dysfunction by regulating mitochondrial biogenesis through the PGC1α–NRF1–TFAM signaling pathway in poultry. In addition, the morphology and structure of mitochondria are essential for mitochondrial function and cell homeostasis [32]. The results of this study showed that knockdown of GHR and the accompanying low expression of IGF1 could reduce the number of mitochondria and alter the structure of mitochondria, as observed in DF-1 cells as increased mitochondrial vacuolation, an absence of dense matrix granules and the disappearance of mitochondria cristae. Our results are in agreement with a previous study showing that a change in IGF1 signaling could alter the morphology and structure of mitochondria in mice Leydig cells [10]. In conclusion, we demonstrated for the first time that the function of mitochondria was impaired in SLD chicken skeletal muscle and GHR knockdown cells, and knockdown of GHR can reduce mitochondrial number and alter the structure of mitochondria in DF-1 cells. We conclude that the GHR gene is essential for chicken mitochondrial function both in vivo and in vitro.

4. Materials and Methods

4.1. Ethics Statement All animal experiments in this study were performed according to the protocols approved by the South China Agriculture University Institutional Animal Care and Use Committee (approval number: SCAU#0015). All animal procedures followed the regulations and guidelines established by this committee and minimized the suffering of animals.

4.2. Animals In this study, we used 7-week-old female SLD chickens in strain N301 as the experimental group, and this strain is characterized by a T354C mutation in exon 5 of GHR as previously described [14]. Meanwhile, we used normal 7-week-old female chickens in strain N202 as a control group, which have a wild-type GHR gene. Here, we used gastrocnemius muscle in SLD chickens to assess the role of GHR Int. J. Mol. Sci. 2019, 20, 1608 9 of 13 in chicken mitochondrial function in vivo. All chickens were obtained from the WenShi Group Co., Ltd. (Guangdong, China).

4.3. Cell Culture and RNA Interference In this study, we interfered with the expression of GHR using a chicken embryo fibroblast (DF-1) cell line to assess the role of GHR in chicken mitochondrial function in vitro. The DF-1 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) with 10% fetal bovine serum (Hyclone, UT, USA) and 0.2% penicillin/streptomycin (Invitrogen, CA, USA). The DF-1 cells were plated on a culture plate and incubated overnight prior to the transfection experiment. The siRNAs used for the knockdown of GHR were synthesized by Guangzhou RiboBio (Guangzhou, China). In our preliminary experiments, we designed four siRNA to interfere with GHR and used the si-GHR with the highest interference efficiency. The sequence of si-GHR was 50-CCUCGAUUUGGAUACCAUA-30. si-GHR and si-NC were transfected in DF-1 cells to a final concentration of 20 nM using Lipofectamine 3000 reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol, and cells were analyzed at 48 h after transfection. The GHR inhibition efficiencies were detected by the fluorescence intensity of FAM siRNA and qRT-PCR.

4.4. Quantitative Real-Time PCR Total RNA was extracted from gastrocnemius muscle or cells with RNAiso reagent (Takara, Japan) according to the manufacturer’s protocol. The RNA integrity and concentration were determined using 1.5% agarose gel electrophoresis and a Nanodrop 2000c spectrophotometer (Thermo, USA), respectively. cDNA was synthesized using PrimeScript RT Reagent Kit (Takara, Japan) for qRT-PCR. The MonAmp™ ChemoHS qPCR Mix (Monad Co., LTD Guangzhou, China) was used for quantitative real-time PCR (qRT-PCR) in a Bio-Rad CFX96 Real-Time Detection instrument (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. Relative gene expression was measured by qRT-PCR twice for each reaction and the nuclear gene β-actin was used as a control. The primers used in qRT-PCR are shown in Table S1.

4.5. Transmission Electron Microscopy TEM was used to determine the morphology and structure of mitochondria. Cells were fixed in 2.5% glutaraldehyde for 4 h at 4 ◦C and then cultured as previously described [16]. A transmission electron microscope (Hitachi HT7700, Tokyo, Japan) was used to examine and photograph mitochondria, and five randomly selected areas were photographed at 2500× magnification and counted as previously reported [33].

4.6. MitoTracker Green Staining and Hoechst 33342 Staining MitoTracker Green staining and Hoechst 33342 staining was used to label the mitochondria and nuclei in DF-1 cells, respectively. Cells were washed twice with PBS and incubated with MitoTracker Green (Beyotime, Shanghai, China) for 30 min at 48 h after transfection. Cells were then suspended in PBS and 10 µL of Hoechst 33342 dye was added (Beyotime, Shanghai, China). After being washed in PBS twice, a fluorescence microscope (Nikon TE2000-U, Tokyo, Japan) was used to capture five randomly selected fields and analyzed with NIS-Elements software.

4.7. Mitochondria Isolation and Mitochondrial Protein Concentration Measurement The mitochondria of gastrocnemius muscle and DF-1 cells were isolated using mitochondrial extraction kits (C3606, C3601; Beyotime, Shanghai, China) according to the manufacturer’s protocol, as previously described [34]. Mitochondrial protein concentration was measured by BCA protein assay to normalize the protein content as previously described [35]. Int. J. Mol. Sci. 2019, 20, 1608 10 of 13

4.8. Measurement of Enzymatic Activity of Mitochondrial OXPHOS Complexes Gastrocnemius muscle was dissected and immediately frozen in liquid nitrogen, and then stored at −80 ◦C[36]. We used commercial assay kits (BC0515, BC3235, BC3245, BC0945; Solarbio, Beijing, China) to measure the enzyme activity of mitochondrial OXPHOS complexes of gastrocnemius muscle and DF-1 cells according to the manufacturer’s protocol. Complex I enzyme activity was determined by the change in absorbance of NADH as measured at 340 nm. Complex II enzyme activity was determined by the change in absorbance of DCIP as measured at 600 nm. The enzyme activity of complex III and complex IV was determined by the change in absorbance of reduced cytochrome c as measured at 550 nm. Absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) according to the manufacturer’s protocol. Data were normalized to the control group and expressed as a percentage of control levels.

4.9. Measurement of Adenosine Triphosphate Level The ATP level was measured using an ATP assay kit (S0026; Beyotime, Shanghai, China) according to the manufacturer’s protocol. A Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) was used to determine the ATP level in gastrocnemius muscle and cells as previously described [37]. Data were normalized to the control group and expressed as a percentage of control levels.

4.10. Measurement of Malondialdehyde Level The MDA level was measured using an MDA assay kit (S0131; Beyotime, Shanghai, China) according to the manufacturer’s protocol. The supernatants of gastrocnemius muscle mitochondria and cell lysis were incubated with MDA reagent for 40 min at 95 ◦C. Absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA). Data were normalized to the control group and expressed as a percentage of control levels.

4.11. Measurement of Mitochondrial Membrane Potential Mitochondrial membrane potential was measured using a JC-1 kit (C2005; Beyotime, Shanghai, China) according to the manufacturer’s protocol. Gastrocnemius muscle mitochondria were fixed with JC-1. The fluorescence was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) and DF-1 cells were incubated with JC-1 for 20 min at 37 ◦C. After washing in PBS twice, the fluorescence was determined using a flow cytometer (BD Biosciences, San Jose, CA, USA), and 10 µM rotenone was used as standard inhibitor of ∆Ψm. The ∆Ψm of mitochondria were represented as the ratio of aggregated and monomeric JC-1, and data were normalized to the control group and expressed as a percentage of control levels.

4.12. Measurement of Mitochondrial Swelling Mitochondrial swelling was measured by the absorbance at 540 nm, as previously described [38]. A decrease in absorbance indicates an increase in mitochondrial swelling. Mitochondria were freshly isolated from gastrocnemius muscle and DF-1 cells, and absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA). Data were normalized to the control group and expressed as a percentage of control levels.

4.13. Measurement of Reactive Oxygen Species Production Reactive oxygen species production of gastrocnemius muscle mitochondria was determined using the rate of NBT reduction at 595 nm, as previously described [39]. Absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA). Reactive oxygen species production in the mitochondria of DF-1 cells was measured using a ROS Assay Kit (S0033; Beyotime, Shanghai, China) according to the manufacturer’s protocol. Cells were incubated with 10 mM DCFH-DA probes at 37 ◦C for 20 min and washed twice with PBS. Dichlorofluorescein (DCF) fluorescence was Int. J. Mol. Sci. 2019, 20, 1608 11 of 13 determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA), and the images of cells were taken by a fluorescence microscope (Nikon TE2000-U, Japan). Data were normalized to the control group and expressed as a percentage of control levels.

4.14. Measurement of Mitochondrial Respiratory Control Ratio Mitochondrial respiratory control ratio was measured using an RCR kit (GMS10097; GenMed Scientiﬁcs Inc., MA, USA) according to the manufacturer’s protocol, as previously described [29]. Oxygen consumption of mitochondria protein was measured using a Clarke-type oxygen electrode (Hansatech Oxytherm, Norfolk, UK). The RCR was represented as the ratio of state III to state IV respiration rate. Data were normalized to the control group and expressed as a percentage of control levels.

4.15. Statistical Analysis All experiments were performed at least three times. The data were presented as means ± standard error of the mean (SEM), the statistical analyses were performed using Student’s t-test, and the signiﬁcance was represented by p-values. p < 0.05 was considered to be statistically signiﬁcant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/7/ 1608/s1. Author Contributions: B.H. designed the study, wrote the paper, carried out experiments, and analyzed data. S.H. participated in data collection and interpretation, and helped with performing some of the manuscript’s experiments. M.Y., Z.L. helped with performing some of the manuscript’s experiments. Q.L., D.Z. helped by providing useful discussion and language correction. H.L., X.Z. participated in the design, manuscript writing and final approval of the manuscript. All authors read and approved the final manuscript. Funding: This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31401046), the China Agriculture Research System (CARS-41-G03), and Guangdong Youth Talent Project. Conflicts of Interest: The authors declare that they have no conflict of interest.

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