diabetes research and clinical practice 102 (2013) 43–52
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Skeletal myoblast transplantation on gene expression profiles of insulin signaling pathway and mitochondrial biogenesis and function in skeletal muscle Jian-Hua Ma a, Li-ping Su b, Jian Zhu a, Peter K. Law c, Kok-Onn Lee d, Lei Ye d,e, Zi-Zheng Wang f,* a
Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China Department of Bioengineering, National University Hospital, Singapore c Cell Therapy Institute, Wuhan, China d Department of Medicine, National University of Singapore, Singapore e Department of Medicine, University of Minnesota, MN, USA f Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing, China b
article info
abstract
Article history:
Aim: The study aims to investigate the gene expression profiling of insulin signaling
Received 28 May 2013
pathway and mitochondrial biogenesis and function in the skeletal muscle of KK mice.
Received in revised form
Methods: KK mice were divided into the following groups: KK control group, basal medium
1 August 2013
(M199) only; KK fibroblast group, with human fibroblast transplantation; KK myoblast group,
Accepted 13 August 2013
with human skeletal myoblast transplantation. C57BL mice received hSkM transplantation
Available online 19 August 2013
as a normal control. Cells were transplanted into mice hind limb skeletal muscle. All animals were treated with cyclosporine for 6 weeks only. The mice were sacrificed in a fasting state at
Keywords:
12 weeks after treatment. Hind limb skeletal muscle was harvested and used for study of
Type 2 diabetes mellitus
gene expression profiling.
Insulin signaling
Results: hSkMs survived extensively in mice skeletal muscle at 12 weeks after cell trans-
Mitochondrion
plantation. Glucose tolerance test showed a significant decrease of blood glucose in the mice
Gene array
of KK myoblast group compared to the KK control and fibroblast groups. Transcriptional patterns of insulin signaling pathway showed alterations in KK myoblast as compared with KK control group (23 genes), KK fibroblast group (7 genes), and C57BL group (8 genes). Transcriptional patterns of mitochondrial biogenesis and function also had alterations in KK myoblast as compared with KK control group (27 genes), KK fibroblast group (9 genes), and C57BL group (6 genes). Conclusions: These data demonstrated for the first time that hSKM transplantation resulted in a change of gene transcript in multiple genes involved in insulin signaling pathway and mitochondrial biogenesis and function. # 2013 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Department of Nuclear Medicine Nanjing First Hospital Nanjing Medical University, 210006 Nanjing, China. E-mail address:
[email protected] (Z.-Z. Wang). 0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.08.006
44
1.
diabetes research and clinical practice 102 (2013) 43–52
Introduction
Diabetes is one of the leading causes of kidney failure and non-traumatic lower-limb amputations among adults in the world. In 2010, the United States was estimated to have spent $198 billion on diabetes treatment [1]. In adults, it was estimated that there were 285 million people with type 2 diabetes making up about 90% of diabetes cases in 2010 [2]. It affects 25% of western populations with a steadily increasing incidence [3]. It is an important cardiovascular disease risk factor [4]. Epidemiological and twin studies have clearly indicated a major polygenetic factor in the development of insulin resistance, a key feature of type 2 diabetes mellitus, that was influenced by environmental factors [5,6]. Insulin resistance is a clinical disorder of glucose metabolism caused by an inability of insulin to promote sufficient glucose uptake into adipose tissue and striated muscle [7]. In a healthy person, as blood sugar concentration rises, insulin is secreted into the blood stream by the pancreatic beta cell of the endocrine pancreas. Insulin stimulates glucose uptake into fat and muscle to promote the storage of glucose as intracellular triglycerides and glycogen in fat and muscle. In addition, insulin inhibits the production and release of glucose from the liver (gluconeogenesis and glycogenolysis) [8]. Previous studies have demonstrated the importance of skeletal muscles in the development of insulin resistance [9– 11]. Mice with muscle-specific Glut-4 knockout were insulin resistant and glucose-intolerant from an early age [9]. An isolated defect in protein kinase C-l in muscle would induce abdominal obesity and other metabolic abnormalities [10]. By contrast, muscle-specific LKB1 (a serine/threonine kinase that is a negative regulator of insulin sensitivity) knockout increased insulin sensitivity and improved glucose homeostasis [11]. These studies suggest that defects in skeletal muscle glucose transport may be key factors in the development of insulin resistance. We had demonstrated that attenuated hyperglycemia and hyperinsulinemia, and improved glucose tolerance of KK mouse could be achieved by xeno-transplantation of human skeletal myoblasts (hSkMs) [12]. Skeletal myoblasts are mononucleated precursor cells of skeletal muscle cells that can fuse with different muscle fiber types and adopt their phenotype [13]. Through fusion with KK mouse skeletal muscle, hSkM transplantation formed hybrid muscle fibers in KK mouse. In the present study, we hypothesized that insulin signaling pathway and mitochondrial biogenesis and function gene profiles would be changed in mouse skeletal muscle after hSkM transplantation.
2.
Materials and methods
All animals were kept and maintained by Research Animal Resources of National University of Singapore (NUS), Singapore and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of NUS.
2.1.
Animals and diets
KK mice were purchased from Jax Lab, Maine, USA. It is a genetically obese animal model of T2DM and is characterized with hyperglycemia, hyperinsulinemia and glucose intolerance [14]. They were about 34% heavier than C57 BL mouse at 14–16 weeks of age [12]. Mice were housed individually in plastic cages in an air-conditioned room at 25 8C with a 12-h light and 12-h dark cycle (light: 9:00 am to 9:00 pm) and free access to food (5K52 PMI Nutrition International LLC, USA) and water (tap water). Mouse at 14–16 weeks old were screened for hyperglycemia and used for experiment. KK mice which met the following criteria were used: (1) fasting blood glucose > 6.5 mmol/L and (2) blood glucose > 20 mmol/L at 30 min or 60 min, and > 11 mmol/L at 2 h during GTT [12].
2.2.
Glucose tolerance test (GTT)
All mice used in the study had GTT. After overnight fasting (about 16 h), the mouse was injected intra-peritoneally (i.p.) with 1 g/kg body weight of glucose diluted in distilled water (100 mg/mL). Blood samples from the tail vein were collected at 0 (before glucose injection), 30, 60 and 120 min after glucose injection. The blood glucose concentration was determined by Accu-Chek Advantage glucometer (Roche, Germany).
2.3.
Culture of hSkM and human fibroblasts
Human skeletal myoblasts were purchased from Bioheart Inc., USA. They were cultured and propagated in 225 mm2 tissue culture flasks and maintained with M199 supplemented with 10% fetal bovine serum, 10 ng/mL FGF (10% M199) at 37 8C in 5% CO2 incubator until confluent. The purity and uniformity of hSkM culture was assessed for desmin and CD56 expression as described earlier [12,15]. Human fibroblasts were kindly given by Associate Professor Toan Thang Phan, Department of Surgery, NUS, Singapore.
2.4.
Cell labeling and transplantation
Cells were labeled with 4,6-diamidino-2-phenylindole (DAPI) (Sigma, USA) overnight before cell transplantation [12]. KK mice meeting the criteria for diabetes were randomly assigned into three groups: KK control group (n = 8): receiving 1.5 mL M199 only, KK fibroblast group (n = 8): receiving 1.5 mL M199 containing 3 107 human fibroblasts, and KK myoblast group (n = 8): receiving 1.5 mL M199 containing 3 107 hSkMs. KK fibroblast group was used in the study to determine whether the improved metabolic parameters were due to the weight loss from immune reaction or a direct effect of the hSkMs on muscle metabolism. C57BL mouse was used as a normal control to determine any side effects related to hSKM transplantation and received 1.5 mL M199 containing 3 107 hSkMs. A total of 20 injections were injected into the bilateral muscle of the anteromedial aspect of the thigh, muscles of the posterior aspect of the leg, and muscles of the gluteal region of mice under anesthesia. After injection, mice were returned to their cage for recovery. All animals received cyclosporine treatment (10 mg/kg/day) for 3 days before treatment until
diabetes research and clinical practice 102 (2013) 43–52
6 weeks after treatment. After blood sampling, animals were sacrificed at 12 weeks.
2.5.
Immunohistochemical studies
The DAPI+ cryo-sections from mouse skeletal muscle that was explanted at 12 weeks after cell transplantation were immunostained for expression of dystrophin to determine the integration of hSkM nuclei into mouse skeletal muscle fibers [12]. Briefly, the tissue sections were fixed in 100% methanol at 20 8C for 20 min followed by incubation with 0.1% Triton-100 for 10 min at 4 8C. After blocking, the primary antibody solutions containing rabbit anti-dystrophin (Sc15376, Santa Cruz, USA) antibody at 1:50 dilution was applied on the samples and incubated for 1 h at room temperature. Following this, goat anti-rabbit IgG conjugated with Fluorescein isothiocyanate (FITC) (F-4890, Sigma, USA) at 1:200 dilution was applied for 1 h. After thorough wash, the samples were counter-stained with propidium iodine and observed under Olympus BX41 (Olympus, Japan) fluorescent microscope and images were recorded using a digital camera with Olympic Micro Image (Olympus, Japan).
2.6.
Cell engraftment rate
Cell engraftment was evaluated by counting DAPI+ hSkM nuclei; 4 legs each from KK myoblast and KK fibroblast groups were evaluated at 12 weeks after cell transplantation. After sacrifice, one leg form each mouse was harvested to have femur removed. Muscles were embedded for cryo-sections and sequentially cut into 8 mm thick sections. DAPI+ nuclei were counted in slides from every 50 sections from top to bottom, and evaluations were averaged at low magnification (100). A total of 15–20 slides were evaluated from each leg to calculate the mean DAPI+ cell number (DAPI+ cells per section area) for each leg. The DAPI+ cell number per leg was estimated as the mean of DAPI+ cell density per section area fold, where the fold was derived from the weight of thigh muscle/each section weight. Section weight was calculated as section area by multiply section thickness and density (1 g/cm3). The DAPI+ cell engraftment rate was calculated as the total number of remaining survived cells divided by the total number of transplanted cells (3 107 hSkMs).
2.7.
Isolation of muscle tissues and total RNA preparation
After sacrificing, mouse skeletal muscles of the anteromedial aspect of the thigh were immediately separated, collected and stored in liquid nitrogen. Total RNA was extracted from frozen muscle samples with Trizol reagent (Invitrogen, USA) according to manufacturer’s instructions. The concentration and purity of RNA was determined by Nanodrop. DNase I (Fermentas, USA) was used to remove DNA contamination from total RNA. cDNA was synthesized using Maxima1 First Strand cDNA Synthesis Kit (Fermentas, USA) from 1 mg total RNA. 1 mL (1 mg) cDNA was used for one array plate. The realtime PCR cycle was performed according to RT2 ProfilerTM PCR Array User Manual as per instruction. The gene expression level changed 2 folds would be considered significantly
45
increased between groups, while the gene expression level changed 0.5 fold would be considered as significantly reduced between groups. All the significantly changed genes were validated and quantified by quantitative PCR (QPCR). The gene expression level that had significantly changed based on array results would be further validated using the same cDNA from each group. To confirm gene expression level, Maxima1 SYBR Green qPCR Master Mix (2X) (Fermentas, USA) was used. Primers were designed using Primer Premier 5 software (Premier Biosoft, USA). Sequences were able to be obtained upon request. The QPCR thermal cycling protocol for 40 cycles was programmed as following: 1 cycle of initial denaturation for 10 min, then denaturation at 95 8C for 15 s, annealing for 30 s and extension at 72 8C for 30 s. PCR array kits for genes expression profiles in skeletal muscle of mouse insulin pathway and mitochondrial biogenesis and function were purchased from SABiosciences, USA. The Mouse Insulin Signaling Pathway RT2 ProfilerTM PCR Array (Cat PAMM-030, QIAGEN-SAbiosciences, USA) profiles the expression of 84 genes related to the role of insulin-responsive genes. Insulin mediates a wide spectrum of biological responses associated with glucose uptake, glycogen, lipid and protein synthesis, transcriptional activation, cell growth and differentiation. The Mouse Mitochondria RT2 ProfilerTM PCR Array (PAMM087, QIAGEN-SAbiosciences, USA) profiles the expression of 84 genes involved in the biogenesis and function of the cell’s powerhouse organelle. The genes monitored by this array include regulators and mediators of mitochondrial molecular transport of not only the metabolites needed for the electron transport chain and oxidative phosphorylation, but also the ions required for maintaining the mitochondrial membrane polarization and potential important for ATP synthesis. Metabolism and energy production are not the only functions of mitochondria. Intrinsic apoptosis pathway genes activated by intracellular damage signaling are also included in this array.
2.8.
Statistical analysis
Statistical analysis was performed using SPSS 20. All data are presented as the mean the standard error meaning. The area under the curve of the 4-point glucose measurements was used to compare the difference among three groups by ANOVA. A probability value of p < 0.05 was considered a significant difference between groups.
3.
Results
3.1.
Body weight
The mean body weights of the four animal groups at 12 weeks after treatment were: KK control group = 32.3 1.3 g, KK fibroblast = 33.1 1.7 g, and KK myoblast group = 30.4 1.2 g, g, and C57BL group = 22.9 0.5 g. The body weight of KK mice was significantly heavier than that of C57BL mice ( p < 0.05 for all). Although there was a tendency of a decreased body weight in the KK myoblast group, no significant difference was found between any two KK groups.
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diabetes research and clinical practice 102 (2013) 43–52
KK control KK fibroblast KK myoblast C57BL
12 weeks
A
30
6
*
HbA1c (%)
5
& 4 3 2 1
Blood glucose (mmol/L)
7
B
25 20 15 10 5 0
0 KK control
KK fibroblast
KK myoblast
C57 BL
Groups
0
0.5
1
2
Hour (s)
Fig. 1 – Glucose homeostasis of mouse at 12 weeks after treatment. (A) HbA1c of KK myoblast group was significantly reduced as compared with KK control and KK fibroblast. However, it was still significantly higher than that of C57BL group, which served as a normal control. (B) GTT showed that KK myoblast group had significantly reduced plasma glucose concentration during GTT and similar to that of C57BL group. (* vs. KK control and fibroblast p < 0.05, & vs. any KK group: p < 0.05).
3.2.
Improvement of HbA1c and glucose tolerance test
Glycosylated hemoglobin assay demonstrated that the KK myoblast group mice achieved significantly better glucose control after hSkM transplantation (Fig. 1A). At 12 weeks after treatment, HbA1c of KK myoblast group was significant lower than those of KK control and fibroblast groups ( p < 0.05 for both). HbA1c of C57BL group was significantly lower than any KK group ( p < 0.05 for all). A significant decrease in blood glucose concentration was observed in the KK myoblast group. At 12 weeks after transplantation, KK control and fibroblast group animals
had severe hyperglycemia and glucose intolerance (Fig. 1B). By contrast, KK myoblast group mice showed a significant reduction in blood glucose as compared with KK control and fibroblast groups ( p < 0.05 for both), and reached to almost similar level as C57BL group ( p > 0.05).
3.3. Survival and integration of hSkMs in host mouse skeletal muscle The labeling efficiency of hSkMs was 100% for DAPI (Fig. 2A). Extensive survival of hSkM shown as DAPI+ nuclei was found in mouse skeletal muscle at 12 weeks after cell transplantation
Fig. 2 – hSkM nuclei integrated into host muscle fibers. (A) DAPI labeling of hSkMs showing 100% labeling efficiency. (B) Survival of DAPI+ hSkMs in mouse skeletal muscle immunostaing for dystrophin protein expression. (C) The same tissue was counter-stained with propidium iodine to show all nuclei. (D) Overlay pictures B and C to show the integration of hSkM nuclei into mouse skeletal muscle. (Bar = 200 mm).
47
diabetes research and clinical practice 102 (2013) 43–52
Table 1 – Comparison of insulin signaling pathway gene transcript levels in the KK hSkM, KK control, KK fibroblast, and C57BL groups at fasting state. Genes
Accession no.
Acaca Acox1
NM_133360 NM_015729
Aebp1 Braf
NM_009636 NM_139294
Cebpa
Categories
Fold change A
B
C
D
Target genes for SREBP1 Lipid Metabolism, Target genes for PPARg Transcription Factors and Regulators MAPK Pathway
2.06" 11.23"
2.87" 1.09
0.94 1.84
0.72 7.66"
9.06" 2.5"
2.01" 0.84
1.71 2.25"
4.5" 2.6"
NM_007678
Cell Growth and Differentiation Protein Metabolism Transcription Factors and Regulators
2.64"
1.47
1.36
1.79
2.27"
4.29"
1
0.53
1.25
2.81"
8.82"
Cfd
NM_013459
Target genes for PPARg
Frap1
NM_020009
PI-3 Kinase Pathway Protein Metabolism
Frs2
NM_177798
Cell Growth and Differentiation Insulin Receptor-associated Proteins Protein Metabolism
4.2"
0.91
0.79
4.6"
Gab1
NM_021356
Insulin Receptor-associated Proteins MAPK Pathway Protein Metabolism
5.62"
0.7
0.86
8.06"
Gpd-1
NM_010271
Carbohydrate Metabolism Target genes for PPARg
9.38"
2.41"
2.51"
3.89"
Hras1
NM_008284
Cell Growth and Differentiation MAPK Pathway Protein Metabolism
3.03"
1.05
1.92
2.87"
Igf2
NM_010514
Cell Growth and Differentiation
1.92
3.51"
10.27"
Irs2
NM_001081212
Cell Growth and Differentiation Insulin Receptor-associated Proteins
2.48"
1.23
1.09
2.23"
Jun
NM_010591
Cell Growth and Differentiation Primary Target Genes for Insulin Signaling Protein Metabolism Transcription Factors and Regulators
2.93"
2.01"
1.05
1.45
Leptin
NM_008493
Lipid metabolism Carbohydrate Metabolism Protein Metabolism Primary Target Genes for Insulin Signaling Cell Growth and Differentiation
2.03"
1.95
30.3"
1.04
Pparg
NM_011146
Target genes for PPARg, Cell Growth and Differentiation Transcription Factors and Regulators
2.03"
0.84
1.06
2.43"
Ppp1ca
NM_031868
Carbohydrate Metabolism Insulin Receptor-associated Proteins Protein Metabolism
4.59"
1.54
2.08"
2.99"
Ptpn1
NM_011201
Insulin Receptor-associated Proteins Protein Metabolism
2.62"
4.66"
1.65
0.56
Raf1
NM_029780
Cell Growth and Differentiation Protein Metabolism, Transcription Factors and Regulators
2.51"
0.97
0.8
2.58"
Shc1
NM_011368
Cell Growth and Differentiation Insulin Receptor-associated Proteins Lipid Metabolism Protein metabolism MAPK Pathway
3.53"
1.11
1.64
3.16"
Glut-1
NM_011400
Carbohydrate Metabolism
2.25"
0.98
0.48#
2.28"
Rps6ka1
NM_009097
MAPK Pathway, PI-3 Kinase Pathway Protein Metabolism
2.58"
1.56
1.31
1.66
11"
19.7"
48
diabetes research and clinical practice 102 (2013) 43–52
Table 1 (Continued ) Genes
UCP1
Accession no.
NM_009463
Categories
Fold change
MAPK Pathway, PI-3 Kinase Pathway Protein Metabolism
A
B
C
D
8.4"
4.76"
4.44"
1.78
" and #: significant upregulation and downregulation in the KK myoblast group compared with KK control group (A), KK fibroblast group (B), and C57BL group (C). (D) KK fibroblast group compared with KK control group.
(Fig. 2B). The mean engraftment rate was 26.3 5.2% at 12 weeks after hSkM transplantation and 1/2 of survival cells integrated into skeletal muscle, while only 3.5 0.6% of human fibroblasts was found in KK mice skeletal muscle at 12 weeks after human fibroblast transplantation. Immunostaining of DAPI+ tissue for dystrophin expression showed that hSkM nuclei were co-localized with host nuclei in the same muscle fibers, suggesting that hSkM integrated into host skeletal muscle fibers to form hybrid muscle fibers (Fig. 2B–D) as dystrophin lineated the boundary of skeletal muscle fibers.
3.4. Array analysis of gene expression of insulin signaling pathway in anteromedial muscle of the thigh
3.5. Array analysis of gene expression of mitochondrial biogenesis and function in anteromedial muscle of the thigh 3.5.1.
3.5.2. 3.4.1.
KK myoblast group versus KK control group
Mice in KK myoblast group compared with KK control group had 23 gene transcripts increased, which was 27.4% of 84 genes screened (Table 1). Most of the 23 increased genes belonged to protein metabolism (13), cell growth and differentiation (10), MAPK pathway (6), and insulin receptorassociated proteins (6), transcription factors and regulators (5), carbohydrate metabolism (4) functional groups.
3.4.2.
KK myoblast group versus KK fibroblast group
Mice in KK myoblast group compared with KK fibroblast group had 7 (8.3% of 84) gene transcripts increased (Table 1). The 7 genes belonged to protein metabolism (3), cell growth and differentiation (1), insulin receptor-associated proteins (1), transcription factors and regulators (2) and target genes for PPARg (2) functional groups.
3.4.3.
KK myoblast group versus C57BL group
Compared with C57BL group, mice in KK myoblast group had 8 gene transcripts changed (Table 1). Among these, 7 gene transcripts increased and 1 decreased. Most of the 7 increased genes belonged to protein metabolism (4), MAPK pathway (2), cardbohydrate metabolism (3), and PI-3 kinase pathway (2) functional groups. The only gene that had decreased transcription was Glut-1. There was no change in the PPARy gene transcipt.
3.4.4.
KK myoblast group versus KK control group
Compared with KK control group, KK myoblast group had 27 (32.1% of 84) gene transcripts increased (Table 2). Most of the 27 genes belonged to small molecule transport (7), mitochondrial transport (7), inner membrane translocation (5), targeting proteins to mitochondria (4), mitochondrion protein import (4), mitochondrial fission and fusion (4), mitochondrial localization (3), and out membrane translocation (2), and membrane polarization & potential (2) functional groups.
KK myoblast group versus KK fibroblast group
Compared with KK fibroblast group, KK myoblast group had only 9 (10.7% of 84) gene transcripts increased (Table 2). Most of the 9 genes belonged to small molecule transport (2), mitochondrial transport (4), inner membrane translocation (1), targeting proteins to mitochondria (1), mitochondrion protein import (2), mitochondrial fission and fusion (1), out membrane translocation (1), and membrane polarization & potential (2) functional groups. No gene that belonged to mitochondrial localization functional group showed any significant difference between KK myoblast and KK fibroblast groups.
3.5.3.
KK myoblast group versus C57BL group
Compared with C57BL group, KK myoblast group had less mitochondrial gene transcripts changed (Table 2). There were 5 gene transcripts increased and 1 gene transcript decreased. The 5 genes belonged to small molecule transport (1), mitochondrial transport (2), inner membrane translocation (1), mitochondrial fission and fusion (1), mitochondrial localization (1), and membrane polarization & potential (1) functional groups. The only gene that had decreased transcription was Slc25a25, which is a small molecular transport gene. No gene that belongs to targeting proteins to mitochondria, mitochondrion protein import and out membrane translocation etc., functional groups showed any significant difference between KK myoblast and C57BL groups.
KK fibroblast group versus KK control group
Compared with KK control group, mice in KK fibroblast group had 15 gene transcripts changed (Table 1). Eight genes, including Acaca, Cebpa, Cfd, Jun, Leptin, Ptpn1, Rps6ka1, and UPC1 which changed significantly in KK myoblast group, did not change in KK fibroblast group as compared to KK control group.
3.5.4.
KK fibroblast group versus KK control group
Compared with KK control group, mice in KK fibroblast group had only 14 gene transcripts changed (Table 2). Thirteen genes, including Aifm2, Aip, Cox10, Fis1, Mipep, Slc25a22, Slc25a25, Stard3, Timm17b, Timm22, Timm44, Tomm40, and UCP1 which changed significantly in KK myoblast group, did not
49
diabetes research and clinical practice 102 (2013) 43–52
Table 2 – Comparison of mitochondria gene transcript levels in the KK hSkM, KK control, KK fibroblast, and C57BL groups at fasting state. Genes
Accession no.
Aifm2 Aip
NM_178058 NM_016666
Apoptotic Genes Mitochondrial Transport Targeting Proteins to Mitochondria Mitochondrion Protein Import
Bcl2l1
NM_009743
Apoptotic Genes Mitochondrial Transport Membrane Polarization & Potential
Cox10
NM_178379
Mitochondrion Protein Import Mitochondrial Fission & Fusion
Cpt1b
NM_009948
Fis1 Grpel1
Categories
Fold change A
B
C
D
1.52 1.21
1.32 0.85
1.61 1.93
3.68"
3.03"
3.63"
2.31"
2.52"
1.1
0.91
Mitochondrial Transport Targeting Proteins to Mitochondria Mitochondrion Protein Import
16.33"
2.06"
1.72
7.94"
NM_025562 NM_024478
Mitochondrial Fission & Fusion Mitochondrial Transport Targeting Proteins to Mitochondria Mitochondrion Protein Import
2.5" 5.39"
1.24 1.15
1.39 1.14
1.88 4.69"
Mfn1
NM_024200
Mitochondrial Fission & Fusion, Mitochondrial Localization
2.03"
0.95
0.86
2.14"
Mipep
NM_027436
Mitochondrial Transport, Targeting Proteins to Mitochondria
2.83"
1.43
1.46
1.97
Opa1
NM_133752
Inner Membrane Translocation, Mitochondrial Localization Mitochondrial Fission & Fusion
5.13"
0.95
2.13"
5.39"
Rhot2 Sfn Slc25a15 Slc25a16 Slc25a17 Slc25a20 Slc25a22 Slc25a25 Slc25a27 Stard3 Taz Timm17b Timm22 Timm44 Tomm34 Tomm40
NM_145999 NM_018754 NM_181325 NM_175194 NM_011399 NM_020520 NM_026646 NM_146118 NM_028711 NM_021547 NM_181516 NM_011591 NM_019818 NM_011592 NM_025996 NM_016871
Mitochondrial Localization Apoptotic Genes Small Molecule Transport Small Molecule Transport Small Molecule Transport Small Molecule Transport Small Molecule Transport Small Molecule Transport Small Molecule Transport Mitochondrial Transport Inner Membrane Translocation Inner Membrane Translocation Inner Membrane Translocation Inner Membrane Translocation Outer Membrane Translocation Outer Membrane Translocation
2.64" 2.14" 2.55 " 3.01 " 3.16 " 3.31 " 3.16 " 2.53 " 4.26 " 2.87 " 4.89 " 3.39 " 3.46 " 2.31 " 3.27 " 5.13 "
1.06 0.77 0.86 1.23 0.89 1.13 2.01" 2.91" 0.92 2.23 " 1.17 2.19 " 1.82 1.09 1.56 3.46"
1.23 3.39" 0.91 0.76 1.22 0.59 2.07" 0.46# 1.2 1.31 1.3 1.17 1.05 1.4 1.16 1.59
2.48" 2.53" 2.97" 2.45" 3.56" 2.95" 1.57 0.87 4.63" 1.28 4.17" 1.55 1.91 1.83 2.1" 1.48
UCP1
NM_009463
Membrane Polarization & Potential Mitochondrial Transport
8.4 "
4.76 "
4.44 "
1.78
2.45" 2.33"
13.4"
" and #: significant upregulation and downregulation in the KK myoblast group compared with KK control group (A), KK fibroblast group (B), and C57BL group (C). (D) KK fibroblast group compared with KK control group.
change in KK fibroblast group as compared to KK control group.
4.
Discussion
We have shown that intra-muscular transplantation of hSkM into KK mouse hind limb skeletal muscle reduced hyperglycemia, and improved glucose tolerance and insulin resistance [12]. In the present study, we have demonstrated for the first time that gene profiles of insulin signaling pathway and mitochondrial biogenesis and function were changed in
skeletal muscle of KK mouse after hSkM transplant, and have documented these changes in gene transcripts. There are 23 (27.4%) and 27 (32.1%) gene transcripts, of insulin signaling pathway and mitochondrial biogenesis and function, respectively, changed in skeletal muscle of KK mouse after hSkM transplantation as compared with KK control mouse. However, the gene number reduced to 7 (8.3%) and 9 (10.7%) as compared with KK fibroblast group, and 8 (9.5%) and 6 (7.1%) as compared with C57BL group. These suggest that hSkM transplantation into KK mouse skeletal muscle could change the expression level of genes involved in insulin signaling pathway and mitochondrial biogenesis and function, which
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may contribute to the reduced hyperglycemia and improved glucose tolerance. Decreased glucose uptake in skeletal muscle plays an important role in development of type 2 diabetes mellitus [16,17], since it is a principal glucose-consuming tissue and is responsible for about 75% of whole body glucose metabolism [17]. Skeletal muscle myofibers are developed by the fusion of mononucleated SkMs with neighboring mature myofibers throughout life, aiding in regeneration after injury to muscle tissue [18,19]. It was found that about 26.3 5.2% of 3 107 hSkM survived in KK mice skeletal muscle at 12 weeks, while 1/2 of surviving cells integrated into skeletal muscle. A nucleus of a SkM contains normal genes that determine its normality and cell characteristics [19]. Thus, it is possible that through natural fusion between donor SkM and host skeletal muscles, the donor nuclei will be in a position to supplement the genes that have been defective or impaired in glucose metabolism in skeletal muscle. The hSkM engraftment rate in the current study was pretty high and may be related to the proliferation of donor hSkM in mice skeletal muscle in 12 weeks, as Lee-Pullen et al. [20] demonstrated that donor SkM cell number can increase by more than 2-fold by 3 months through proliferation. Our previous and current studies demonstrate that hSkM transplantation achieved better glucose homeostasis and improved glucose tolerance, which were accompanied with survival of hSkMs in KK mouse skeletal muscle at 12 weeks after cell transplantation using a transient immune-suppression treatment. The hSkM nuclei integrated into host skeletal muscle fibers. The fusion between donor nuclei with host skeletal muscle fibers formed hybrid muscle fibers. This could help the donor hSkM to escape host immuno-rejection after withdrawal of cyclosporine treatment as mature skeletal muscle fibers do not express mayor histocompatibility complex class 1 antigens [21]. The donor nuclei may also co-express exogenous genes together with host genes in the hybrid muscle fibers. The current study further demonstrated that implantation of normal hSkM could increase expression level of 23 genes involved in insulin signaling pathway and 27 genes involved in mitochondrial biogenesis and function. It was found that 23 genes of insulin signaling pathway increased in KK myoblast group as compared with KK control group. Except secondary effector target genes for insulin signaling functional group, all other functional groups have genes significantly changed. Prominently, half of these genes (56.55% of 23 genes) are involved in protein metabolism. Most of them are multi-functional and are involved in other functional groups. For example, Cebpa, Frs2, Hras1, Jun, Leptin, Raf, and Shc1 are not only involved in protein metabolism, but also cell growth and differentiation. Compared with the KK fibroblast group, 7 gene transcripts were increased in KK myoblast group: Acaca, Aebp1, Cfd, Gpd-1, Jun, Ptpn1, and UCP1. Compared with C57BL group, 7 gene transcripts increased and 1 decreased in KK myoblast group: Braf, Frap1, Gpd-1, Igf2, Leptin, Ppp1ca, and UCP1 increased, while Glut-1 decreased. We found that only KK mouse received hSkM transplantation had significantly improved hyperglycemia, glucose tolerance and insulin resistance as compared with not only KK control mouse,
but also KK mouse group received human fibroblast transplantation. However KK myoblast group did not reach the same glucose homeostasis as the C57BL group. These suggest that Acaca, Aebp1, Cfd, Gpd-1, Jun, Ptpn1, and UCP1 might be more important genes than other remaining 16 genes contributing to glucose homeostasis, since their transcript levels were similar between KK myoblast and KK fibroblast groups. Several of them, such as Acaca, Abbp1, Cfd, Gpd-1, are involved in fat metabolism, storage, and adipogenesis. Acaca is involved in fat metabolism through sterol receptor element binding protein-1 (SREBP1), which is a key transcript factor that regulates glucose metabolism and fat storage [22]. Aebp1 is a regulator in adipose tissue and energy homeostasis [23]. Cfd (also called as Adipsin) is involved in adipogenesis through PPARg, which regulates glucose metabolism and fatty acid storage [24]. Gpd-1 protein plays a critical role in carbohydrate and lipid metabolism [25]. Jun is involved in cell proliferation, cell death, inflammation, and DNA repair in response to a variety of extracellular stimuli [26]. Ptpn1 encodes PTPB1 protein that is a negative regulator of both insulin and leptin signaling and is involved in the control of glucose homeostasis and energy expenditure [27]. UCP1 serves as a proton transporter in mitochondria inner membrane which uncouples oxidative metabolism from ATP synthesis [28]. UCP1 has been reported to play important roles for energy homeostasis in rodents and neonate of larger mammals including human. We found 27 genes of mitochondrial biogenesis and function increased in KK myoblast group as compared with KK fibroblast group. All functional groups had genes that significantly changed in the KK myoblast group. Half of the genes (51.9% of 27) were involved in small molecule transport (7) and mitochondrial transport (7). Except for genes of small molecule transport, inner and outer membrane translocations, most others are also multi-functional and are involved in other functional groups. For example, Aip, Cpt1b, and Grpel1 are not only involved in mitochondrial transport, but are also involved in mitochondrial protein import and targeting proteins to mitochondria. Compared with KK fibroblast group, 9 gene transcripts increased in KK myoblast group: Bcl2l1, Cox10, Cpt1b, Slc25a22, Slc25a25, Stard3, Timm17b, Tomm40, and UCP1. Compared with C57BL group, 5 gene transcripts increased and 1 decreased in C57BL group: Bcl2l1, Opa1, Sfn, Slc25a22, and UCP1 increased, while Slc25a25 decreased. We only found that KK mice which received hSkM transplantation had significantly improved hyperglycemia, glucose tolerance and insulin resistance as compared with KK control and KK fibroblast groups, which did not reach the same glucose homeostasis as C57BL group. These suggest that Bcl2l1, Cox10, Cpt1b, Slc25a22, Slc25a25, Stard3, Timm17b, Tomm40, and UCP1 might be more important genes than other remaining 18 genes contributing to glucose homeostasis. As the remaining 18 gene transcript levels were similar between KK myoblast and KK fibroblast groups. Bcl2L1 is a potent inhibitor of cell death and is located at outer membrane of mitochondria and regulates mitochondrial membrane channel opening, which regulates mitochondrial membrane potential, and thus controls the production of reactive oxygen species and release of cytochrome C by mitochondria, both of which are the potent
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inducers of cell apoptosis [29]. Cox10 is a component of the mitochondrial respiratory chain, catalyzes the electron transfer from reduced cytochrome C to oxygen [30]. Cpt1b is an isoform of carnitine palmitoyltransferase I (CPT1), which is associated with the outer mitochondrial membrane and mediates the transport of long-chain fatty acids across the membrane [30]. Thus, CPT1 play important roles in many metabolic disorders, including diabetes [30]. Slc25a22 and Slc25a25 are mitochondrial carriers that transport glutamate and phosphate across the membranes of the mitochondria, respectively [31], while Stard3 can cause cholesterol transport to the mitochondria [32]. Timm17b and Tomm40 are subunits of mitochondrial inner membrane translocase and mitochondrial outer membrane translocase, respectively. The inner membrane translocase facilitates the translocation of proteins across the inner membrane and into the matrix, while the outer membrane translocase is required for the movement of proteins into mitochondria [33,34]. The expression levels of multi-genes involved in insulin signaling pathway and mitochondria biogenesis and function changed, and these changes probably contributed to enhanced glucose transportation and metabolism in skeletal muscle. This, in turn, achieves reduced hyperglycemia and hyperinsulinemia and ameliorated the diabetic phenotype of the KK mice. In conclusion, our present study indicates that xenotransplantation of normal hSkM into limb skeletal muscle of KK mouse up-regulated multi-genes involved in impaired insulin signaling pathway and mitochondria biogenesis and function. This is accompanied with improved glucose tolerance and reduced hyperglycemia in the KK mouse. Therefore, normal hSkM transplantation could regulate expression level of genes that play important roles in pathogenesis of skeletal muscle insulin resistance.
5.
Conflict of interest
None.
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