KPC1 dishomeostasis-mediated by sympathetic nervous system over-excitement involved in myofiber types alteration and insulin resistance through NF-KB signaling pathway

KPC1 dishomeostasis-mediated by sympathetic nervous system over-excitement involved in myofiber types alteration and insulin resistance through NF-KB signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article KPC1 dishomeostasis-mediated by sympathetic nervous system over-excitement involved in myofiber types alteration and insulin resistance through NF-KB signaling pathway Zhong-yu Wang, Shi-jun Yi, Wei Xu, Li Xiang, Ye-peng Peng, Jing Yue, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8438697/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Whether sympathetic over-activity involved in skeletal muscle insulin resistance and development of type 2 diabetes (T2DM) through affecting the differentiation and fusion of skeletal muscle satellite cells and changes in muscle fiber types, is still unknown. KPC1 deficiency deteriorated norepinephrine (NE)-induced loss of slow-twitch type I myofibers by micro osmotic pump (MP) and increased insulin resistance in mice. Continuous single dose (CS) and MP administration of NE and epinephrine (E) have been created, showing the inhibitory effects on C2C12 myoblast cells differentiation/fusion and type I slow-twitch fiber formation and glucose transporter type 4, glucose transport and uptake in vitro , matching with the changes of skeletal muscle fiber types in early and late T2DM in vivo , respectively. Notably, KPC1 expression showed the unique expression mode that it was gradually increased, reaching peak value on the fourth day, and then gradually decreased during the process of myoblast cells differentiation/fusion and myofiber formation. Furthermore, over-expression of KPC1 on third day, not zero day, showed the almost complete reversal effect on the inhibitory role of NE in myoblast cells differentiation/fusion and type I slow-twitch fiber formation, indicating KPC1’ specific time and dosage mode of action. Mechanistic studies revealed that NE reduced KPC1 and NF-KBp50/p65 protein levels, especially in the NF-KBp50 nucleation distribution. Curiously, E increased KPC1 and NF-KBp50 protein levels while unchanged NF-KBp65 levels, but showing the similar inhibitory effects on myoblast cells differentiation/fusion and slow-twitch type I myofiber formation. NE or E-mediated inhibitory role in myoblast differentiation/fusion could be worsen by NF-KBp50 inhibitor SN50, but partially abolished by NF-KBp65 blocker PDTC. Taken together, over-excitation of the sympathetic nervous system disturbed the balance between KPC1's time and dosage to cause the common inhibitory role in myoblast differentiation/fusion and slow-twitch fiber formation and glucose utilization, which were associated with abnormal matching of NF-KBp50/p65 signaling. Excessive activation of sympathetic nervous system myoblast differentiation/fusion myofiber types KPC1 NF-KB T2DM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Type 2 diabetes (T2DM) is on the increase, which seriously threatens the health of people. T2DM is closely related to insulin resistance. And skeletal muscle is the main site of peripheral insulin resistance [ 1 ]. Actually, in the early stage of diabetes, slow muscle fibers were significantly reduced, while fast muscle fibers were significantly increased, and skeletal muscle atrophy occurred in the late stage of diabetes [ 4 ]. Previous studies have often attributed changes in skeletal muscle fibers to the damaging effects of high blood sugar. Of note, T2DM is often accompanied by autonomic nervous dysfunction characterized by increased sympathetic activity [ 2 ], which further worsens diabetes or further impairs glucose metabolism [ 3 ]. However, apart from harmful changes of sympathetic nerves following T2DM, whether sympathetic over-activity involved in skeletal muscle insulin resistance and development of T2DM through affecting the differentiation and fusion of skeletal muscle satellite cells and changes in muscle fiber types, is still unknown. Actually, transient increase in sympathetic activity and abnormal glucose tolerance in the body may be a protective response from temporary danger, cold, exercise, staying up late and stress [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. However, it is still unclear whether the intermittent, continuous increase in sympathetic activity leads to a transition from protective response to compensatory to damaging changes, especially whether it preferentially affects the differentiation and fusion of skeletal muscle satellite cells and changes in muscle fiber types, and participates in skeletal muscle insulin resistance and T2DM. It is worth noting that when the sympathetic nervous system is over-activated, not only does it release more norepinephrine (NE) from its terminals, but the activated sympathetic nervous system also activates the adrenal medulla to synthesize and release NE and epinephrine (E), further enhancing the effect of sympathetic over-activation and involving β-adrenergic receptors (β-AdR ) and α-AdR [ 14 ]. Considering that NE mainly activates α1-AdR, but also activates β1-AdR; And E mainly activates β1-AdR, but also activates α1-AdR [ 15 ]. To better prevent and treat T2DM in the early stage, revealing the role of different forms of sympathetic nervous system over-excitation in skeletal muscle insulin resistance and the development of T2DM has become a key issue that must be addressed. Looking back at previous studies, single dose administration with a high dose of 5 × 10 − 5 M/L were used to induce skeletal muscle atrophy [ 16 ]. However, this administration method in vitro was difficult to truly reflect and embody the impact of over-excitation of the sympathetic nervous system on skeletal muscle in vivo . Therefore, it is necessary to create new cell models that can more accurately reflect and simulate the relationship between different pattern of over-excitation of the sympathetic nervous system and changes in the characteristics of skeletal muscle satellite cells. Considering that the process of differentiation and fusion of myoblasts to form myotubes and their muscle fibers involves protein synthesis and degradation, among which the ubiquitin proteasome pathway is one of the most important protein degradation pathways, which participates in maintaining the balance between protein production and degradation [ 14 ]. Kip1 ubiquitination-promoting complex 1 (KPC1) is an important regulatory factor in the ubiquitin proteasome system, and its most amazing function is ubiquitination of NF-kB p105, which is degraded and cleaved by proteases to form active p50[ 17 ]. Over-expression of KPC1 produces excessive p50 while down-regulating NF-kBp65, promoting tumor suppressor signal expression and limiting tumor growth [ 18 , 19 ]. Unfortunately, previous reports on the role of NF-kB in the differentiation and fusion of myoblasts to form myotubes and muscle fibers have only focused on NF-kBp65, and researchers have attributed the inhibitory effect of NF-kBp65 on myoblast differentiation to its promotion of myoblast proliferation [ 20 ]. However, it is still unclear whether excessive sympathetic nervous system excitation regulates the differentiation and fusion of myoblasts to form myotubes and muscle fibers by affecting KPC1 and its NF-kB. In the study, for the first time, the excitation pattern of sympathetic nervous system will be divided into at least four types based on the degree, frequency, and duration of excitation: transient, intermittent transient, continuous transient and sustained transient. Our results showed that the difference in the release patterns of NE and E not only affected the differentiation and fusion of skeletal muscle cells and the formation of myotubes, but also the type of muscle fibers, which are involved in the early reduction of slow muscle fibers and the increase of fast muscle fibers mediated by skeletal muscle insulin resistance in T2DM, as well as muscle atrophy in the late stage of T2DM. Methods Animals Based on a C57BL/6 background, KPC1 gene knockout (KPC1-/-) mice was generated through CRISPR-Cas9 technology (deletion of exons 1, 2, 8, 9 of NM_ 84585) on Cyagen Biosciences Inc., Suzhou, China (Supplementary Fig. 1). Genotyping with genomic DNA extracted from mouse tails was identified by using PCR technology. The primers (Sangon Biotech Co., Ltd., Shanghai, China) used were as follows: F1: 5’-TCAACCCTTGGACAGTATCCTC-3’; R1: 5’-CCTGTCACCCTTCTCCTTGAAC-3’; R2: 5’-GTCTGCTGTTTCTTACCCTC AC-3’. KPC1 +/− , KPC1 −/− and KPC1 +/+ (Wild-type, WT) male mice from the same litter of mice were used in all studies unless otherwise stated. All the mice were housed in SPF (Specific Pathogen Free) class Laboratory Animal Center of Hubei University of Medicine, with a 12 h light/dark cycle and disinfected water. All mouse experiments were performed following the protocols approved by the Laboratory Animal Care and Ethics Committee of Hubei University of Medicine, China. The ethics committee number is 2024-S-169. The work has been reported in line with the ARRIVE guidelines 2.0. Reagents and chemicals SN-50 and PDTC were purchased from Selleck Chemicals LLC (Houston, Texas, USA), and prepared for the following experimental according to the manufacturer's instructions. Immunofluorescence double staining of skeletal muscle tissue The mice were anesthetized with isopentane, and then blood samples were collected. The mice were sacrificed by carbon dioxide and skeletal muscles were removed. The dissected tibialis anterior muscles, and soleus muscles from WT and KPC1-/- mice with or without osmotic pump administration of norepinephrine (NE) were fixed for 48 hours in 4% paraformaldehyde (BL539A, biosharp®, Beijing Labgic Technology Co., Ltd., Beijing, China), and embedded in paraffin (80200-0017, Citotest Labware Manufacturing Co., Ltd., Haimen, China). The 5-micrometer-thick slice of the above samples were cut out and deparaffinised. Antigen retrieval were performed for these sections following the treatment of sodium citrate (pH 6.0) (BL604A, biosharp®, Beijing Labgic Technology Co., Ltd., Beijing, China) at 95°C for 5 minutes. One hour after optimal non-specific antigen blocking with 5% goat serum (ANT052, AntGene Biotechnology Co., Ltd., Wuhan, China) at room temperature. And then, the section were incubated with primary antibodies against type I fibers (1:200, ab234431, Abcam Corporation, Cambridge, England) and type II fibers (1:200, ab51263, Abcam Corporation, Cambridge, England) at 4°C overnight. Finally, appropriate horseradish peroxidase (HRP)-labelled secondary antibodies were used to show the fibers, and hematoxylin counterstaining of cell nuclei were performed. These images were captured under a microscope (BX53 + DP74, Olympus Corporation, Japan) [ 21 ]. Glucose tolerance test (GTT) and insulin tolerance test (ITT) For the GTT, 20% glucose (A100188-0500, Sangon Biotech Co., Ltd., Shanghai, China) solution (1 mL/kg body weight) were intraperitoneally injected into the mice 16 hours after fasting. After injection, blood glucose was measured at the indicated time (0, 15, 30, 60, 90 and 120 min). For the ITT, insulin (BDPH-0036-A, biosharp®, Beijing Labgic Technology Co., Ltd., Beijing, China) (0.5 U/kg body weight) were subcutaneously injected into the mice 4 hours after fasting, and then blood glucose were measured at the indicated time (0, 15, 30, 45, 60 and 90 min) [ 22 ]. To control the order of injection and measurements, a fixed ear tag numbers was applied. Myoblast culture and differentiation C2C12 myoblast cells (SCSP-505) were purchased from the Cell Resource Center of the Shanghai Academy of Life Science, affiliated to the Chinese Academy of Sciences. The culture medium with high-glucose Dulbecco’s modified Eagle medium (DMEM, C11995500BT, Gibco®, Grand Island, New York State, USA) containing 10% fetal bovine serum (FBS, SA211.02, CellMax Co., Ltd., Beijing, China) and 1% v/v penicillin/streptomycin (SV30010, HyClone, Logan, Utah, USA), were used to culture C2C12 myoblast cells in an incubator with 5% CO 2 at 37°C. Myoblast differentiation was performed following our previously reported method [ 22 ]. Briefly, the cells were cultured under the above medium and reached 75% confluence, and then incubated with replaced differentiation medium (DM) containing high-glucose DMEM and 2% horse serum (BI 04-124-1A, Sigma, St. Louis, Missouri, USA) for the indicated times, were observed daily by an inverted microscope (CKX53, Olympus Corporation, Japan). At the specified time points, differentiated myoblasts were observed and stained with the mature muscle fiber marker myosin heavy chain (MyHC) (1:200, sc-20641, Santa Cruz, Dallas, Texas, USA), type I fibers (1:200, ab234431, Abcam Corporation, Cambridge, England) and type II fibers (1:200, ab51263, Abcam Corporation, Cambridge, England), and imaged under a fluorescence microscope (COOLSHOT80i, Nikon, Japan). Fluorescence imaging was performed with the following filter sets: DAPI (excitation 325–375 nm, emission 435–485 nm), FITC (excitation 460–500 nm, emission 510–560 nm), and TRITC (excitation 530–560 nm, emission 570–620 nm) [ 22 ]. Methods for continuous single-dose or osmotic pump administration of NE and E in vitro To match the traits of long-term physiological or pathological levels NE or E in the tissues and plasma of people with health, sub-health and T2DM [ 13 , 23 , 24 , 25 , 26 ], we created a novel method of osmotic pump administration of NE and E in vitro , apart from continuous single-dose administration in vitro . Briefly, osmotic pump administration of NE or E were treated at zero day of myoblast differentiation with replacing differentiation medium (DM) containing high-glucose DMEM and 2% horse serum for 6 days, different single doses were loaded as indicated (10 − 9 Mol/L, 10 − 8 Mol/L,10 − 7 Mol/L and10 − 6 Mol/L). Continuous single-dose administration of NE or E were treated at zero day of myoblast differentiation, and then added with NE or E once a day for 6 days. Different single doses, as indicated (10 − 8 Mol/L, 10 − 7 Mol/L,10 − 6 Mol/L and10 − 5 Mol/L), were added to the DM at 7–8 PM each day when the DM was replaced. Detection of myoblast differentiation/fusion and myotube morphology by immunofluorescence Immunofluorescence staining was performed as our previously described [ 22 ]. Briefly, at the indicated time, after washed with PBS, the cells were fixed with 4% paraformaldehyde (BL539A, biosharp®, Beijing Labgic Technology Co., Ltd., Beijing, China), and incubated with the primary antibody MyHC (myotube marker, 1:200, sc-376157(FITC), sc-376157 (TRITC), sc-20641, Santa Cruz, Dallas, Texas, USA) and appropriate fluorescence-labelled secondary antibodies. The nuclei were stained with DAPI (D9542, Sigma-Aldrich®, Merck KGaA, Germany). Only MyHC positive myoblasts with 3 or more nuclei within a cellular structure were defined as myotubes, the other were excluded as differentiated cells without mutual fusion to myotubes. Two double-blind individuals evaluated all images by using Image J. The numbers of myotubes with more than 5 nuclei were calculated and analyzed. Differentiated myoblasts were stained for Glut4, MEF2C, MyHC, NOQ or My32 using the polyclonal primary antibodies Glut4 (1:150,ab33780, Abcam Corporation, Cambridge, England), MEF2C (1:200, 5030S, Cell Signaling Technology Inc., Danvers, Massachusetts, USA), MyHC (1:200, sc-376157-FITC), slow skeletal myosin (NOQ7.5.4D, NOQ, 1:200, ab11083, Abcam Corporation, Cambridge, England) or fast myosin skeletal heavy chain (My32, ab51263, 1:200, Abcam Corporation, Cambridge, England) and the appropriate fluorescence-conjugated secondary antibodies. Images were captured under a microscope (IX53 + DP73, Olympus Corporation, Japan). The fluorescence microscope equipped with filter sets was used under the following conditions: a DAPI filter set (excitation: 325–375 nm, emission: 435–485 nm), a FITC filter set (excitation: 460–500 nm, emission: 510–560 nm), and a TRITC filter set (excitation: 530–560 nm, emission: 570–620 nm)[ 22 ]. RT-qPCR Based on the expression of myosin heavy chain (MyHC) isoform, Skeletal muscle fibers are generally classified into four types: MyHC-1 (slow-twitch oxidative), MyHC-2a (fast-twitch oxidative), MyHC-2b (fast-twitch glycolytic), and MyHC-2x (fast-twitch oxidative-glycolytic) [ 22 ]. Their expression were detected on a CFX96™ real-time PCR detection system (Bio-Rad Inc., California, USA) using HiScript® III RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme Biotech Co., Ltd., Nanjing, China). The relative quantification 2 −ΔΔCt method was used to evaluate the data, and expressed as mean ± SD. The four types myofibers primer sequences were shown in Supplementary Table 1. Western blot Western blot C2C12 myoblast cells were lysed in RIPA buffer (MA0151, MeilunBio®, Dalian, China) supplemented with PMSF (MB3800-1, MeilunBio®, Dalian, China). Proteins were separated by SDS-PAGE gel and transferred to a PVDF membrane (ISEQ00010, Merck Millipore, Merck KGaA, Germany). After the membrane was blocked with 5% skim milk (P0216, Beyotime Biotechnology Inc., Shanghai, China), the membrane was incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies (IgG). The primary antibodies used were as follows: α-tubulin (1:10000, ab7291, Abcam Corporation, Cambridge, England), KPC1 (1:1000, sc-101122, Santa Cruz, Dallas, Texas, USA), NF-kBp50 (1:1000, 13586S, Cell Signaling Technology Inc., Danvers, Massachusetts, USA), NF-kBp65 (1:1000, sc-109, Santa Cruz, Dallas, Texas, USA). Protein expression was detected with a ChemiDoc™ system (Bio-Rad Inc., California, USA), and gray level analysis was carried out with ImageJ software. Adenoviral vector construction, transfection and myotubes types assay KPC1-knockdown (Ad-shKPC1) adenoviral vectors used in our lab were constructed as described previously [ 21 ]. And KPC1-overexpressing (Ad-KPC1) adenoviral vectors were purchased from WZ Biosciences Inc (Jinan, China). To study the role of KPC1 in myoblast differentiation, Ad-shKPC1 or Ad-KPC1 (1 × 10 9 pfu) was added to the DM in zero or third day of myoblast cells differentiation. Meanwhile, continuous single-dose administration of NE (10 − 5 Mol/L) were treated at third day of myoblast differentiation, and then added with NE once a day for 6 days. At last, MyHC (1:200, sc-376157(FITC), sc-376157 (TRITC), sc-20641, Santa Cruz, Dallas, Texas, USA), slow skeletal myosin (MyHC-I, NOQ7.5.4D, NOQ, 1:200, ab11083, Abcam Corporation, Cambridge, England) or fast myosin skeletal heavy chain (MyHC-II, My32, ab51263, 1:200, Abcam Corporation, Cambridge, England), and appropriate fluorescence-labelled secondary antibodies were used to perform immunofluorescence staining. The nuclei were stained with DAPI (D9542, Sigma-Aldrich®, Merck KGaA, Germany). MyHC, MyHC-I or MyHC-II positive myoblasts with 3 or more nuclei within a cellular structure were defined as myotubes, the other were excluded as differentiated cells without mutual fusion to myotubes. The numbers of myotubes with more than 5 nuclei were calculated and analyzed. Over-expression and knockdown of KPC1 were identificatied by RP-PCR in myoblast cells after transfection (Supplementary Fig. 2). Immunofluorescence staining for NF-κB p50 C2C12 myoblast cells were cultured under the above medium and reached 75% confluence, and then incubated with replaced differentiation medium (DM) containing high-glucose DMEM and 2% horse serum (BI 04-124-1A, Sigma, St. Louis, Missouri, USA). Subsequently, continuous single-dose administration of NE (10 − 5 Mol/L) were treated at third day of myoblast differentiation, and then added with NE once a day for 6 days, and Ad-KPC1 was added to the DM in third day of myoblast cells differentiation. Finally, the differentiated myoblasts were observed and stained with NF-kBp50 (1:1000, 13586S, Cell Signaling Technology Inc., Danvers, Massachusetts, USA), and imaged under a fluorescence microscope (COOLSHOT80i, Nikon, Japan). Fluorescence imaging was performed with the following filter sets: DAPI (excitation 325–375 nm, emission 435–485 nm), FITC (excitation 460–500 nm, emission 510–560 nm) [ 22 ]. Glucose uptake assay Four days After C2C12 myoblasts differentiation in 24-well plates, glucose uptake assays were done by using the Glucose Uptake-Glo™ Assay Kit (J1341, Promega Corporation, Madison, Wisconsin, USA) following the manufacturer's instructions [ 22 ]. RNA-seq analysis Tibialis anterior muscles (TA), and soleus muscles (SL) from WT mice were sent to OE Biotech Co., Ltd. (Shanghai, China) for RNA extraction, library preparation, and sequencing. 150-bp paired-end reads were generated. Low quality reads were removed from the raw reads, and the clean reads were mapped to the Mus musculus reference genome sequence. Read counts for each gene were calculated using htseq software. Fragments per kilobase of exon per million mapped fragments (FPKM) were used to assess gene expression. Differentially expressed genes were identified using the following criteria: corrected P 1[ 22 ]. Transmission electron microscopy 28 days after, Mitochondrial function were evaluated by using transmission electron microscopy in soleus muscles (SL) of mice with or without osmotic pump administration of NE. Statistical analysis Experimental data were analyzed and plotted by GraphPad Prism 8.0 software, and all the data are expressed as mean ± SD (standard deviation). Two groups were compared using the two-tailed Student's t-test and the confidence level is 95%, and more than two groups were compared using the One-way ANOVA test if the data normally distributed. The nonparametric test was used if the data was not normally distributed. Results Osmotic pump administration of norepinephrine increased blood glucose levels while reduced numbers and mitochondrial integrity of type I muscle fibers To observe the effect of osmotic pump administration of norepinephrine on myoblast differentiation, fusion, myotube formation and specialization, we firstly uesed RNAseq to confirm the difference between the soleus muscles (SL, major MyHC-I) and tibialis anterior muscles (TA, major MyHC-II), we found that adrenergic receptors (AdR) were more different that SOM majorly expressed Adra1a, Adra2b, Adra2c, Adrb1, Adrb2 and Adrb3 while TA expressed Adra2a (Figure.1A). After treatment of osmotic pump administration of norepinephrine, MyHC-I positive myofibers numbers obviously decreased in the SL compared with physiological saline treatment (Figure.1B-1C). Meanwhile, typical changes such as mitochondrial fragmentation and membrane rupture were found in the SL with osmotic pump administration of norepinephrine (Figure.1D), and blood glucose levels were higher than physiological saline treatment (Figure.1D). These indicated that NE-induced blood glucose imbalance could be associated with the type I muscle fibers numbers and mitochondrial integrity. The effects of simulated in vitro administration of three modes of sympathetic nervous system excitation on myoblast differentiation, fusion, and myotube formation To mimic the the at least three pattern of sympathetic nerves over-excitation such transient, continuous transient and sustained transient, apart from physiological excitation, single dose (S), continuous single dose (CS), and micro osmotic pump (MP) administration of norepinephrine (NE) have created and used to treat C2C12 myoblast cells under differentiated condition. After 6 days, the fusion and myotube characteristics were analyzed by MyHC (myosin heavy chain) immunofluorescence staining under microscopy. Compared with single dose administration, continuous single and micro osmotic pump treatments significantly inhibited the differentiation, fusion, and myotube formation of C2C12 myoblasts cells, especially the micro osmotic pump (Supplemental Figure.3A-3G). NE reduced the numbers of type I muscle fiber than type II muscle fiber To further confirm the role of continuous single dose (CS), and micro osmotic pump continuous (MP) administration of norepinephrine (NE) on myoblast differentiation, fusion, myotube formation and specialization, different dosages NE were used, as shown in Supplemental Fig. 4–6, we found that continuous single dose administration of NE inhibited myoblast differentiation, fusion, myotube formation and specialization of type I and II myofiber in dosage-dependent manner, however, resulting in the increases of type II myofiber. The difference is that, micro osmotic pump administration of NE showed a stronger inhibitory effect on the aforementioned observations. More importantly, under similar total dosage conditions, micro osmotic pump administration of NE inhibited more obviously myoblast differentiation, fusion, myotube formation and specialization of type I and II myofibers while continuous single dose administration of NE obviously reduced numbers of type I myofibers (Figure.2A-2E, Figure.3A-3B), and increased the expressions of MyHC-IIb and MyHC-IIX mRNA (Figure.2A-2D). These results indicated that different release patterns of NE could be associated with the formation of different muscle fiber types. NE reduced the glucose transport uptake of myotubes To further confirm if NE-induced myofibers types changes involved in the alteration of the glucose transport uptake, we detected the expressions of Glut4 and IRS-1, finding that continuous single or micro osmotic pump administration of NE could inhibit the expressions of Glut4 and IRS-1 in dosage-dependent manner (Supplemental Figure.7A-7D). Furthermore, micro osmotic pump continuous administration of NE more obviously decreased expression of Glut4 and IRS-1 compared with continuous single dose administration (Figure.3C-3E). Meanwhile, the glucose transport uptake in myotubes were more significantly reduced following micro osmotic pump continuous administration of NE than continuous single dose administration (Figure.3F). These results indicated that different release patterns of NE could be associated with the glucose transport uptake mediated by the altered muscle fiber types. KPC1 involved in specialization of muscle fiber types and blood glucose homeostasis Since the altered myofibers types involved in degradation of proteins, the RING finger protein family (RNFs) performed the E3 ubiquitin ligase to affect the development of tumors, immune and neurodegenerative diseases [ 27 ]. We firstly used RNAseq to confirm the difference of RNFs between the soleus muscles (SL) and tibialis anterior muscles (TA), we found that Rnf123 (KPC1) showed lower expressions in the SL compared with TA (Figure.4A). Importantly, we found that knockout of KPC1 reduced the numbers of MyHC-I positive myofibers in the SL, compared with wide-type (WT) mice (Figure.4B-4C). In line with the reduced MyHC-I positive myofibers, blood glucose tolerance and insulin sensitivity were slightly decreased in WT mice with treatment of NE (Figure.4H-4I). To confirm the similarity of altered muscle fiber types between NE and KPC1 during myoblast cells differentiation, fusion and myotubes formation, we firstly detected the changes of KPC1 during the myoblast cells differentiation, and found that KPC1 mRNA expressions were gradually increased, reaching peak value on the fourth day of myoblast cells differentiation, and then gradually decreased during the whole 6 days of differentiation, fusion and myotubes formation (Figure.4D). Furthermore, knockdown of KPC1 inhibited myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-I positive myofiber formation (Figure.4F-4G). These results indicated that KPC1-mediated myofibers types changes could involve in regulation of blood glucose homeostasis. KPC1 involved in the formation of muscle fibers in time and dosage-dependent manner To confirm the characteristic of KPC1 on myoblast cells differentiation, fusion and myotubes formation, KPC1 over-expression medated by adeno virus (Ad-KPC1) were added to transfect into myoblast cells on the zero or third day of myoblast cells differentiation, results showed that KPC1 over-expression following the transfection of Ad-KPC1 at the zero day inhibited myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-I positive myotubes, compared with third day transfection. Furthermore, the difference is that KPC1 over-expression following the transfection of Ad-KPC1 at the third day slightly promoted myoblast cells differentiation, fusion and myotubes formation of MyHC-I and MyHC-II (Figure.5A-5E). These results indicated that KPC1 involved in the formation of muscle fibers in time and dosage-dependent manner. KPC1 reversed the inhibitory effects of NE on the reduced type I muscle fibers numbers in time and dosage-dependent manner To further confirm if NE-mediated muscle fibers types alteration involved in KPC1, KPC1 expressions were detected following NE treatment, results showed that NE reduced KPC1 mRNA and protein levels in dosage-dependent manner (Figure.6A-6B). Furthermore, knockout of KPC1 further reduced type I muscle fibers numbers in the SOM following the micro osmotic pump administration of NE (Figure.6C-6D). On the contrary, the addition of Ad-KPC1 on the zero day of myoblast cells differentiation, partially reversed the inhibitory effect of NE on myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-II positive myotubes. However, the addition of Ad-KPC1 on the third day could almost completely recovered the NE-mediated inhibition into the relative normal levels of myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-I positive myotubes (Figure.6F-6H, and Supplemental Figure.8). These results indicated that KPC1 could reverse the inhibitory effects of NE on the reduced type I muscle fibers numbers in time and dosage-dependent manner. KPC1 reversed the inhibitory effects of NE-induced myoblast cells differentiation and myotubes formation through NF-KBp50 Since KPC1 acted as E3 ubiquitin ligase to promote NF-KBp105 degradation into NF-KBp50 [ 17 , 18 , 19 ]. As shown in Figure.7A-7B, NE reduced the levels of NF-KBp50 and NF-KBp65 following NE-induced KPC1 decrease (Fig. 6 A- 6 B), and the number of NF-KBp50 positive cell nuclei, especially in reduction of trend of nucleation distribution of NF-KBp50 (Figure.7C-7D). However, the addition of Ad-KPC1 on the zero day of myoblast cells differentiation, partially reversed the NE-mediated effects. Meanwhile, NF-KBp50 inhibitor SN50 deteriorated the inhibitory role of NE in myoblast cells differentiation, fusion and myotubes formation while NF-KBp65 inhibitor PDTC partially blocked the NE-mediated effects (Figure.7E). These results indicated that the inhibitory effects of NE could be related with the reduced NF-KBp50 accompanied by the decreased KPC1 levels. KPC1 reversed the inhibitory effects of epinephrine-induced myoblast cells differentiation and myotubes formation through NF-KB Since the activated sympathetic nervous system also activates the adrenal medulla to synthesize and release NE and epinephrine (E) [ 14 , 15 ], as shown in Supplemental Fig. 9–12, we found that continuous single or micro osmotic pump administration of epinephrine (E) or isoproterenol (ISO) showed the inhibitory effects of myoblast cells differentiation, myotubes fusion, MyHC-I, and MyHC-II positive muscle fibers formation similar to NE administration. However, unlike NE, both E and ISO increased the levels of KPC1 proteins, resulting in the increased levels of NF-KBp50 and NF-KBp65 proteins in dosage-dependent manner (Figure.8A-8H). Similarly, SN50 deteriorated the inhibitory role of E or ISO in myoblast cells differentiation, fusion and myotubes formation while PDTC partially blocked the E or ISO-mediated effects (Figure.8I). These results indicated that the inhibitory effects of E or ISO could be related with the increased NF-KBp65 accompanied by the increased KPC1 levels. Discussion In this study, we have identified KPC1 as a novel factor for blood glucose homeostasis, acting as a linker between skeletal muscle insulin resistance and sympathetic nervous system (SNS) over-excitation in vivo . KPC1 deficiency disrupts normal skeletal muscle types, leading to significant increases in skeletal muscle insulin resistance. Since sympathetic nervous system (SNS) over-excitation involved in insulin resistance and developing T2DM, unbalanced KPC1 may be an essential factor causing overactivated SNS-related T2DM. Thus, KPC1−/− mice may serve as a valuable model for dissecting molecular mechanisms underlying over-activated SNS-related T2DM. Indeed, we have created a novel myoblast differentiation, fusion and special myofiber formation model in vitro that matched the traits of myofiber types with early and late T2DM in vivo , identifying a novel mechanism underlying over-activated SNS-linked T2DM; i.e., decreased type I myofiber types due to decreased expression of NF-KBp50 in myoblast cells by KPC1 deficiency. Factors such as danger, cold, staying up late, lack of sleep, stress, and intense exercise often cause abnormal SNS excitation [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 23 , 24 , 25 , 26 ], based on its degree, frequency, and duration, SNS excitation pattern is for the first time divided into at least four types: transient, intermittent, continuous and sustained. In our previous study, matching with transient, intermittent, continuous excitation pattern of SNS, single dose, interval single dose and continuous single dose administration of ISO were created, finding that continuous single dose administration of ISO significantly inhibited C2C12 myoblast differentiation, fusion and myotube formation [ 28 , 29 ]. Herein, in order to better simulate the relationship between the sustained increase activity of SNS in vivo and the functional changes of skeletal muscle satellite cells, a novel model was created to treat C2C12 myoblasts by releasing norepinephrine (NE) or epinephrine (E) through an osmotic pump. We found that continuous single administration of NE or E significantly inhibited the differentiation, fusion, and myotube formation of C2C12 myoblast cells, mainly decreasing MyHC-Ⅰ positive myofibers formation while relatively increasing MyHC-II positive myofibers numbers, in line with the changes of skeletal muscle fiber types in early T2DM. Excitingly, osmotic pump release of NE or E not only prominently blocked the differentiation, fusion, and myotube formation of C2C12 myoblast cells, but also reducing the formation and numbers of MyHC-Ⅰ and MyHC-II positive myofibers, which could consistent with the changes of skeletal muscle fiber types in advanced T2DM. These results suggested that two unique models of myoblast cells in vitro that matched the traits of myofiber types with early and late T2DM in vivo , were created for future research. Due to the association between skeletal muscle insulin resistance and T2DM with reduced expression of Glut4, IRS-1, etc.[ 30 , 31 ], this study found that continuous single administration or osmotic pump release of NE or E concentration dependently reduced the expression of Glut4 and IRS-1, accompanied by similar effects of the inhibited myoblast cells differentiation/fusion, and decreased MyHC-Ⅰ positive myofibers. Further analysis of the differences between the two modes revealed that, under the same total concentration, the NE or E mode of osmotic pump release more dramatically down-regulated Glut4 and IRS-1 expression compared to the continuous single dose mode. At the same time, it reduced the transport and uptake of glucose. Herein, these results indicated that the created administration patterns of NE or E in vitro matching with the continuous and sustained increase in SNS excitation in vivo were respectively involved in the abnormal increase of blood glucose in the early and late stages of T2DM, which were associated with functional changes of myoblast cells and myofiber types. Although previous results showed that excess of E could trigger glucose intolerance largely by damaged insulin secretion and excess of NE could trigger glucose intolerance largely by induced insulin resistance [ 32 ], our present finding were consistent with abnormalities of carbohydrate metabolism in patients with SNS over-excitation [ 33 , 34 , 35 , 36 , 37 ]. In a word, relationship between SNS over-excitation related diseases including hypertension and subtle and overt abnormalities of carbohydrate metabolism should be a real existence, furthermore, both NE and E could regulate blood glucose homeostasis through our newly identified common skeletal muscle target organs, particularly inhibition of myoblast differentiation/fusion and slow muscle reduction. Considering the obvious difference of RNFs targeting the ubiquitin proteasome system between slow and fast muscle fibers, especially in RNF123 (KPC1), in order to intervene early in the changes of skeletal muscle fibers and blood glucose imbalance mediated by SNS over-activation, myoblast cells model with continuous single administration of NE or E were used to reveal the relationship between SNS and KPC1. Unlike the role of KPC1 in mediating tumor occurrence and development [ 17 , 18 , 19 ], we firstly found that the expression of KPC1 was gradually increased, reached the highest on the fourth day, and then falling and maintaining a high levels during the differentiation and fusion of myoblasts cells to form myotubes and myofiber, showing a time- and dose-dependent traits. To further unlock its functional characteristics, we administered KPC1 over-expression treatment on the 0th and 3rd day of myoblasts cells differentiation, and found that day 0 treatment significantly inhibited myoblast differentiation/fusion, type I and II myofibers formation, which were consistent with the increased expression of KPC1 induced by ISO or E. Meanwhile, KPC1 knockdown treatment on the 0th and 3rd day of myoblasts cells differentiation obviously reduced myoblast differentiation/fusion and type I and II myofibers numbers. Surprisingly, KPC1 over-expression treatment on the 3rd day of myoblasts cells differentiation almost completely abolished the NE-mediated inhibitory effects. These results indicated that at the appropriate time, the appropriate expression of KPC1 should exhibit unique temporal biological characteristics in skeletal muscle regeneration and repair, providing a new target for the precise treatment of skeletal muscle atrophy and related diseases such as T2DM in the future. Of note, although NE decreased KPC1 expression, while both E and ISO increased its expression, both NE, E and ISO exhibited same inhibitory effects on myoblast cells differentiation/fusion and myofibers formation, which was related to their binding to different receptors. For example, α-AdR agonist phenylephrine and β2-AdR agonist could promote the myoblast cells differentiation/fusion and myofibers formation [ 28 , 29 , 38 , 39 ]. In addition, NE, E and ISO could desensitize β2-AdR and α1-AdR, and then bind other receptors to perform its effects [ 40 , 41 , 42 , 43 , 44 ]. Considering the unique function of KPC1 in the process of myoblast cells differentiation, the same inhibitory effects mediated by NE, E and ISO could be associated with the altered expression time and level of KPC1, indicating that KPC1 could be a common target for the abnormal glucose regulation of patients with the excess of NE and/or E. The most amazing function of KPC1 is its ubiquitination regulation of NF-kB1 p105, which is degraded and cleaved by proteases to form active p50. Over-expression of KPC1 produces excessive p50 while down-regulating NF-kBp65, promoting tumor suppressor signal expression and limiting tumor growth [ 17 , 18 , 19 ]. Similarly, we found that NE reduced KPC1 expression and NF-kBp50 and p65 levels; however, E increased KPC1 expression and NF-kBp50, but p65 levels were not changed. Previous reports had only focused on NF-kBp65 and attributed its inhibitory effect on myoblast differentiation to its promotion of myoblast proliferation [ 20 , 45 , 46 ]. Unfortunately, the role of NF-kBp50 in the differentiation and fusion of myoblasts cells to form myofibers is still unknown. Herein, we unexpectedly discovered that the NF-kBp50 inhibitor SN50 not only worsened NE but also worsened the inhibitory effect of E or ISO-mediated myoblast differentiation and fusion. NF-kBp65 inhibitor PDTC partially cancelled the inhibitory effects mediated by NE, E or ISO. In a word, NF-kBp50 could play a crucial role in the differentiation and fusion of myoblasts and the formation of myofibers, apart from the interaction with NF-kBp65. Conclusion In summary, the pattern differences of SNS over-activation may be involved in the occurrence and development of skeletal muscle insulin resistance and T2DM by triggering KPC1 ‌dis-homeostasis, disrupting the balance of NF-kBp50/p65, leading to inhibition of myoblast cells differentiation and fusion, changes in myofiber types, and providing new strategies for early intervention of skeletal muscle insulin resistance to prevent and treat T2DM(Fig. 9 ). Abbreviations AdR adrenergic receptor T2DM Type 2 diabetes mellitus NE norepinephrine E epinephrine ISO isoproterenol GA gastrocnemius muscle TA tibialis anterior SL soleus muscle Ad Adenovirus MyHC Myosin heavy chain NOQ Slow skeletal myosin heavy chain My32 Fast myosin skeletal heavy chain MEF2C Myocyte-specific enhancer factor 2c MyoG Myogenin Glut4 Glucose transporter protein 4 RNFs RING finger protein family KPC1 Kip1 ubiquitination-promoting complex 1 SNS sympathetic nervous system MEF2C Myocyte-specific enhancer factor 2C MyoD Myogenic differentiation 1 M Mol/L ShRNA Short hairpin RNA Declarations Supplementary information Supplementary information for this study is available in the online Supplementary materials. Consent for publication No. Clinical trial number Not applicable. Competing interests All the authors declare that they have no conflicts of interest. Funding This work was supported by National Natural Science Foundation of China (82270299 to J.M.T), the Hubei Provincial Natural Science Foundation (2025CSA046 to J.M.T, 2022CFB005 to L.C, 2024AFC024 to Y.W ), the Educational Commission Fund of Hubei Provincial (D20222107 to L.C ), Open Project of Hubei Provincial Clinical Medical Research Center for Umbilical Cord Blood Hematopoietic Stem Cell Therapy(2024SCOF003, 2025SCOF015 to J.M. T). Author Contribution 1. M. T conceived and initiated the project; Z.Y.W, L.X, W.X and S.J.Y performed the mouse experiments; Z.Y.W, L.X, W.X, S.J.Y and Y.P.P performed the immunofluorescence experiments; Z.Y.W, J.Y and J.Y.W conducted the Western blotting experiments; Y.L, Y.Z, X.L, and S.N helped prepare the experimental materials;L.L.S and L.C acquired the data; K.G and F.L.Z helped analyse the data; W.Z.W and S.J.C drafted the manuscript; J.M. T, S.L.P, W.Z.W and S.J.C revised the paper and checked the proofs. 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Gong","suffix":""},{"id":581477626,"identity":"8d5579e8-ad95-4d2f-9088-e514c929256d","order_by":14,"name":"Fuli Zhang","email":"","orcid":"","institution":"Hubei University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fuli","middleName":"","lastName":"Zhang","suffix":""},{"id":581477627,"identity":"5ed96e93-b0f9-4b29-95f8-56fbd748cd37","order_by":15,"name":"Ying-ming Li","email":"","orcid":"","institution":"Hubei University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ying-ming","middleName":"","lastName":"Li","suffix":""},{"id":581477630,"identity":"96860685-a3f4-4c64-b408-5bf6500ff669","order_by":16,"name":"Wu-Zhou Wu","email":"","orcid":"","institution":"Hubei University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wu-Zhou","middleName":"","lastName":"Wu","suffix":""},{"id":581477631,"identity":"79ff6f23-0107-4b33-a09d-91b4173cc264","order_by":17,"name":"Shao-juan Chen","email":"","orcid":"","institution":"Hubei University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shao-juan","middleName":"","lastName":"Chen","suffix":""},{"id":581477634,"identity":"4624cee6-1996-47a0-8d0b-953c38eb3cd0","order_by":18,"name":"Shang-ling Pan","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shang-ling","middleName":"","lastName":"Pan","suffix":""},{"id":581477638,"identity":"7e4d758c-706f-4cf3-abeb-127dc8c44264","order_by":19,"name":"Jun-ming Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYLACCQY2Hn72xsaHH4hRzQPRwicj2XO42ViCaC0MDHI2BjPS2wR4iNFiz3728AvLHDMeA8mHbUDr7OR0GwjZwpOXZiG5LY3HXDqx7UEBQ7Kx2QGCDssxM5DcdozHcnZiu4EEw4HEbQS18L8BafnPY3DzYJsED1FaJHKMH0huY+MxuMFIrJYbb8wYQFokexKBgWxAhF/Y+3OMPwO12POzH3/48EOFnRxBLUDAJo2IQQPCykGA+SNR6WQUjIJRMApGLgAATYI8UvBxhZAAAAAASUVORK5CYII=","orcid":"","institution":"Hubei University of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jun-ming","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2025-12-24 04:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8438697/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8438697/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101350596,"identity":"29a6c6b4-c460-498e-b619-fcb7f1b15722","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3309706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNE increased blood glucose levels while reduced numbers and mitochondrial integrity of type I muscle fiber\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A).RNA sequence results of tibialis anterior muscles (TAM) and soleus muscles (SOM) from WT mice showed that alpha and beta adrenergic receptors (AdR) was different; (B)Typical images of type I (slow) skeletal myosin heavy chain (MyHC-I) immunohistochemical staining in the SOM of WT mice with or without osmotic pump (MP) administration of norepinephrine (NE) for 28 days by using NOQ7.5.4D clone antibody; (C) Brown colour indicated MyHC-I positive slow muscle fiber. (C) Quantitative analysis of MyHC-I positive muscle fibers numbers. *P\u0026lt;0.05, vs. WT;\u0026nbsp; (D) Typical images of transmission electron microscopy for mitochondrial change in the SOM of WT mice with or without MP administration of NE for 28 days. Green arrow indicated the normal mitochondrion, red arrow indicated the mitochondrion with membrane rupture; (E) Blood glucose levels was higher in the WT mice with MP administration of NE than the WT mice with PBS administration.\u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. WT.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/9b671b9076e5e99615883808.png"},{"id":101350599,"identity":"7bb69d61-6776-4421-953d-4fcea08d5f36","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4931256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNE reduced the numbers of type I muscle fiber than type II muscle fiber\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D). Real-time PCR of MyHC-I, MyHC-IIa, MyHC-IIb and MyHC-IIx in the differentiated myoblast cells with MP or CS administration of NE for six days. *P\u0026lt;0.05, vs. CS group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. Ctrl group. Ctrl: control group, MP:osmotic pump, and CS:continuous single; (E). Typical images of skeletal myosin heavy chain (MyHC, sc-20641) and type I (slow) skeletal myosin heavy chain (MyHC-I, NOQ7.5.4D clone antibody) immunofluorescence staining in the differentiated myoblast cells with MP or CS administration of NE for six days. Green fluorescence: MyHC; red fluorescence: MyHC-I; DAPI: nuclei; (F). Typical images of MyHC and type II (fast) skeletal myosin heavy chain (MyHC-II, MY-32 antibody) immunofluorescence staining in the differentiated myoblast cells with MP or CS administration of NE for six days. Green fluorescence: MyHC, red fluorescence: MyHC-II; DAPI: nuclei.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/709323c5a9d9b6e6e86c998c.png"},{"id":101397799,"identity":"5d43c27d-7e33-4c15-acd3-4455f9c83c7a","added_by":"auto","created_at":"2026-01-29 09:37:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2214521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNE reduced the glucose transport uptake of myotubes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B). Quantitative analysis of MyHC-I or MyHC-II positive myotubes numbers in\u0026nbsp; in the differentiated myoblast cells with MP or CS administration of NE for six days; (C-D). Real-time PCR of Glut4 and IRS-1 in the differentiated myoblast cells with MP or CS administration of NE for six days. *P\u0026lt;0.05, vs. Ctrl group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. CS group; (E). Typical images of immunofluorescence staining for Glut4 and MyHC-I in the differentiated myoblast cells with MP or CS administration of NE for six days. Green fluorescence: MyHC-I; red fluorescence: Glut4; DAPI: nuclei; (F). Typical images of immunofluorescence staining for Glut4 and MyHC-I in the differentiated myoblast cells with MP or CS administration of NE for six days. Green fluorescence: MyHC-II; red fluorescence: Glut4; DAPI: nuclei; (G). Relative glucose uptake assay in the differentiated myoblast cells with MP or CS administration of NE for six days.\u0026nbsp; *P\u0026lt;0.05, vs Ctrl+2-DG group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. CS+2-DG group; \u003csup\u003e\u0026amp;\u003c/sup\u003eP\u0026lt;0.05, vs. Ctrl+2-DG+Insulin group; \u003csup\u003e@\u003c/sup\u003eP\u0026lt;0.05, vs. CS+2-DG+Insulin group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/ac620e2e61291ebdc85f11fc.png"},{"id":101397926,"identity":"be0e8899-18b7-4059-ae7d-e9db38dd8e71","added_by":"auto","created_at":"2026-01-29 09:38:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1448668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKPC1 involved in\u0026nbsp; specialization of\u0026nbsp; muscle fiber types and blood glucose homeostasis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A).RNA sequence results of tibialis anterior muscles (TAM) and soleus muscles (SOM) from WT mice showed that the RING finger protein family were different; (B-C). KPC1 (RNF123) knockout (KPC1\u003csup\u003e-/-\u003c/sup\u003e) obviously decreased the MyHC-I positive muscle fibers numbers of SOM. (B). Typical images of MyHC-I ( NOQ7.5.4D clone antibody) immunofluorescence staining in the WT and KPC1-/- mice. (C). Quantitative analysis of MyHC-I positive muscle fibers numbers. *P\u0026lt;0.05, vs. WT;\u0026nbsp; (D). Real-time PCR of KPC1 in the differentiated myoblast cells with the indicated times. *P\u0026lt;0.05, vs. other days; (E-G). KPC1 (RNF123) knockdown (Ad-shKPC1) obviously decreased the MyHC-I or MyHC-II positive myotubes numbers in the differentiated myoblast cells. (E). Experimental design framework diagram; (F). Quantitative analysis of MyHC-I or MyHC-II positive myotubes numbers. *P\u0026lt;0.05, vs. Ctrl; (G). Typical images of MyHC-I ( NOQ7.5.4D clone antibody) or MyHC-II (MY-32 antibody) immunofluorescence staining in the differentiated myoblast cells with or without treatment of Ad-shKPC1, Red fluorescence: MyHC-I or MyHC-II; DAPI: nuclei. (H-I). KPC1 (RNF123) knockout (KPC1\u003csup\u003e-/-\u003c/sup\u003e) decreased the glucose tolerance (H) and insulin sensitivity (I). *P\u0026lt;0.05, vs. WT mice.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/1ff828cc63b318565f173eeb.png"},{"id":101350597,"identity":"5993513e-ad51-481b-9d09-758403777101","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4074565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKPC1 involved in the formation of muscle fibers in time and dosage-dependent manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A).Experimental design framework diagram of Ad-KPC1 transfection into myoblast cells during myoblast cells differentiation and fusion and myotubes formation. (B).Typical images of MyHC (sc-20641) and MyHC-I ( NOQ7.5.4D clone antibody) immunofluorescence staining in the differentiated myoblast cells with or without treatment of Ad-KPC1, Green fluorescence:MyHC, Red fluorescence: MyHC-I; DAPI: nuclei. (C). Quantitative analysis of MyHC-I positive myotubes numbers. *P\u0026lt;0.05, vs. Ctrl; \u003csup\u003e#\u003c/sup\u003eP\u0026gt;0.05, vs. Ad-KPC1 on third day; (D).Typical images of MyHC (sc-20641) and MyHC-II (MY-32 antibody) immunofluorescence staining in the differentiated myoblast cells with or without treatment of Ad-KPC1, Green fluorescence:MyHC, Red fluorescence: MyHC-II; DAPI: nuclei. (E). Quantitative analysis of MyHC-II positive myotubes numbers. *P\u0026lt;0.05, vs. Ctrl; \u003csup\u003e#\u003c/sup\u003eP\u0026gt;0.05, vs. Ad-KPC1 on third day.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/9992e2d74463c0e4ff4b15ac.png"},{"id":101398655,"identity":"b0510138-3474-45df-8290-8b6982b92bcd","added_by":"auto","created_at":"2026-01-29 09:43:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3630494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKPC1 reversed the inhibitory effects of NE on the reduced type I and II muscle fibers numbers in time-dependent manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Real-time PCR of KPC1 in the differentiated myoblast cells with CS treatment of NE in the indicated dosages. *P\u0026lt;0.05, vs. 0 or 10\u003csup\u003e-8\u003c/sup\u003e Mol/L NE ; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. 0, 10\u003csup\u003e-8\u003c/sup\u003e or 10\u003csup\u003e-7\u003c/sup\u003e Mol/L NE; (B). Western Blot showed that CS treatment of NE decreased KPC1 protein levels in the differentiated myoblast cells with CS treatment of NE in the indicated dosages. (C-D). KPC1 knockout deteriorated the decrease in the MyHC-I positive muscle fibers numbers of SOM in mice with MP administration of NE for 28 days. (C). Typical images of MyHC-I ( NOQ7.5.4D clone antibody) immunofluorescence staining in WT or KPC1 knockout mice with MP administration of NE for 28 days. (D). Quantitative analysis of MyHC-I positive muscle fibers numbers. *P\u0026lt;0.05, vs. WT+NE. (E-H). Over-expression of KPC1 abolished the inhibitory effects of NE on the reduced type I and II myotubes numbers in time-dependent manner. (E). Experimental design framework diagram of Ad-KPC1 transfection and NE (10\u003csup\u003e-5\u003c/sup\u003e Mol/L) addition during myoblast cells differentiation and fusion and myotubes formation. (F). Typical images of MyHC-I immunofluorescence staining in the differentiated myoblast cells with treatment of NE (10\u003csup\u003e-5\u003c/sup\u003e Mol/L) in the indicated group. (G-H). Quantitative analysis of MyHC-I positive myotubes numbers. *P\u0026gt;0.05, vs. NE group;\u0026nbsp; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. NE or NE+Ad-KPC1 on zero day; \u003csup\u003e\u0026amp;\u003c/sup\u003eP\u0026gt;0.05, vs Ctrl group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/5eea12bfa19d6e9c4f48f11d.png"},{"id":101350602,"identity":"d7e50a2e-ddde-4b37-b06f-f8750b3e5b8b","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6960261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKPC1 inversed the inhibitory effects of NE-induced myoblast cells differentiation and myotubes formation through NF-kBp50\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B).Western Blot showed that CS treatment of NE decreased NF-kBp50 and p65 levels in the differentiated myoblast cells. (A). Typical western blot results. (B). Semi-quantitative analysis of NF-kBp50 and p65. *P\u0026lt;0.05, vs. 0, 10\u003csup\u003e-8\u003c/sup\u003e or 10\u003csup\u003e-7 \u003c/sup\u003eMol/L NE group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. other NE group. (C). Typical images in nucleation distribution of NF-kBp50 immunofluorescence staining in the differentiated myoblast cells with treatment of NE (10\u003csup\u003e-5\u003c/sup\u003e Mol/L) and Ad-KPC1 in the indicated group. (D). NF-kBp50 positive numbers of nucleation distribution. *P\u0026lt;0.05, vs. 10\u003csup\u003e-5 \u003c/sup\u003eMol/L NE group; \u003csup\u003e#\u003c/sup\u003eP\u0026gt;0.05, vs. Ctrl group. (E). Typical images of MyHC immunofluorescence staining in the differentiated myoblast cells with CS treatment of NE (10\u003csup\u003e-5\u003c/sup\u003e Mol/L), NF-kBp50 inhibitor SN50, or NF-kBp65 inhibitor PDTC.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/5866720fe779a7eb05b5237c.png"},{"id":101350603,"identity":"3289ee2e-a8ec-4b7a-b884-27977b164be9","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4005955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKPC1 inversed the inhibitory effects of ISO or E-induced myoblast cells differentiation and myotubes formation through NFKB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B). KPC1, NF-kBp50 and p65 protein levels in the differentiated myoblast cells with CS treatment of epinephrine (E) as determined by Western Blot. (A). Typical Western Blot results. (B). Semi-quantitative analysis of KPC1, NF-kBp50 and p65. \u003csup\u003e\u0026amp;\u003c/sup\u003eP\u0026lt;0.05, vs. 0 Mol/L E group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. other E group. (C-D). KPC1, NF-kBp50 and p65 protein levels in the differentiated myoblast cells with CS treatment of isoproterenol (ISO) as determined by Western Blot. (C). Typical western blot results. (D). Semi-quantitative analysis of KPC1, NF-kBp50 and p65. \u003csup\u003e\u0026amp;\u003c/sup\u003eP\u0026lt;0.05, vs. 0 Mol/L ISO group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, vs. 10\u003csup\u003e-8\u003c/sup\u003e Mol/L ISO group; \u003csup\u003e@\u003c/sup\u003eP\u0026gt;0.05, vs. 10\u003csup\u003e-8\u003c/sup\u003e, 10\u003csup\u003e-7\u003c/sup\u003e or 10\u003csup\u003e-6\u003c/sup\u003e Mol/L ISO group. (E). Typical images of MyHC immunofluorescence staining in the differentiated myoblast cells with CS treatment of E (10\u003csup\u003e-5\u003c/sup\u003e Mol/L) or ISO (10\u003csup\u003e-5\u003c/sup\u003e Mol/L) following the addition of NF-kBp50 inhibitor SN50, or NF-kBp65 inhibitor PDTC.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/980510a05460c0e4bd61159b.png"},{"id":101350601,"identity":"35fe9792-0f0d-4268-881a-6323f4e6e29e","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":695131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of this work.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) KPC1 acts as a bridge between skeletal muscle type and sympathetic over-activity-induced insulin resistance and T2DM \u003cem\u003ein vivo\u003c/em\u003e. (B) KPC1 affected myoblast cells differentiation/fusion and type I slow-twitch fiber formation in the specific time and dosage mode of action. (C)Two unique models of myoblast cells \u003cem\u003ein vitro\u003c/em\u003e that matched the traits of myofiber types with early and late T2DM mediated by different pattern of SNS over-activation \u003cem\u003ein vivo, \u003c/em\u003ewhich was related with KPC1 ‌dis-homeostasis, disrupting the balance of NF-kBp50/p65.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/f27804d3c11d0eb6c9ef2c26.png"},{"id":102963654,"identity":"39a67697-bdcf-423c-86ae-87385f379bab","added_by":"auto","created_at":"2026-02-19 04:19:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29581997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/fe985a17-4408-404d-b5c9-800647074e37.pdf"},{"id":101350594,"identity":"997312a6-d0be-4844-a078-c7b6b5dd81df","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":24008,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/e00c7918202021dcc169cd2f.docx"},{"id":101350605,"identity":"51d0872c-4de9-4c5f-896b-5e30a335bea8","added_by":"auto","created_at":"2026-01-28 18:33:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14119993,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-8438697/v1/60c94cc423c3378195a6e10c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"KPC1 dishomeostasis-mediated by sympathetic nervous system over-excitement involved in myofiber types alteration and insulin resistance through NF-KB signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eType 2 diabetes (T2DM) is on the increase, which seriously threatens the health of people. T2DM is closely related to insulin resistance. And skeletal muscle is the main site of peripheral insulin resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Actually, in the early stage of diabetes, slow muscle fibers were significantly reduced, while fast muscle fibers were significantly increased, and skeletal muscle atrophy occurred in the late stage of diabetes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous studies have often attributed changes in skeletal muscle fibers to the damaging effects of high blood sugar. Of note, T2DM is often accompanied by autonomic nervous dysfunction characterized by increased sympathetic activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], which further worsens diabetes or further impairs glucose metabolism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, apart from harmful changes of sympathetic nerves following T2DM, whether sympathetic over-activity involved in skeletal muscle insulin resistance and development of T2DM through affecting the differentiation and fusion of skeletal muscle satellite cells and changes in muscle fiber types, is still unknown.\u003c/p\u003e \u003cp\u003eActually, transient increase in sympathetic activity and abnormal glucose tolerance in the body may be a protective response from temporary danger, cold, exercise, staying up late and stress [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, it is still unclear whether the intermittent, continuous increase in sympathetic activity leads to a transition from protective response to compensatory to damaging changes, especially whether it preferentially affects the differentiation and fusion of skeletal muscle satellite cells and changes in muscle fiber types, and participates in skeletal muscle insulin resistance and T2DM.\u003c/p\u003e \u003cp\u003eIt is worth noting that when the sympathetic nervous system is over-activated, not only does it release more norepinephrine (NE) from its terminals, but the activated sympathetic nervous system also activates the adrenal medulla to synthesize and release NE and epinephrine (E), further enhancing the effect of sympathetic over-activation and involving β-adrenergic receptors (β-AdR ) and α-AdR [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Considering that NE mainly activates α1-AdR, but also activates β1-AdR; And E mainly activates β1-AdR, but also activates α1-AdR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To better prevent and treat T2DM in the early stage, revealing the role of different forms of sympathetic nervous system over-excitation in skeletal muscle insulin resistance and the development of T2DM has become a key issue that must be addressed.\u003c/p\u003e \u003cp\u003eLooking back at previous studies, single dose administration with a high dose of 5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M/L were used to induce skeletal muscle atrophy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, this administration method \u003cem\u003ein vitro\u003c/em\u003e was difficult to truly reflect and embody the impact of over-excitation of the sympathetic nervous system on skeletal muscle \u003cem\u003ein vivo\u003c/em\u003e. Therefore, it is necessary to create new cell models that can more accurately reflect and simulate the relationship between different pattern of over-excitation of the sympathetic nervous system and changes in the characteristics of skeletal muscle satellite cells.\u003c/p\u003e \u003cp\u003eConsidering that the process of differentiation and fusion of myoblasts to form myotubes and their muscle fibers involves protein synthesis and degradation, among which the ubiquitin proteasome pathway is one of the most important protein degradation pathways, which participates in maintaining the balance between protein production and degradation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Kip1 ubiquitination-promoting complex 1 (KPC1) is an important regulatory factor in the ubiquitin proteasome system, and its most amazing function is ubiquitination of NF-kB p105, which is degraded and cleaved by proteases to form active p50[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Over-expression of KPC1 produces excessive p50 while down-regulating NF-kBp65, promoting tumor suppressor signal expression and limiting tumor growth [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Unfortunately, previous reports on the role of NF-kB in the differentiation and fusion of myoblasts to form myotubes and muscle fibers have only focused on NF-kBp65, and researchers have attributed the inhibitory effect of NF-kBp65 on myoblast differentiation to its promotion of myoblast proliferation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, it is still unclear whether excessive sympathetic nervous system excitation regulates the differentiation and fusion of myoblasts to form myotubes and muscle fibers by affecting KPC1 and its NF-kB.\u003c/p\u003e \u003cp\u003eIn the study, for the first time, the excitation pattern of sympathetic nervous system will be divided into at least four types based on the degree, frequency, and duration of excitation: transient, intermittent transient, continuous transient and sustained transient. Our results showed that the difference in the release patterns of NE and E not only affected the differentiation and fusion of skeletal muscle cells and the formation of myotubes, but also the type of muscle fibers, which are involved in the early reduction of slow muscle fibers and the increase of fast muscle fibers mediated by skeletal muscle insulin resistance in T2DM, as well as muscle atrophy in the late stage of T2DM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eBased on a C57BL/6 background, KPC1 gene knockout (KPC1-/-) mice was generated through CRISPR-Cas9 technology (deletion of exons 1, 2, 8, 9 of NM_ 84585) on Cyagen Biosciences Inc., Suzhou, China (Supplementary Fig.\u0026nbsp;1). Genotyping with genomic DNA extracted from mouse tails was identified by using PCR technology. The primers (Sangon Biotech Co., Ltd., Shanghai, China) used were as follows: F1: 5\u0026rsquo;-TCAACCCTTGGACAGTATCCTC-3\u0026rsquo;; R1: 5\u0026rsquo;-CCTGTCACCCTTCTCCTTGAAC-3\u0026rsquo;; R2: 5\u0026rsquo;-GTCTGCTGTTTCTTACCCTC AC-3\u0026rsquo;. KPC1\u003csup\u003e+/\u0026minus;\u003c/sup\u003e, KPC1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and KPC1\u003csup\u003e+/+\u003c/sup\u003e (Wild-type, WT) male mice from the same litter of mice were used in all studies unless otherwise stated. All the mice were housed in SPF (Specific Pathogen Free) class Laboratory Animal Center of Hubei University of Medicine, with a 12 h light/dark cycle and disinfected water. All mouse experiments were performed following the protocols approved by the Laboratory Animal Care and Ethics Committee of Hubei University of Medicine, China. The ethics committee number is 2024-S-169. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents and chemicals\u003c/h3\u003e\n\u003cp\u003eSN-50 and PDTC were purchased from Selleck Chemicals LLC (Houston, Texas, USA), and prepared for the following experimental according to the manufacturer's instructions.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence double staining of skeletal muscle tissue\u003c/h3\u003e\n\u003cp\u003eThe mice were anesthetized with isopentane, and then blood samples were collected. The mice were sacrificed by carbon dioxide and skeletal muscles were removed. The dissected tibialis anterior muscles, and soleus muscles from WT and KPC1-/- mice with or without osmotic pump administration of norepinephrine (NE) were fixed for 48 hours in 4% paraformaldehyde (BL539A, biosharp\u0026reg;, Beijing Labgic Technology Co., Ltd., Beijing, China), and embedded in paraffin (80200-0017, Citotest Labware Manufacturing Co., Ltd., Haimen, China). The 5-micrometer-thick slice of the above samples were cut out and deparaffinised. Antigen retrieval were performed for these sections following the treatment of sodium citrate (pH 6.0) (BL604A, biosharp\u0026reg;, Beijing Labgic Technology Co., Ltd., Beijing, China) at 95\u0026deg;C for 5 minutes. One hour after optimal non-specific antigen blocking with 5% goat serum (ANT052, AntGene Biotechnology Co., Ltd., Wuhan, China) at room temperature. And then, the section were incubated with primary antibodies against type I fibers (1:200, ab234431, Abcam Corporation, Cambridge, England) and type II fibers (1:200, ab51263, Abcam Corporation, Cambridge, England) at 4\u0026deg;C overnight. Finally, appropriate horseradish peroxidase (HRP)-labelled secondary antibodies were used to show the fibers, and hematoxylin counterstaining of cell nuclei were performed. These images were captured under a microscope (BX53\u0026thinsp;+\u0026thinsp;DP74, Olympus Corporation, Japan) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGlucose tolerance test (GTT) and insulin tolerance test (ITT)\u003c/h3\u003e\n\u003cp\u003eFor the GTT, 20% glucose (A100188-0500, Sangon Biotech Co., Ltd., Shanghai, China) solution (1 mL/kg body weight) were intraperitoneally injected into the mice 16 hours after fasting. After injection, blood glucose was measured at the indicated time (0, 15, 30, 60, 90 and 120 min). For the ITT, insulin (BDPH-0036-A, biosharp\u0026reg;, Beijing Labgic Technology Co., Ltd., Beijing, China) (0.5 U/kg body weight) were subcutaneously injected into the mice 4 hours after fasting, and then blood glucose were measured at the indicated time (0, 15, 30, 45, 60 and 90 min) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To control the order of injection and measurements, a fixed ear tag numbers was applied.\u003c/p\u003e\n\u003ch3\u003eMyoblast culture and differentiation\u003c/h3\u003e\n\u003cp\u003eC2C12 myoblast cells (SCSP-505) were purchased from the Cell Resource Center of the Shanghai Academy of Life Science, affiliated to the Chinese Academy of Sciences. The culture medium with high-glucose Dulbecco\u0026rsquo;s modified Eagle medium (DMEM, C11995500BT, Gibco\u0026reg;, Grand Island, New York State, USA) containing 10% fetal bovine serum (FBS, SA211.02, CellMax Co., Ltd., Beijing, China) and 1% v/v penicillin/streptomycin (SV30010, HyClone, Logan, Utah, USA), were used to culture C2C12 myoblast cells in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Myoblast differentiation was performed following our previously reported method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, the cells were cultured under the above medium and reached 75% confluence, and then incubated with replaced differentiation medium (DM) containing high-glucose DMEM and 2% horse serum (BI 04-124-1A, Sigma, St. Louis, Missouri, USA) for the indicated times, were observed daily by an inverted microscope (CKX53, Olympus Corporation, Japan). At the specified time points, differentiated myoblasts were observed and stained with the mature muscle fiber marker myosin heavy chain (MyHC) (1:200, sc-20641, Santa Cruz, Dallas, Texas, USA), type I fibers (1:200, ab234431, Abcam Corporation, Cambridge, England) and type II fibers (1:200, ab51263, Abcam Corporation, Cambridge, England), and imaged under a fluorescence microscope (COOLSHOT80i, Nikon, Japan). Fluorescence imaging was performed with the following filter sets: DAPI (excitation 325\u0026ndash;375 nm, emission 435\u0026ndash;485 nm), FITC (excitation 460\u0026ndash;500 nm, emission 510\u0026ndash;560 nm), and TRITC (excitation 530\u0026ndash;560 nm, emission 570\u0026ndash;620 nm) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods for continuous single-dose or osmotic pump administration of NE and E\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo match the traits of long-term physiological or pathological levels NE or E in the tissues and plasma of people with health, sub-health and T2DM [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we created a novel method of osmotic pump administration of NE and E \u003cem\u003ein vitro\u003c/em\u003e, apart from continuous single-dose administration \u003cem\u003ein vitro\u003c/em\u003e. Briefly, osmotic pump administration of NE or E were treated at zero day of myoblast differentiation with replacing differentiation medium (DM) containing high-glucose DMEM and 2% horse serum for 6 days, different single doses were loaded as indicated (10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e Mol/L, 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e Mol/L,10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e Mol/L and10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e Mol/L). Continuous single-dose administration of NE or E were treated at zero day of myoblast differentiation, and then added with NE or E once a day for 6 days. Different single doses, as indicated (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e Mol/L, 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e Mol/L,10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e Mol/L and10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e Mol/L), were added to the DM at 7\u0026ndash;8 PM each day when the DM was replaced.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection of myoblast differentiation/fusion and myotube morphology by immunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed as our previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, at the indicated time, after washed with PBS, the cells were fixed with 4% paraformaldehyde (BL539A, biosharp\u0026reg;, Beijing Labgic Technology Co., Ltd., Beijing, China), and incubated with the primary antibody MyHC (myotube marker, 1:200, sc-376157(FITC), sc-376157 (TRITC), sc-20641, Santa Cruz, Dallas, Texas, USA) and appropriate fluorescence-labelled secondary antibodies. The nuclei were stained with DAPI (D9542, Sigma-Aldrich\u0026reg;, Merck KGaA, Germany). Only MyHC positive myoblasts with 3 or more nuclei within a cellular structure were defined as myotubes, the other were excluded as differentiated cells without mutual fusion to myotubes. Two double-blind individuals evaluated all images by using Image J. The numbers of myotubes with more than 5 nuclei were calculated and analyzed.\u003c/p\u003e \u003cp\u003eDifferentiated myoblasts were stained for Glut4, MEF2C, MyHC, NOQ or My32 using the polyclonal primary antibodies Glut4 (1:150,ab33780, Abcam Corporation, Cambridge, England), MEF2C (1:200, 5030S, Cell Signaling Technology Inc., Danvers, Massachusetts, USA), MyHC (1:200, sc-376157-FITC), slow skeletal myosin (NOQ7.5.4D, NOQ, 1:200, ab11083, Abcam Corporation, Cambridge, England) or fast myosin skeletal heavy chain (My32, ab51263, 1:200, Abcam Corporation, Cambridge, England) and the appropriate fluorescence-conjugated secondary antibodies. Images were captured under a microscope (IX53\u0026thinsp;+\u0026thinsp;DP73, Olympus Corporation, Japan). The fluorescence microscope equipped with filter sets was used under the following conditions: a DAPI filter set (excitation: 325\u0026ndash;375 nm, emission: 435\u0026ndash;485 nm), a FITC filter set (excitation: 460\u0026ndash;500 nm, emission: 510\u0026ndash;560 nm), and a TRITC filter set (excitation: 530\u0026ndash;560 nm, emission: 570\u0026ndash;620 nm)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRT-qPCR\u003c/h3\u003e\n\u003cp\u003eBased on the expression of myosin heavy chain (MyHC) isoform, Skeletal muscle fibers are generally classified into four types: MyHC-1 (slow-twitch oxidative), MyHC-2a (fast-twitch oxidative), MyHC-2b (fast-twitch glycolytic), and MyHC-2x (fast-twitch oxidative-glycolytic) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Their expression were detected on a CFX96\u0026trade; real-time PCR detection system (Bio-Rad Inc., California, USA) using HiScript\u0026reg; III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (R323-01, Vazyme Biotech Co., Ltd., Nanjing, China). The relative quantification 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to evaluate the data, and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The four types myofibers primer sequences were shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot\u003c/div\u003e \u003cp\u003eC2C12 myoblast cells were lysed in RIPA buffer (MA0151, MeilunBio\u0026reg;, Dalian, China) supplemented with PMSF (MB3800-1, MeilunBio\u0026reg;, Dalian, China). Proteins were separated by SDS-PAGE gel and transferred to a PVDF membrane (ISEQ00010, Merck Millipore, Merck KGaA, Germany). After the membrane was blocked with 5% skim milk (P0216, Beyotime Biotechnology Inc., Shanghai, China), the membrane was incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies (IgG). The primary antibodies used were as follows: α-tubulin (1:10000, ab7291, Abcam Corporation, Cambridge, England), KPC1 (1:1000, sc-101122, Santa Cruz, Dallas, Texas, USA), NF-kBp50 (1:1000, 13586S, Cell Signaling Technology Inc., Danvers, Massachusetts, USA), NF-kBp65 (1:1000, sc-109, Santa Cruz, Dallas, Texas, USA). Protein expression was detected with a ChemiDoc\u0026trade; system (Bio-Rad Inc., California, USA), and gray level analysis was carried out with ImageJ software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAdenoviral vector construction, transfection and myotubes types assay\u003c/h2\u003e \u003cp\u003eKPC1-knockdown (Ad-shKPC1) adenoviral vectors used in our lab were constructed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. And KPC1-overexpressing (Ad-KPC1) adenoviral vectors were purchased from WZ Biosciences Inc (Jinan, China). To study the role of KPC1 in myoblast differentiation, Ad-shKPC1 or Ad-KPC1 (1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e pfu) was added to the DM in zero or third day of myoblast cells differentiation. Meanwhile, continuous single-dose administration of NE (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e Mol/L) were treated at third day of myoblast differentiation, and then added with NE once a day for 6 days. At last, MyHC (1:200, sc-376157(FITC), sc-376157 (TRITC), sc-20641, Santa Cruz, Dallas, Texas, USA), slow skeletal myosin (MyHC-I, NOQ7.5.4D, NOQ, 1:200, ab11083, Abcam Corporation, Cambridge, England) or fast myosin skeletal heavy chain (MyHC-II, My32, ab51263, 1:200, Abcam Corporation, Cambridge, England), and appropriate fluorescence-labelled secondary antibodies were used to perform immunofluorescence staining. The nuclei were stained with DAPI (D9542, Sigma-Aldrich\u0026reg;, Merck KGaA, Germany). MyHC, MyHC-I or MyHC-II positive myoblasts with 3 or more nuclei within a cellular structure were defined as myotubes, the other were excluded as differentiated cells without mutual fusion to myotubes. The numbers of myotubes with more than 5 nuclei were calculated and analyzed. Over-expression and knockdown of KPC1 were identificatied by RP-PCR in myoblast cells after transfection (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining for NF-κB p50\u003c/h2\u003e \u003cp\u003eC2C12 myoblast cells were cultured under the above medium and reached 75% confluence, and then incubated with replaced differentiation medium (DM) containing high-glucose DMEM and 2% horse serum (BI 04-124-1A, Sigma, St. Louis, Missouri, USA). Subsequently, continuous single-dose administration of NE (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e Mol/L) were treated at third day of myoblast differentiation, and then added with NE once a day for 6 days, and Ad-KPC1 was added to the DM in third day of myoblast cells differentiation. Finally, the differentiated myoblasts were observed and stained with NF-kBp50 (1:1000, 13586S, Cell Signaling Technology Inc., Danvers, Massachusetts, USA), and imaged under a fluorescence microscope (COOLSHOT80i, Nikon, Japan). Fluorescence imaging was performed with the following filter sets: DAPI (excitation 325\u0026ndash;375 nm, emission 435\u0026ndash;485 nm), FITC (excitation 460\u0026ndash;500 nm, emission 510\u0026ndash;560 nm) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGlucose uptake assay\u003c/h2\u003e \u003cp\u003eFour days After C2C12 myoblasts differentiation in 24-well plates, glucose uptake assays were done by using the Glucose Uptake-Glo\u0026trade; Assay Kit (J1341, Promega Corporation, Madison, Wisconsin, USA) following the manufacturer's instructions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eTibialis anterior muscles (TA), and soleus muscles (SL) from WT mice were sent to OE Biotech Co., Ltd. (Shanghai, China) for RNA extraction, library preparation, and sequencing. 150-bp paired-end reads were generated. Low quality reads were removed from the raw reads, and the clean reads were mapped to the \u003cem\u003eMus musculus\u003c/em\u003e reference genome sequence. Read counts for each gene were calculated using htseq software. Fragments per kilobase of exon per million mapped fragments (FPKM) were used to assess gene expression. Differentially expressed genes were identified using the following criteria: corrected \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log 2 (fold change)\u0026thinsp;\u0026gt;\u0026thinsp;1[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003e28 days after, Mitochondrial function were evaluated by using transmission electron microscopy in soleus muscles (SL) of mice with or without osmotic pump administration of NE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperimental data were analyzed and plotted by GraphPad Prism 8.0 software, and all the data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation). Two groups were compared using the two-tailed Student's t-test and the confidence level is 95%, and more than two groups were compared using the One-way ANOVA test if the data normally distributed. The nonparametric test was used if the data was not normally distributed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOsmotic pump administration of norepinephrine increased blood glucose levels while reduced numbers and mitochondrial integrity of type I muscle fibers\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo observe the effect of osmotic pump administration of norepinephrine on myoblast differentiation, fusion, myotube formation and specialization, we firstly uesed RNAseq to confirm the difference between the soleus muscles (SL, major MyHC-I) and tibialis anterior muscles (TA, major MyHC-II), we found that adrenergic receptors (AdR) were more different that SOM majorly expressed Adra1a, Adra2b, Adra2c, Adrb1, Adrb2 and Adrb3 while TA expressed Adra2a (Figure.1A). After treatment of osmotic pump administration of norepinephrine, MyHC-I positive myofibers numbers obviously decreased in the SL compared with physiological saline treatment (Figure.1B-1C). Meanwhile, typical changes such as mitochondrial fragmentation and membrane rupture were found in the SL with osmotic pump administration of norepinephrine (Figure.1D), and blood glucose levels were higher than physiological saline treatment (Figure.1D). These indicated that NE-induced blood glucose imbalance could be associated with the type I muscle fibers numbers and mitochondrial integrity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effects of simulated\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eadministration of three modes of sympathetic nervous system excitation on myoblast differentiation, fusion, and myotube formation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo mimic the the at least three pattern of sympathetic nerves over-excitation such transient, continuous transient and sustained transient, apart from physiological excitation, single dose (S), continuous single dose (CS), and micro osmotic pump (MP) administration of norepinephrine (NE) have created and used to treat C2C12 myoblast cells under differentiated condition. After 6 days, the fusion and myotube characteristics were analyzed by MyHC (myosin heavy chain) immunofluorescence staining under microscopy. Compared with single dose administration, continuous single and micro osmotic pump treatments significantly inhibited the differentiation, fusion, and myotube formation of C2C12 myoblasts cells, especially the micro osmotic pump (Supplemental Figure.3A-3G).\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNE reduced the numbers of type I muscle fiber than type II muscle fiber\u003c/h2\u003e \u003cp\u003eTo further confirm the role of continuous single dose (CS), and micro osmotic pump continuous (MP) administration of norepinephrine (NE) on myoblast differentiation, fusion, myotube formation and specialization, different dosages NE were used, as shown in Supplemental Fig.\u0026nbsp;4\u0026ndash;6, we found that continuous single dose administration of NE inhibited myoblast differentiation, fusion, myotube formation and specialization of type I and II myofiber in dosage-dependent manner, however, resulting in the increases of type II myofiber. The difference is that, micro osmotic pump administration of NE showed a stronger inhibitory effect on the aforementioned observations. More importantly, under similar total dosage conditions, micro osmotic pump administration of NE inhibited more obviously myoblast differentiation, fusion, myotube formation and specialization of type I and II myofibers while continuous single dose administration of NE obviously reduced numbers of type I myofibers (Figure.2A-2E, Figure.3A-3B), and increased the expressions of MyHC-IIb and MyHC-IIX mRNA (Figure.2A-2D). These results indicated that different release patterns of NE could be associated with the formation of different muscle fiber types.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNE reduced the glucose transport uptake of myotubes\u003c/h2\u003e \u003cp\u003eTo further confirm if NE-induced myofibers types changes involved in the alteration of the glucose transport uptake, we detected the expressions of Glut4 and IRS-1, finding that continuous single or micro osmotic pump administration of NE could inhibit the expressions of Glut4 and IRS-1 in dosage-dependent manner (Supplemental Figure.7A-7D). Furthermore, micro osmotic pump continuous administration of NE more obviously decreased expression of Glut4 and IRS-1 compared with continuous single dose administration (Figure.3C-3E). Meanwhile, the glucose transport uptake in myotubes were more significantly reduced following micro osmotic pump continuous administration of NE than continuous single dose administration (Figure.3F). These results indicated that different release patterns of NE could be associated with the glucose transport uptake mediated by the altered muscle fiber types.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eKPC1 involved in specialization of muscle fiber types and blood glucose homeostasis\u003c/h2\u003e \u003cp\u003eSince the altered myofibers types involved in degradation of proteins, the RING finger protein family (RNFs) performed the E3 ubiquitin ligase to affect the development of tumors, immune and neurodegenerative diseases [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We firstly used RNAseq to confirm the difference of RNFs between the soleus muscles (SL) and tibialis anterior muscles (TA), we found that Rnf123 (KPC1) showed lower expressions in the SL compared with TA (Figure.4A). Importantly, we found that knockout of KPC1 reduced the numbers of MyHC-I positive myofibers in the SL, compared with wide-type (WT) mice (Figure.4B-4C). In line with the reduced MyHC-I positive myofibers, blood glucose tolerance and insulin sensitivity were slightly decreased in WT mice with treatment of NE (Figure.4H-4I).\u003c/p\u003e \u003cp\u003eTo confirm the similarity of altered muscle fiber types between NE and KPC1 during myoblast cells differentiation, fusion and myotubes formation, we firstly detected the changes of KPC1 during the myoblast cells differentiation, and found that KPC1 mRNA expressions were gradually increased, reaching peak value on the fourth day of myoblast cells differentiation, and then gradually decreased during the whole 6 days of differentiation, fusion and myotubes formation (Figure.4D). Furthermore, knockdown of KPC1 inhibited myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-I positive myofiber formation (Figure.4F-4G). These results indicated that KPC1-mediated myofibers types changes could involve in regulation of blood glucose homeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eKPC1 involved in the formation of muscle fibers in time and dosage-dependent manner\u003c/h2\u003e \u003cp\u003eTo confirm the characteristic of KPC1 on myoblast cells differentiation, fusion and myotubes formation, KPC1 over-expression medated by adeno virus (Ad-KPC1) were added to transfect into myoblast cells on the zero or third day of myoblast cells differentiation, results showed that KPC1 over-expression following the transfection of Ad-KPC1 at the zero day inhibited myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-I positive myotubes, compared with third day transfection. Furthermore, the difference is that KPC1 over-expression following the transfection of Ad-KPC1 at the third day slightly promoted myoblast cells differentiation, fusion and myotubes formation of MyHC-I and MyHC-II (Figure.5A-5E). These results indicated that KPC1 involved in the formation of muscle fibers in time and dosage-dependent manner.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKPC1 reversed the inhibitory effects of NE on the reduced type I muscle fibers numbers in time and dosage-dependent manner\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further confirm if NE-mediated muscle fibers types alteration involved in KPC1, KPC1 expressions were detected following NE treatment, results showed that NE reduced KPC1 mRNA and protein levels in dosage-dependent manner (Figure.6A-6B). Furthermore, knockout of KPC1 further reduced type I muscle fibers numbers in the SOM following the micro osmotic pump administration of NE (Figure.6C-6D). On the contrary, the addition of Ad-KPC1 on the zero day of myoblast cells differentiation, partially reversed the inhibitory effect of NE on myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-II positive myotubes. However, the addition of Ad-KPC1 on the third day could almost completely recovered the NE-mediated inhibition into the relative normal levels of myoblast cells differentiation, fusion and myotubes formation, especially in MyHC-I positive myotubes (Figure.6F-6H, and Supplemental Figure.8). These results indicated that KPC1 could reverse the inhibitory effects of NE on the reduced type I muscle fibers numbers in time and dosage-dependent manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eKPC1 reversed the inhibitory effects of NE-induced myoblast cells differentiation and myotubes formation through NF-KBp50\u003c/h2\u003e \u003cp\u003eSince KPC1 acted as E3 ubiquitin ligase to promote NF-KBp105 degradation into NF-KBp50 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As shown in Figure.7A-7B, NE reduced the levels of NF-KBp50 and NF-KBp65 following NE-induced KPC1 decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and the number of NF-KBp50 positive cell nuclei, especially in reduction of trend of nucleation distribution of NF-KBp50 (Figure.7C-7D). However, the addition of Ad-KPC1 on the zero day of myoblast cells differentiation, partially reversed the NE-mediated effects. Meanwhile, NF-KBp50 inhibitor SN50 deteriorated the inhibitory role of NE in myoblast cells differentiation, fusion and myotubes formation while NF-KBp65 inhibitor PDTC partially blocked the NE-mediated effects (Figure.7E). These results indicated that the inhibitory effects of NE could be related with the reduced NF-KBp50 accompanied by the decreased KPC1 levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eKPC1 reversed the inhibitory effects of epinephrine-induced myoblast cells differentiation and myotubes formation through NF-KB\u003c/h2\u003e \u003cp\u003eSince the activated sympathetic nervous system also activates the adrenal medulla to synthesize and release NE and epinephrine (E) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], as shown in Supplemental Fig.\u0026nbsp;9\u0026ndash;12, we found that continuous single or micro osmotic pump administration of epinephrine (E) or isoproterenol (ISO) showed the inhibitory effects of myoblast cells differentiation, myotubes fusion, MyHC-I, and MyHC-II positive muscle fibers formation similar to NE administration. However, unlike NE, both E and ISO increased the levels of KPC1 proteins, resulting in the increased levels of NF-KBp50 and NF-KBp65 proteins in dosage-dependent manner (Figure.8A-8H). Similarly, SN50 deteriorated the inhibitory role of E or ISO in myoblast cells differentiation, fusion and myotubes formation while PDTC partially blocked the E or ISO-mediated effects (Figure.8I). These results indicated that the inhibitory effects of E or ISO could be related with the increased NF-KBp65 accompanied by the increased KPC1 levels.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we have identified KPC1 as a novel factor for blood glucose homeostasis, acting as a linker between skeletal muscle insulin resistance and sympathetic nervous system (SNS) over-excitation \u003cem\u003ein vivo\u003c/em\u003e. KPC1 deficiency disrupts normal skeletal muscle types, leading to significant increases in skeletal muscle insulin resistance. Since sympathetic nervous system (SNS) over-excitation involved in insulin resistance and developing T2DM, unbalanced KPC1 may be an essential factor causing overactivated SNS-related T2DM. Thus, KPC1\u0026minus;/\u0026minus; mice may serve as a valuable model for dissecting molecular mechanisms underlying over-activated SNS-related T2DM. Indeed, we have created a novel myoblast differentiation, fusion and special myofiber formation model \u003cem\u003ein vitro\u003c/em\u003e that matched the traits of myofiber types with early and late T2DM \u003cem\u003ein vivo\u003c/em\u003e, identifying a novel mechanism underlying over-activated SNS-linked T2DM; i.e., decreased type I myofiber types due to decreased expression of NF-KBp50 in myoblast cells by KPC1 deficiency.\u003c/p\u003e \u003cp\u003eFactors such as danger, cold, staying up late, lack of sleep, stress, and intense exercise often cause abnormal SNS excitation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], based on its degree, frequency, and duration, SNS excitation pattern is for the first time divided into at least four types: transient, intermittent, continuous and sustained. In our previous study, matching with transient, intermittent, continuous excitation pattern of SNS, single dose, interval single dose and continuous single dose administration of ISO were created, finding that continuous single dose administration of ISO significantly inhibited C2C12 myoblast differentiation, fusion and myotube formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Herein, in order to better simulate the relationship between the sustained increase activity of SNS \u003cem\u003ein vivo\u003c/em\u003e and the functional changes of skeletal muscle satellite cells, a novel model was created to treat C2C12 myoblasts by releasing norepinephrine (NE) or epinephrine (E) through an osmotic pump. We found that continuous single administration of NE or E significantly inhibited the differentiation, fusion, and myotube formation of C2C12 myoblast cells, mainly decreasing MyHC-Ⅰ positive myofibers formation while relatively increasing MyHC-II positive myofibers numbers, in line with the changes of skeletal muscle fiber types in early T2DM. Excitingly, osmotic pump release of NE or E not only prominently blocked the differentiation, fusion, and myotube formation of C2C12 myoblast cells, but also reducing the formation and numbers of MyHC-Ⅰ and MyHC-II positive myofibers, which could consistent with the changes of skeletal muscle fiber types in advanced T2DM. These results suggested that two unique models of myoblast cells \u003cem\u003ein vitro\u003c/em\u003e that matched the traits of myofiber types with early and late T2DM \u003cem\u003ein vivo\u003c/em\u003e, were created for future research.\u003c/p\u003e \u003cp\u003eDue to the association between skeletal muscle insulin resistance and T2DM with reduced expression of Glut4, IRS-1, etc.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], this study found that continuous single administration or osmotic pump release of NE or E concentration dependently reduced the expression of Glut4 and IRS-1, accompanied by similar effects of the inhibited myoblast cells differentiation/fusion, and decreased MyHC-Ⅰ positive myofibers. Further analysis of the differences between the two modes revealed that, under the same total concentration, the NE or E mode of osmotic pump release more dramatically down-regulated Glut4 and IRS-1 expression compared to the continuous single dose mode. At the same time, it reduced the transport and uptake of glucose. Herein, these results indicated that the created administration patterns of NE or E \u003cem\u003ein vitro\u003c/em\u003e matching with the continuous and sustained increase in SNS excitation \u003cem\u003ein vivo\u003c/em\u003e were respectively involved in the abnormal increase of blood glucose in the early and late stages of T2DM, which were associated with functional changes of myoblast cells and myofiber types. Although previous results showed that excess of E could trigger glucose intolerance largely by damaged insulin secretion and excess of NE could trigger glucose intolerance largely by induced insulin resistance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], our present finding were consistent with abnormalities of carbohydrate metabolism in patients with SNS over-excitation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In a word, relationship between SNS over-excitation related diseases including hypertension and subtle and overt abnormalities of carbohydrate metabolism should be a real existence, furthermore, both NE and E could regulate blood glucose homeostasis through our newly identified common skeletal muscle target organs, particularly inhibition of myoblast differentiation/fusion and slow muscle reduction.\u003c/p\u003e \u003cp\u003eConsidering the obvious difference of RNFs targeting the ubiquitin proteasome system between slow and fast muscle fibers, especially in RNF123 (KPC1), in order to intervene early in the changes of skeletal muscle fibers and blood glucose imbalance mediated by SNS over-activation, myoblast cells model with continuous single administration of NE or E were used to reveal the relationship between SNS and KPC1. Unlike the role of KPC1 in mediating tumor occurrence and development [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], we firstly found that the expression of KPC1 was gradually increased, reached the highest on the fourth day, and then falling and maintaining a high levels during the differentiation and fusion of myoblasts cells to form myotubes and myofiber, showing a time- and dose-dependent traits. To further unlock its functional characteristics, we administered KPC1 over-expression treatment on the 0th and 3rd day of myoblasts cells differentiation, and found that day 0 treatment significantly inhibited myoblast differentiation/fusion, type I and II myofibers formation, which were consistent with the increased expression of KPC1 induced by ISO or E. Meanwhile, KPC1 knockdown treatment on the 0th and 3rd day of myoblasts cells differentiation obviously reduced myoblast differentiation/fusion and type I and II myofibers numbers. Surprisingly, KPC1 over-expression treatment on the 3rd day of myoblasts cells differentiation almost completely abolished the NE-mediated inhibitory effects. These results indicated that at the appropriate time, the appropriate expression of KPC1 should exhibit unique temporal biological characteristics in skeletal muscle regeneration and repair, providing a new target for the precise treatment of skeletal muscle atrophy and related diseases such as T2DM in the future.\u003c/p\u003e \u003cp\u003eOf note, although NE decreased KPC1 expression, while both E and ISO increased its expression, both NE, E and ISO exhibited same inhibitory effects on myoblast cells differentiation/fusion and myofibers formation, which was related to their binding to different receptors. For example, α-AdR agonist phenylephrine and β2-AdR agonist could promote the myoblast cells differentiation/fusion and myofibers formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In addition, NE, E and ISO could desensitize β2-AdR and α1-AdR, and then bind other receptors to perform its effects [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Considering the unique function of KPC1 in the process of myoblast cells differentiation, the same inhibitory effects mediated by NE, E and ISO could be associated with the altered expression time and level of KPC1, indicating that KPC1 could be a common target for the abnormal glucose regulation of patients with the excess of NE and/or E.\u003c/p\u003e \u003cp\u003eThe most amazing function of KPC1 is its ubiquitination regulation of NF-kB1 p105, which is degraded and cleaved by proteases to form active p50. Over-expression of KPC1 produces excessive p50 while down-regulating NF-kBp65, promoting tumor suppressor signal expression and limiting tumor growth [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarly, we found that NE reduced KPC1 expression and NF-kBp50 and p65 levels; however, E increased KPC1 expression and NF-kBp50, but p65 levels were not changed. Previous reports had only focused on NF-kBp65 and attributed its inhibitory effect on myoblast differentiation to its promotion of myoblast proliferation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Unfortunately, the role of NF-kBp50 in the differentiation and fusion of myoblasts cells to form myofibers is still unknown. Herein, we unexpectedly discovered that the NF-kBp50 inhibitor SN50 not only worsened NE but also worsened the inhibitory effect of E or ISO-mediated myoblast differentiation and fusion. NF-kBp65 inhibitor PDTC partially cancelled the inhibitory effects mediated by NE, E or ISO. In a word, NF-kBp50 could play a crucial role in the differentiation and fusion of myoblasts and the formation of myofibers, apart from the interaction with NF-kBp65.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the pattern differences of SNS over-activation may be involved in the occurrence and development of skeletal muscle insulin resistance and T2DM by triggering KPC1 \u0026zwnj;dis-homeostasis, disrupting the balance of NF-kBp50/p65, leading to inhibition of myoblast cells differentiation and fusion, changes in myofiber types, and providing new strategies for early intervention of skeletal muscle insulin resistance to prevent and treat T2DM(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAdR adrenergic receptor\u003c/p\u003e\u003cp\u003eT2DM Type 2 diabetes mellitus\u003c/p\u003e\u003cp\u003eNE norepinephrine\u003c/p\u003e\u003cp\u003eE epinephrine\u003c/p\u003e\u003cp\u003eISO isoproterenol\u003c/p\u003e\u003cp\u003eGA gastrocnemius muscle\u003c/p\u003e\u003cp\u003eTA tibialis anterior\u003c/p\u003e\u003cp\u003eSL soleus muscle\u003c/p\u003e\u003cp\u003eAd Adenovirus\u003c/p\u003e\u003cp\u003eMyHC Myosin heavy chain\u003c/p\u003e\u003cp\u003eNOQ Slow skeletal myosin heavy chain\u003c/p\u003e\u003cp\u003eMy32 Fast myosin skeletal heavy chain\u003c/p\u003e\u003cp\u003eMEF2C Myocyte-specific enhancer factor 2c\u003c/p\u003e\u003cp\u003eMyoG Myogenin\u003c/p\u003e\u003cp\u003eGlut4 Glucose transporter protein 4\u003c/p\u003e\u003cp\u003eRNFs RING finger protein family\u003c/p\u003e\u003cp\u003eKPC1 Kip1 ubiquitination-promoting complex 1\u003c/p\u003e\u003cp\u003eSNS sympathetic nervous system\u003c/p\u003e\u003cp\u003eMEF2C Myocyte-specific enhancer factor 2C\u003c/p\u003e\u003cp\u003eMyoD Myogenic differentiation 1\u003c/p\u003e\u003cp\u003eM Mol/L\u003c/p\u003e\u003cp\u003eShRNA Short hairpin RNA\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eSupplementary information\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eSupplementary information for this study is available in the online Supplementary materials.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNo.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll the authors declare that they have no conflicts of interest.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by National Natural Science Foundation of China (82270299 to J.M.T), the Hubei Provincial Natural Science Foundation (2025CSA046 to J.M.T, 2022CFB005 to L.C, 2024AFC024 to Y.W ), the Educational Commission Fund of Hubei Provincial (D20222107 to L.C ), Open Project of Hubei Provincial Clinical Medical Research Center for Umbilical Cord Blood Hematopoietic Stem Cell Therapy(2024SCOF003, 2025SCOF015 to J.M. T).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e1. M. T conceived and initiated the project; Z.Y.W, L.X, W.X and S.J.Y performed the mouse experiments; Z.Y.W, L.X, W.X, S.J.Y and Y.P.P performed the immunofluorescence experiments; Z.Y.W, J.Y and J.Y.W conducted the Western blotting experiments; Y.L, Y.Z, X.L, and S.N helped prepare the experimental materials;L.L.S and L.C acquired the data; K.G and F.L.Z helped analyse the data; W.Z.W and S.J.C drafted the manuscript; J.M. T, S.L.P, W.Z.W and S.J.C revised the paper and checked the proofs. All the authors approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the members of Biomedical Research Institute of Hubei University of Medicine for assisting in osmotic pump administration. The authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eCorrespondence and requests for materials and data should be addressed to Jun-ming Tang.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKitada, M., \u0026amp; Koya, D. (2021). Autophagy in metabolic disease and ageing. \u003cem\u003eNature Reviews. Endocrinology\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(11), 647\u0026ndash;661.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTobaldini, E., Costantino, G., Solbiati, M., et al. (2017). Sleep, sleep deprivation, autonomic nervous system and cardiovascular diseases. \u003cem\u003eNeuroscience And Biobehavioral Reviews\u003c/em\u003e, \u003cem\u003e74\u003c/em\u003e(Pt B), 321\u0026ndash;329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKittnar, O. (2015). 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Total Flavonoids Extracted from Oxytropis falcata Bunge Improve Insulin Resistance through Regulation on the IKKβ/NF-κB Inflammatory Pathway. \u003cem\u003eEvid Based Complement Alternat Med\u003c/em\u003e, \u003cem\u003e2017\u003c/em\u003e, 2405124. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2017/2405124\u003c/span\u003e\u003cspan address=\"10.1155/2017/2405124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Excessive activation of sympathetic nervous system, myoblast differentiation/fusion, myofiber types, KPC1, NF-KB, T2DM","lastPublishedDoi":"10.21203/rs.3.rs-8438697/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8438697/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhether sympathetic over-activity involved in skeletal muscle insulin resistance and development of type 2 diabetes (T2DM) through affecting the differentiation and fusion of skeletal muscle satellite cells and changes in muscle fiber types, is still unknown. KPC1 deficiency deteriorated norepinephrine (NE)-induced loss of slow-twitch type I myofibers by micro osmotic pump (MP) and increased insulin resistance in mice. Continuous single dose (CS) and MP administration of NE and epinephrine (E) have been created, showing the inhibitory effects on C2C12 myoblast cells differentiation/fusion and type I slow-twitch fiber formation and glucose transporter type 4, glucose transport and uptake \u003cem\u003ein vitro\u003c/em\u003e, matching with the changes of skeletal muscle fiber types in early and late T2DM \u003cem\u003ein vivo\u003c/em\u003e, respectively. Notably, KPC1 expression showed the unique expression mode that it was gradually increased, reaching peak value on the fourth day, and then gradually decreased during the process of myoblast cells differentiation/fusion and myofiber formation. Furthermore, over-expression of KPC1 on third day, not zero day, showed the almost complete reversal effect on the inhibitory role of NE in myoblast cells differentiation/fusion and type I slow-twitch fiber formation, indicating KPC1\u0026rsquo; specific time and dosage mode of action. Mechanistic studies revealed that NE reduced KPC1 and NF-KBp50/p65 protein levels, especially in the NF-KBp50 nucleation distribution. Curiously, E increased KPC1 and NF-KBp50 protein levels while unchanged NF-KBp65 levels, but showing the similar inhibitory effects on myoblast cells differentiation/fusion and slow-twitch type I myofiber formation. NE or E-mediated inhibitory role in myoblast differentiation/fusion could be worsen by NF-KBp50 inhibitor SN50, but partially abolished by NF-KBp65 blocker PDTC. Taken together, over-excitation of the sympathetic nervous system disturbed the balance between KPC1's time and dosage to cause the common inhibitory role in myoblast differentiation/fusion and slow-twitch fiber formation and glucose utilization, which were associated with abnormal matching of NF-KBp50/p65 signaling.\u003c/p\u003e","manuscriptTitle":"KPC1 dishomeostasis-mediated by sympathetic nervous system over-excitement involved in myofiber types alteration and insulin resistance through NF-KB signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 18:33:29","doi":"10.21203/rs.3.rs-8438697/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4f75e459-08d6-484f-b805-18814ac40e7b","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-18T05:54:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 18:33:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8438697","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8438697","identity":"rs-8438697","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}