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Niclosamide ethanolamine ameliorates diabetes-related muscle wasting by inhibiting autophagy

Niclosamide ethanolamine ameliorates diabetes-related muscle wasting by inhibiting autophagy Background: Diabetes-related muscle wasting is one of the devastating complications of diabetes, which is associated with muscle autophagy due to insulin-mediated glucose starvation. However, treatment for diabetes- related muscle wasting is limited. Our previous study already found that niclosamide ethanolamine salt has the therapeutic effects on insulin deficiency of type 1 diabetes mice and muscle wasting induced by doxorubicin. Therefore, we aim to investigate the therapeutic effects of niclosamide ethanolamine salt on diabetes-induced muscle wasting and to explore whether the mechanism is associated with muscle autophagy. Methods: Type 1 diabetes mice were induced by intraperitoneal injection of streptozotocin, then were fed with regular diet supplemented with 10 g/kg niclosamide ethanolamine salt. The whole experiment lasted for 8 weeks. At the end of the study, grip strength, weights of tibialis anterior, gastrocnemius, soleus, and extensor digitorum longus muscle were measured. Tibialis anterior muscles stained with PAS were used for evaluating the fiber cross sectional area. Immunofluorescence analysis of myosin heavy chain expression in extensor digitorum longus and soleus muscle was used for determining the composition of the muscle fiber type. Electronic microscopy was applied to observe the autophagy in the atrophied muscle. Serum insulin levels and fasting blood glucose were also measured. Tissues of gastrocnemius muscle were used for detecting the expression of the proteins related to autophagy. Results: In this study, we found that niclosamide ethanolamine salt could ameliorate muscle atrophy in the type 1 diabetes mice as well, such as enhancing the declined grip strength, improving limb weight and increasing the numbers of glycolytic muscle fiber. Electron microscopy also confirmed that there did exist abundant autophagic vacuoles in the atrophied muscle of the type 1 diabetes mice. Specifically, niclosamide ethanolamine salt could reduce the over expression of autophagy-related proteins, including p-AMPK (Thr172), FoxO3a, p-ULK1 (Ser555), LC3B II, and p- p38 in gastrocnemius muscle of the type 1 diabetes mice. * Correspondence: smm026@163.com; sunhuili2011@126.com Yuchun Cai and Hongyue Zhan contributed equally to this work. Department of Pathology, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian District, Shenzhen 518033, Guangdong, China Department of Nephrology, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian District, Shenzhen 518033, Guangdong, China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cai et al. Skeletal Muscle (2021) 11:15 Page 2 of 10 Conclusion: Niclosamide ethanolamine salt could ameliorate muscle wasting. The mechanisms underlying might be associated with inhibition of muscle autophagy. Keywords: Niclosamide ethanolamine salt, Diabetes-related muscle wasting, Autophagy Background regulations. Male C57BL/6J mice were purchased Global diabetes prevalence has been increased rapidly in from Guangdong Medical Laboratory Animal Center recent decades. It was estimated that there were 463 and were housed in the Laboratory Animal Center of million people suffering from diabetes in 2019, and the Shenzhen Graduate School of Peking University. The type number is assumed to reach as many as 700 million in 1 diabetes (T1D) mice were induced by the administration 2045 [1]. In the past years, researches on the complica- of multiple low doses of STZ (Sigma-Aldrich, St. Louis, tions of diabetes have mostly focused on vascular dis- MO, USA) dissolved in citrate buffer via intra-peritoneal eases [2, 3]. Accumulating evidences indicated that injection (55 mg/kg body weight per day) for 5 consecu- accelerated loss of muscle mass and strength is also a tive days. Normal control (T1D-ctrl) mice were intra- devastating complication of diabetes [4],which might peritoneally injected with an equal volume of citrate lead to slow movements, unstable gait, and even fre- buffer. The T1D mice were randomly allocated into T1D quent falls. What is more, alterations of the biomechan- group and T1D + NEN group according to the fasting ics of the feet caused by muscle atrophy might increase blood glucose at the 9th day after the last injection of the risk of developing a foot ulcer in diabetes [5, 6]. STZ. Mice in T1D-ctrl and T1D groups were fed with Therefore, studies investigating the pathogenesis and ex- regular diet as before, while the T1D + NEN group were ploring new medications for diabetes-related muscle at- fed with a regular diet supplemented with 10 g/kg NEN. rophy are in an urgent need. The whole treatment lasted for 8 weeks. In diabetes, muscle atrophy might take place due to inflammation, hyperglycemia, insulin deficiency, autoph- Grip strength test agy activation, and ubiquitin-proteasome degradation. Mice’s limb grip strength was measured by using a dyna- However, protein degradation with a net loss of muscle mometer for mice (ZH-YLS-13A, Anhui Zhenghua Bio- mass is the crucial feature in atrophic muscle. The logical Instrument Equipment Co. Ltd., Huaibei, China) autophagy-lysosome systems are one of the major pro- the day before being sacrificed. Limb grip strength was tein degradation pathways and proved to contribute to performed according to the manufacturer’s instructions. muscle atrophy [7]. Insulin, which is key in the process The PC interface software automatically sensed com- of glucose uptake, plays a crucial role in protein synthe- pression or tension and recorded the peak value (in sis and degradation in muscle [8]. Accumulating evi- mV). Calibrate factor was measured by using standard dences showed that the glucose starvation by insulin weight (1.98 N). Limb strength (in Newton) was calcu- deficiency might trigger the muscle autophagy. lated by peak value (in mV) × calibrate factor. For this Our previous study found that niclosamide ethanol- assay, three measurements were performed for each amine salt (NEN), a classic anthelmintic drug approved mouse and average of the results was used for analyzing. by FDA, can improve the declined insulin level and body weight of streptozotocin (STZ)-induced diabetic mice Blood glucose and body weight measurements [9]. More important, NEN can improve the muscle wast- Every 2 weeks, each mouse was weighed and blood sam- ing induced by doxorubicin [10]. However, the effects of ples of them were obtained by tail vein puncture for NEN on diabetes-related muscle atrophy are not yet blood glucose measurements by using a blood glucose clear. Therefore, this study aims to investigate the thera- meter (Roche, Basel, Switzerland). peutic effects of NEN on diabetes-induced muscle atro- phy and to explore whether the mechanism is associated with muscle autophagy. Tissue preparation At the end of the experiment, mice were sacrificed, then the tibialis anterior (TA), soleus (SOL), extensor digi- Methods torum longus (EDL), and gastrocnemius (GAs) were dis- Animal model sected and blotted on paper, and then weighed Animal studies were approved by the Guangzhou immediately. TA muscle tissues were fixed in 10% for- University of Chinese Medicine Institutional Animal malin for fiber cross-sectional area determination. EDL Care and Use Committee and were performed under muscle tissues (sized 1 mm ) were fixed in 2.5% glutaral- protocols in accordance with relevant guidelines and dehyde and then were post-fixed in 1% osmic acid for Cai et al. Skeletal Muscle (2021) 11:15 Page 3 of 10 transmission electronic microscopy (TEM) examination. polyvinylidenedifluoride (PVDF) membranes (Merck The GAs muscle tissues were immediately snap-frozen Millipore, Danvers, MA, USA). After blocking in TBS in liquid nitrogen and stored at − 80 °C for later analysis. buffer containing 5% non-fat dry milk for 1 h at room The SOL muscles and EDL muscles were firstly embed- temperature, the membranes were incubated and gently ded in O.C.T. compound (Tissue-Tek, Sakura Finetek, shaken overnight at 4 °C with primary antibodies. After USA), then frozen in liquid nitrogen-cooled isopentane, washing with TBS, the membranes were incubated with and lastly stored at – 80 °C for fiber type determination. secondary antibodies for 1 h at room temperature with shaking. After washing, the protein bands were detected Fiber cross-sectional area and fiber size distribution and analyzed by a ChemiDoc™ MP Imaging System (Bio- determination Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate de- The muscles were photographed by digital camera. hydrogenase (GAPDH) was used as the loading control. Paraffin-embedded TA muscle sections were stained The results are expressed as the integrated optical density with periodic acid-Schiff (PAS). At least 40% of all fibers relative to GAPDH. Primary antibodies against GAPDH within a muscle cross section (about 550–1500 fibers) were used as the loading control. The results are expressed were outlined to evaluate muscle fiber cross-sectional as the integrated optical density relative to GAPDH. area and the fiber size distribution by using ImageJ Soft- Primary antibodies against p-ULK1(ser555), ULK1, p- ware (National Institutes of Health, Bethesda, MD, AMPK (Thr172), AMPK, LC3B, p-p38 MAPK(Thr180/ USA). Tyr182), p38 MAPK, and FoxO3a were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary Fiber type determination antibody against GAPDH was from Proteintech Group, Inc. The prepared SOL and EDL tissues were cut into 10- (Chicago, IL, USA). μm-thick cryo-sections with a cryostat (CM1950, Leica, Germany) maintained at − 20 °C, then immunofluores- Statistical analysis cence analysis of MHC expression was performed in the Data were expressed as mean ± SD. Statistical differ- procedures as described previously [10]. Primary anti- ences between two groups were analyzed using unpaired bodies against MHC-I (BA-F8), MHC-IIa (SC-71) and Student’s t tests. Repeated measures analyses of variance MHC-IIb (BF-F3) were purchased from the Develop- (ANOVA) were conducted for the blood glucose and mental Studies Hybridoma Bank (University of Iowa, body weight data, the effects being group (T1D-ctrl vs. National Institutes of Health, USA), whereas secondary T1D, T1D vs T1D + NEN) and week (week 0, 2, 4, 6, 8, antibodies were purchased from Invitrogen (USA). The 10). Post hoc testing was performed using Bonferroni. resulting images were visualized and were captured on a Statistical analysis was performed using SPSS statistical confocal microscopy (LSM710, Carl Zeiss, Oberkochen, software, version 16.0, and P < 0.05 was considered Germany). Individual images were taken across the en- statistically significant. tire cross-section, then were assembled into a composite panoramic image with Photoshop 7.0 (Adobe, USA). All Results fibers within the entire image were characterized for NEN prevented muscle weakness in the T1D mice fiber type analysis. In order to detect whether the muscle function of the T1D mice is affected by NEN, mice were subjected to Electronic microscopy grip strength assessment. It turned out that the T1D TEM images were photographed by JEM-1400(JEOL, mice exhibited declined grip strength, while NEN treat- Tokyo, Japan). Autophagic vacuoles in inter-myofibrillar ment could enhance the grip strength of the T1D mice area and sub-sarcolemmal area were observed and (Fig. 1). photographed. NEN restored body weight and improved limb muscle Enzyme-linked immunosorbent assay (ELISA) atrophy in the T1D mice Enzyme-linked immunosorbent assay kits were used to In addition to enhancing muscle strength, NEN also had measure serum insulin (Merck Millipore, Danvers, MA, therapeutic effect on skeletal muscle atrophy in theT1D USA) according to the manufacturer’s instructions. mice. The T1D mice exhibited decreased bodyweight significantly compared with normal control mice from Immunoblotting analysis the 9th day after the injection of STZ (P = 0.000 < Snap-frozen GAs muscle tissues were homogenized in 0.001). Following the treatment with NEN, body weight lysis buffer and prepared in sample loading buffer (Bio- of the T1D mice were increasing gradually (P = 0.015< Rad, Hercules, CA, USA). Lysate proteins were separated 0.05) (Fig. 2a). At the end of experiment, hindlimb mus- on a 10% SDS-PAGE gels and then transferred to cles of the T1D mice were smaller than those of the Cai et al. Skeletal Muscle (2021) 11:15 Page 4 of 10 ### *** T1D-Ctrl T1D T1D+NEN *** Fig. 1 NEN improved the limb grip strength of the T1D mice. Bar graph of limb grip strength. n = 6 per group. P < 0.001 vs. the T1D-Ctrl ### group. P < 0.001 vs. the T1D group a c 0.06 T1D-Ctrl 0.05 p=0.06 T1D T1D+NEN 0.04 *** 0.03 ### # ### 0.02 T1D-Ctrl T1D T1D+NEN 0.20 p=0.06 0.15 *** *** *** *** 0.10 p<0.001(T1D-Ctrl VS. T1D) *** *** p=0.015(T1D VS. T1D+NEN) 0.05 0 2 468 10 0.00 T1D-Ctrl T1D T1D+NEN Weeks post treatment 0.015 p=0.200 TA GAs EDL SOL 0.010 *** 0.005 0.000 T1D-Ctrl T1D T1D+NEN 0.010 0.008 ** 0.006 0.004 0.002 0.000 T1D-Ctrl T1D T1D+NEN Fig. 2 NEN restored body weight and muscle mass in the T1D mice. a Body weight of mice was measured every 2 weeks; Repeated measures analyses of variance (ANOVA) were conducted, then post hoc testing was performed using Bonferroni; the T1D mice exhibited decreased bodyweight significantly compared with normal control mice from the 9th day after the injection of STZ (P = 0.000 < 0.001); following the treatment with NEN, body weight of theT1D mice were increasing gradually (P = 0.015 < 0.05). b Representative images of muscle. c Weight of ** *** # TA muscle. d Weight of GAs muscle. e Weight of EDL muscle. f Weight of SOL muscle. P < 0.01 and P < 0.001 vs. the T1D-Ctrl group. P < ### 0.05 and P < 0.001 vs. the T1D group. n = 6 in each group T1D+NEN T1D T1D-Ctrl Body Weight (g) LimbGrip Strength(N) SOL Muslce Weight(g) EDL Muscle weight(g) GAs Muscle Weight(g) TA Muslce Weight(g) Cai et al. Skeletal Muscle (2021) 11:15 Page 5 of 10 control mice, but were improved by the treatment with NEN improved insulin deficiency and energy shortage in NEN (Fig. 2b). The analysis of individual low hindlimb T1D mice muscles revealed that the weights of TA, GAs, EDL, and Our previous study suggested that the protective effects SOL were decreased in theT1D mice group compared of NEN on the diabetic mice might be due to improving with control group significantly, while NEN could in- β-cell function or inducing the α-to-β-like cell conver- crease the muscle mass of TA, GAs and SOL (Fig. 2c–f). sion [11, 12]. Consistent with previous studies, the serum insulin level of the T1D mice was significantly lower than that of the control group and following NEN NEN increased theT1D mice’s muscle fiber size treatment could raise it (Fig. 5a). In consequence, T1D + We further measured cross-sectional area of skeletal NEN group showed lower blood glucose than the T1D muscle fibers in the mice. Likewise, the mean cross- group (P = 0.000 < 0.001) (Fig. 5b). The expression of p- sectional area of TA muscle in the T1D mice had AMPK (Thr172) increased remarkably as a consequence shrunken remarkably, and NEN did increase the mean of insulin deficiency in T1D mice’s muscle, which indi- cross-sectional area of TA muscle fiber (Fig. 3a, c). In cated that there was short of energy supply. NEN could addition, the cross-sectional area distribution of TA decrease the expression of p-AMPK (Thr172) (Fig. 7a, b, muscle in the T1D mice developed a shift towards c). smaller fibers, and NEN could normalized this change as well (Fig. 3b). NEN suppressed glucose starvation-induced muscle NEN restored glycolytic muscle fiber in the T1D mice autophagy in T1D mice As shown in Fig. 4a, c, e, the numbers of type II fiber Electron microscopy revealed that autophagic vacuoles were decreased in SOL muscle obviously but no change were abundant within inter-myofibrillar and sub- in EDL muscle in theT1D mice, and NEN could increase sarcolemmal area (Fig. 6a–h). Accordingly, we next in- the numbers of type II fiber in SOL muscle. Further- vestigated the expression of autophagy-related proteins more, the composition of type II glycolytic fibers was an- to see if there was excessive autophagy in the muscle of alyzed, and it showed that the fiber subtypes were the T1D mice. It turned out that the protein expressions altered in the T1D mice. Type IIa fibers decreased in the of FoxO3a, p-ULK1 (Ser555), LC3B-II, and p-p38 MAPK SOL muscle (Fig. 4b, e), while type IIb fibers were less in (Thr180/Tyr182) were greatly elevated in the T1D mice. the EDL muscle (Fig. 4d, e). Interestingly, NEN could re- Moreover, the excessive expressions of the above pro- store these fiber subtypes (Fig. 4b, d, e). teins in the T1D mice were diminished in T1D + NEN a b 2000 30 T1D-Ctrl T1D T1D+NEN *** ~250 ~500 ~750 ~1000 ~1250 ~1500 ~1750 ~2000 ~2250 ~2500 T1D-Ctrl T1D T1D+NEN Fiber Cross section area(um ) T1D-Ctrl T1D T1D+NEN Fig. 3 NEN improved the muscle fiber cross-sectional area. a TA muscle fiber cross-sectional area in each group. b Frequency histograms showing the distribution of cross-sectional area of TA muscle fibers. c Representative images of fiber size alteration (PAS stained. scale bar, 20 *** # μm). P < 0.001 vs. the T1D-Ctrl group. P < 0.05 vs. the T1D group. n = 6 in each group TA Muscle Fiber CSA(?m ) TA Muscle CSA Distribution Population (%) Cai et al. Skeletal Muscle (2021) 11:15 Page 6 of 10 SOL Muscle EDL Muscle a c Fiber Type Composition Fiber Type Composition T1D-Ctrl 1.0 1.005 T1D-Ctrl T1D T1D T1D+NEN 0.8 T1D+NEN 1.000 *** 0.6 *** 0.995 0.010 0.4 0.008 0.006 0.2 0.004 0.002 0.0 0.000 III III Muscle Fiber Type Muscle Fiber Type b d 0.8 1.00 ### ** 0.75 *** 0.6 0.50 *** ** 0.4 0.25 ## 0.010 0.2 0.005 0.0 0.000 I IIa IIx IIb I IIa IIx IIb Muscle Fiber Type Muscle Fiber Type Type I Type IIa Type IIb Type IIx laminin Fig. 4 Muscle fiber type composition of SOL and EDL with immunofluorescence analysis. a, b SOL muscle fiber type composition. c, d EDL ** *** muscle fiber type composition. e Representative images of muscle fiber type composition (scale bar, 50 μm). n = 6 per group. P < 0.01, and P # ## ### < 0.001 vs. the T1D-Ctrl group. P < 0.05, P < 0.01 and P < 0.001 vs. the T1D group Population (%) Population(%) Population (%) Population(%) Cai et al. Skeletal Muscle (2021) 11:15 Page 7 of 10 ab 2.5 T1D-Ctrl *** T1D *** 2.0 *** T1D+NEN *** 1.5 1.0 *** ### ### ### ### *** ### 0.5 *** 0.0 T1D-Ctrl T1D T1D+NEN p<0.001(T1D-Ctrl VS. T1D) p<0.001(T1D VS. T1D+NEN) 02468 10 Weeks post treatment Fig. 5 Serum insulin and blood glucose levels of mice from each group. a Serum insulin levels at 12 weeks post-treatment with NEN. b Fasting blood glucose was measured every 2 weeks in the whole experiment; repeated measures analyses of variance (ANOVA) were conducted, then post hoc testing was performed using Bonferroni; the T1D mice exhibited increased blood glucose significantly compared with normal control mice through the whole experiment (P = 0.000 < 0.001); following the treatment with NEN, blood glucose of the T1D mice started to decrease at *** ### the 4th week (P = 0.000 < 0.001). P < 0.001 vs. the T1D-Ctrl group. P < 0.001 vs. the T1D group. n = 6 in each group group. These indicated that NEN could ameliorate strength. It suggested that the effects of NEN on muscle muscle autophagy (Fig. 7a–i). wasting might benefit from increasing the weight of T1D mice muscle. Discussion Skeletal muscle fibers are characterized as one type of This study indicates that NEN prevents muscle wasting slow-twitch fiber (type I) and three types of fast-twitch in the T1D mice and the mechanism underlying might fibers (type IIa, type IIx/d, and type IIb), of which type be associated with the inhibition of muscle autophagy IIb fibers are primarily glycolytic. In diabetes, fibers induced by the glucose starvation. might change from fast-twitch type to slow-twitch type It is reported that weight gains have benefit effects on with preferential atrophy of type II fiber [14, 15], as type the enhancement of muscle strength [13]. Consistent II fiber were more vulnerable to nutritional deficiencies with our previous study [10], this study showed that [16]. Loss of glycolytic muscles might lead to grip NEN could counteract the decrease of body weight, strength declined [17]. In this study, the fibers of TA muscle mass along with enhancing hindlimb grip muscle, which was almost composed of type II fibers, ab c d ef g h Fig. 6 Ultra-structure of autophagic vacuoles in EDL muscle in T1D mice. a, b Early autophagic vacuole in inter-myofibrillar area. c, d Early autophagic vacuole in sub-sarcolemmal area. e, f Late autophagic vacuole in inter-myofibrillar area. g, h Late autophagic vacuoles in sub- sarcolemmal area. The arrowheads indicate the two limiting membranes in autophagic vacuoles. Scale bar 500 nm for a, c, e, g, 200 nm for b, d, f, h Serum insulin(ng/mL) Blood Glucose (mmol/L) Cai et al. Skeletal Muscle (2021) 11:15 Page 8 of 10 T1D-Ctrl T1D T1D+NEN p-AMPK (Thr 172) AMPK GAPDH FoxO3a GAPDH p-ULK1 (Ser555) ULK1 GAPDH LC3B II GAPDH p-p38 p38 GAPDH p=0.09 bc 4 d 4 2.5 ** 2.0 3 3 1.5 2 2 1.0 1 1 0.5 0 0 0.0 T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN ef 3 2.5 g 2.0 ** 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0 0.0 0.0 T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN hi 3 2.5 ** ** 2.0 1.5 ## ## 1.0 0.5 0 0.0 T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN Fig. 7 Autophagy-related proteins expression in GAs muscle tissues. a Western blot images of autophagy-related proteins in GAs muscle tissues of each group. Bar graphs showing the fold change of p-AMPK/GAPDH (b), p-AMPK/AMPK (c), FoxO3a/GAPDH (d), p-ULK1(ser555)/GAPDH (e), p- * ** ULK1(ser555)/ULK1(f), LC3B II/GAPDH (g), p-p38/GAPDH (h), and p-p38/p38 MAPK (i) expression in GAs muscle tissues. P < 0.05 and P < 0.01 vs. # ## the T1D-Ctrl group. P < 0.05 and P < 0.01 vs. the T1D group. n = 4 in each group were atrophy obviously in the T1D mice. We also found production rate [19]. Interestingly, autophagy can acti- that the fibers of fast/glycolytic were decreased in SOL vate bulk protein degradation to harvest amino acids as muscle and EDL muscle. Taken together, we implied a fuel for ATP production through the tricarboxylic acid that the protection of NEN on diabetes-related muscle (TCA) cycle to maintain the energy balance [20]. There- wasting might be partly due to the restoration of type II fore, in insulin deficiency T1D mice, autophagy plays a fiber. crucial role in muscle atrophy, which might be activated Skeletal muscle is the prominent organ for insulin- by the energy shortage to use amino acid as a substitute mediated glucose uptake [18]. Glucose starvation in skel- for glucose. AMPK is a sensor of intracellular energy, etal muscle caused by insulin deficiency could result in which can be activated by any mechanisms that disrupt significant reduction in muscle mitochondrial ATP ATP generation [21]. Studies showed that AMPK p-ULK1(ser555)/GAPDH fold change P-AMPK/GAPDH fold change p-p38 MAPK/GAPDH fold change p-ULK1(ser555)/ULK1 fold change p-AMPK/AMPK fold change p-p38 MAPK /p38 MAPK fold change LC3B II/GAPDH FoxO3a/GAPDH fold change fold change Cai et al. Skeletal Muscle (2021) 11:15 Page 9 of 10 activation can promote skeletal muscle cells autophagy by Declarations activating FoxO3a and ULK1 [22, 23]. Accumulating evi- Ethics approval and consent to participate dences indicate that FoxO3a is the main transcriptional All procedures of animal study were approved by the Guangzhou University regulator of autophagy by controlling a broad range of of Chinese Medicine Institutional Animal Care and Use Committee and in accordance with relevant guidelines and regulations. atrophy-related genes, including Fbxo32 (Atrogin-1) and Trim63 (MuRF-1), and other autophagy genes [7, 11]. Consent for publication ULK1 is one of the essential inductors of the autophagy Not applicable. pathway, which initiates the formation of the autophago- some [24]. Our study showed that T1D mice treated with Competing interests NEN did develop less expression of p-AMPK (Thr172), The authors declare that they have no competing interests. FoxO3a, and p-ULK1 (Ser555) than T1D mice. P38 MAPK is also known to regulate autophagy in Author details Department of Nephrology, Shenzhen Traditional Chinese Medicine skeletal muscle [25, 26] due to the over phosphorylation Hospital, The Fourth Clinical Medical College of Guangzhou University of in skeletal muscle under variety of cellular stresses, in- Chinese Medicine, 1 Fuhua Road, Futian District, Shenzhen 518033, cluding endurance exercise and fasted state [27]. Our Guangdong, China. Department of Critical Care Medicine, Shantou Hospital of Traditional Chinese Medicine, Shantou, China. Department of Pathology, previous study also found that NEN can prevent muscle Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical atrophy by inhibition of p38 MAPK/FoxO3a activation College of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian in mice exposed to doxorubicin [10]. Similarly, as the District, Shenzhen 518033, Guangdong, China. above results showed, NEN treatment also inhibited the Received: 9 December 2020 Accepted: 26 May 2021 over-activation of p38MAPK in T1D mice. The level of LC3BII, a marker of autophagosome presence [28], was reduced by NEN treatment in T1D mice in this study. References The result further prompted that autophagy was sup- 1. Harding JL, Pavkov ME, Magliano DJ, Shaw JE, Gregg EW. Global trends in pressed by NEN. diabetes complications: a review of current evidence. Diabetologia. 2019; 62(1):3–16. https://doi.org/10.1007/s00125-018-4711-2. 2. Williams R, Karuranga S, Malanda B, Saeedi P, Basit A, Besançon S, et al. Global and regional estimates and projections of diabetes-related health Conclusion expenditure: results from the International Diabetes Federation Diabetes In summary, we concluded that NEN could ameliorate Atlas, 9th edition. Diabetes Res Clin Pract. 2020;162:108072. 3. Gregg EW, Sattar N, Ali MK. 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Annu Rev Cell Dev Biol. 2011;27(1):107–32. muscle. Yuchun Cai, Hongyue Zhan, and Huili Sun were responsible for https://doi.org/10.1146/annurev-cellbio-092910-154005. drafting the manuscript. All authors read and reviewed the manuscript. 9. O'Neill BT, Bhardwaj G, Penniman CM, Krumpoch MT, Suarez Beltran PA, Klaus K, et al. FoxO transcription factors are critical regulators of diabetes- related muscle atrophy. Diabetes. 2019;68(3):556–70. https://doi.org/10.2337/ Funding db18-0416. This study was supported by grants from the National Natural Science 10. Zhan H, Wang M, Han P, Yu X, Wang Y, Weng W, et al. Niclosamide Foundation of China (81673794,82004156), Shenzhen Science and ethanolamine prevents muscle wasting by inhibiting p38 MAPK-FoxO3a Technology Project (JCYJ20190812183603627), and the Sanming Project of activation in mice exposed to doxorubicin. Int J Clin Exp Med. 2020;13(2):10. Medicine in Shenzhen (SZSM201512040). 11. Han P, Shao M, Guo L, Wang W, Song G, Yu X, et al. 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Lancet Diabetes Endocrinol. 2014;2(10):819–29. https://doi.org/10.1016/S2213-8587(14)70034-8. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Niclosamide ethanolamine ameliorates diabetes-related muscle wasting by inhibiting autophagy

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Abstract

Background: Diabetes-related muscle wasting is one of the devastating complications of diabetes, which is associated with muscle autophagy due to insulin-mediated glucose starvation. However, treatment for diabetes- related muscle wasting is limited. Our previous study already found that niclosamide ethanolamine salt has the therapeutic effects on insulin deficiency of type 1 diabetes mice and muscle wasting induced by doxorubicin. Therefore, we aim to investigate the therapeutic effects of niclosamide ethanolamine salt on diabetes-induced muscle wasting and to explore whether the mechanism is associated with muscle autophagy. Methods: Type 1 diabetes mice were induced by intraperitoneal injection of streptozotocin, then were fed with regular diet supplemented with 10 g/kg niclosamide ethanolamine salt. The whole experiment lasted for 8 weeks. At the end of the study, grip strength, weights of tibialis anterior, gastrocnemius, soleus, and extensor digitorum longus muscle were measured. Tibialis anterior muscles stained with PAS were used for evaluating the fiber cross sectional area. Immunofluorescence analysis of myosin heavy chain expression in extensor digitorum longus and soleus muscle was used for determining the composition of the muscle fiber type. Electronic microscopy was applied to observe the autophagy in the atrophied muscle. Serum insulin levels and fasting blood glucose were also measured. Tissues of gastrocnemius muscle were used for detecting the expression of the proteins related to autophagy. Results: In this study, we found that niclosamide ethanolamine salt could ameliorate muscle atrophy in the type 1 diabetes mice as well, such as enhancing the declined grip strength, improving limb weight and increasing the numbers of glycolytic muscle fiber. Electron microscopy also confirmed that there did exist abundant autophagic vacuoles in the atrophied muscle of the type 1 diabetes mice. Specifically, niclosamide ethanolamine salt could reduce the over expression of autophagy-related proteins, including p-AMPK (Thr172), FoxO3a, p-ULK1 (Ser555), LC3B II, and p- p38 in gastrocnemius muscle of the type 1 diabetes mice. * Correspondence: smm026@163.com; sunhuili2011@126.com Yuchun Cai and Hongyue Zhan contributed equally to this work. Department of Pathology, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian District, Shenzhen 518033, Guangdong, China Department of Nephrology, Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian District, Shenzhen 518033, Guangdong, China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cai et al. Skeletal Muscle (2021) 11:15 Page 2 of 10 Conclusion: Niclosamide ethanolamine salt could ameliorate muscle wasting. The mechanisms underlying might be associated with inhibition of muscle autophagy. Keywords: Niclosamide ethanolamine salt, Diabetes-related muscle wasting, Autophagy Background regulations. Male C57BL/6J mice were purchased Global diabetes prevalence has been increased rapidly in from Guangdong Medical Laboratory Animal Center recent decades. It was estimated that there were 463 and were housed in the Laboratory Animal Center of million people suffering from diabetes in 2019, and the Shenzhen Graduate School of Peking University. The type number is assumed to reach as many as 700 million in 1 diabetes (T1D) mice were induced by the administration 2045 [1]. In the past years, researches on the complica- of multiple low doses of STZ (Sigma-Aldrich, St. Louis, tions of diabetes have mostly focused on vascular dis- MO, USA) dissolved in citrate buffer via intra-peritoneal eases [2, 3]. Accumulating evidences indicated that injection (55 mg/kg body weight per day) for 5 consecu- accelerated loss of muscle mass and strength is also a tive days. Normal control (T1D-ctrl) mice were intra- devastating complication of diabetes [4],which might peritoneally injected with an equal volume of citrate lead to slow movements, unstable gait, and even fre- buffer. The T1D mice were randomly allocated into T1D quent falls. What is more, alterations of the biomechan- group and T1D + NEN group according to the fasting ics of the feet caused by muscle atrophy might increase blood glucose at the 9th day after the last injection of the risk of developing a foot ulcer in diabetes [5, 6]. STZ. Mice in T1D-ctrl and T1D groups were fed with Therefore, studies investigating the pathogenesis and ex- regular diet as before, while the T1D + NEN group were ploring new medications for diabetes-related muscle at- fed with a regular diet supplemented with 10 g/kg NEN. rophy are in an urgent need. The whole treatment lasted for 8 weeks. In diabetes, muscle atrophy might take place due to inflammation, hyperglycemia, insulin deficiency, autoph- Grip strength test agy activation, and ubiquitin-proteasome degradation. Mice’s limb grip strength was measured by using a dyna- However, protein degradation with a net loss of muscle mometer for mice (ZH-YLS-13A, Anhui Zhenghua Bio- mass is the crucial feature in atrophic muscle. The logical Instrument Equipment Co. Ltd., Huaibei, China) autophagy-lysosome systems are one of the major pro- the day before being sacrificed. Limb grip strength was tein degradation pathways and proved to contribute to performed according to the manufacturer’s instructions. muscle atrophy [7]. Insulin, which is key in the process The PC interface software automatically sensed com- of glucose uptake, plays a crucial role in protein synthe- pression or tension and recorded the peak value (in sis and degradation in muscle [8]. Accumulating evi- mV). Calibrate factor was measured by using standard dences showed that the glucose starvation by insulin weight (1.98 N). Limb strength (in Newton) was calcu- deficiency might trigger the muscle autophagy. lated by peak value (in mV) × calibrate factor. For this Our previous study found that niclosamide ethanol- assay, three measurements were performed for each amine salt (NEN), a classic anthelmintic drug approved mouse and average of the results was used for analyzing. by FDA, can improve the declined insulin level and body weight of streptozotocin (STZ)-induced diabetic mice Blood glucose and body weight measurements [9]. More important, NEN can improve the muscle wast- Every 2 weeks, each mouse was weighed and blood sam- ing induced by doxorubicin [10]. However, the effects of ples of them were obtained by tail vein puncture for NEN on diabetes-related muscle atrophy are not yet blood glucose measurements by using a blood glucose clear. Therefore, this study aims to investigate the thera- meter (Roche, Basel, Switzerland). peutic effects of NEN on diabetes-induced muscle atro- phy and to explore whether the mechanism is associated with muscle autophagy. Tissue preparation At the end of the experiment, mice were sacrificed, then the tibialis anterior (TA), soleus (SOL), extensor digi- Methods torum longus (EDL), and gastrocnemius (GAs) were dis- Animal model sected and blotted on paper, and then weighed Animal studies were approved by the Guangzhou immediately. TA muscle tissues were fixed in 10% for- University of Chinese Medicine Institutional Animal malin for fiber cross-sectional area determination. EDL Care and Use Committee and were performed under muscle tissues (sized 1 mm ) were fixed in 2.5% glutaral- protocols in accordance with relevant guidelines and dehyde and then were post-fixed in 1% osmic acid for Cai et al. Skeletal Muscle (2021) 11:15 Page 3 of 10 transmission electronic microscopy (TEM) examination. polyvinylidenedifluoride (PVDF) membranes (Merck The GAs muscle tissues were immediately snap-frozen Millipore, Danvers, MA, USA). After blocking in TBS in liquid nitrogen and stored at − 80 °C for later analysis. buffer containing 5% non-fat dry milk for 1 h at room The SOL muscles and EDL muscles were firstly embed- temperature, the membranes were incubated and gently ded in O.C.T. compound (Tissue-Tek, Sakura Finetek, shaken overnight at 4 °C with primary antibodies. After USA), then frozen in liquid nitrogen-cooled isopentane, washing with TBS, the membranes were incubated with and lastly stored at – 80 °C for fiber type determination. secondary antibodies for 1 h at room temperature with shaking. After washing, the protein bands were detected Fiber cross-sectional area and fiber size distribution and analyzed by a ChemiDoc™ MP Imaging System (Bio- determination Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate de- The muscles were photographed by digital camera. hydrogenase (GAPDH) was used as the loading control. Paraffin-embedded TA muscle sections were stained The results are expressed as the integrated optical density with periodic acid-Schiff (PAS). At least 40% of all fibers relative to GAPDH. Primary antibodies against GAPDH within a muscle cross section (about 550–1500 fibers) were used as the loading control. The results are expressed were outlined to evaluate muscle fiber cross-sectional as the integrated optical density relative to GAPDH. area and the fiber size distribution by using ImageJ Soft- Primary antibodies against p-ULK1(ser555), ULK1, p- ware (National Institutes of Health, Bethesda, MD, AMPK (Thr172), AMPK, LC3B, p-p38 MAPK(Thr180/ USA). Tyr182), p38 MAPK, and FoxO3a were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary Fiber type determination antibody against GAPDH was from Proteintech Group, Inc. The prepared SOL and EDL tissues were cut into 10- (Chicago, IL, USA). μm-thick cryo-sections with a cryostat (CM1950, Leica, Germany) maintained at − 20 °C, then immunofluores- Statistical analysis cence analysis of MHC expression was performed in the Data were expressed as mean ± SD. Statistical differ- procedures as described previously [10]. Primary anti- ences between two groups were analyzed using unpaired bodies against MHC-I (BA-F8), MHC-IIa (SC-71) and Student’s t tests. Repeated measures analyses of variance MHC-IIb (BF-F3) were purchased from the Develop- (ANOVA) were conducted for the blood glucose and mental Studies Hybridoma Bank (University of Iowa, body weight data, the effects being group (T1D-ctrl vs. National Institutes of Health, USA), whereas secondary T1D, T1D vs T1D + NEN) and week (week 0, 2, 4, 6, 8, antibodies were purchased from Invitrogen (USA). The 10). Post hoc testing was performed using Bonferroni. resulting images were visualized and were captured on a Statistical analysis was performed using SPSS statistical confocal microscopy (LSM710, Carl Zeiss, Oberkochen, software, version 16.0, and P < 0.05 was considered Germany). Individual images were taken across the en- statistically significant. tire cross-section, then were assembled into a composite panoramic image with Photoshop 7.0 (Adobe, USA). All Results fibers within the entire image were characterized for NEN prevented muscle weakness in the T1D mice fiber type analysis. In order to detect whether the muscle function of the T1D mice is affected by NEN, mice were subjected to Electronic microscopy grip strength assessment. It turned out that the T1D TEM images were photographed by JEM-1400(JEOL, mice exhibited declined grip strength, while NEN treat- Tokyo, Japan). Autophagic vacuoles in inter-myofibrillar ment could enhance the grip strength of the T1D mice area and sub-sarcolemmal area were observed and (Fig. 1). photographed. NEN restored body weight and improved limb muscle Enzyme-linked immunosorbent assay (ELISA) atrophy in the T1D mice Enzyme-linked immunosorbent assay kits were used to In addition to enhancing muscle strength, NEN also had measure serum insulin (Merck Millipore, Danvers, MA, therapeutic effect on skeletal muscle atrophy in theT1D USA) according to the manufacturer’s instructions. mice. The T1D mice exhibited decreased bodyweight significantly compared with normal control mice from Immunoblotting analysis the 9th day after the injection of STZ (P = 0.000 < Snap-frozen GAs muscle tissues were homogenized in 0.001). Following the treatment with NEN, body weight lysis buffer and prepared in sample loading buffer (Bio- of the T1D mice were increasing gradually (P = 0.015< Rad, Hercules, CA, USA). Lysate proteins were separated 0.05) (Fig. 2a). At the end of experiment, hindlimb mus- on a 10% SDS-PAGE gels and then transferred to cles of the T1D mice were smaller than those of the Cai et al. Skeletal Muscle (2021) 11:15 Page 4 of 10 ### *** T1D-Ctrl T1D T1D+NEN *** Fig. 1 NEN improved the limb grip strength of the T1D mice. Bar graph of limb grip strength. n = 6 per group. P < 0.001 vs. the T1D-Ctrl ### group. P < 0.001 vs. the T1D group a c 0.06 T1D-Ctrl 0.05 p=0.06 T1D T1D+NEN 0.04 *** 0.03 ### # ### 0.02 T1D-Ctrl T1D T1D+NEN 0.20 p=0.06 0.15 *** *** *** *** 0.10 p<0.001(T1D-Ctrl VS. T1D) *** *** p=0.015(T1D VS. T1D+NEN) 0.05 0 2 468 10 0.00 T1D-Ctrl T1D T1D+NEN Weeks post treatment 0.015 p=0.200 TA GAs EDL SOL 0.010 *** 0.005 0.000 T1D-Ctrl T1D T1D+NEN 0.010 0.008 ** 0.006 0.004 0.002 0.000 T1D-Ctrl T1D T1D+NEN Fig. 2 NEN restored body weight and muscle mass in the T1D mice. a Body weight of mice was measured every 2 weeks; Repeated measures analyses of variance (ANOVA) were conducted, then post hoc testing was performed using Bonferroni; the T1D mice exhibited decreased bodyweight significantly compared with normal control mice from the 9th day after the injection of STZ (P = 0.000 < 0.001); following the treatment with NEN, body weight of theT1D mice were increasing gradually (P = 0.015 < 0.05). b Representative images of muscle. c Weight of ** *** # TA muscle. d Weight of GAs muscle. e Weight of EDL muscle. f Weight of SOL muscle. P < 0.01 and P < 0.001 vs. the T1D-Ctrl group. P < ### 0.05 and P < 0.001 vs. the T1D group. n = 6 in each group T1D+NEN T1D T1D-Ctrl Body Weight (g) LimbGrip Strength(N) SOL Muslce Weight(g) EDL Muscle weight(g) GAs Muscle Weight(g) TA Muslce Weight(g) Cai et al. Skeletal Muscle (2021) 11:15 Page 5 of 10 control mice, but were improved by the treatment with NEN improved insulin deficiency and energy shortage in NEN (Fig. 2b). The analysis of individual low hindlimb T1D mice muscles revealed that the weights of TA, GAs, EDL, and Our previous study suggested that the protective effects SOL were decreased in theT1D mice group compared of NEN on the diabetic mice might be due to improving with control group significantly, while NEN could in- β-cell function or inducing the α-to-β-like cell conver- crease the muscle mass of TA, GAs and SOL (Fig. 2c–f). sion [11, 12]. Consistent with previous studies, the serum insulin level of the T1D mice was significantly lower than that of the control group and following NEN NEN increased theT1D mice’s muscle fiber size treatment could raise it (Fig. 5a). In consequence, T1D + We further measured cross-sectional area of skeletal NEN group showed lower blood glucose than the T1D muscle fibers in the mice. Likewise, the mean cross- group (P = 0.000 < 0.001) (Fig. 5b). The expression of p- sectional area of TA muscle in the T1D mice had AMPK (Thr172) increased remarkably as a consequence shrunken remarkably, and NEN did increase the mean of insulin deficiency in T1D mice’s muscle, which indi- cross-sectional area of TA muscle fiber (Fig. 3a, c). In cated that there was short of energy supply. NEN could addition, the cross-sectional area distribution of TA decrease the expression of p-AMPK (Thr172) (Fig. 7a, b, muscle in the T1D mice developed a shift towards c). smaller fibers, and NEN could normalized this change as well (Fig. 3b). NEN suppressed glucose starvation-induced muscle NEN restored glycolytic muscle fiber in the T1D mice autophagy in T1D mice As shown in Fig. 4a, c, e, the numbers of type II fiber Electron microscopy revealed that autophagic vacuoles were decreased in SOL muscle obviously but no change were abundant within inter-myofibrillar and sub- in EDL muscle in theT1D mice, and NEN could increase sarcolemmal area (Fig. 6a–h). Accordingly, we next in- the numbers of type II fiber in SOL muscle. Further- vestigated the expression of autophagy-related proteins more, the composition of type II glycolytic fibers was an- to see if there was excessive autophagy in the muscle of alyzed, and it showed that the fiber subtypes were the T1D mice. It turned out that the protein expressions altered in the T1D mice. Type IIa fibers decreased in the of FoxO3a, p-ULK1 (Ser555), LC3B-II, and p-p38 MAPK SOL muscle (Fig. 4b, e), while type IIb fibers were less in (Thr180/Tyr182) were greatly elevated in the T1D mice. the EDL muscle (Fig. 4d, e). Interestingly, NEN could re- Moreover, the excessive expressions of the above pro- store these fiber subtypes (Fig. 4b, d, e). teins in the T1D mice were diminished in T1D + NEN a b 2000 30 T1D-Ctrl T1D T1D+NEN *** ~250 ~500 ~750 ~1000 ~1250 ~1500 ~1750 ~2000 ~2250 ~2500 T1D-Ctrl T1D T1D+NEN Fiber Cross section area(um ) T1D-Ctrl T1D T1D+NEN Fig. 3 NEN improved the muscle fiber cross-sectional area. a TA muscle fiber cross-sectional area in each group. b Frequency histograms showing the distribution of cross-sectional area of TA muscle fibers. c Representative images of fiber size alteration (PAS stained. scale bar, 20 *** # μm). P < 0.001 vs. the T1D-Ctrl group. P < 0.05 vs. the T1D group. n = 6 in each group TA Muscle Fiber CSA(?m ) TA Muscle CSA Distribution Population (%) Cai et al. Skeletal Muscle (2021) 11:15 Page 6 of 10 SOL Muscle EDL Muscle a c Fiber Type Composition Fiber Type Composition T1D-Ctrl 1.0 1.005 T1D-Ctrl T1D T1D T1D+NEN 0.8 T1D+NEN 1.000 *** 0.6 *** 0.995 0.010 0.4 0.008 0.006 0.2 0.004 0.002 0.0 0.000 III III Muscle Fiber Type Muscle Fiber Type b d 0.8 1.00 ### ** 0.75 *** 0.6 0.50 *** ** 0.4 0.25 ## 0.010 0.2 0.005 0.0 0.000 I IIa IIx IIb I IIa IIx IIb Muscle Fiber Type Muscle Fiber Type Type I Type IIa Type IIb Type IIx laminin Fig. 4 Muscle fiber type composition of SOL and EDL with immunofluorescence analysis. a, b SOL muscle fiber type composition. c, d EDL ** *** muscle fiber type composition. e Representative images of muscle fiber type composition (scale bar, 50 μm). n = 6 per group. P < 0.01, and P # ## ### < 0.001 vs. the T1D-Ctrl group. P < 0.05, P < 0.01 and P < 0.001 vs. the T1D group Population (%) Population(%) Population (%) Population(%) Cai et al. Skeletal Muscle (2021) 11:15 Page 7 of 10 ab 2.5 T1D-Ctrl *** T1D *** 2.0 *** T1D+NEN *** 1.5 1.0 *** ### ### ### ### *** ### 0.5 *** 0.0 T1D-Ctrl T1D T1D+NEN p<0.001(T1D-Ctrl VS. T1D) p<0.001(T1D VS. T1D+NEN) 02468 10 Weeks post treatment Fig. 5 Serum insulin and blood glucose levels of mice from each group. a Serum insulin levels at 12 weeks post-treatment with NEN. b Fasting blood glucose was measured every 2 weeks in the whole experiment; repeated measures analyses of variance (ANOVA) were conducted, then post hoc testing was performed using Bonferroni; the T1D mice exhibited increased blood glucose significantly compared with normal control mice through the whole experiment (P = 0.000 < 0.001); following the treatment with NEN, blood glucose of the T1D mice started to decrease at *** ### the 4th week (P = 0.000 < 0.001). P < 0.001 vs. the T1D-Ctrl group. P < 0.001 vs. the T1D group. n = 6 in each group group. These indicated that NEN could ameliorate strength. It suggested that the effects of NEN on muscle muscle autophagy (Fig. 7a–i). wasting might benefit from increasing the weight of T1D mice muscle. Discussion Skeletal muscle fibers are characterized as one type of This study indicates that NEN prevents muscle wasting slow-twitch fiber (type I) and three types of fast-twitch in the T1D mice and the mechanism underlying might fibers (type IIa, type IIx/d, and type IIb), of which type be associated with the inhibition of muscle autophagy IIb fibers are primarily glycolytic. In diabetes, fibers induced by the glucose starvation. might change from fast-twitch type to slow-twitch type It is reported that weight gains have benefit effects on with preferential atrophy of type II fiber [14, 15], as type the enhancement of muscle strength [13]. Consistent II fiber were more vulnerable to nutritional deficiencies with our previous study [10], this study showed that [16]. Loss of glycolytic muscles might lead to grip NEN could counteract the decrease of body weight, strength declined [17]. In this study, the fibers of TA muscle mass along with enhancing hindlimb grip muscle, which was almost composed of type II fibers, ab c d ef g h Fig. 6 Ultra-structure of autophagic vacuoles in EDL muscle in T1D mice. a, b Early autophagic vacuole in inter-myofibrillar area. c, d Early autophagic vacuole in sub-sarcolemmal area. e, f Late autophagic vacuole in inter-myofibrillar area. g, h Late autophagic vacuoles in sub- sarcolemmal area. The arrowheads indicate the two limiting membranes in autophagic vacuoles. Scale bar 500 nm for a, c, e, g, 200 nm for b, d, f, h Serum insulin(ng/mL) Blood Glucose (mmol/L) Cai et al. Skeletal Muscle (2021) 11:15 Page 8 of 10 T1D-Ctrl T1D T1D+NEN p-AMPK (Thr 172) AMPK GAPDH FoxO3a GAPDH p-ULK1 (Ser555) ULK1 GAPDH LC3B II GAPDH p-p38 p38 GAPDH p=0.09 bc 4 d 4 2.5 ** 2.0 3 3 1.5 2 2 1.0 1 1 0.5 0 0 0.0 T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN ef 3 2.5 g 2.0 ** 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0 0.0 0.0 T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN hi 3 2.5 ** ** 2.0 1.5 ## ## 1.0 0.5 0 0.0 T1D-Ctrl T1D T1D+NEN T1D-Ctrl T1D T1D+NEN Fig. 7 Autophagy-related proteins expression in GAs muscle tissues. a Western blot images of autophagy-related proteins in GAs muscle tissues of each group. Bar graphs showing the fold change of p-AMPK/GAPDH (b), p-AMPK/AMPK (c), FoxO3a/GAPDH (d), p-ULK1(ser555)/GAPDH (e), p- * ** ULK1(ser555)/ULK1(f), LC3B II/GAPDH (g), p-p38/GAPDH (h), and p-p38/p38 MAPK (i) expression in GAs muscle tissues. P < 0.05 and P < 0.01 vs. # ## the T1D-Ctrl group. P < 0.05 and P < 0.01 vs. the T1D group. n = 4 in each group were atrophy obviously in the T1D mice. We also found production rate [19]. Interestingly, autophagy can acti- that the fibers of fast/glycolytic were decreased in SOL vate bulk protein degradation to harvest amino acids as muscle and EDL muscle. Taken together, we implied a fuel for ATP production through the tricarboxylic acid that the protection of NEN on diabetes-related muscle (TCA) cycle to maintain the energy balance [20]. There- wasting might be partly due to the restoration of type II fore, in insulin deficiency T1D mice, autophagy plays a fiber. crucial role in muscle atrophy, which might be activated Skeletal muscle is the prominent organ for insulin- by the energy shortage to use amino acid as a substitute mediated glucose uptake [18]. Glucose starvation in skel- for glucose. AMPK is a sensor of intracellular energy, etal muscle caused by insulin deficiency could result in which can be activated by any mechanisms that disrupt significant reduction in muscle mitochondrial ATP ATP generation [21]. Studies showed that AMPK p-ULK1(ser555)/GAPDH fold change P-AMPK/GAPDH fold change p-p38 MAPK/GAPDH fold change p-ULK1(ser555)/ULK1 fold change p-AMPK/AMPK fold change p-p38 MAPK /p38 MAPK fold change LC3B II/GAPDH FoxO3a/GAPDH fold change fold change Cai et al. Skeletal Muscle (2021) 11:15 Page 9 of 10 activation can promote skeletal muscle cells autophagy by Declarations activating FoxO3a and ULK1 [22, 23]. Accumulating evi- Ethics approval and consent to participate dences indicate that FoxO3a is the main transcriptional All procedures of animal study were approved by the Guangzhou University regulator of autophagy by controlling a broad range of of Chinese Medicine Institutional Animal Care and Use Committee and in accordance with relevant guidelines and regulations. atrophy-related genes, including Fbxo32 (Atrogin-1) and Trim63 (MuRF-1), and other autophagy genes [7, 11]. Consent for publication ULK1 is one of the essential inductors of the autophagy Not applicable. pathway, which initiates the formation of the autophago- some [24]. Our study showed that T1D mice treated with Competing interests NEN did develop less expression of p-AMPK (Thr172), The authors declare that they have no competing interests. FoxO3a, and p-ULK1 (Ser555) than T1D mice. P38 MAPK is also known to regulate autophagy in Author details Department of Nephrology, Shenzhen Traditional Chinese Medicine skeletal muscle [25, 26] due to the over phosphorylation Hospital, The Fourth Clinical Medical College of Guangzhou University of in skeletal muscle under variety of cellular stresses, in- Chinese Medicine, 1 Fuhua Road, Futian District, Shenzhen 518033, cluding endurance exercise and fasted state [27]. Our Guangdong, China. Department of Critical Care Medicine, Shantou Hospital of Traditional Chinese Medicine, Shantou, China. Department of Pathology, previous study also found that NEN can prevent muscle Shenzhen Traditional Chinese Medicine Hospital, The Fourth Clinical Medical atrophy by inhibition of p38 MAPK/FoxO3a activation College of Guangzhou University of Chinese Medicine, 1 Fuhua Road, Futian in mice exposed to doxorubicin [10]. Similarly, as the District, Shenzhen 518033, Guangdong, China. above results showed, NEN treatment also inhibited the Received: 9 December 2020 Accepted: 26 May 2021 over-activation of p38MAPK in T1D mice. The level of LC3BII, a marker of autophagosome presence [28], was reduced by NEN treatment in T1D mice in this study. References The result further prompted that autophagy was sup- 1. Harding JL, Pavkov ME, Magliano DJ, Shaw JE, Gregg EW. Global trends in pressed by NEN. diabetes complications: a review of current evidence. Diabetologia. 2019; 62(1):3–16. https://doi.org/10.1007/s00125-018-4711-2. 2. Williams R, Karuranga S, Malanda B, Saeedi P, Basit A, Besançon S, et al. Global and regional estimates and projections of diabetes-related health Conclusion expenditure: results from the International Diabetes Federation Diabetes In summary, we concluded that NEN could ameliorate Atlas, 9th edition. Diabetes Res Clin Pract. 2020;162:108072. 3. Gregg EW, Sattar N, Ali MK. 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Lancet Diabetes Endocrinol. 2014;2(10):819–29. https://doi.org/10.1016/S2213-8587(14)70034-8. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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