Muscle Of Normal Rats Research Articles

Transcriptome sequencing and bioinformatics analysis of gastrocnemius muscle in type 2 diabetes mellitus rats

ObjectiveType 2 diabetes mellitus (T2DM) is one of the high risk factors for sarcopenia. However, the pathogenesis of diabetic sarcopenia has not been fully elucidated. This study obtained transcriptome profiles of gastrocnemius muscle in normal and T2DM rats based on high-throughput sequencing technology, which may provide new ideas for exploring the pathogenesis of diabetic sarcopenia.MethodsTwelve adult male Sprague-Dawley rats were randomly divided into Control group and T2DM group, and gastrocnemius muscle tissue was retained for transcriptome sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) 6 months later. Screening differentially expressed genes (DEGs), Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Gnomes (KEGG) functional annotation and enrichment analysis were performed for DEGs. Six DEGs related to apoptosis were selected for qTR-PCR verification.ResultsTranscriptomic analysis showed that there were 1016 DEGs between the gastrocnemius muscle of T2DM and normal rats, among which 665 DEGs were up-regulated and 351 DEGs were down-regulated. GO analysis showed that the extracellular matrix organization was the most enriched in biological processes, with 26 DEGs. The extracellular matrix with 35 DEGs was the most abundant cellular component. The extracellular matrix structural constituent, with 26 DEGs, was the most enriched in molecular functions. The highest number of DEGs enriched in biological processes, cellular components and molecular functions were positive regulation of transcription by RNA polymerase II, nucleus and metal ion binding, respectively. There were 78, 230 and 89 DEGs respectively. KEGG pathway enrichment analysis showed that ECM-receptor interaction, PI3K-Akt signaling pathway and TGF-β signaling pathway(p < 0.001) had higher enrichment degree and number of DEGs. qRT-PCR results showed that the fold change of Map3k14, Atf4, Pik3r1, Il3ra, Gadd45b and Bid were 1.95, 3.25, 2.97, 2.38, 0.43 and 3.6, respectively. The fold change of transcriptome sequencing were 3.45, 2.21, 2.59, 5.39, 0.49 and 2.78, respectively. The transcriptional trends obtained by qRT-PCR were consistent with those obtained by transcriptome sequencing.ConclusionsTranscriptomic analysis was used to obtain the “gene profiles” of gastrocnemius muscle of T2DM and normal rats. qRT-PCR verification showed that the genes related to apoptosis were differentially expressed. These DEGs and enrichment pathways may provide new ideas for exploring the pathogenesis of diabetic sarcopenia.

Direct-Acting Antiviral Drug Modulates the Mitochondrial Biogenesis in Different Tissues of Young Female Rats.

(1) Background: Hepatitis C virus (HCV) infection is endemic in Egypt, with the highest prevalence rate worldwide. Sofosbuvir (SOF) is a nucleos(t)ide analog that specifically inhibits HCV replication. This study aimed to explore the possible effects of the therapeutic dose of SOF on the mitochondrial biogenesis and functions of the liver, muscle, and ovarian tissues of young normal female rats. (2) Methods: This study was conducted on 20 female Wistar rats, classified into two groups, the control group and the exposed group; the latter was orally supplemented with 4 mg/kg/day of SOF for 3 months. (3) Results: The exposure to SOF impairs mitochondrial biogenesis via mitochondrial DNA copy number decline and suppressed mitochondrial biogenesis-regulated parameters at mRNA and protein levels. Also, SOF suppresses the DNA polymerase γ (POLG) expression, citrate synthase activity, and mitochondrial NADH dehydrogenase subunit-5 (ND5) content, which impairs mitochondrial functions. SOF increased lipid peroxidation and oxidative DNA damage markers and decreased tissue expression of nuclear factor erythroid 2-related factor 2 (Nfe2l2). (4) Conclusions: The present findings demonstrate the adverse effects of SOF on mitochondrial biogenesis and function in different tissues of young female rats, which mostly appeared in ovarian tissues.

Melatonin Supplement Plus Exercise Effectively Counteracts the Challenges of Isoproterenol-Induced Cardiac Injury in Rats.

To explore the combined effects of exercise and melatonin supplement against the challenges of isoproterenol-induced cardiac oxidative stress and injury in rats., the expression of peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α), mitochondrial biogenesis, and adenosine triphosphate (ATP) was up-regulated in cardiac muscle in normal rats and in a melatonin and exercise regimented group. Cardiac injury was induced by two subcutaneous injections of isoproterenol in the rats. The combination of exercise and melatonin supplement successfully counteracted the isoproterenol-induced cardiac injury, which is reflected by the improved hemodynamic parameters, reduction in oxidative stress markers, and cardiac injury serum markers (cardiac troponin-I and creatine kinase-MB). The cardiac tissue level of ATP, expression of PGC-1α and mitochondrial biogenesis-related genes, mitochondrial membrane potential, and the activities of typical antioxidants (glutathione, superoxide dismutase) were preserved, whereas the levels of reactive oxygen species, lipid peroxidation, and inflammatory cytokines were suppressed in the melatonin and exercise regimented (MEI) group compared to the group treated with isoproterenol alone. Furthermore, the expression of endoplasmic reticular stress- and apoptosis-related proteins (Bax, Bcl2, and caspase-3) was also effectively suppressed in the MEI group. Therefore, the present study suggests that melatonin supplement in combination with exercise prevents cardiac injury, possibly through the preservation of mitochondrial function and inhibition of oxidative stress in rats.

Curcumin Loaded Nanocarriers with Varying Charges Augmented with Electroporation Designed for Colon Cancer Therapy.

(1) Background: The size and surface charge are the most significant parameters of nanocarriers that determine their efficiency and potential application. The poor cell uptake of encapsulated drugs is the main limitation in anticancer treatment. The well-defined properties of nanocarriers will enable to target specific tissue and deliver an active cargo. (2) Methods: In the current study, poly(D,L -lactide) (PLA) nanocarriers loaded with curcumin (CUR) and differing surface charge were evaluated for transport efficacy in combination with electroporation (EP) in dependence on the type of cells. The obtained CUR-loaded nanoparticles with diameters ranging from 195 to 334 nm (derived from dynamic light scattering (DLS)) were characterized by atomic force microscopy (AFM) (morphology and shape) and Doppler electrophoresis (ζ-potential) as well as UV-vis spectroscopy (CUR encapsulation efficiency (about 90%) and photobleaching rate). The drug delivery properties of the obtained PLA nanocarriers enhanced by electroporation were assessed in human colon cancer cells (LoVo), excitable normal rat muscle cells (L6), and free of voltage-gated ion channels cells (CHO-K1). CLSM studies, viability, and ROS release were performed to determine the biological effects of nanocarriers. (3) Results: The highest photodynamic activity indicated anionic nanocarriers (1a) stabilized by C12(COONa)2 surfactant. Nanocarriers were cytotoxic for LoVo cells and less cytotoxic for normal cells. ROS release increased in cancer cells with the increasing electric field intensity, irradiation, and time after EP. Muscle L6 cells were less sensitive to electric pulses. (4) Conclusions: EP stimulation for CUR-PLA nanocarriers transport was considered to improve the regulated and more effective delivery of nanosystems differing in surface charge.

Exercise effects on γ3-AMPK activity, Akt substrate of 160 kDa phosphorylation, and glucose uptake in muscle of normal and insulin-resistant female rats.

Previous studies demonstrated that acute exercise can enhance glucose uptake (GU), γ3-AMP-activated protein kinase (AMPK) activity, and Akt substrate of 160 kDa (AS160) phosphorylation in skeletal muscles from low-fat diet (LFD)- and high-fat diet (HFD)-fed male rats. Because little is known about exercise effects on these outcomes in females, we assessed postexercise GU by muscles incubated ± insulin, delta-insulin GU (GU of muscles incubated with insulin minus GU uptake of paired muscles incubated without insulin), and muscle signaling proteins from female rats fed a LFD or a brief HFD (2 wk). Rats were sedentary (LFD-SED, HFD-SED) or swim exercised. Immediately postexercise (IPEX) or 3 h postexercise (3hPEX), epitrochlearis muscles were incubated (no insulin IPEX; ±insulin 3hPEX) to determine GU. Muscle γ3-AMPK activity (IPEX, 3hPEX) and phosphorylated AS160 (pAS160; 3hPEX) were also assessed. γ3-AMPK activity and insulin-independent GU of IPEX rats exceeded sedentary rats without diet-related differences in either outcome. At 3hPEX, both GU by insulin-stimulated muscles and delta-insulin GU exceeded their respective diet-matched sedentary controls. GU by insulin-stimulated muscles, but not delta-insulin GU for LFD-3hPEX, exceeded HFD-3hPEX. LFD-3hPEX versus LFD-SED had greater γ3-AMPK activity and greater pAS160. HFD-3hPEX exceeded HFD-SED for pAS160 but not for γ3-AMPK activity. pAS160 and γ3-AMPK at 3hPEX did not differ between diet groups. These results revealed that increased γ3-AMPK activity at 3hPEX was not essential for greater GU in insulin-stimulated muscle or greater delta-insulin GU in HFD female rats. Similarly elevated γ3-AMPK activity in LFD-IPEX versus HFD-IPEX and pAS160 in LFD-3hPEX versus HFD-3hPEX may contribute to the comparable delta-insulin GU at 3hPEX in both diet groups.NEW & NOTEWORTHY Glucose uptake (GU) and phosphorylated AS160 (pAS160) by insulin-stimulated muscles at 3 h postexercise (3hPEX) exceeded diet-matched controls in female low-fat diet-fed (LFD) or high-fat diet-fed (HFD) rats. GU with insulin for LFD-3hPEX exceeded HFD-3hPEX, whereas pAS160 was similar between these groups. γ3-AMPK immediately postexercise (IPEX) was similarly elevated in LFD and HFD, but only LFD-3hPEX had increased γ3-AMPK. These results suggest that greater γ3-AMPK at IPEX and pAS160 at 3hPEX may contribute to elevated GU with insulin, but greater γ3-AMPK at 3hPEX was dispensable for female HFD rats.

Acetyl-CoA carboxylase 2 inhibition reduces skeletal muscle bioactive lipid content and attenuates progression of type 2 diabetes in Zucker diabetic fatty rats

Intramyocellular lipid (IMCL) accumulation in skeletal muscle is closely associated with development of insulin resistance. In particular, diacylglycerol and ceramide are currently considered as causal bioactive lipids for impaired insulin action. Recently, inhibition of acetyl-CoA carboxylase 2 (ACC2), which negatively modulates mitochondrial fatty acid oxidation, has been shown to reduce total IMCL content and improve whole-body insulin resistance. This study aimed to investigate whether ACC2 inhibition-induced compositional changes in bioactive lipids, especially diacylglycerol and ceramide, within skeletal muscle contribute to the improved insulin resistance. In skeletal muscle of normal rats, treatment of the ACC2 inhibitor compound 2e significantly decreased both diacylglycerol and ceramide levels while having no significant impact on other lipid metabolite levels. In skeletal muscle of Zucker diabetic fatty (ZDF) rats, which exhibited greater lipid accumulation than that of normal rats, compound 2e significantly decreased diacylglycerol and ceramide levels corresponding to reduced long chain acyl-CoA pools. Additionally, in the lipid metabolomics study, ZDF rats treated with compound 2e also showed improved diabetes-related metabolic disturbance, as reflected by delayed hyperinsulinemia as well as upregulated gene expression associated with diabetic conditions in skeletal muscle. These metabolic improvements were strongly correlated with the bioactive lipid reductions. Furthermore, long-term treatment of compound 2e markedly improved whole-body insulin resistance, attenuated hyperglycemia and delayed insulin secretion defect even at severe diabetic conditions. These findings suggest that ACC2 inhibition decreases diacylglycerol and ceramide accumulation within skeletal muscle by enhancing acyl-CoA breakdown, leading to attenuation of lipid-induced insulin resistance and subsequent diabetes progression.

29-LB: Exercise Effects on ?3-AMPK Activity, Akt Substrate of 160 kDa Phosphorylation, and Glucose Uptake in Muscle of Normal and Insulin-Resistant Female Rats

Background: Previous studies demonstrated that exercise can enhance insulin-stimulated glucose uptake (ISGU) concomitant with greater Akt Substrate of 160 kDa (AS160) phosphorylation in muscles from normal or insulin resistant, male rats. However, little is known about exercise effects on ISGU in insulin-resistant muscle from female rats. Our goal was to assess exercise effects on ISGU and key signaling proteins in muscles from female rats fed a low-fat or high-fat diet. Methods: Female rats were randomly assigned to 2 diet-groups: standard chow (low-fat diet, LFD: 14% kCal fat) or high-fat diet (HFD: 60% kCal fat). After 2 weeks, rats from each diet-group either remained sedentary (LFD-SED or HFD-SED) or exercised (2h duration swim). At 3h post-exercise (3hPEX), paired epitrochlearis muscles were isolated and incubated with [3H]-2-deoxyglucose ±insulin to measure GU. Muscles were also assessed for γ3-AMPK activity and phosphorylated AS160 (pAS160; Ser588 and Thr642). Results: HFD-SED vs. LFD-SED rats had lower ISGU. At 3hPEX in each diet group, ISGU exceeded their diet-matched, sedentary controls. ISGU for LFD-3hPEX was greater than HFD-3hPEX. LFD-3hPEX vs. LFD-SED had greater γ3-AMPK activity and insulin-stimulated pAS160 (Ser588 and Thr642). For HFD-3hPEX vs. HFD-SED, insulin-stimulated pAS160 Thr642 was increased. However, γ3-AMPK activity was not different between HFD-SED and 3hPEX. Insulin-stimulated pAS160 Ser588 and Thr642 at 3hPEX did not differ between diet-groups, but γ3-AMPK for LFD-3hPEX exceeded HFD-3hPEX. Conclusions: These results indicated that prior exercise can enhance ISGU for LFD and HFD female rats, and suggested that the exercise-increase in ISGU for each diet-group may be related to greater pAS160. The elevated γ3-AMPK activity for LFD-3hPEX vs. LFD-SED, but not HFD-3hPEX vs. HFD-SED, raises the possibility that this difference may be relevant for the greater ISGU in LFD-3hPEX vs. HFD-3hPEX. Disclosure H. Wang: None. E. B. Arias: None. A. Zheng: None. S. Kwak: None. J. Zhao: None. G. Dong: None. G. D. Cartee: None. Funding National Institutes of Health (DK71771)

Acid mine drainage in an Indian high-sulfur coal mining area: Cytotoxicity assay and remediation study

Opencast mining causes significant environmental concern due to acid mine drainage (AMD) caused by the oxidation of pyrites and other sulfur-bearing minerals. The present study intends to determine the seasonal variability of AMD in the affected area of the Ledo opencast mining, the cytotoxicity of the AMD, and the AMD remediation process. The physicochemical properties of the collected samples were analyzed by using laboratory-based methods and sophisticated instrumental tools. The cytotoxicity study of AMD water was performed by using different cell lines such as normal rat muscle and human carcinoma cells. The study demonstrates that the mine water samples have high conductivity (1.30–2.49 ms cm−1) with high total dissolved solids (1068−1339 ppm) which can change the ionic composition of water. The concentration level of trace elements are also found to be higher than the permissible limit during monsoon season. A simple laboratory-based remediation process of AMD has been carried out in the current study by using size segregated pulverized limestone and the process reveals the decrease in elemental concentrations of AMD water. This study will be useful to develop a remediation technique to minimize the concentration levels of hazardous elements and ions in the AMD water.

Role of different nicotinic acetylcholine receptor subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats: neuroelectrophysiology

Objective To evaluate the role of different nicotinic acetylcholine receptor (nAChR) subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats by neuroelectrophysiology. Methods SPF adult male Sprague-Dawley rats, weighing 200-220 g, aged 2-3 months, were studied.Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized rats.Twelve portions of sciatic nerve-tibialis anterior muscles taken at day 3 after successful establishment of the sepsis model served as group S. Another 12 portions of sciatic nerve-tibialis anterior muscles of normal rats served as group C. The endplate potential (EPP) was monitored by intracellular microelectrode recording.Six portions of nerve-muscle samples were incubated with the adult nAChR (e-nAChR) specific antagonist waglerin-1 at a concentration of 1 μmol/L, the fetal nAChR (γ-nAChR) specific antagonist αA-OIVA at a concentration of 250 nmol/L and neuronal nAChR (α7-nAChR) antagonist methyllycaconitine (MLA) at a final concentration of 6 nmol/L.The amplitude of EPP was recorded at 15 min of incubation, and the difference in the amplitude of EPP (ΔEPP) and changing rate of EPP (ΔEPP%) were calculated.Another 6 portions of samples were incubated for 15 min with the Kreb mixture of waglerin-1 and 250 nmol/L αA-OIVA at a final concentration of 1 μmol/L, the Kreb mixture of waglerin-1 and 6 nmol/L MLA at a final concentration of 1 μmol/L, and the Kreb mixture of αA-OIVA and 6 nmol/L MLA at a final concentration of 250 nmol/L, and then the amplitude of EPP was recorded.Washout was performed with antagonist free Kreb solution for 15 min between treatments with different blockers. Results Compared with group C, ΔEPP and ΔEPP% were significantly decreased after antagonizing e-nAChR, ΔEPP% was decreased after antagonizing γ-nAChR, ΔEPP and ΔEPP% were increased after antagonizing α7-nAChR, the amplitude of EPP was increased after antagonizing e-nAChR+ γ-nAChR and e-nAChR+ α7-nAChR, and the amplitude of EPP was decreased after antagonizing γ-nAChR+ α7-nAChR (P<0.05). ΔEPP and ΔEPP% were significantly higher after antagonizing α7-nAChR than after antagonizing γ-nAChR in group S (P<0.05). The EPP was significantly lower after antagonizing e-nAChR+ α7-nAChR than after antagonizing e-nAChR+ γ-nAChR (P<0.05). Conclusion Weakened function of e-nAChR and enhanced function of α7-nAChR in the membrane of the end-plate in skeletal muscle are the main reasons for sepsis-related nerve-muscle dysfunction in septic rats, and the role is comparable between them. Key words: Sepsis; Neuromuscular diseases; Receptors, cholinergic

Study on denervated rat skeletal muscle by 7.0 T 31P-MR phosphorus spectroscopy

Objective To investigate the changes of 31P-MRS in denervated skeletal muscle at 7.0 T MR system. Methods In the experiment group, a total of 18 male Sprague-Dawley rats aged 6-8 weeks old and weighing 200-250 g were obtained. The right posterior femoral nerve were transected, and the proximal stumps were ligated by using 5-0 nylon stitches to preclude spontaneous repair. A sham surgery (incision and exploration of the nerve) was performed at the same time (n=6). Before rat model established and at varying times after initial surgery (3 d, and 1, 2, 4, 6, and 8 weeks). 31P-MR spectra of rat quadriceps femoris were acquired on 7.0 T Agilent small animal MR imaging system. We quantified the phosphocreatine (PCr), adenosine triphosphate(β-ATP), and inorganic phosphate (Pi) using Creatine phosphate disodium salt(10 mmol/L) as an external standard. The ratios, include PCr/Pi, Pi/β-ATP, PCr/β-ATP, PCr/(PCr+Pi) and Pi/(PCr+Pi) were quantified in jMRUI. All data were analyzed by one way analysis of variance (ANOVA) with posttest inter-group comparisons using Bonferroni test. Comparative analysis methods between the experimental and control group were performed via independent samples t test. P<0.05 was considered as statistically significance. Results The average measured concentrations of β-ATP, PCr, and Pi in control groups were (6.654±0.178) μmol/g, (25.656±0.738) μmol/g, and (1.594±0.096) μmol/g, respectively. There were significant statistically differences in β-ATP, PCr, Pi, PCr/Pi, PCr/PCr+Pi,Pi/PCr+Pi between the study and control group(P<0.05) at any measurement time point after denervation. Pi/β-ATP,PCr/β-ATP and pHi in the experimental group significantly statistically differed from those of the control group (all P<0.05)at each time point except on day 3 after operation. The total concentrations of β-ATP and PCr were reduced by 11.5% and 19.7% respectively on day 3. Thereafter, β-ATP and PCr declined rapidly by 63.1% and 68.8% at week 4 respectively, then decreased slowely by 74.0%和82.3% till week 10. The change of PCr/Pi is similar to β-ATP and PCr, but more remarkable. Pi, Pi/PCr+Pi, Pi/ATP showed a progressively increase till week 10. The intra-cellular pH (pHi) of normal rat muscles was 7.033±0.017, While the pHi gently in experiment rat muscles decreased during the entire experiment. Conclusion 31P-MRS with 7.0 T can quantify the temporal changes of energy metabolism and pHi in normal and denervated rat muscles. It shows that the dysfunction of energy metabolism are progressive with time and that they begin within a short period following the nerve section, the change of β-ATP, PCr, PCr/Pi take place primarily within 4 weeks after denervation. β-ATP, PCr, and PCr/Pi may be potential biomarkers of energy metabolism in the evaluation of denervated muscle atrophy. Key words: Magnetic resonance spectroscopy; Muscular disorders, atrophic; Denervation; Energy metabolism; Intracellular pH

Dietary Ginger Extracts Enhanced Glucose Uptake by Muscle and Adipose of Normal and Diabetic Rats via Mimicry of Insulin Action

The mechanism for the blood glucose lowering effect of different ginger extracts is yet to be clearly understood, that of raw ginger extracts has been sparsely explored while the mechanism for the cooked ginger extract, the form in which it is mostly consumed, has not been delved into. This study was therefore designed to determine the effect of raw and cooked ginger (Zingiber officinale Roscoe) extracts on glucose uptake by the muscle and adipose of normal, streptozocin-induced and high-fat diet-induced diabetic rats. Fresh ginger rhizomes were washed, peeled, milled and sieved without adding water to obtain the raw extract. A portion of this was boiled for 1 hour to give the cooked extract. Matured male albino rats (140) were divided into two major groups with 70 rats in each group. Group A was further divided into 7 sub-groups (n=10) 40 of which were rendered diabetic with intraperitoneal injection of streptozocin (60mg/kg body weight) to mimic Type 1 diabetes while in Group B Type 2 diabetes was induced with a 12 week consumption of High-Fat Diet (HFD). The remaining 30 rats in each group were left to remain non-diabetic/ normal. Ginger extracts were administered as a daily oral dose for 4 weeks after diabetes induction. The animals were the sacrificed and glucose uptake by the muscle and adipose tissue was determined using standard analytical procedure. Mean data were compared using Least Significant Difference at p≤ 0.05. It was clearly evidenced that both raw and cooked ginger extracts significantly enhanced and increased glucose uptake by the muscle and adipose tissues even in the absence of insulin, hence, mimicking insulin action. Ginger in both raw and cooked forms may therefore be beneficial in the prevention and management of diabetes mellitus. Human trial is hereby recommended.

The effects and mechanism of electrical stimulation on esophageal muscle in rat: an experimental study

Objective To explore the correlation between smooth muscle contraction and stimulation parameters, and mechanism of electrical stimulation on the esophageal body muscle strips in normal rats. Methods A segment of lower part esophagus of 20 healthy male WISTAR rats was obtained. Single phase square wave(width 25 ms)was used and the electrical stimulation lasted for 3 s with varied electrical parameter(current: 1.5 V, 3.0 V, 5.0 V, 8.0 V; frequency: 5 Hz, 10 Hz, 20 Hz). The correlation between tension and stimulation parameters was studied.Fifty healthy male rats were divided randomly into control group(n=10), ATR group(n=10), PHT group(n=10), L-NNA group(n=10), TTX group(n=10). Respective drugs were given 10 minutes before lower part of esophagus was stimulated with single phase square wave(5.0 V; 20 Hz; width 25 ms), the tension was recorded and compared among groups. Results At frequency 20 Hz and width 25 ms, the peak value of contraction in 1.5 V group was 0 g. The peak value of contraction in 5.0 V group was more than 3.0 V group and less than 8.0 V group(P 0.1, P 0.1, P 0.05). Conclusion The electrical stimulation of esophageal muscle in normal rats could cause effective contraction immediately. The peak value of contraction is correlated positively with volgage and frequency of electrical stimulation. The effect of electrical stimulation on esophageal muscle is not influenced by nurontransmeters. Key words: Achalasia; Mechanism; Neurotransmitter; Electrical stimulus; Esophageal muscle

Vestibular-evoked myogenic potentials recorded from miniature pigs and rats

ObjectiveTo report vestibular evoked myogenic potentials from different recording sites (neck extensor or masseter muscles) in miniature pigs and rats. MethodsPotentials were recorded using 1000 Hz tone bursts from the neck extensor muscle or masseter muscle in normal adult Bama miniature pigs and rats anesthetized with 3% pentobarbital sodium and Sumianxin II. ResultsAt 80 dB SPL, the first positive wave (P wave) of VEMPs was recognizable in 58% of rats with a latency of 6.45 ± 0.23 ms and an amplitude of 1.45 ± 0.49 μV when recorded from the neck extensor muscle, and in 50% of rats with a latency of 6.38 ± 0.34 ms and an amplitude of 1.57 ± 0.35 μV when recorded from the masseter muscle. In miniature pigs, at the same stimulus intensity, P wave was recognizable in 58% of the animals with a latency of 7.65 ± 0.64 ms and an amplitude of 1.66 ± 0.34 μV when recorded from the neck extensor muscle, and in 50% of the animals with a latency of 7.65 ± 0.64 ms and an amplitude of 0.31 ± 0.28 μV when recorded from the masseter muscle. ConclusionVEMP can be induced from both neck extensor and masseter muscles in the miniature pig and rat. For a given species, the site of recording affects P wave induction rate and amplitude but not latency. Consistency and repeatability analysis suggests that the masseter muscle is a better recording site in miniature pigs while the cervical extensor is a better recording site in rats. For a given recording site, both latency and amplitude of the P wave are slightly greater in miniature pigs than in rats.

Effect of Exercise Training on Signaling of Interleukin-6 in Skeletal Muscles of Type 2 Diabetic Rats.

Diabetes and exercise training have been shown to involve interleukin 6 (IL-6) signaling in muscle. However, the relationship between the actions of these two stimuli on muscle IL-6 and their downstream components is still unknown. Thus, the effect of endurance training on the key components of muscle IL-6 signaling transduction was investigated in a rat model of type 2 diabetes. Diabetes was induced by streptozotocin (STZ) in male Wistar rats fed a high-fat diet, with normal rats acting as controls. The animals were left to conduct their normal activities or assigned to endurance training in a treadmill. At the end of 8 weeks, blood biochemical profiles, exercise performance, muscle oxidative capacity, glucose transporter 4 (GLUT4) protein distribution, and expressions of IL-6 and its downstream proteins were determined. Blood biochemical profiles of the diabetic rats were altered compared to normal rats, whereas endurance training improved blood chemistry and exercise performance. It also increased muscle oxidative capacity, and promoted GLUT4 subcellular localization to the membrane in muscles. Furthermore, protein expression of IL-6 receptor (IL-6Rα) was increased in both normal and diabetic rats after endurance training, but no significant changes in IL-6, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), or suppressor of cytokine signaling 3 (SOC3) were observed in muscles of normal and diabetic rats. IL-6 signaling pathway mediating muscle response to endurance training was conserved in type 2 diabetes. There was no link between training-induced IL-6 downstream targets in skeletal muscles and IL-6-induced type 2 diabetes.

Histopathological changes in rat pancreas and skeletal muscle associated with high fat diet induced insulin resistance

The effects of a high fat diet on the development of diabetes mellitus, insulin resistance and secretion have been widely investigated. We investigated the effects of a high fat diet on the pancreas and skeletal muscle of normal rats to explore diet-induced insulin resistance mechanisms. Forty-four male Wistar rats were divided into six groups: a control group fed standard chow, a group fed a 45% fat diet and a group fed a 60% fat diet for 3 weeks to measure acute effects; an additional three groups were fed the same diet regimens for 8 weeks to measure chronic effects. The morphological effects of the two high fat diets were examined by light microscopy. Insulin in pancreatic islets was detected using immunohistochemistry. The homeostasis model assessment of insulin resistance index and insulin staining intensity in islets increased significantly with acute administration of high fat diets, whereas staining intensity decreased with chronic administration of the 45% fat diet. Islet areas increased significantly with chronic administration. High fat diet administration led to islet degeneration, interlobular adipocyte accumulation and vacuolization in the pancreatic tissue, as well as degeneration and lipid droplet accumulation in the skeletal muscle tissue. Vacuolization in the pancreas and lipid droplets in skeletal muscle tissue increased significantly with chronic high fat diet administration. We suggest that the glucolipotoxic effects of high fat diet administration depend on the ratio of saturated to unsaturated fatty acid content in the diet and to the total fat content of the diet.

Participation of Calcitonin Gene Related Peptide Released via Axon Reflex in the Local Increase in Muscle Blood Flow following Manual Acupuncture

The purpose of this study was to determine how calcitonin gene related peptide (CGRP) via axon reflex participates in increasing local muscle blood flow (MBF) following manual acupuncture (MA). Male Sprague-Dawley rats (N=56, 270-350 g) were used. We examined (1) the effects of MA on MBF in the tibialis anterior (TA) muscle in normal rats; (2) the effects of MA on MBF in the TA injected with saline or hCGRP8-37 (low: 2×10(-4) mol/litre; high: 2×10(-3) mol/litre), a competitive CGRP receptor antagonist, in rats; and (3) the effects of MA on MBF in the TA in capsaicin-treated rats. The capsaicin-treated rats were injected with capsaicin dissolved in an ethanol solution within 24 h after birth (50 mg/kg subcutaneously). MA was applied to the right TA for 1 min. (51)Cr-labelled microspheres (15 μm in diameter) were used to measure MBF. MA significantly increased MBF without changing arterial blood pressure in normal rats (p<0.05). MA also significantly increased MBF in saline-injected, low hCGRP8-37-injected and high hCGRP8-37-injected rats (p<0.001, 005 and 0.05, respectively). The increases in low and high hCGRP8-37-injected rats were lower than those in saline-injected rats, but the difference was not significant. However, MA did not significantly increase MBF in capsaicin-treated rats (p=0.38). We obtained conflicting results, suggesting that the participation of CGRP released via axon reflex may be limited to a local increase in MBF following MA.

Effect of sera from seronegative myasthenia gravis patients on neuromuscular junctions

About 85% of patients with generalized myasthenia gravis (MG) have anti-nicotinic acetylcholine receptor (nAChR) antibodies in their sera (seropositive MG; SPMG). The other 15% (seronegative MG; SNMG) are also considered to have antibody-mediated disease, but the nature of the antibodies in SNMG is not fully understood. We investigated the effect of sera from patients with MG on spontaneous muscle action potentials and acetylcholine (ACh)-induced potentials, and we examined the localization of epitopes recognized by SPMG sera or SNMG sera. SPMG sera and SNMG sera inhibited spontaneous muscle action potentials and ACh-induced potentials in the spinal-muscle co-culture system. However, spontaneous muscle action potentials and ACh-induced potentials in the neuromuscular junctions were strongly blocked by SPMG serum, whereas they were weakly blocked by SNMG serum. Both types of sera reacted strongly with the neuromuscular junctions in normal rat muscles, as shown by double immunostaining with serum and α-bungarotoxin. The SPMG epitope remained in the neuromuscular junctions, whereas that of SNMG disappeared after denervation of the sciatic nerve. Therefore, we suggest that the skeletal muscle weakness in SNMG may be due to an interaction with presynaptic neuromuscular transmission and nAChR.

A Novel Method to Measure Glucose Uptake by Single Skeletal Muscle Fibers Reveals a Similar Level of Insulin Resistance for Type IIA, IIB, IIX and IIB/X Fibers from Obese Zucker Rat Epitrochlearis Muscle

We have previously developed a method to measure glucose uptake (GU) by single fibers from rat skeletal muscle, showing in normal Wistar rat muscle, insulin‐stimulated GU was significantly greater in fibers expressing IIA vs. all other measured fiber types (IIA&gt; IIB, IIX, IIB/X). Our aim for this study was to use this method to gain insights into insulin resistance. For this purpose we measured glucose uptake by single fibers from Obese Zucker (OZ; a widely used model of insulin resistance) vs. Lean Zucker (LZ) rats. Epitrochlearis muscles were incubated ex vivo with [3H]‐2‐ deoxy‐D‐glucose, with or without insulin, before isolation of ~10–30 single fibers from each muscle. Fiber type (myosin heavy chain, MHC, isoform) and GU were determined for each fiber. Basal GU values by single fibers were not significantly different between OZ and LZ, regardless of MHC isoform expression. For GU by single fibers from insulin‐stimulated muscles, there was a significant main effect of genotype, and post‐hoc analysis indicated that insulin‐stimulated GU values for each fiber type were significantly greater for LZ vs. OZ rats. However, the calculated relative decrement for insulin‐stimulated GU between fibers from OZ vs. LZ did not differ significantly among the fiber types (IIA= 44%, IIB=58%, IIX=52%, IIB/X =52%). These results provide unique information about skeletal muscle GU for a fuller understanding insulin resistance.

Vascular Gene Transfer of SDF-1 Promotes Endothelial Progenitor Cell Engraftment and Enhances Angiogenesis in Ischemic Muscle

Gene therapy approaches to enhance endothelial progenitor cell (EPC) homing may augment cell engraftment to ischemic tissue and lead to a greater therapeutic response. Therefore, we assessed the effects of ultrasound-mediated (UM) transfection of the chemokine stromal cell-derived factor-1 (SDF-1) on homing and engraftment of intravenously administered EPCs and the subsequent angiogenic response in chronically ischemic skeletal muscle. Bone marrow-derived EPCs were isolated from donor Fisher 344 rats, cultured and labeled in preparation for injection into recipient animals via a jugular vein. Using a model of chronic hindlimb ischemia in rats, we demonstrated that UM destruction of intravenous carrier microbubbles loaded with SDF-1 plasmid DNA resulted in targeted transfection of the vascular endothelium within ischemic muscle and greater local engraftment of EPCs. The combination of SDF-1gene therapy and EPCs lead to the greatest increase in tissue perfusion and microvascular density within ischemic muscle, compared to no treatment or either monotherapy alone. Our results demonstrate that UM transfection of SDF-1 improves EPC targeting to chronically ischemic tissue, enhancing vascular engraftment and leading to a more robust neovascularization response.

Natriuretic Peptide-Dependent cGMP Signal Pathway Potentiated the Relaxation of Gastric Smooth Muscle in Streptozotocin-Induced Diabetic Rats

A common gastrointestinal complication of diabetes is gastroparesis, and patients with gastroparesis may present with early satiety, nausea, vomiting, bloating, postprandial fullness, or upper abdominal pain. However, the pathogenesis is not clear yet. A recent study indicated that atrial natriuretic peptide (ANP) was secreted from the gastric mucosa and the ANP family plays an inhibitory role in the regulation of gastrointestinal motility, but the effect of the natriuretic peptide signal pathway on diabetic gastroparesis has not been reported. The study investigated the effect of C-type natriuretic peptide (CNP) particulate guanylyl cyclase (pGC) cyclic guanosine monophosphate (cGMP) signaling on gastroparesis in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats; 4 weeks after induction, the experiments were performed. The spontaneous contraction of gastric smooth muscle strips was recorded by using physiographs in control and diabetic rats. The pGC activity in response to CNP and cGMP production in gastric smooth muscle were measured by using radioimmunoassay (RIA) in normal and diabetic rats. CNP induced a longer lasting relaxation of gastric antral circular smooth muscle strips in STZ-induced diabetic rats. The inhibitory effect of CNP on spontaneous contraction revealed a dose-dependency, and the inhibitory percentages were 25.5 +/- 1.7%, 43.6 +/- 3.2%, 85.1 +/- 2.5% in diabetic rats and 20.5 +/- 1.5%, 31.1 +/- 1.7%, 58.9 +/- 3.7% in the control group at the concentrations of 0.01, 0.03, and 0.1 mumol/l, respectively. The cGMP production and pGC activity in response to CNP in gastric muscle tissues were significantly potentiated in STZ-induced diabetic rats. Natriuretic peptide receptor type B (NPR-B) gene was expressed in the gastric smooth muscles of normal and diabetic rats, and the expression was increased in diabetic rats. The results suggest that natriuretic peptide-dependent pGC-cGMP signal is upregulated and may contribute to diabetic gastroparesis in STZ-induced diabetic rats.