Lean and Obese Zucker Rat Extensor Digitorum Longus Muscle high-frequency electrical stimulation (HFES) Data: Regulation of MAPKs Associated Proteins

Anaerobic exercise has been advocated as a prescribed treatment for the management of diabetes: however, alterations in exercise-induced signaling remain largely unexplored in the diabetic muscle. Here, we compare the basal and the in situ contraction-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK 1/2, p38, and JNK in the lean and obese (fa/fa) Zucker rat extensor digitorum longus (EDL) muscle following a single bout of contractile stimuli. This article represents data associated with prior publications from our (Katta et al., 2009a, 2009b, 2008) [1–3] and concurrent Data in Brief articles (Ginjupalli et al., 2017a, 2017b; Rice et al., 2017a, 2017b) [4–7].


a b s t r a c t
Anaerobic exercise has been advocated as a prescribed treatment for the management of diabetes: however, alterations in exerciseinduced signaling remain largely unexplored in the diabetic muscle. Here, we compare the basal and the in situ contractioninduced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK 1/2, p38, and JNK in the lean and obese (fa/fa) Zucker rat extensor digitorum longus (EDL) muscle following a single bout of contractile stimuli. This article represents data associated with prior publications from our (Katta et al., 2009a(Katta et al., , 2009b(Katta et al., , 2008 [1][2][3] Contents lists available at ScienceDirect  (Ginjupalli et al., 2017a(Ginjupalli et al., , 2017b; Rice et al., 2017aRice et al., , 2017b [4][5][6][7]. & 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area Diabetic skeletal muscle response to exercise Type of data Graph, figure How data was acquired Immunoblotting

Data format Analyzed Experimental factors
A high-frequency electrical stimulation (HFES) was used to produce 10 sets of 6 contractions over a 22-min period. Tissues were collected and protein was then isolated from tissue for western blot analysis. Experimental features EDL obtained from Lean and Obese male Zucker rats were used in this experiment

Value of the data
The data presented in this Brief is vital to understanding the effect of diabetes on skeletal muscle mechanotransduction.
This data gives insight into the how diabetes alters tissue response to stimuli. This data provides a more thorough understanding of the MAPKs involvement in exercise mediated signaling in both diabetic and non-diabetic muscle tissue.
To determine the effect of HFES on EDL from OSXZ and LNZ animals we evaluated the expression of p38 alpha and gamma. EDL basal p38 alpha content demonstrated no significant difference in the OSXZ when compared to LNZ. HFES resulted in an increase in p38 alpha in the LNZ EDL (43. 8 7 8.8%, at 3 h, p o 0.05) when compared to LNZ contralateral control (Fig. 2C). However, HFES did not elicit a response in the OSZX EDL when compared to contralateral OXSZ control (Fig. 2C). EDL basal p38 gamma content demonstrated no significant difference in the OSXZ when compared to LNZ. HFES resulted in an increase in p38 gamma in the LNZ EDL (14.1 7 2.9%, at 0 h, p o 0.05) when compared to LNZ contralateral control (Fig. 2D). However HFES resulted in an decrease in p38 gamma (8.0 7 1.8%, 14.4 7 0.9%, and 29.2 7 1.4%, at 0, 1,and 3 h, p o 0.05) in the OSZX EDL when compared to contralateral OXSZ control (Fig. 2D). To determine the effect of HFES on EDL from OSXZ and LNZ animals we evaluated the phosphorylation of p38 alpha and gamma thr 180/tyr 182 to total p38 alpha and gamma. EDL basal phosphorylation of p38 alpha thr 180/tyr 182 to total p38 alpha was higher (41.5 7 2.4%, p o 0.05) in the OSXZ when compared to LNZ (Fig. 2E). HFES resulted in an decrease in phosphorylation of p38 alpha thr180/tyr 182 to total p38 alpha in the LNZ EDL (19.4 7 0.6%, 36.2 7 2.2%, and 41.5 7 2.4%, at 0, 1,and 3 h, p o 0.05) when compared to LNZ contralateral control (Fig. 2E). HFES resulted in an increase in phosphorylation of p38 thr 180/tyr 182 to total p38 alpha in the OSXZ EDL (12.3 7 3.9% and 13.9 7 3.8%, at 1 and 3 h, p o 0.05) when compared to OSXZ contralateral control (Fig. 2E). EDL basal phosphorylation of p38 gamma thr 180/tyr 182 to total p38 gamma demonstrated a significant decrease (52.5 7 4.2%, p o 0.05) in the OSXZ when compared to LNZ (Fig. 2F). HFES did not elicit a response in phosphorylation of p38 gamma thr 180/tyr 182 to total p38 gamma in the LNZ EDL when compared to LNZ contralateral control (Fig. 2F). HFES resulted in an increase in phosphorylation of p38 gamma thr 180/tyr 182 total p38 in the OSXZ EDL (49.3 7 4.2%, 54.2 7 4.7%, and 87.8 7 6.7%, at 0, 1, and 3 h, p o 0.05) when compared to OSXZ contralateral control (Fig. 2F).

Animals
All procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals as approved by the Council of the American Physiological Society and the Animal Use Review Board of Marshall University. Young (10 week, n ¼ 12) male lean Zucker (non-diabetic) (LNZ) and young (10 week, n ¼ 12) male obese syndrome-X Zucker (diabetic) (OSXZ) rats were obtained from the Charles River Laboratories and barrier housed one per cage in an AAALAC approved vivarium. Housing conditions consisted of a 12H: 12H dark-light cycle and the temperature was maintained at 22 7 2°C. Animals were provided food and water ad libitum. Rats were allowed to recover from shipment for at least two weeks before the commencement of experimentation during which time the animals were carefully observed and weighed weekly.

Contractile stimulation of skeletal muscles
The high-frequency electrical stimulation (HFES) model has been previously described [8] and was chosen on the basis of its efficacy in stimulating protein translation and muscle hypertrophy in vivo [9]. The HFES model used in the present study produced 10 sets of 6 contractions with an overall protocol time of 22 min. Animals were killed by a lethal dose of pentobarbital sodium at baseline, immediately following, 1 h or 3 h (n ¼ 6 normal, n ¼ 6 diabetic for 0, 1, and 3 h) after HFES. Once excised, muscles were blotted dry, trimmed of visible fat and tendon projections, weighed, immediately frozen in liquid nitrogen, and stored at − 80°C.

Immunoblot analysis
Skeletal muscles were snap-frozen in liquid nitrogen at the end of each experiment. Protein isolates were prepared from the muscles by pulverizing the samples under liquid nitrogen using a mortar and pestle before washing (3 × 5 min) with ice-cold phosphate buffered saline (PBS). T-PER (2 mL/1 g tissue weight) (Pierce, Rockford, IL) supplemented with 100 mM NaF, 1 mM Na 3 VO 4 , 2 mM PMSF 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepsatin was used to extract proteins as detailed by the manufacturer. After centrifugation (1000g × 10 min), the supernatant was collected and the protein concentration of the homogenates was determined in triplicate using the Bradford method (Pierce) with bovine serum albumin as a standard. Samples were diluted to a concentration of 1.5 mg/mL in SDS-loading buffer and boiled for 5 min before loading thirty micrograms of total protein for separation on 10% or 15% SDS-PAGE gels. After electrophoresis, proteins were transferred onto Hybond nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ) using standard conditions and stained with Ponceau S to verify transfer and equal loading of lanes.
Membranes were blocked in buffer (5% nonfat dry milk in TBST) for 1 h at room temperature, washed (TBST, 3 × 5 min), and incubated in primary antibody overnight at 4°C. Unbound antibody was removed by washing the membranes (TBST, 3 × 5 min) and the membranes were incubated in horseradish peroxidase (HRP)-linked secondary antibodies for 1 h at room temperature and rewashed (TBST, 3 × 5 min).
The exposure time was adjusted to keep the integrated optical densities within a linear and nonsaturated range. Molecular weight markers (Cell Signaling) were used as mass standards and NIH 3T3 cell lysates were included as positive controls. Membranes were stripped using the Restore western blot stripping buffer as detailed by the manufacturer. The absence of antibody binding after stripping was confirmed using the ECL reagent before washing (TBST, 3 × 5 min) the membranes and reprobing. Experimental error associated with membrane stripping and reprobing was minimized by randomizing the antibody incubations between experiments.

Data analysis
Data were analyzed using Sigma Stat 3.0 statistical software and the results are presented as mean 7 SEM. Two-way ANOVA followed by the Student-Newman-Keuls post-hoc testing to determine differences between groups. The level of significance accepted a priori was o 0.05.