Modulation of Skeletal Muscle Performance and SERCAby Exercise and Adiponectin Gene Therapy

in Insulin-Resistant Rat

Yasmeen Safwat,1 Nadia Yassin,1,2 Maha Gamal El Din,1,2 and Lobna Kassem1,2

This study addresses the potential application of adiponectin gene therapy and exercise in protection againstskeletal muscle dysfunction in type 2 diabetes mellitus (T2DM) while focusing on the role of sarco and endo-plasmic reticulum Ca + 2 ATPase (SERCA) and Glut4. 50 rats were divided into five groups: control, T2DM,T2DM treated with either adiponectin gene or exercise or a combination of both. Serum glucose, insulin, HOMAindex, triglycerides, and cholesterol were measured. Weight gain%, muscle contractile parameters {(peak twitchtension (Pt), peak tetanic tension (PTT), half relaxation time (HRT)}, and gene expression of SERCA, Glut4, andadiponectin were assessed in gastrocnemius muscle. Diabetic rats treated with either adiponectin gene or ex-ercise showed significant reduction in all serum parameters and wt gain%. There was significant elevation in Ptand PTT with shortening in HRT. Furthermore, a significant increase in SERCA, Glut4, and adiponectin geneexpression was noticed in both groups. Combination therapy caused marked gene expression of SERCA,GLUT4, and greater improvement in muscle contractility than either of the monotherapies. Skeletal muscledysfunction in T2DM is mediated via impaired SERCA and Glut 4. Combination therapy offered best protectionagainst muscle dysfunction and provides a novel promising strategy for a complete cure of muscle dysfunctionin T2DM.

Introduction

Type 2 diabetes mellitus (T2DM) is the most commonmetabolic disorder worldwide with a prevalence that

rises markedly with increasing degrees of obesity and asedentary lifestyle (Sullivan, 2005). Insulin resistance is as-sociated with a primary cellular defect in insulin action and acompensatory increase in insulin secretion (Kashyap andDefronzo, 2007).

Adiponectin is a 244–amino acid collagen-like protein thatis mainly secreted by adipocytes and acts as a hormone withanti-inflammatory and insulin sensitizing properties (Kado-waki et al., 2006). Findings from animal studies and meta-bolic studies in humans suggest several mechanisms throughwhich adiponectin may decrease the risk of T2DM. Theseinclude suppression of hepatic gluconeogenesis, stimulationof fatty acid oxidation in the liver and skeletal muscle,stimulation of glucose uptake in skeletal muscle, and stim-ulation of insulin secretion (Kadowaki et al., 2006). It wassuggested that higher adiponectin levels are associated witha lower risk of T2DM (Li et al., 2009).

Exercise has many well-recognized beneficial effects inhealthy as well as in diabetic subjects (Thompson, 2003;

Boule et al., 2005; Pedersen, 2006). Regular exercise was re-corded to improve insulin sensitivity in healthy individuals(Duncan et al., 2003; Boule et al., 2005). It also improves bloodglucose control, and may prevent T2DM in high-risk indi-viduals (Knowler et al., 2002; Sigal et al., 2004). It was shownthat even with impaired insulin action, exercise-induced in-crease in skeletal muscle glucose uptake seemed intact(Martin et al., 1995).

Sarco and endoplasmic reticulum Ca + 2 ATPase (SERCAs)are responsible for the maintenance of Ca2 + ion concentra-tions within the endoplasmic reticulum (MacLennan et al.,1985).Many studies linked insulin resistance in T2DM toimpaired SERCA activity. Other studies reported severalcommon dysfunctions of Ca2 + handling and alteration inSERCA activity in response to glucose in NIDDM model.This indicates a role of SERCA in the poor insulin secretion(Marie et al., 2001). Moreover, many novel links betweenSERCA and exercise were established. In mammalian skel-etal muscle, SERCA activity has been shown to be acutelyreduced after exhaustive exercise bouts (Byrd et al., 1989;Gollnick et al., 1991). Acute exercise training also has beenfound to improve Ca2 + sequestering performance of trained-striated muscle (Viru, 1994). In addition, previous studies

1Department of Physiology, Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City, Egypt.2Department of Physiology, Kasr Al Aini-Faculty of Medicine, Cairo University, New Cairo City, Egypt.

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showed that the impairment of sarcoplasmic reticulum (SR)function in diabetic cardiomyopathy is caused by reducedactivity of SERCA2a which is due primarily to a decrease inSERCA2a expression (Wold et al., 2005).

In the present study, we have employed type 2 diabeticrats with skeletal muscle weakness to determine whetheradiponectin gene therapy and regular swimming exerciseregimen are able to counter the progression of skeletalmuscle dysfunction-related T2DM in this commonly em-ployed animal model. We also investigated the role of SER-CA and adiponectin in the pathogenesis of skeletal muscledysfunction in this model. In addition, the links betweenSERCA, adiponectin, and GLUT4 were studied.

Materials and Methods

The present experiment was reviewed and approved by theCommittee of Ethics of Animal Experiments and conductedaccording to the Guidelines for Animal Experiments, GermanUniversity in Cairo, Faculty of Pharmacy and Biotechnology.

Experimental animals and protocols

Eight-week-old albino male rats were obtained from theanimal house of National Research Center, Cairo, Egypt. Theanimals were housed in wire mesh cages at room tempera-ture. They were fed their respective diets with free access towater. Animals were randomly divided into five groups, andeach group consisted of 8 rats: control group (CG), diabeticgroup (DG), DG treated by adiponectin gene (DAG), DGtreated by swimming exercise (DEG), and DG treated by acombination of both (DAEG). T2DM was induced by theadministration of a high fat diet (fat content 42% of energy),based on lard (HF-L), olive oil (HF-O), coconut fat (HF-C), orfish oil (derived from cod liver, HF-F) for a 12 week dietcourse (Buettner et al., 2006) with a single intraperitonealinjection of 45 mg/kg of streptozotocin (Sigma). Control ratswere injected with the same volume of citrate buffer. Dia-betes was confirmed on the second day post-STZ adminis-tration if serum glucose concentration was at least 250 mg/dL. At the end of the experimental, protocol rats were se-dated with an intraperitoneal injection of thiopental Sodiumbefore surgery. The left gastrocnemius was isolated from itsdistal insertion, keeping nerve and vasculature intact for theassessment of the physiological muscle contraction parame-ters, which include Peak Twitch Tension (Pt), Peak TetanicTension (PTT), and Half Relaxation Time (HRT).

Preparation and transfection of adiponectin gene

Adiponectin gene was taken from a muscle tissue to beamplified by RT-PCR and purified by using Qiagen Kit,where the purification solution is slightly acidic, containingguanine chloride, and this enhances DNA negative charge.DNA was washed to remove small pieces of DNA. This wasachieved using Ethanol and Tris buffer. This solution so-lublizes smaller pieces of DNA. Lipofectamine� 2000 (In-vitrogen) was used as transfection kit, and it is a 3: 1 (W/W)liposome formulation of the polycationic lipid 2, 3-dioley-loxy-N-[2(sperminecarboxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutrallipdioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water. Transfection activity was enhanced by using

Plus� Reagent (Cat. No 11514-015) to precomplex the DNA(Shih et al, 1997; Hawley-Nelson et al., 1993), where 0.2–0.4mgDNA was diluted in 25mL of Opti-MEM� I Reduced Serummedium and mixed gently. Lipofectamine� was mixed gentlybefore use, and then, 0.5–5mL was diluted in 25mL of Opti-MEM� I Reduced Serum medium and mixed gently. The di-luted DNA was combined with diluted Lipofectamine�,mixed gently, and incubated for 15–45 min at room temper-ature. For each transfection, 0.15 mL of Opti-MEM� I Mediumwas added to the tube containing the complexes and mixedgently. Finally, 0.2 mL of the diluted complexes was added byan intramuscular injection.

Exercise protocol

Exercise training was carried out by long-term swimtraining, over a period of 9 weeks (Reynolds et al., 1997).Animal training was done in a group swimming method(Ueno et al., 1997) in a half-filled cylindrical plastic con-tainer, containing water at room temperature, in a session of10 min, 5 days/week. The rats were acclimated to swim-ming by gradually increasing the swimming time and ses-sions as follows: 1st 2 weeks, 10 min, 1 session, 2nd 2 weeks,20 min, 2 sessions, 3rd 2 weeks, 30 min, 3 sessions, last 3weeks, 40 min, and 4 sessions. Sessions were separated by1 h rest (Ploug et al., 1990; Peijie et al., 2004; de Lemos et al.,2007).

Biochemical analysis

Plasma glucose was assayed by an automated glucoseoxidase procedure using a Beckman Glucose Analyzer II(Beckman Instruments) (Gault et al., 2002). Plasma insulinwas determined using ELISA kit for the measurement of rat/mouse insulin (Linco Research). Determination of homeo-stasis model assessment of insulin resistance (HOMA-IR):HOMA-IR was calculated as the product of fasting insulin(microunits/mL) multiplied by fasting glucose (mM) dividedby 22.5 (Matthews et al., 1985). Plasma triacylglycerol andcholesterol levels were measured using a TriglycerideQuantification Kit (BioVision Research) (Wahlefeld, 1974)and a Cholesterol Assay Kit (BioAssay System) (Roeschlauet al., 1974). All analyses were carried out according to themanufacturer’s instructions.

Assessment of muscle physiological parameters

Gastrocnemius muscle was isolated from its distal in-sertion, keeping nerve and vasculature intact, and securedto an MLT0210/D force-displacement transducer by a smallmetal hook fastened to the calcaneal tendon. The leg wassecured by screws tightened onto the medial and lateralcondyles of the femur. Using a MLA0320 Stimulator, themuscle was stimulated through twin platinum electrodesthat were applied directly to the surface of the muscle.Optimal voltage was determined by generating singletwitch contractions at increasing voltages until no increasein single-twitch force production was observed. Optimalmuscle length was determined in a similar manner. Speci-fically, muscle length was manipulated, and single-twitchforce production was observed. The length and voltage atwhich a single twitch produced the greatest force were usedthroughout the stimulation protocol. For Pt assessment, the

ADIPONECTIN GENE THERAPY IN DIABETIC 2 RAT MODEL 379

stimulating voltage was set to produce a maximum con-traction using a gradual increase in the square-wave pulsesfrom 0 to 10 V until producing maximum contraction for1 ms duration and 20 Hz frequency. Recording was dis-played at sampling rate 1K/sec. For PTT assessment, theleft gastrocnemius muscles were stimulated by 15 supra-maximal pulses, 1 ms duration with a gradual increase infrequency (from 20 to 50 Hz) until the muscle was com-pletely tetanized, and HRT was assessed from the maxi-mum to half the maximum peak tension at muscle optimallength during the relaxation phase of the twitch. Muscletemperature was monitored and maintained at 35�C–37�C,while the muscle was kept hydrated with saline. Data werecollected through an AD Instruments Bridge Amp andPowerlab 4/25 and were analyzed with Chart5 PowerLabsoftware for Windows. The tissues were immediately col-lected and either snap frozen or mounted on cork usingmounting medium and quick frozen in isopentane cooledby liquid nitrogen. Samples were maintained at - 80�C untilfurther analysis for detection of gene expression.

PCR analyses for RT-PCR

30 mg of gastrocnemius muscle was collected at 67 daysafter vector infusion and snap frozen in liquid nitrogen.Total RNA was extracted using SV Total RNA IsolationSystem (Promega) and reverse transcripted. Optimal primerand cDNA template concentrations were determined bytitration. Amplification efficiencies for each primer set weredetermined to be similar. Data were represented as differ-ences between threshold cycle values (DCT) for the tran-scripts of interest and the internal standard, b-actin. Theexpression of the muscle genes encoding for SERCA,

GLUT4, and adiponectin was examined using semi-quan-titative RT-PCR. Oligonucleotide sequences are available onrequest.

Statistical analyses

Differences between groups were determined by F test,Anova, and Post hoc test. Data are expressed as means –standard errors (SEM).

Results

Effect of Adiponectin gene transfer therapyand/or exercise on diabetic state

The use of a high fat diet and streptozotocin resulted in aninsulin-resistant diabetic model (DG group) that exhibitedhyperglycemia, hyperinsulinemia, and increased levels of tri-glycerides and cholesterol in comparison to control rats.Treatment with either adiponectin gene or exercise or bothresulted in a significant decrease in all these parameters com-pared with the DG group. However, the treatment with eithertherapy did not improve the values of these parameters to thecontrol values. With regard to HOMA-IR, serum triglycerides,and cholesterol levels, the combination between adiponectingene and swimming exercise resulted in a significant reductionin these parameters compared with DEG and in only serumcholesterol level when compared with DAG. (Table 1).

Effect of Adiponectin gene transfer therapy and/orexercise on wt gain%

Induction of T2DM led to a significant increase in wtgain% compared with CG. Adiponectin gene therapy or

Table 1. Serum Glucose, Insulin, Homeostasis Model Assessment of Insulin Resistance, Serum Triglycerides,

and Cholesterol Levels of the Five Studied Rat Groups at the End of the Experimental Protocol

Parameters CG DG DAG DEG DAEG

Serum glucose (mM) 3.42 – 1.20 11.57 – 1.88* 7.84 – 1.21*# 8.97 – 0.927*# 7.38 – 0.958*#

Serum insulin (mIU/mL) 11.35 – 1.368 33.38 – 4.636* 22.16 – 2*# 24.88 – 5.02*# 21.43 – 3.009*#

HOMA-IR 1.75 – 0.778 17.1 – 3.146* 7.68 – 1.129*# 9.84 – 1.86*# 7.023 – 1.22*#x

Serum triglycerides (mg/dL) 67.89 – 6.336 122.0 – 2.549* 78.09 – 5.088*# 95.88 – 7.085*#k 69.74 – 7.636#x

Serum cholesterol (mg/dL) 125.7 – 5.77 191.6 – 8.974* 143.4 – 5.96*# 159.5 – 7.568*#k 129.3 – 6.254#kx

Data are means – SEM.*( p < 0.05) versus CG.#( p < 0.05) versus DG.k( p < 0.05) versus DAG.x( p < 0.05) versus DEG.Control, nondiabetic male albino rats; CG, control group; DG, type 2 diabetic male albino rats; DAG, type 2 diabetic male albino rats

treated by adiponectin gene therapy; DEG; type 2 diabetic male albino rats following a regular swimming exercise regimen; DAEG, type 2diabetic male albino rats treated by adiponectin gene therapy and following a regular swimming exercise regimen.

Table 2. Wt Gain% at the End of the Experimental Protocol in the Five Studied Rat Groups

Wt gain% before sacrification CG DG DAG DEG DAEG

Mean – SD 41.5 – 6.392 59.70 – 5.523* 36.14 – 9.633# 47.72 – 2.716#k 30.06 – 5.619*#x

Data are means – SEM.*( p < 0.05) versus CG.#( p < 0.05) versus DG.k( p < 0.05) versus DAG.x( p < 0.05) versus DEG.

380 SAFWAT ET AL.

exercise resulted in a significant reduction in wt gain%compared with DG but still not reaching the control values.Meanwhile, the combination therapy resulted in a significantdecrease in wt gain% compared with the control. (Table 2).

Effect of Adiponectin gene transfer therapyand/or exercise on muscle contraction parameters

Pt and PTT were significantly decreased in DG with aprolongation of HRT compared with CG. Treatment witheither adiponectin gene or performing swimming exerciseelevated both Pt and PTT with shortening of HRT comparedwith DG. However, the present results elucidated that adi-ponectin gene therapy exerted a better protective role thanexercise. Furthermore, the cotreatment using both therapiesnot only caused a significant increase in Pt and shortening ofHRT compared with DG but also resulted in a greater sig-

nificant increase in Pt and more shortening in HRT than CG.Therefore, the current data showed that the best protectiveeffect was offered by the combination therapy. (Figs. 1–3).

Effect of Adiponectin gene transfer therapyand/or exercise on muscle gene expression

SERCA, GLUT4, and adiponectin muscle gene expressionswere significantly lower in DG than in CG. Either adipo-nectin gene therapy and/or exercise significantly increasedexpression of these genes compared with DG. Nevertheless,combination therapy proved to have a better protective roleconcerning SERCA and GLUT4 than the use of adiponectingene as monotherapy. (Figs. 4–6).

Discussion

T2DM is one of the most common metabolic disorders,representing a major health issue worldwide (Narayan et al.,

FIG. 1. Peak Twitch Tension (Pt) in five studied rat groups.Pt in five studied rat groups. DG was impaired in Pt com-pared with the control. Both Adiponectin gene therapy andregular swimming exercise regimen restored Pt in diabeticrats. However, adiponectin gene therapy provided a betterPt compared with regular swimming exercise regimen.Combined therapy restored Pt to the control values. Data aremeans – SEM, *(p < 0.05) versus CG, #(p < 0.05) versus DG,k(p < 0.05) versus DAG, $(p < 0.05) versus DEG.

FIG. 2. Peak Tetanic Tension (PTT) in five studied ratgroups. DG had impaired in PTT compared with the control.Both Adiponectin gene therapy and regular swimming exer-cise regimen restored PTT in diabetic rats. However, adipo-nectin gene therapy provided a better PTT compared withregular swimming exercise regimen. Combined therapy re-stored PTT to the control values. Data are means – SEM,*(p < 0.05) versus CG, #(p < 0.05) versus DG, k(p < 0.05) versusDAG, $(p < 0.05) versus DEG.

FIG. 3. Half Relaxation Time (HRT) in five studied ratgroups. HRT in five studied rat groups. DG had prolongedin HRT compared with the control. Both Adiponectin genetherapy and regular swimming exercise regimen shortenedHRT in diabetic rats. Combined therapy shortened HRT tothe control values. Data are means – SEM, *(p < 0.05) versusCG, #(p < 0.05) versus DG, k(p < 0.05) versus DAG, x(p < 0.05)versus DEG.

FIG. 4. SERCA muscle gene expression in five studiedrat groups. Data are means – SEM, *( p < 0.05) versus CG,#( p < 0.05) versus DG, k( p < 0.05) versus DAG, x( p < 0.05)versus DEG.

ADIPONECTIN GENE THERAPY IN DIABETIC 2 RAT MODEL 381

2000). Since skeletal muscles are the primary sites of insulin-dependent glucose disposal (Ferrannini et al., 1999), thus,resistance of the skeletal muscles to insulin-dependent glu-cose uptake may be an early step in the development ofT2DM (Cline et al., 1999).

In the present study, the administration of a high fat dietand STZ injection in DG produced a significant increase inserum glucose, insulin, HOMA-IR, serum triglycerides, andcholesterol levels compared with CG, indicating the devel-opment of insulin resistance.

In the present study, the muscle mechanical contractileperformance was assessed. Pt and PTT were recorded. Ptreflects the strength of the force and the contractile activity ofthe muscle, whereas PTT signifies that impairment in forcegeneration in fibers on removal of external Ca2 + is due toimpaired SR Ca2 + release (Payne et al., 2004). DG showed asignificant decrease in Pt and PTT to control values. Thereduction in Pt and PTT is in agreement with several studies,including that of Paulus and Grossie (1983), who recorded a

marked decrease in twitch and tetanic forces in uncontrolleddiabetic rats (Paulus and Grossie, 1983). Several mechanismswere involved to explain this deterioration. The polyolpathway was implicated and was driven by both hy-poinsulinemia and hyperglycemia (Cameron et al., 1989,1990). In addition, the hypoinsulinemia was considered asecondary factor causing atrophy, particularly of fast mus-cles (Cameron et al., 1990).

In the present work, HRT was also measured. HRT de-pends on the uptake of Ca2 + by the SR (Gollnick et al., 1991),the rate of dissociation of cross-bridges (Crow and Kush-merick, 1983; de Haan et al., 1989), and the free energy ofhydrolysis of ATP (Dawson et al., 1980; Russell et al., 1984).The current results showed that HRT was prolonged in theuntreated diabetic rats. Cotter and his co-workers (1993) re-ported similar results. They attributed the prolongation ofHRT to the polyol accumulation that causes swelling of SRtubules and SR damage (Cotter et al., 1993).Current resultsrevealed a significant decrease in the gastrocnemius muscleSERCA gene expression level in DG. Therefore, such an al-teration in SERCA gene expression and the resulting de-crease in Ca2 + uptake by SR could explain the prolongationof HRT in DG in this study.

Depletion of GLUT4 in either adipose tissue or skeletalmuscle causes insulin resistance (Zisman et al., 2000; Abelet al., 2001). Therefore, in the present work, the reduction inthe muscle GLUT4 gene expression in DG confirmed theinsulin resistance and maybe one of the causes of the re-duction in Pt and PTT, as it is associated with a decrease inglucose uptake, a reduction in the energy and fuel in themuscle, resulting in the weak contraction obtained.

Treatment with adiponectin gene therapy resulted in animprovement in the diabetic state. However, these groupvalues did not reach the control values, suggesting partialimprovement in these parameters. Another study showedsimilar results to our own. Adiponectin administration inboth wild-type (WT) and diabetic animal models revealed anenhancement in insulin sensitivity and corrected metabolicabnormalities (Yoon et al., 2006). Yamauchi et al. (2002)proved that these adiponectin effects are mediated throughAMPK activation. Activation of PPAR-a, a further importantfactor, that regulates the expression of genes involved inglucose and lipid metabolism was also identified to mediatethe insulin sensitizing action of adiponectin (Kadowaki andYamauchi, 2005).

With regard to the gastrocnemius muscle contractile per-formance, DG treated with the adiponectin gene showed asignificant increase in Pt and PTT compared with the un-treated DG, with no significant difference with the controlvalues, suggesting complete improvement in the muscleforce after gene therapy. The present study is in agreementwith the work of Krause and his co-workers (2008), whoassessed the muscle contractile properties, where PTT was50% lower in adiponectin-KO (Ad-KO) mice compared withWT mice. Furthermore, this study revealed a shortening inHRT in DG on gene therapy, suggesting an increase in Ca2 +

uptake by the SR (Krause et al., 2008) (Table 3).In addition, the present study proved an increase in the

muscle SERCA, GLUT4, and adiponectin gene expressions inthe treated DG with the adiponectin gene. Thus, the short-ening of HRT can be explained by the significant increase inthe SERCA gene expression and, consequently, the increase

FIG. 5. GLUT4 muscle gene expression in five studiedrat groups. Data are means – SEM, *( p < 0.05) versus CG,#( p < 0.05) versus DG, k( p < 0.05) versus DAG, x( p < 0.05)versus DEG.

FIG. 6. Adiponectin muscle gene expression in five studiedrat groups. Data are means – SEM, *( p < 0.05) versus CG,#( p < 0.05) versus DG, x( p < 0.05) versus DEG.

382 SAFWAT ET AL.

in the Ca2 + uptake by SR. It has been postulated that adi-ponectin may attribute to the enhanced glucose uptake inskeletal muscle cells (Berg et al., 2001) via increasing GLUT4expression and translocation (Ceddia et al., 2005) (Table 4).

In the present study, we examined the effects of a 9 weeksdaily swimming exercise regimen on T2DM rats. DG fol-lowing the swimming exercise regimen showed a significantreduction in the measured serum parameters compared withDG. However, this same group showed a significant eleva-tion in all these measured parameters compared with CG,suggesting a partial protection.

Pt and PTT showed a significant increase in the trainedDG compared with the untrained DG and a significant de-crease compared with CG. Moreover, this same groupshowed a shortening in HRT compared with DG (Table 3).

Moreover, the trained DG revealed a significant increasein the muscle SERCA, GLUT4, and adiponectin gene ex-pressions. This coincides with other studies which indicatedthat both moderate- and high-intensity exercise significantlyincrease the SERCA2a expression in the gastrocnemius(Kubo et al., 2003). Exercise training has profound effects onPPAR-g activation in rats (Petridou et al., 2007). Activation ofPPAR-g affects GLUT4 (Berger and Moller, 2002), activatesAMPK (Saha et al., 2004), and improves insulin action (Saltieland Olefsky, 1996). Since PPAR-g activation is also associ-ated with increased adiponectin production (Tsuchida et al.,2005), activation of PPAR-g with exercise represents a pos-sible mechanism that links the insulin sensitizing effects ofexercise with adiponectin (Table 4).

Furthermore, the combination of adiponectin gene therapyand exercise resulted in a significant reduction in all the se-rum parameters.

The present work also revealed a significant elevation inPt and PTT with a shortening in HRT in DG in the combined

therapies compared with both DG and CG, suggesting fullimprovement in the muscle contractile performance by thecombination therapy (Table 3).

Finally, the combination therapy group showed a signifi-cant increase in the gastrocnemius muscle SERCA, GLUT4,and adiponectin gene expressions. An interesting observa-tion in the previous studies was that both exercise and adi-ponectin shared somehow similar insulin sensitizing effects.Both promoted skeletal muscle glucose uptake, increasedfatty acid oxidation, and modulated GLUT4 translocationand AMPK activity (Ren et al., 1994; Tomas et al., 2002;Wright et al., 2004; Ceddia et al., 2005) (Table 4).

Conclusions

To our knowledge, this is the first study linking SERCAand skeletal muscle contractile performance in a T2DM in-sulin resistant animal model. There was alteration in SERCA,GLUT4, and adiponectin genes expression in the gastrocne-mius muscle associated with contractile dysfunction. Adi-ponectin gene therapy or swimming exercise, by increasingthe expression of these genes, exerted a partial protectiveeffect on the insulin resistance state and the skeletal musclecontractile performance. However, the use of a combinationtherapy resulted in the best protection against the skeletalmuscle deterioration. Taken together, these data support thepotential use of such a combination as a promising strategyin the treatment of T2DM muscle dysfunction.

Recommendations

Further investigations on the role of adiponectin genetherapy and exercise training in other pathophysiologicpathways involved in diabetic skeletal muscle dysfunctionare recommended. In addition, the trial of various types of

Table 3. Pt, PTT, and HRT of the Five Studied Rat Groups at the End of the Experimental Protocol

Parameters CG DG DAG DEG DAEG

Pt (N) 0.405 – 0.018 0.106 – 0.025* 0.384 – 0.013# 0.215 – 0.015*#@ 0.448 – 0.012*#@x

PTT (N) 0.752 – 0.073 0.179 – 0.103* 0.695 – 0.041# 0.554 – 0.047*#@ 0.796 – 0.029#@x

HRT (sec) 0.040 – 0.0024 0.085 – 0.001* 0.056 – 0.0015*# 0.058 – 0.0015*# 0.03651 – 0.0016*#@x

Data are means – SEM.*( p < 0.05) versus CG.#( p < 0.05) versus DG.@( p < 0.05) versus DAG.x( p < 0.05) versus DEG.Pt, peak twitch tension; PTT, peak tetanic tension; HRT, half relaxation time.

Table 4. Muscle SERCA Gene Expression, Muscle GLUT4 Gene Expression, and Muscle Adiponectin

Gene Expression of the Five Studied Rat Groups at the End of the Experimental Protocol

Parameters CG DG DAG DEG DAEG

Muscle SERCA gene expression 10.80 – 2.19 4.641 – 1.388* 9.516 – 0.8228# 8.583 – 0.4386*# 10.94 – 1.501#x

Muscle GLUT4 gene expression 1.65 – 0.624 0.702 – 0.28* 1.79 – 0.2587 1.55 – 0.466# 2.541 – 0.28*#@x

Muscle adiponectin gene expression 1.845 – 0.691 0.41 – 0.1929* 2.113 – 0.6402# 1.718 – 0.517# 2.83 – 0.3709*#x

Data are means – SEM.*( p < 0.05) versus CG.#( p < 0.05) versus DG.@( p < 0.05) versus DAG.x( p < 0.05) versus DEG.SERCA, sarco and endoplasmic reticulum Ca + 2 ATPase.

ADIPONECTIN GENE THERAPY IN DIABETIC 2 RAT MODEL 383

exercise protocols is recommended in order to select the onethat provides the best protection available.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Yasmeen Safwat, MS

Department of PhysiologyFaculty of Pharmacy and Biotechnology

German University in CairoMain Entrance Al Tagamoa Al Khames

New Cairo City 11341Egypt

E-mail: yasmeen_safwat@hotmail.com

Received for publication November 27, 2012; received inrevised form April 9, 2013; accepted April 9, 2013.

ADIPONECTIN GENE THERAPY IN DIABETIC 2 RAT MODEL 385