Acta Diabetol (2014) 51:963–972 DOI 10.1007/s00592-014-0645-4

ORIGINAL ARTICLE

Enhanced insulin sensitivity and acute regulation of metabolic genes and signaling pathways after a single electrical or manual acupuncture session in female insulin-resistant rats Anna Benrick • Manuel Maliqueo • Julia Johansson Miao Sun • Xiaoke Wu • Louise Mannera˚s-Holm • Elisabet Stener-Victorin



Received: 9 July 2014 / Accepted: 25 August 2014 / Published online: 14 September 2014 Ó Springer-Verlag Italia 2014

Abstract Aim To compare the effect of a single session of acupuncture with either low-frequency electrical or manual stimulation on insulin sensitivity and molecular pathways in the insulin-resistant dihydrotestosterone-induced rat polycystic ovary syndrome (PCOS) model. Both stimulations cause activation of afferent nerve fibers. In addition, electrical stimulation causes muscle contractions, enabling us to differentiate changes induced by activation of sensory afferents from contraction-induced changes. Materials and Methods Control and PCOS rats were divided into no-stimulation, manual-, and electrical stimulation groups and insulin sensitivity was measured by euglycemic hyperinsulinemic clamp. Manually stimulated needles were rotated 180° ten times every 5 min, or lowfrequency electrical stimulation was applied to evoke muscle twitches for 45 min. Gene and protein expression were analyzed by real-time PCR and Western blot. Results The glucose infusion rate (GIR) was lower in

PCOS rats than in controls. Electrical stimulation was superior to manual stimulation during treatment but both methods increased GIR to the same extent in the poststimulation period. Electrical stimulation decreased mRNA expression of Adipor2, Adrb1, Fndc5, Erk2, and Tfam in soleus muscle and increased ovarian Adrb2 and Pdf. Manual stimulation decreased ovarian mRNA expression of Erk2 and Sdnd. Electrical stimulation increased phosphorylated ERK levels in soleus muscle. Conclusions One acupuncture session with electrical stimulation improves insulin sensitivity and modulates skeletal muscle gene and protein expression more than manual stimulation. Although electrical stimulation is superior to manual in enhancing insulin sensitivity during stimulation, they are equally effective after stimulation indicating that it is activation of sensory afferents rather than muscle contraction per se leading to the observed changes. Keywords PCOS  DHT  Insulin resistance  Glucose infusion rate  Electroacupuncture  Manual stimulation

Managed by Antonio Secchi. A. Benrick  M. Maliqueo  J. Johansson  M. Sun  E. Stener-Victorin (&) Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Box 434, 405 30 Go¨teborg, Sweden e-mail: [email protected] M. Sun  X. Wu  E. Stener-Victorin Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, China L. Mannera˚s-Holm Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Go¨teborg, Sweden

Introduction The metabolic syndrome is a constellation of obesity-related metabolic risk factors that are associated with the development of diabetes and cardiovascular disease. Components of the syndrome are increased waist circumference, high blood pressure, impaired fasting glucose and disturbed blood lipids including LDL-cholesterol, total cholesterol and triglycerides. However, no existing definition of the metabolic syndrome meets the criteria as a syndrome [1], which is a pre-morbid condition rather than a clinical diagnosis [2]. Polycystic ovary syndrome (PCOS) is often referred to as the female metabolic syndrome and is besides

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the metabolic derangements a disorder of irregular menses, hyperandrogenism and/or polycystic ovary morphology [3]. PCOS is the most common endocrine disorder among women and is associated with increased prevalence of impaired glucose tolerance and type 2 diabetes (T2D), independent of body mass index [4]. With a prevalence of insulin resistance (IR) and compensatory hyperinsulinemia between 65 and 80 % in lean and up to 90 % in obese women with PCOS, it is important to determine the pathophysiological mechanisms of the disease and the effects of interventions [5, 6]. There is an evident genetic component in the etiology of PCOS, and THADA and DENND1A have been pointed out as PCOS susceptibility genes [7]. Although there is a high rate of T2D in women with PCOS and family members of women with PCOS, T2Dassociated risk variants are not important risk variants for the development of PCOS [8]. Changes in diet and lifestyle are the first choice for improving and preventing IR and T2D in women with PCOS, and metformin is prescribed to those with impaired glucose tolerance who do not respond to lifestyle changes [3]. Metformin is a safe and effective treatment for T2D but is associated with severe gastrointestinal side effects [9]. It is well known that physical exercise has beneficial effects on metabolic dysfunctions—including improved energy balance, reduced adiposity, and regulation of key signaling molecules in skeletal muscle and adipose tissue— but the mechanisms behind these effects are largely unknown. Increasing evidence suggests that acupuncture with low-frequency (2 Hz) electrical stimulation of the needles (electroacupuncture) with repetitive muscle contraction improves glucose metabolism by modifying insulin sensitivity in a manner similar to physical exercise [10–12]. Manual manipulation of the acupuncture needles by rotation is another common method of stimulation [13]. Whether manual stimulation increases glucose uptake to the same extent as electrical stimulation has not previously been investigated. In dihydrotestosterone (DHT)-induced PCOS rats that exhibit obesity and IR similar to that in human PCOS [14, 15], we found that repeated low-frequency electrical stimulation of abdominal and hind limb muscles for 5 weeks restores whole-body insulin sensitivity to the same extent as exercise and that this does not alter adipose tissue mass and cellularity [10, 16]. This effect might involve regulation of adipose and skeletal muscle tissue signaling pathways because electrical stimulation and exercise each partly restore divergent gene and protein expression associated with IR, obesity, and inflammation [10, 16, 17]. Repeated electrical stimulation also improves diet- and prednisolone-induced IR in male rats [12, 18] and restores impaired glucose tolerance in db/db mice [19] and a genetic model of T2D [20]. A single bout of exercise has been shown to induce rapid changes in skeletal muscle and adipose tissue, and these

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effects have been attributed to muscle contractions. However, increased insulin sensitivity following low-frequency electrical stimulation of skeletal muscles was abolished after transection of afferent nerves [21]. This indicates that the response is mediated by activation of afferent nerves rather than by muscle contractions per se. Manual manipulation of the acupuncture needles also activates the afferent nerve fibers [13]. Based on repeated stimulation studies [10, 16, 17], and the fact that electrical stimulation is continuous whereas manual stimulation is intermittent (every 5th minute), we hypothesize that electrical stimulation causing muscle contractions improves insulin sensitivity to a larger extent than manual stimulation and that the effect is mediated via different molecular pathways. The aim of the present study was to compare the effect of a single session of acupuncture with either electrical or manual stimulation on changes in insulin sensitivity in rats with and without DHT-induced PCOS. Both stimulations cause activation of afferent nerve fibers. In addition, electrical stimulation causes muscle contractions. Thus, we have the potential to separate if it is the activation of sensory afferents or the muscle contractions that cause changes in glucose uptake. We also aimed to investigate gene and protein expression of molecules that regulate glucose uptake in skeletal muscle and adipose tissue.

Materials and methods Animals Female Wistar rats were purchased from Charles River (Sulzfeld, Germany). Animals were fed ad libitum with standard rat chow (Harlan Teklad Global Diet, Harlan, Germany) and had free access to water. They were cared for according to the principles of the Guide to the Care and Use of Experimental Animals (www.sjv.se), and the Animal Ethics Committee at the University of Gothenburg approved the study. Study design The study outline is shown in Fig. 1. Pups at 14–15 days of age arrived with lactating dams, and a slow-releasing DHTpellet (7.5 mg, 8.3 lg/day, Innovative Research of America, Sarasota, USA) was implanted subcutaneously in the neck of the pups at 21 days of age to induce PCOS. The pellet was inserted under light anesthesia with isoflurane (2 % Isoba vetÒ, Schering-Plough AB, Stockholm, Sweden). At 14–25 weeks of age, control and PCOS rats were each randomly divided into the following three experimental groups: no-stimulation (Controls and PCOS; n = 9 and n = 7, respectively), manual stimulation (n = 19 and n = 19), and electrical stimulation (n = 12 and n = 21).

Acta Diabetol (2014) 51:963–972

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Fig. 1 Outline of study design. DHT dihydrotestosterone, wk weeks

Experiment (14-15 wk of age)

Euglycemic hyperinsulinemic clamp Implantation of DHT pellet (21-day-old rats)

Steady state

PCOS

Controls

Stimulation

Post-stimulation

Electrical stimulation Manual stimulation No stimulation

Electrical stimulation (2 Hz) Manual stimulation

Vaginal smear The stage of cyclicity in control rats was determined daily before the euglycemic hyperinsulinemic clamp by microscopic analysis of the predominant cell types in vaginal smears. Clamp studies were performed in the estrus phase in control rats, and DHT-induced PCOS rats were studied irrespective of cycle day because they were acyclic [14]. Euglycemic hyperinsulinemic clamp and acupuncture stimulation At 14–15 weeks of age, all rats were subjected to surgery and a euglycemic hyperinsulinemic clamp as previously described [14]. In brief, all rats were anesthetized with thiobutabarbital sodium (130 mg/kg i.p.; Inactin; SigmaAldrich St. Louis, USA) during surgery, the insulin clamp, and following sensory stimulation. After cannulation of vena jugularis and arteria carotis, they rested for at least 10 min before clamp. Clamp started with a bolus of insulin and was thereafter infused at 8 mU min-1 kg-1. The plasma glucose concentration was analyzed every 5 min and maintained at 6 mM with a 20 % glucose solution. The mean glucose infusion rate (GIR) was normalized to body weight. After steady state, which was reached after approximately 45 min and kept for another 30 min, acupuncture needles (HEGU Svenska AB, Landsbro, Sweden) were placed in the rectus abdominis muscle and in the triceps surae muscle from the medial side bilaterally in somatic segments corresponding to the innervation of the pancreas and ovaries. Two needles were placed in abdominal muscle and corresponded to the acupuncture

points ST 27, ST28, and ST29 and four needles in the leg muscle corresponded to SP6 and SP9 on each side. For electrical stimulation, the needles were attached to an electrical stimulator (CEFAR ACU II; Cefar-Compex Scandinavia, Malmo¨, Sweden). The stimulation was of low frequency (2 Hz), and the intensity was high enough to evoke muscle twitches and varied from 0.8 mA to 2.2 mA due to receptor adaptation. For manual stimulation, the needles were rotated 180° 10 times every 5 min during the treatment. The treatment lasted for 45 min for both stimulations, and animals in the no-stimulation group were kept at steady state without intervention for the same length of time. After treatment, all rats were kept at steady state and followed for another 45 min. Tissue collection, RNA preparation, and real-time PCR At the end of the clamp, 45 min after stimulation, animals were decapitated and skeletal muscle, adipose tissue, and ovaries were dissected and stored at -80 °C. RNA isolation from the ovaries, soleus muscle, and mesenteric fat was performed using commercial kits (74704 and 74804 for fat, Qiagen, Germantown, USA). cDNA was prepared using SuperScript VILO (Life Technologies, Paisley, UK) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with TaqMan low-density array cards (Applied Biosystems, Life Technologies) and the 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol. TaqMan gene expression assay numbers, GenBank accession numbers, and mean Ct-values are given in Table 1. Data were analyzed using SDS 1.4 software (Applied Biosystems). Out of five

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966 Table 1 Gene list for TaqMan low-density array and TaqMan gene expression assay numbers, GenBank accession numbers, and mean Ct-values in all samples

Acta Diabetol (2014) 51:963–972

Gene symbol

TaqMan assay ID no.

GenBank accession no.

18S

Hs99999901_s1

X03205.1

Actb

Rn00667869_m1

Hprt1 Ppia Gapdh

Endogenous controls

Mean Ct-value Fat

Ovary

Muscle

8.6

8.3

8.5

NM_031144.2

18.4

18.0

21.0

Rn01527840_m1

NM_012583.2

24.0

22.7

23.2

Rn00690933_m1

NM_017101.1

20.3

19.2

22.3

Rn01775763_g1

NM_017008.3

21.1

20.6

18.2

Sympathetic and adrenergic pathway Th

Rn00562500_m1

NM_012740.3

36.8

35.0

Undet.

Adrb1

Rn00824536_s1

NM_012701.1

26.3

29.2

30.8

Adrb2

Rn00560650_s1

NM_012492.2

25.6

26.3

26.2

Adrb3

Rn01478698_g1

NM_013108.1

28.5

34.8

Undet.

Ngf

Rn01533872_m1

XM_227525.5

29.7

28.7

30.0

Ngfr

Rn00561634_m1

NM_012610.2

30.0

29.4

32.2

Ntrk1

Rn00572130_m1

NM_021589.1

31.5

31.7

33.2

Adcy3 Adcy4

Rn00590729_m1 Rn00570644_m1

NM_130779.2 NM_019285.2

26.4 25.0

24.9 26.3

26.8 27.4

Insulin receptor pathway Tbc1d1

Rn01413271_m1

XM_341215.4

26.7

26.3

27.4

Gsk3b

Rn00583429_m1

NM_032080.1

24.3

23.3

23.4

Pik3r1

Rn00564547_m1

NM_013005.1

21.0

22.1

22.1

Pik3cb

Rn00585107_m1

NM_053481.1

24.6

24.5

27.2

Pten

Rn00477208_m1

NM_031606.1

21.8

22.0

23.2

Slc2a4

Rn01752377_m1

NM_012751.1

23.8

25.9

20.0

Nr4a1

Rn01533237_m1

NM_024388.1

25.8

23.5

23.6

Nr4a3

Rn00569312_g1

NM_031628.1

25.0

25.5

25.5

Pdpk1

Rn00579366_m1

NM_031081.1

24.6

24.1

24.2

Akt2

Rn00690901_m1

NM_017093.1

23.6

22.9

22.6

Adipor1

Rn01483784_m1

NM_207587.1

23.8

22.9

23.4

Adipor2

Rn01463173_m1

NM_001037979.1

23.2

22.3

21.7

G6pc

Rn00689876_m1

NM_013098.2

Undet.

Undet.

Undet.

Erk 2 Erk 1

Rn00587719_m1 Rn00820922_g1

NM_053842.1 NM_017347.2

24.7 25.3

23.6 23.5

24.2 25.2

Mtor

Rn00571541_m1

NM_019906.1

25.6

24.2

24.5

Tfam

Rn00580051_m1

NM_031326.1

24.6

23.8

23.2

Ppargc1a

Rn00580241_m1

NM_031347.1

30.3

29.1

24.8

Pparg

Rn00440945_m1

NM_001145366.1

24.4

26.3

29.0

Atp5 h

Rn00820986_g1

NM_019383.2

23.1

22.7

21.3

Sdhd

Rn01644331_g1

NM_198788.1

22.0

21.7

19.7

Cox7c

Rn01493939_g1

NM_001134705.1

21.8

20.6

19.2

Nrf1

Rn01455958_m1

NM_001100708.1

24.9

24.9

25.8

Sirt1 (rCG60587)

Rn01428094_m1

CH473988.1

31.1

32.0

33.1

Mitochondrial function

Muscle insulin resistance

123

Pdk4

Rn00585577_m1

NM_053551.1

27.8

24.9

21.4

Cpt1b

Rn00682395_m1

NM_013200.1

27.6

28.9

22.0

Acox1 Ucp3

Rn01460628_m1 Rn00565874_m1

NM_017340.2 NM_013167.2

22.9 27.8

23.1 31.6

23.1 25.1

Fndc5

Rn01519161_m1

XM_002729542.2

31.0

28.8

22.2

Gys1

Rn01476417_m1

NM_001109615.1

25.6

24.8

20.8

Acta Diabetol (2014) 51:963–972 Table 1 continued

967

Gene symbol

TaqMan assay ID no.

GenBank accession no.

Mean Ct-value

Rn01759072_m1

NM_001007617.1

27.5

25.6

27.5

Ucp2

Rn01754856_m1

NM_019354.2

22.7

20.9

25.0

Fabp4

Rn00670361_m1

NM_053365.1

17.0

23.8

21.2

Slc27a1

Rn00585821_m1

NM_053580.2

26.9

24.9

27.1

Nuak2 Fat oxidation

putative reference genes (Table 1), the combination of beta actin (Actb) and peptidylprolyl-isomerase A (Ppia) was selected as reference gene for the muscle and ovary samples and Actb and hypoxanthine phosphoribosyl transferase (Hprt) for fat tissue [22]. Gene expression values were calculated using the 2 DDCt method and expressed as fold changes compared with unstimulated control rats. Immunoblotting For immunoblotting, we used antibodies against adiponectin receptor 2 (ADIPOR2) (sc-46755; Santa Cruz Biotechnology, Santa Cruz, USA), phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) (Thr202/Tyr204) (#9101; Cell Signaling, Beverly, USA), and GAPDH (sc-25778; Santa Cruz Biotechnology). Protein preparation from soleus muscle was performed as described [16]. Supernatants were collected and protein concentration was determined using a Direct DetectTM spectrometer (Millipore, Billerica, USA). Total protein (* 20 lg) was separated on precast 4–12 % Bis–Tris gels (Invitrogen, Life Technologies) or 12 % precast polyacrylamide gel (TGXTM Gel, Bio-Rad Laboratories AB, Solna, Sweden) and transferred onto PVDF membranes using the iBlotTM dry blotting system (Invitrogen) or Trans-BlotÒ TurboTM Transfer System (Bio-Rad). Membranes were first blocked in 5 % nonfat dry milk or 5 9 animal-free blocker (Vector Laboratories, Burlingame, USA) and then incubated with primary antibody. The blots were then washed and incubated with secondary antibody. Protein bands were developed with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and photographed with a ChemiDoc MP imaging system (Bio-Rad). GAPDH was used as a loading control, and normalization was performed with the Image Lab software (Bio-Rad). Values are expressed in arbitrary densitometric units of relative abundance in relation to the mean of the unstimulated controls. Statistical analyses Values are reported as the mean ± SEM, and P B 0.05 was considered significant. Statistical analyses were performed with SPSS software (version 21.0; SPSS, Inc., Chicago, IL). Differences between controls and DHT-induced PCOS rats for mean GIR. The final GIR value during the steady state

was set to 1 to calculate the fold changes during treatment and the post-treatment period. Group differences in fold change in GIR during and after stimulation were analyzed by repeated measures ANOVA with the Bonferroni post hoc test. The trapezoidal rule was used to calculate the area under the curve (AUC) at steady state, during stimulation, and post-stimulation. The AUC for the steady state period was calculated on GIR during the last 30 min before the start of treatment. The treatment period AUC was based on GIR measured during the 45 min of treatment, and the AUC for the post-stimulation period was calculated on GIR measured for 45 min. AUC was expressed as AUC/min. Changes in the AUC/min from steady state to stimulation and from steady state to post-stimulation were calculated, and the differences between stimulation and groups (controls vs. PCOS) were analyzed by two-way ANOVA with the Tukey post hoc test. Gene expression was not normally distributed, so we performed a Kruskal–Wallis test followed by the Mann–Whitney U test. Protein expression was normally distributed and was analyzed with one-way ANOVA followed by Dunnett’s post hoc test.

Results DHT-induced PCOS versus controls The mean GIR at steady state was lower in DHT-induced PCOS rats than in controls (Fig. 2), demonstrating decreased systemic insulin sensitivity. 25

Glucose infusion rate (mg min-1 kg-1)

Undet. undetermined

20

***

15 10 5

n = 33

n = 47

Control

PCOS

0

Fig. 2 Mean GIR at steady state. Values are mean ± SEM. Statistical significance is according to Student’s t test; ***P \ 0.001 versus controls

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Effect of manual and electrical stimulations on insulin sensitivity

a greater fold change in GIR during and after stimulation compared with no-stimulation in both control (Fig. 3e) and PCOS rats (Fig. 3f). Further, electrical stimulation had a higher fold change in GIR during stimulation than manual in both control (Fig. 3e) and PCOS rats (Fig. 3f). However, after stimulation, there was no significant difference in GIR between electrical and manual stimulations in control (Fig. 3e) and PCOS rats (Fig. 3f).

GIR in the no-stimulation groups did not change during the clamp (Fig. 3a, b), suggesting that the anesthesia per se had a minimal effect on GIR over time. Manual stimulation tended to increase GIR during and after stimulation compared with no-stimulation in control and PCOS rats (Fig. 3c, d). Electrical stimulation resulted in

b

Controls

-35 -30 -25 -20-15-10 -5 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 min Steady state No-stimulation Post-stimulation

-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 min Steady state No-stimulation Post-stimulation

d (P = 0.567 vs no stimulation)

(P = 0.089 vs no stimulation)

1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7

Steady state

Post-stimulation

f

1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7

P = 0.001 vs no stimulation P = 0.001 vs no stimulation P = 0.021 vs manual stimulation (P = 0.187 vs manual stimulation)

GIR (fold change)

GIR (fold change)

e

Manual stimulation

1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7

5 4 3

*** +++ *** +++

Control during stimulation PCOS during stimulation Control post -stimulation PCOS post-stimulation Group effect P > 0.05 Treatment effect P < 0.001

*** ***

GIR ( AUC/min)

6

P = 0.003 v vs no stimulation P < 0.001 vvs no stimulation vs manual stim i ulat l ion) P < 0.001 vs v manual stimulation ((P = 0.267 v

**

7

Post-stimulation

**

8

Electrical stimulation

-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 min Electrical stimulation Steady state Post-stimulation

-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 min Steady state Post-stimulation Electrical stimulation

g

(P = 0.078 vs no stimulation)

-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 min

-35-30-25-20-15-10 -5 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 Min Steady state

(P = 0.124 vs no stimulation)

***

1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7

GIR (fold change)

GIR (fold change)

c

DHT-induced PCOS

1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7

***

1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7

GIR (fold change)

GIR (fold change)

a

2 1 0 -1

No stimulation

Manual stimulation

Fig. 3 Fold change in GIR at steady state, during stimulation and after stimulation in controls and PCOS rats with a and b nostimulation, c and d manual stimulation, e and f electrical stimulation. g Differences in the area under the curve (DAUC/min) in controls and PCOS rats. Values are expressed as the mean ± SEM. The

123

Electrical stimulation

differences in fold changes in GIR were determined with repeated measurement ANOVA with Bonferroni post hoc test. Changes in the AUC/min were analyzed by two-way ANOVA with the Tukey post hoc test. **P \ 0.01; ***P \ 0.001 versus no-stimulation; ???P \ 0.001 versus manual stimulation

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When GIR was calculated as changes in AUC/min (DAUC/min) and analyzed by two-way ANOVA taking both type of stimulation and group into consideration, electrical stimulation had higher DAUC/min during and after stimulation compared with no-stimulation, with no difference between control and PCOS rats (Fig. 3g). The DAUC/min was higher during and after manual stimulation compared with no-stimulation, with no difference between control and PCOS rats (Fig. 3g). Electrical stimulation had higher DAUC/min during stimulation compared with manual, but there was no difference in DAUC/min between stimulations in the post-stimulation period (Fig. 3g). Effect of manual and electrical stimulations on gene expression The mRNA expression of 43 target genes related to IR, mitochondrial function, steroidogenesis, and sympathetic nerve activity was measured in the soleus muscle, mesenteric adipose tissue, and the ovary (Table 1). In the soleus muscle, the mRNA expression of Adipor2, Adrb1, Fndc5, Erk2, and Tfam was higher in PCOS rats than in controls (Table 2). In PCOS rats, the mRNA expression of Adipor2, Adrb1, Fndc5, Erk2, Ngf, and Tfam was lower in the electrical stimulation group than in the nostimulation group (Table 2). The manual stimulation group had higher expression of Ngf and Pten than the no-stimulation group (Table 2). In the mesenteric adipose tissue of PCOS rats, the mRNA expression of Pik3cb was higher in the electrical stimulation group than in the no-stimulation group (Table 2). In the ovary, the mRNA expression of Adrb2 and Pdk4 was lower and the mRNA expression of Erk2 and Sdhd was Table 2 Relative gene expression in soleus muscle, mesenteric adipose tissue depot, and the ovary

Presented values are 2 DDCt (mean ± SEM) relative to the control group NS no-stimulation, MS manual stimulation, ES electrical stimulation P values were determined with the Kruskal–Wallis test followed by the Mann–Whitney U test.   P \ 0.05,    P \ 0.01 versus Control NS. * P \ 0.05, ** P \ 0.01, *** P \ 0.001 versus PCOS NS

Gene

Control NS

higher in PCOS rats than in controls (Table 2). In PCOS rats, the expression of Adrb2 and Pdk4 was higher after electrical stimulation than in the no-stimulation group, and expression of Erk2 and Sdhd was lower in the manual stimulation group compared with the no-stimulation group (Table 2). Effect of manual and electrical stimulations on muscle protein expression As skeletal muscle is the major regulator of glucose homeostasis, we determined whether changes in gene expression in the soleus muscle were associated with changes in protein expression. The analysis of protein expression was limited to ADIPOR2 and pERK1/2 in the soleus muscle. ADIPOR2 levels were significantly altered based on one-way ANOVA (P = 0.005). ADIPOR2 was significantly increased in the soleus muscle after manual stimulation compared with no-stimulation in PCOS rats and tended to be higher after electrical stimulation (P = 0.086) (Fig. 4a). Although not significant using one-way ANOVA (P = 0.09), pERK1/2 was significantly increased after electrical stimulation compared with no-stimulation in PCOS rats with Dunnett’s post hoc test (Fig. 4b). Manual stimulation of the acupuncture needles did not affect pERK1/2 in PCOS rats (P = 0.81), which is in line with the gene expression data.

Discussion This is the first study demonstrating that one treatment of acupuncture with electrical stimulation is a stronger PCOS NS

PCOS MS

PCOS ES

Soleus muscle Adipor2

1.00 ± 0.08

1.39 ± 0.07 

1.23 ± 0.11

0.99 ± 0.05***

Adrb1

1.00 ± 0.18

1.80 ± 0.23 

1.27 ± 0.12

1.04 ± 0.16*

Fndc5

1.00 ± 0.08

1.30 ± 0.06 

1.47 ± 0.15

1.08 ± 0.03*

Erk2

1.00 ± 0.13

1.48 ± 0.08 

1.32 ± 0.09

1.09 ± 0.11**

Ngf

1.00 ± 0.08

1.18 ± 0.10

1.86 ± 0.23*

1.52 ± 0.14*

Pten

1.00 ± 0.08

0.96 ± 0.07

1.23 ± 0.10*

0.94 ± 0.03

1.00 ± 0.08

1.40 ± 0.10 

1.38 ± 0.07

1.15 ± 0.06*

1.00 ± 0.14

1.15 ± 0.43

1.30 ± 0.46

1.53 ± 0.17*

Tfam Mesenteric fat Pik3cb Ovary Adrb2

1.00 ± 0.10

0.62 ± 0.06  

0.86 ± 3.20

1.01 ± 0.09**

Erk2

1.00 ± 0.07

1.50 ± 0.20 

1.01 ± 0.05*

1.36 ± 0.15

Pdk4

1.00 ± 0.26

0.30 ± 0.08  

0.49 ± 0.12

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inducer of GIR increase than with manual stimulation in both controls and insulin-resistant PCOS rats. Although electrical stimulation is superior to manual stimulation in enhancing insulin sensitivity during stimulation, they are equally effective post-stimulation. Acupuncture with low-frequency electrical stimulation and repetitive muscle contraction activates physiological processes similar to those resulting from physical exercise [23–26]. Acute muscle contraction is a potent stimulator of glucose transport even in states of severe IR [23, 24]. Like exercise, electrical acupuncture stimulation has been shown to stimulate glucose transport in skeletal muscle and to increase the insulin sensitivity of glucose transport in rats [18, 20, 25, 26]. Furthermore, both long-term exercise training and acupuncture with low-frequency electrical stimulation lead to increased insulin sensitivity and GLUT4 protein expression in rats [10, 16, 17, 27]. The acute effects of electrical stimulation in male rat models of IR/T2D include improved glucose tolerance and insulin sensitivity as well as increased GLUT4 levels [18, 20, 26]. Acupuncture with low-frequency electrical stimulation lowers plasma glucose and increases plasma insulin in normal and T2D rats but not in T1D rats and can therefore be considered insulin dependent [21, 26]. Furthermore, electrical stimulation increases the response of plasma glucose to exogenous insulin in both T1D and T2D rats [18, 21]. However, there are no previous data elucidating whether manual stimulation could influence insulin glucose homeostasis in the same manner as electrical stimulation.

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Fig. 4 Protein content of a ADIPOR2 and b phosphorylated ERK1/2 (Tyr202/Tyr 204) in controls and PCOS rat soleus muscle as determined by Western blot. Top representative immunoblots from independent samples are shown. Bottom densitometric analysis of protein expression. Statistics were analyzed with one-way ANOVA with Dunnet’s post hoc test. Values are mean ± SEM. NS nostimulation, MS manual stimulation, ES electrical stimulation

Acta Diabetol (2014) 51:963–972

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Here, we demonstrate that a single session of acupuncture with either manual or electrical stimulation increases GIR in an insulin-resistant rat model of PCOS and in female controls. Both manual and electrical stimulations of acupuncture needles activate sensory afferents (A-delta fibers and C-fibers) [13, 21] with the main difference being that low-frequency electrical stimulation also induces muscle contractions. The novel finding in the present study is that we have been able to separate the contraction-induced increase in glucose uptake and changes in gene expression from the effects induced by needle stimulation of the afferent nerves. During treatment, electrical stimulation of the needles was superior to manual stimulation in increasing GIR, and we propose that the difference is due to insulinindependent contraction-induced glucose uptake. Poststimulation, there was no difference between electrical and manual stimulations, and we propose that this is related to an insulin-dependent glucose uptake mediated by activation of somatic afferents. Muscle tissue was strongly affected by manual and electric stimulations, and we found the regulation of seven genes to be significantly altered in soleus muscle compared to only one gene in mesenteric fat. Electrical stimulation altered more genes than manual stimulation in skeletal muscle (six vs. two) suggesting that it is a more potent stimulus. Erk2 gene expression was significantly upregulated in both muscle and ovary tissues in PCOS rats compared with controls, and it was downregulated in soleus muscle after

Acta Diabetol (2014) 51:963–972

electrical stimulation and in the ovary after manual stimulation. As skeletal muscle is the main regulator of wholebody glucose uptake, protein levels were measured in the soleus muscle. The downstream signaling pathway of ERK1/2 activation involves increased serine phosphorylation of insulin receptor substrate (IRS)-1. This results in a subsequent decrease in tyrosine phosphorylation of IRS-1 and inhibition of phosphoinositide 3-kinase, a critical step in GLUT4 translocation [28]. We here show that insulin stimulation by hyperinsulinemic clamp does not alter pERK1/2 levels in PCOS rats. We did find increased levels of pERK1/2 in PCOS rats receiving acupuncture with electrical stimulation, and this is in line with the fact that ERK1/2 phosphorylation is a muscle contraction-induced metabolic regulator that increases after exercise and electrical stimulation [29, 30]. As pERK1/2 levels were unchanged by manual stimulation of the needles, we propose that this effect is mediated by muscle contractions. However, this does not explain the insulin sensitizing effect of acupuncture because ERK1/2 activation might actually contribute to resistance against insulin’s metabolic effects. Adiponectin is an adipose tissue-derived hormone that plays a pivotal role in the regulation of glucose and lipid metabolism. A recent meta-analysis confirmed that circulating adiponectin levels are decreased in women with PCOS [31] and in DHT-induced PCOS mice [32]. AdipoR1 and AdipoR2 serve as receptors for adiponectin and mediate an increase in the activity of AMP-activated protein kinases and peroxisome proliferator-activated receptors. Although acute exercise does not cause major changes in plasma adiponectin concentrations [33, 34], the mRNA expression of AdipoR1 increase and AdipoR2 decrease in rat skeletal muscle after exercise [33]. AdipoR2 expression in muscle was increased in PCOS rats compared with controls, and electrical stimulation decreased AdipoR2 expression in a manner similar to what is previously observed in response to exercise [33]. Protein levels of ADIPOR2 were unchanged between PCOS and control rats without stimulation, but the levels increased in PCOS rats after acupuncture with both manual and electrical stimulations. These findings suggest that acupuncture affects the expression of adiponectin receptors, possibly leading to improved insulin sensitivity by regulating adiponectin action in the muscle. Interestingly, PCOS rats had decreased ovarian Adrb2 gene expression compared with controls that underwent the clamp during estrus, and this decrease was normalized to controls after electrical stimulation. Adrb2 gene expression in skeletal muscle was not significantly different, but tended to be higher, in PCOS rats compared with controls (P = 0.12) while Adrb1 expression was increased. This is in line with data from women with PCOS showing an increase in sympathetic nerve activity [35].

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This paper had two major findings. The first was that acupuncture with electrical stimulation is a stronger stimulus for glucose uptake than manual stimulation and that it induces more pronounced changes in molecular pathways and improves insulin sensitivity more rapidly. The second was that both electrical and manual stimulations are equally effective during the post-stimulation period. This suggests that it is the stimulation of sensory afferents rather than the muscle contractions per se that lead to improved insulin sensitivity. Our data also suggest that acupuncture with manual and electrical stimulations mediates their effects on glucose uptake via partly different signaling pathways— including activation of afferent nerves and contractioninduced uptake—but such findings require further investigation before any firm conclusions can be drawn. The ultimate goal is to translate these findings into the clinical situation. Acknowledgments We thank the Genomics Core Facility at the Sahlgrenska Academy, University of Gothenburg, for the use of equipment and support. The genomics facility was funded by a grant from the Knut and Alice Wallenberg Foundation. This work was supported by the Swedish Medical Research Council (Project No. 2008-72VP-15445-01A and K2012-55X-15276-08-3); Wilhelm and Martina Lundgren’s Science Fund; the Hjalmar Svensson Foundation; ˚ ke Wiberg Foundation the Adlerbert Research Foundation; the A (Project No. 226441413); The Royal Society of Arts and Sciences in Gothenburg; and the Swedish federal government under the LUA/ ALF agreement ALFFGBG-136481. Conflict of interest Anna Benrick, Manuel Maliqueo, Julia Johansson, Miao Sun, Xiaoke Wu, Louise Mannera˚s-Holm, and Elisabet Stener-Victorin declare that they have no conflict of interest. Human and animal rights disclosure This article does not contain any studies with human or animal subjects performed by the any of the authors. Statement of informed consent We would like to mention that there are no patients in this study.

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Enhanced insulin sensitivity and acute regulation of metabolic genes and signaling pathways after a single electrical or manual acupuncture session in female insulin-resistant rats.

To compare the effect of a single session of acupuncture with either low-frequency electrical or manual stimulation on insulin sensitivity and molecul...
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