Electrical vs manual acupuncture stimulation in a rat model of polycystic ovary syndrome: different effects on muscle and fat tissue insulin signaling

Abstract

In rats with dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), repeated low-frequency electrical stimulation of acupuncture needles restores whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamp. We hypothesized that electrical stimulation causing muscle contractions and manual stimulation causing needle sensation have different effects on insulin sensitivity and related signaling pathways in skeletal muscle and adipose tissue, with electrical stimulation being more effective in DHT-induced PCOS rats. From age 70 days, rats received manual or low-frequency electrical stimulation of needles in abdominal and hind limb muscle five times/wk for 4-5 wks; controls were handled but untreated rats. Low-frequency electrical stimulation modified gene expression (decreased Tbc1d1 in soleus, increased Nr4a3 in mesenteric fat) and protein expression (increased pAS160/AS160, Nr4a3 and decreased GLUT4) by western blot and increased GLUT4 expression by immunohistochemistry in soleus muscle; glucose clearance during oral glucose tolerance tests was unaffected. Manual stimulation led to faster glucose clearance and modified mainly gene expression in mesenteric adipose tissue (increased Nr4a3, Mapk3/Erk, Adcy3, Gsk3b), but not protein expression to the same extent; however, Nr4a3 was reduced in soleus muscle. The novel finding is that electrical and manual muscle stimulation affect glucose homeostasis in DHT-induced PCOS rats through different mechanisms. Repeated electrical stimulation regulated key functional molecular pathways important for insulin sensitivity in soleus muscle and mesenteric adipose tissue to a larger extent than manual stimulation. Manual stimulation improved whole-body glucose tolerance, an effect not observed after electrical stimulation, but did not affect molecular signaling pathways to the same extent as electrical stimulation. Although more functional signaling pathways related to insulin sensitivity were affected by electrical stimulation, our findings suggest that manual stimulation of acupuncture needles has a greater effect on glucose tolerance. The underlying mechanism of the differential effects of the intermittent manual and the continuous electrical stimulation remains to be elucidated.

Figure 1. Results of OGTT in rats with DHT-induced PCOS after 4–5 wks of treatment.

(A) Glucose concentration in plasma at 0, 15, 30, 60 and 120 min. (B, C) Slope of glucose clearance at 15 min (B) and 30 min (C). Values are mean ± SEM, *P<0.05 manual stimulation vs. controls, **P<0.01 manual stimulation vs. controls. ^# P<0.05 electrical vs. manual stimulation.

Figure 2. Glycogen content in rats with DHT-induced PCOS after 4–5 wks of treatment.

Glycogen content in (A) liver, (B) EDL, and (C) soleus determined with a colorimetric assay. Values are mean ± SEM. (*)P  = 0.074 (Mann-Whitney U test).

Figure 3. Protein levels in soleus muscle and mesenteric adipose tissue detected by western blot after 4–5 wks of treatment in rats with DHT-induced PCOS. GLUT4 (A, E), pAS160/AS160 ratio (B), AS160 (F), TBC1D1 (C, G), and Nr4a3 (D, H).

Representative immunoblots of each protein are shown. Values were normalized to β-actin and are expressed in arbitrary units (A.U.) (mean ± SEM). *P<0.05 vs. controls, **P<0.01 vs. controls, #P<0.05 vs. manual stimulation (Mann-Whitney U test).

Figure 4. Distribution and expression of GLUT4, determined by immunofluorescence staining, in soleus muscle and mesenteric adipose tissue of rats with DHT-induced PCOS.

No visual difference in immunoreactivity is observed in the mesenteric adipose tissue depot (A1–A3). In soleus muscle of control rats, GLUT4 is predominantly localized in the nucleus and cell membrane (C1). Both manual and electrical stimulation increased immunoreactivity in the nucleus, cell membrane, and cytosolic fraction (C2, C3). Staining was notably more intense after electrical stimulation than manual stimulation. Similar results were obtained when the staining was repeated in 3 rats/group for mesenteric adipose tissue and 4 rats/group for soleus muscle. The selected immunofluorescence images are representative of those in randomly selected section from multiple animals. B1–B3 and D1–D3: DAPI staining for nuclei in corresponding rat (A1–A3 and C1–C3). All photographs were taken with a ×20 objective.