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Acupuncture attenuates hyperglycaemia and improves ovarian function in female rats subjected to continuous light exposure
  1. Xuezhi Kang1,
  2. Lina Jia2,
  3. Yaming Li2,
  4. Xu Zhang2
  1. 1 Acupuncture Moxibustion and Tuina Traumatology of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China
  2. 2 Department of Traditional Chinese Medicine, Huadong Hospital Affiliated to Fudan University, Shanghai, China
  1. Correspondence to Dr Lina Jia, Department of Traditional Chinese Medicine, Huadong Hospital Affiliated to Fudan University, No. 221 West YanAn Road, Shanghai, China; jia_ln{at}163.com

Abstract

Objective Exposure to unnatural light cycles is increasingly associated with obesity and the metabolic syndrome. The purpose of this study was to examine the effects of electroacupuncture (EA) on glucose metabolism and ovarian function in female rats subjected to long-term continuous light exposure.

Methods Female Sprague-Dawley rats (n=24) were divided into three experimental groups: an LD group that was maintained under a normal light-dark cycle (healthy control); an LL group that was exposed to continuous light for 21 weeks but remained untreated; and an LL+EA group that received EA at ST36 and SP6 during weeks 17 to 21 of continuous light exposure.

Results Oestrous cycles of female rats kept in a continuously lit environment for 21 weeks were disordered and polycystic ovarian syndrome (PCOS)-like changes occurred, accompanied by increased fasting blood glucose (6.23±0.33 vs 5.27±0.40 mmol/L in week 17, p=0.015) and reduced fasting levels of serum testosterone (0.07±0.018 vs 0.12±0.058 ng/L, p=0.043) and insulin (0.89±0.20 vs 1.43±0.46 ng/L, p=0.006). After 5 weeks of EA treatment at ST36 and SP6, ovarian cycle disruption was mitigated and blood glucose levels showed a gradual decline (5.18±0.37 vs 5.80±0.55 mmol/L, p=0.017; and 5.73±0.31 vs 6.62±0.13 mmol/L, p=0.004; in the fourth and fifth weeks of EA treatment, respectively). EA also attenuated the reductions otherwise seen in serum insulin and testosterone levels.

Conclusion Prolonged exposure to light can lead to a decline in ovarian and pancreatic function. EA at ST36 and SP6 may reduce abnormally elevated blood glucose levels and improve ovarian and pancreatic hormone levels.

  • Electro acupuncture
  • Continuous light exposure
  • Blood glucoses
  • Testosterone
  • Fasting insulin

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Introduction

Human circadian rhythms are synchronised to light/dark cycles. Over time, organisms have adapted to diurnal variations in their physiology and metabolism. These rhythms are regulated by molecular circadian clocks. However, in recent years, humans have shifted away from the naturally occurring solar light cycle in favour of artificial, irregular light schedules produced by electrical lighting. Disruption of the core clock genes secondary to interference with these environmental considerations may lead to an increased risk of cardiometabolic disease.1 2 Genetic disruption of clock genes in mice induces metabolic dysfunction at distinct phases of the sleep/wake cycle.3 Constant exposure to light induces alterations in melatonin levels, food intake, visceral adiposity and circadian rhythms in rats.4 Exposure of adult female rats to continuous light leads to the gradual development of chronic anovulation.5 6 However, it remains unknown how continuous light exposure affects glucose levels in female rats.

As contemporary exposure to artificial light environments is unlikely to change, there may be a potential role for complementary and alternative medicine (CAM) in allowing humans to enjoy the convenience of artificially lit environments, while reducing the potential side effects of light exposure to a minimum. Acupuncture is now widely practised in both East Asian and Western countries as a method of pain relief and as a treatment for a variety of other disorders.7–10 As an important CAM modality, there is evidence to suggest that acupuncture is safe and effective for appetite control,11 weight loss12 and sleep regulation. Furthermore, acupuncture has been reported to have beneficial effects on glucose metabolism and insulin signalling in obese women.13

Many studies have shown that circadian misalignment increases insulin resistance and decreases pancreatic function.14 We have found in previous research that high testosterone levels, similar to those observed in women with polycystic ovarian syndrome (PCOS), have been shown in female rats after 16 weeks of continuous light exposure.6 It is possible that longer durations of light exposure in rats may adversely impact endocrine function and metabolism even further. The aim of this study was to observe the effect of long-term continuous light exposure for 21 weeks on serum concentrations of glucose in rats and to determine if acupuncture has any beneficial effects.

Methods

Experimental animals

This study was approved by the ethics committee of Huadong Hospital, which is affiliated with Fudan University (reference no. #20140306001). The primary outcome measure for this study was the fasting blood glucose level; the secondary outcome measures included serum testosterone, oestradiol and insulin levels. Assuming a moderate effect size at an α level of 0.05% and 80% power, it was estimated that eight animals per group would be required to detect statistically significant differences in the primary outcome.15 Twenty-four 6-week-old female Sprague-Dawley rats were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (SEACCAS; China) and housed at 22–25°C in a customised experimental box6 (Chinese patent no. ZL201420754611.0). Animals were randomly and evenly assigned to one of the following three exposure groups (n=8 each): (1) a control (circadian rhythm) group maintained under a 12/12 hour light/dark cycle with lights on at 08:00 Beijing standard time (LD group) for 21 weeks; (2) a model group exposed to light continuously for 24 hours a day (LL group) for 21 weeks; and (3) a treatment group that received electroacupuncture (EA) therapy for the final 5 weeks of continuous light exposure for 24 hours a day for 21 weeks (LL+EA group). All rats were allowed free access to water and a standard rat diet (prepared by SEACCAS).

Electroacupuncture and fasting blood glucose testing cycle

Rats in the LL+EA group received EA therapy alongside continuous light exposure for the final 5 weeks of the 21-week study period. During the preparatory phase of the experiment, all three groups of rats received 16 weeks of environmental light exposure only (for 12 hours per day in the LD group and 24 hours per day in the LL and LL+EA groups, respectively). EA therapy and blood glucose measurement started from the 17th week onwards and lasted for 5 weeks in total. In the LL+EA group only, EA was given to conscious rats three times a week. Acupuncture points were located according to body-length measurement, as described.16 Sterile, stainless steel needles (0.25 mm in diameter, 30 mm in length, Hwato brand, China) were inserted at locations equivalent to the human acupuncture points ST36 (Zusanli) and SP6 (Sanyinjiao) and advanced to a depth of 3–5 mm. Electrical stimulation was applied using an EA unit (model no. G6805-2, Shanghai Medical Instrument High-tech Co, China) and oscilloscope (model no. XJ4210A; Shanghai Xinjian Instrument and Equipment Co, Ltd, China) at 2 Hz frequency, 3 mA intensity and pulse width 0.2 ms±30%. Rats in the LL+EA group were treated from the 17th to 21 st week alongside the ongoing continuous lighting exposure. In addition, from the 17th week of the experiment, body weight was measured weekly and blood was collected from the caudal vein of all rats at the same time of day and week for the measurement of fasting blood glucose using an ACCU-CHEK glucometer and test paper (Roche Corporation, Ireland). The timing of fasting blood glucose measurement, EA therapy and overall experimental design are shown in figure 1.

Figure 1

Schematic diagram of the experimental design depicting the timing of electroacupuncture (EA) therapy and blood glucose measurement. LD, 12/12 hour light/dark cycle; LL, continuous light exposure. 

Vaginal smears

Vaginal smears were performed on all rats daily and their oestrous cycles were observed at the end of the 21st week of the experiment. A sterile cotton swab was soaked in 0.9% saline and used to sample the lower third of the vaginal wall before being removed and smeared in one direction across a glass slide. The cells were evaluated under light microscopy by investigators kept blind to group allocation, and samples were classified as reflecting one of the following stages of the oestrous cycle: di-oestrus; pro-oestrus; oestrus; or met-oestrus. Any rat that failed to show regularity of the four stages of oestrus was considered to have an indiscriminate oestrous cycle and the proportions of such rats per group were compared at the end of the study.

ELISA

At the end of the 21st week, all rats were terminally anaesthetised with an intraperitoneal injection of 10% chloral hydrate solution (4 mL/kg) and decapitated. Blood samples were obtained from the inferior vena cava and transferred into Vacutainer tubes containing coagulant (coated with frosting and copolymer of α olefins and maleic acid). The serum samples were separated by centrifugation at 2000 g for 10 min and the resultant clear supernatants were kept at −80°C for subsequent ELISA determination of testosterone and oestradiol (catalogue no. 582701 and 582251, respectively, Cayman Chemical, Ann Arbor, MI, USA) and insulin (catalogue no. EZRMI-13K, Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Laboratory workers performing the ELISAs were blinded to treatment allocation during the analysis.

Histology

The uteri and ovaries of all rats were harvested after euthanasia and weighed using a precision balance (Sartorius, BT 125D, Germany). Thereafter, ovarian tissue was sampled, fixed with 4% formaldehyde buffer, embedded in paraffin, sectioned at 4 µm/slide, and stained with haematoxylin-eosin (HE). Sections were photographed under a Nikon microscope (CLIPSE Ti-S, Japan) at 40×, 100×, 200× and 400× magnifications, and examined for the presence of cystic follicles, which were defined according to previously described criteria.17

Data analysis

All data are expressed as mean±SE. After first confirming normality of distribution and homogeneity of variance, serial measurements of body weight and blood glucose were analysed by repeated measurement analysis of variance (ANOVA) followed by Fisher’s test of least significance difference. Group comparisons for ovarian pathology and vaginal smear were made using Fisher’s exact test, and one-way ANOVA was used to compare ovarian weight and levels of testosterone, insulin and oestradiol data between the three groups. Statistical significance was defined as p<0.05 or p<0.01. Data analysis was performed in Origin 8.0 software (OriginLab Corp, Northampton, MA, USA).

Results

Changes in body weight

Figure 2 shows the serial measurements of body weight between weeks 16 and 21 by group. Repeated measures ANOVA revealed a significant difference in body weight between the three groups (p=0.011). Rats in the LL group were significantly lighter than those in the LD group (p<0.01) and demonstrated a reduced ability to gain weight between weeks 17 and 21 while under continuous light exposure. Despite 5 weeks of EA treatment, rats in the LL+EA group similarly weighed significantly less than those in the LD group from week 17 to 21 (p<0.05), and there were no statistically significant differences between the LL and LL+EA groups at any time point.

Figure 2

Serial measurements of body weight (top panel) and blood glucose (bottom panel) in 24 rats kept under a 12/12 hour light/dark cycle (LD group, n=8) or subjected to continuous light exposure (n=16) and left untreated (LL group, n=8) or treated with 5 weeks of electroacupuncture (LL+EA group, n=8). *p<0.05 versus LD group; **p<0.01 versus LD group; #p<0.05 versus LL group.

Changes in fasting blood glucose

Before the start of EA treatment in the 17th week, compared with the LD group, levels of blood glucose were higher in the LL group (6.23±0.33 vs.  5.27±0.40 mmol/L, p=0.015) and the LL+EA group (p=0.001). During the course of the EA treatment, the elevated blood sugar levels in the LL+EA group were noted to drop gradually. Although no statistically significant reduction in blood glucose levels was observed in the LL+EA group during the first 3 weeks of EA therapy, by the last 2 weeks of treatment blood glucose determinations in the LL+EA group had fallen to levels that were significantly lower than the (untreated) LL group (fourth week: 5.18±0.37 mmol/L vs 5.80±0.55 mmol/L, p=0.017; fifth week: 5.73±0.31 mmol/L vs 6.62±0.13 mmol/L, p=0.004). In addition, we found that blood glucose levels remained significantly higher in the LL group than the LD group throughout the experimental process (p<0.05).

Changes in ovarian/uterine weight and fasting insulin/testosterone levels

As shown in figure 3A, both left and right ovarian weights in the LL group were similar to the LD group. Moreover, ovarian weights in the LL+EA group were similar to LD and LL groups (p=0.420 and p=0.578). Uterine weights in all three groups were similar (p=0.420; figure 3B).

Figure 3

Ovarian (A) and uterine (B) weight, and serum concentrations of insulin (C), oestradiol (D) and testosterone (E) in 24 rats kept under a 12/12 hour light/dark cycle (LD group, n=8) or subjected to continuous light exposure (n=16) and left untreated (LL group, n=8) or treated with 5 weeks of electroacupuncture (LL+EA group, n=8). #p<0.05 versus LL group; **p<0.01 versus LD group. LOV, left ovary; ROV, right ovary.

Figure 3C–E shows the differences in fasting levels of insulin, oestradiol and testosterone, respectively, at the end of the 21st week between the three groups. Fasting insulin and testosterone levels in the LL group were both significantly decreased compared with the LD group (insulin: 0.89±0.20 ng/L vs 1.43±0.46 ng/L, p=0.006; testosterone: 0.07±0.018 ng/L vs 0.12±0.058 ng/L, p=0.043). EA treatment appeared to have an effect on both parameters, as fasting serum levels of insulin and testosterone in the LL+EA group were significantly increased relative to the (untreated) LL group (p=0.045 and p=0.044, respectively). However, serum oestradiol did not significantly differ between the three groups (p>0.05; figure 3D).

Ovarian cycle changes

Figure 4 shows representative image sets from the three experimental groups demonstrating oestrous cyclicity, as analysed at the end of the 21st week using daily vaginal smears. Rats in the LD group all exhibited disciplinary transmutation illustrated by the sequence of di-oestrus, pro-oestrus, oestrus and met-oestrus (figure 4A). By contrast, rats in the (untreated) LL group frequently displayed an indiscriminate oestrous cycle, which arrested at the pro-oestrus stage for all 4 days (figure 4B). The proportion of rats exhibiting an indiscriminate oestrous cycle was markedly greater in the LL group versus the LD group (7 of 8 (88%) vs 0 of 8 (0%), p<0.001). Furthermore, fewer rats in the LL+EA group (figure 4C) had an indiscriminate oestrous cycle compared with the LL group (3 of 8 (38%) vs 7 of 8 (88%), p=0.039).

Figure 4

Representative photographs of vaginal smears obtained on 4 consecutive days (a, b, c and d, respectively) in 24 rats kept under a 12/12 hour light/dark cycle (LD group, n=8) or subjected to continuous light exposure (n=16) and left untreated (LL group, n=8) or treated with 5 weeks of electroacupuncture (LL+EA group, n=8). (A) Disciplinary transmutation demonstrated by sequence of pro-oestrus (a), oestrus (b), met-oestrus (c) and dioestrus (d). (B) Indiscriminate oestrous cycle, arrested at pro-oestrus stage for all 4 days. (C) Indiscriminate oestrous cycle demonstrating recovered sequence of oestrus (a), met-oestrus (b), di-oestrus (c) and pro-oestrus (d).

Ovarian histopathological changes

Figure 5 shows representative photographs of histological ovarian tissue sections stained with HE under 40× (a), 100× (b), 200× (c) and 400× (d) magnifications in LD (figure 5A), LL (figure 5B) and LL+EA (figure 5C) groups. Histopathological examination of ovarian tissue samples from the LL and LL+EA group showed polycystic ovarian tissue formation as previously described,6 while the LD group consistently demonstrated normal ovarian tissue histology. The proportions of rats with cystic follicles, defined according to previously described criteria,17 were comparable in the LL and LL+EA groups (7 of 8 (88%) vs 6 of 8 (75%), respectively, p=0.522).

Figure 5

Histological ovarian tissue sections stained with haematoxylin and eosin and photographed under 40× (a), 100× (b), 200× ( c ) and 400× (d) magnifications in 24 rats kept under a 12/12 hour light/dark cycle (LD group, n=8) or subjected to continuous light exposure (n=16) and left untreated (LL group, n=8) or treated with 5 weeks of electroacupuncture (LL+EA group, n=8). (A) Light microscopic analysis shows follicles and corpora lutea in different stages of development and regression, with no structural abnormalities and no ovarian cysts. Theca and granulosa cell layers appear normal. (B) Increased number of follicles, thickened theca cell layer and diminished granulosa cell layer. (C) Increased number of follicles.

Discussion

Prolonged light exposure disrupts ovarian cycles and glucose metabolism in rats

Our previous studies showed that 16 weeks of constant light exposure induces hyperandrogenism and multiple follicular cysts in the ovary in rats.6 We believe that this rodent model can be applied to the study of PCOS. In the present study, illumination time was prolonged from 16 weeks to 21 weeks and the impact of acupuncture on physiological adaptation to these adverse environmental conditions was additionally investigated. Observation using vaginal smears showed that continuous light exposure for 21 (as opposed to 16) weeks still led to disruption of the ovarian cycle in female rats. Interestingly, however, instead of being elevated, androgen levels were significantly lower in untreated LL versus LD groups. With the extended duration of continuous light exposure, it appears that ovarian hypertrophy and hyperandrogenism may have evolved into a state of reduced ovarian volume with low ovarian hormone levels.

In the face of an apparent decline in endocrine function, we hypothesise that sustained light exposure not only induces endocrine dysfunction by way of a circadian rhythm disorder, but also leads to accelerated ageing and degradation. Disruption of circadian rhythms by environmental manipulation or mutations in specific clock genes leads to various age-related pathologies and may reduce lifespan in mice.18 Interruption of androgen receptor effects in granulosa cells can also lead to various infertility-associated conditions19 and low functional ovarian reserve may represent a state of androgen deficiency.20 Androgen deficiency, characterised by low total testosterone levels, is now widely considered to be a precursor condition of premature ovarian ageing.21 From this perspective, the apparent change from a high testosterone state to a low testosterone state with the extended illumination time may reflect premature ovarian failure in the rats, although this was not specifically considered in the design of the present study. Prolonged light exposure can impair the normal function of the endocrine pancreas, which is reflected by high blood glucose and low insulin levels. In experimental diabetes, testosterone accelerates hyperglycaemic decompensation, in which β-cell destruction is induced by oxidative stress or inflammation.22 23 Therefore, inflammation and other mechanisms may be responsible for the observed elevation (at 16 weeks) and reduction (at 21 weeks) in androgen levels.

EA simultaneously improved ovarian cycle and glucose metabolism

In China, acupuncture is widely used to treat irregular menstruation24 and needling at ST36 is classically indicated for resistance against ageing.25 SP6, which is located at a level four finger breadths above the ankle joint on the medial aspect of the lower leg, is commonly stimulated using either acupuncture or acupressure in the setting of gynaecological disorders.26 27 Indeed SP6 acupressure has been considered as a self-management approach to improve women’s general health.28 EA at SP6 has been shown to improve oocyte quality and pregnancy outcome in patients with PCOS,29 and can generate a significant analgesic effect in primary dysmenorrhoea.30 Our experimental results also show that acupuncture (at SP6 and ST36) can significantly improve disorders of the ovarian cycle induced by continuous light exposure. There were no significant changes in histopathological parameters and ovarian weight, which may be related to the length and timing of onset of the acupuncture treatment.

EA treatment has also been shown to have a therapeutic effect on insulin resistance in diabetes mellitus.1 2 EA at ST36, when applied at 15 Hz frequency and 10 mA intensity for 30 min, has been found to lower blood glucose levels.31 EA at bilateral ST36 may improve glucose tolerance by stimulating cholinergic nerves and nitric oxide synthase, and lowering plasma levels of free fatty acids.32 The glucose-lowering effect of EA may also be related to upregulation of obestatin expression in the hypothalamus,33 activation of GLUT4 via upregulation of MAPK expression34 and modulation of cell adhesion molecules.35 The present experiment suggests that changes in blood sugar level may be a gradual process, as 3 weeks of treatment were needed before a significant difference was observed. However, ultimately the effect of acupuncture is likely to depend on the extent to which metabolism is disordered in the particular animal model.

EA at SP6 or ST36 has been shown to normalise insulin sensitivity and ameliorate insulin resistance and hyperinsulinaemia in PCOS rats, probably by regulating the function of β cells in the pancreatic islets and by reducing oxidative stress and free androgen levels.36 EA may play a dual role in the regulation of sex hormones and metabolism. For example, acupuncture can reduce the burden of menopause-related symptoms,37 increase levels of oestradiol,38 treat premature ovarian failure39 and improve diminished ovarian reserve.40 These effects may be related to upregulation of gene and protein expression of components of the PI3K/Akt/mTOR signalling pathway.41 Our study suggests that acupuncture can improve ovarian hormone and oestrous cycle disorders, which is encouraging with respect to the potential role of acupuncture in the treatment of ovarian failure.

Conclusion

Prolonged exposure to light leads to a decline in ovarian and pancreatic function. EA at ST36 and SP6 may reduce abnormally elevated blood glucose levels and improve levels of ovarian and pancreatic hormones.

References

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Footnotes

  • Contributors KXZ and JLN performed all the experiments and wrote the submitted article. LYM and ZX supervised the experiments. All authors approved the final version accepted for publication.

  • Funding This work was supported by NSFC (Natural Science Foundation of China) grant no. 81001569 and 81102657.

  • Competing interests None declared.

  • Patient consent Patient consent detail has been removed from this case description/these case descriptions to ensure anonymity. The editors and reviewers have seen the detailed information available and are satisfied that the information backs up the case the authors are making.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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