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Expression levels of the hypothalamic AMPK gene determines the responsiveness of the rats to electroacupuncture-induced analgesia
© Kim et al.; licensee BioMed Central Ltd. 2014
Received: 3 February 2014
Accepted: 25 June 2014
Published: 30 June 2014
Although electroacupuncture (EA) relieves various types of pain, individual differences in the sensitivity to EA analgesia have been reported, causing experimental and clinical difficulties. Our functional genomic study using cDNA microarray identified that 5’-AMP-activated protein kinase (AMPK), a well-known factor in the regulation of energy homeostasis, is the most highly expressed gene in the hypothalamus of the rats that were sensitive to EA analgesia (“responder”), as compared to the rats that were insensitive to EA analgesia (“non-responder”). In this study, we investigated the causal relationship between the hypothalamic AMPK and the individual variation in EA analgesia.
Sprague-Dawley (SD) rats were divided into the responder and the non-responder groups, based on EA-induced analgesic effects in the tail flick latency (TFL) test, which measures the latency of the tail flick response elicited by radiant heat applied to the tail. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the expression levels of AMPK mRNA in the hypothalamus of the responder and non-responder rats. Further, we examined whether viral manipulation of the AMPK expression in the hypothalamus modulates EA analgesia in rats.
The real-time RT-PCR analysis showed that mRNA expression levels of AMPK in the hypothalamus of the responder rats are significantly higher than those of the non-responder rats, validating the previous microarray results. Microinjection of dominant negative (DN) AMPK adenovirus, which inhibits AMPK activity, into the rat hypothalamus significantly attenuates EA analgesia (p < 0.05), whereas wild type (WT) AMPK virus did not affect EA analgesia (p > 0.05).
The present results demonstrated that levels of AMPK gene expression in the rat hypothalamus determine the individual differences in the sensitivity to EA analgesia. Thus, our findings provide a clinically useful evidence for the application of acupuncture or EA for analgesia.
Acupuncture has been traditionally used for thousands of years in East Asia including China, Korea and Japan to relieve pain and is now viewed as an alternative method of medicine in Western countries [1, 2]. Electroacupuncture (EA) is a modified technique that utilizes electrical stimulation to enhance the analgesic effects of acupuncture [3, 4]. Previous studies have shown that acupuncture or EA stimulation at specific acupoints (e.g. ST36 and HI4) relieves various types of pain including acute thermal, inflammatory and chronic neuropathic pain, which were known to be mediated by activation of the descending pain inhibitory system [3, 5–7]. However, there have been many reports showing individual differences in the sensitivity to EA analgesia, which cause experimental and clinical difficulties: About 30-40% of rats were insensitive to EA in an acute thermal pain test, tail flick latency (TFL) test [5, 8, 9]. The similar results could be observed in the rat models of inflammatory and neuropathic pain [10, 11].
Using cDNA microarray study in the rat hypothalamus, a center of the descending pain inhibitory system, we previously identified several genes that mediate the individual variation in the sensitivity to EA analgesia : The expression levels of 5’-AMP-activated protein kinase (AMPK), dopamine beta-hydroxylase (DBH), acetylcholinesterase T subunit (AChET) in the hypothalamus of the responder rats were significantly higher than those of the non-responder rats. Since cDNA microarray alone could be subject to errors through cross-hybridization, the gene expressions for further study should be validated using new RNA samples . Indeed, our previous study using real-time RT-PCR confirmed that the mRNA expressions of AChET and DBH in the responder group were greater than those in the non-responder one . We also demonstrated that overexpression of AChET  or DBH  in the rat hypothalamus by viral gene transfer significantly potentiates EA analgesia. However, the post-microarray validation of AMPK and its functional role in EA analgesia have not been studied, despite the highest expression of AMPK in the responder rats as compared to the non-responders among the above three genes.
AMPK has a key role in the regulation of energy balance at both the cellular and whole-body levels, placing it at the center stage in studies of metabolic disorders . Recently, AMPK has also been identified as a potential target for therapy of acute and chronic pain [18, 19]. In the present study, we investigated the relationship between the hypothalamic AMPK and the individual variation in EA analgesia by using real-time RT-PCR and genetic manipulation. We report here that the expression levels of AMPK gene in the hypothalamus play an important role in determining the individual differences in the sensitivity to EA analgesia in rats.
Adult male Sprague-Dawley rats (7 weeks old) (Daehan biolink, Chungbuk, Korea) were housed in cages (3-4 rats per cage) with water and food available ad libitum. The room was maintained with a 12 h-light/dark cycle (a light cycle; 08:00-20:00, a dark cycle; 20:00-08:00) and kept at 23 ± 2°C. All animals were acclimated in their cages for 1 week prior to any experiments. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Kyung Hee University [KHUASP(SE)-12-013] and were conducted in accordance with the guidelines of the International Association for the Study of Pain .
Acute thermal pain behavior: TFL test
The rats showing a TFL increase after EA stimulation that was greater than 30% were classified as responders (mean TFL increase ratio = 59.00%, n = 10), whereas the rats showing less than a 20% TFL increase as non-responders (mean TFL increase ratio = 8.25%, n = 8). Since the other subjects (20-30% TFL increase after EA) are ambiguous for a clear classification, those rats were discarded .
Rats in both groups were rapidly sacrificed after EA stimulation and TFL test, and the hypothalamus were separated. RNA was then isolated from the hypothalamus using a Trizol reagent (Invitrogen) according to the manufacturer’s instructions, after which the RNA was quantified using a model ND-1000 apparatus (NanoDrop Technologies, Wilmington, DE, USA). The integrity of the RNA was confirmed by denaturing agarose gel electrophoresis. Single-stranded cDNA was prepared using First Strand cDNA Synthesis Kit (Roche Diagnostics Korea Applied Science, Seoul, Korea). The integrity of the cDNA was confirmed by amplifying GAPDH. The real-time PCR was conducted by a LightCycler 480 (Roche Applied Science, Indianapolis, IN) employing SYBR Green I as the dsDNA-specific binding dye for continuous fluorescence monitoring. The PCR protocol comprised 10 min at 95°C; 45 cycles of 10s at 95°C, 10s at 60°C and 10s at 72°C. After the cycles were finished, the signal of each temperature between 65 and 95°C was also detected to generate a dissociation curve. The sequences of the human primers were AMPK (forward 5’-tgaagccagagaacgtgttg-3’, reverse 5’- ataatttggcgatccacagc-3’) and GAPDH (forward 5’-tgccactcagaagactgtgg-3’, reverse 5’-ttcagctctgggatgacctt-3’). The mRNA levels of AMPK were compared by calculating the crossing point (Cp) value and normalized by the reference genes (GAPDH) using the LightCycler 480 Relative Quantification software (Roche).
Production of adenovirus vector
AMPK wild type α subunit (WT) and a dominant negative form (DN), in which Asp157 was replaced with alanine, were generated by PCR as previously described . The early region 1-deleted recombinant adenoviral vector encoding AMPK α subunit was generated by introducing AMPK cDNA into the shuttle plasmid pAv1 under the transcriptional control of the cytomegalovirus immediately early enhancer/promoter . The recombinant shuttle plasmid was cotransfected with the early region 1-deleted adenovirus serotype 5 genome, pJM17, and amplified in HEK 293 cells. The recombinant adenoviruses were purified by two centrifugation steps on cesium chloride gradients and dialyzed against 10 mM Tris-HCl, pH 8.0, 1 mM MgCl2, and 10% glycerol. The number of viral particles was assessed by measurement of the optical density at 260 nm . The titers of GFP control, WT and DN AMPK viruses were 1.5 × 1012 pfu/ml, 2.0 × 1012 pfu/ml and 2.0 × 1012 pfu/ml, respectively.
Microinjection of adenovirus into the hypothalamus
All the data are presented as mean ± SEM. Statistical analysis was done with Prism 5.0 (Graph Pad Software, USA). The unpaired t-test was used for statistical analysis. In all cases, p < 0.05 was considered significant.
Measurement of AMPK mRNA levels in the rat hypothalamus by real-time RT-PCR
Effects of adenoviral gene transfer of AMPK into the hypothalamus on EA-induced analgesia
Pain is considered both a sensation and an emotion, showing considerable complexity and subjectivity. In clinical and laboratory settings, the perception of pain bears a poor relationship to the intensity of the noxious stimulus . Therefore, strong interest exists in understanding the individual differences in response to pain and analgesics. To elucidate the genetic contributions to such individual variability in animals and humans, researchers are now employing a variety of approaches, such as microarray analysis, epigenetics and human brain imaging [13, 28, 29].
The analgesic effects of EA also show marked individual differences in acute, inflammatory and neuropathic pain rats [5, 8–11]. To identify and characterize the genes that cause these individual differences in response to EA analgesia, we previously conducted cDNA microarray analysis, using the hypothalamus, a main center of EA analgesia and the descending pain inhibitory system . Among several genes that are more abundantly expressed in the responder rats than non-responder rats, AMPK gene is the most differently expressed between the two groups. In the present study, we confirmed this with a real-time RT-PCR (Fiure 3) strongly suggesting that the expression of AMPK in the hypothalamus is closely associated with individual differences in response to EA analgesia. This study further validated the results by using viral gene transfer of AMPK into the hypothalamus (Figure 4). EA-induced analgesic effects were gradually decreased and slightly increased after injection of DN AMPK virus and WT AMPK virus, respectively, producing a significant difference between the two groups at 7 and 14 days post-injection.
The mammalian AMPK is a heterotrimer consisting of an α catalytic subunit and β and γ noncatalytic subunit . Isolation of AMPK to homogenously revealed that the catalytic subunit (α) co-purifies with two other noncatalytic subunit (β and γ). The formation of a trimeric subunit complex is necessary for an optimal AMPK activity and it is known that overexpression of wild type α subunit does not exert any positive effect on an endogenous AMPK activity [24, 26]. Consistent with these reports, there was no significant increase in EA-induced analgesic effect after WT AMPK virus injection. Conversely, the inhibition of AMPK activity by DN AMPK virus injection significantly decreased the EA analgesia (Figure 4).
AMPK is primarily regulated by cellular AMP/ATP and nutrient levels and plays a central role in the regulation of energy homeostasis and metabolic stress . It has emerged as a promising new drug target for treatment metabolic disorders, including obesity, type 2 diabetes and cardiovascular disease . Several studies also suggested that AMPK activation plays a significant role in important neuronal processes, including the regulation of neuronal plasticity and long-term potentiation, and the protection of neurons from neurodegenerative diseases . Although there has been little research on the role of AMPK in nociception, very recent studies demonstrated that AMPK activation significantly alleviates acute, inflammatory and neuropathic pain through the modulation of mammalian target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) signaling in the periphery and spinal cord that are related to pain hypersensitivity [18, 19, 32]. Our data further demonstrated that the hypothalamic AMPK play a role in mediating individual differences in response to EA-mediated analgesia. Thus, these findings not only provide a clinically useful evidence for the application of acupuncture or EA for analgesia, but also suggest an unexpected role of the hypothalamic AMPK in pain modulation.
It is currently unclear how the hypothalamic AMPK plays a role in EA-induced analgesia as shown in this study. One possible explanation is that AMPK might regulate EA analgesia-related neuropeptides that released in the hypothalamus. AMPK activation in the hypothalamus is positively correlated with neuropeptide Y (NPY) expressions  and this hypothalamic NPY has a significant antinociceptive effect . Interestingly, several reports demonstrated that acupuncture or EA stimulation at ST36 decreases NPY levels in the hypothalamus [35, 36]. Thus, we cautiously assumed that the responder rats with high AMPK levels, but not non-responders, might maintain sufficient NPY levels in the hypothalamus to be involved in antinociception, although EA stimulation decreased NPY expressions. In addition to this, further studies to explore the relationship between the AMPK and beta-endorphin in the hypothalamus, a well-known EA analgesia mediator, are required. Also, it would be interesting to examine the analgesic effects of EA on pathological pain, such as neuropathic pain , the mechanism of which is somewhat different from acute pain (e.g. TFL test). Although the individual differences in the sensitivity of acute nociceptive and chronic neuropathic pain to EA in rats were known to be maintained , we believe that studies using pathological pain models could provide a better understanding of EA-induced analgesia and its responsiveness.
In conclusion, we demonstrate that mRNA expression of AMPK in the hypothalamus of the responder rats is significantly higher than the non-responder rats. Furthermore, adenoviral gene transfer of AMPK in the hypothalamus could alter the EA-induced analgesia. Taken together, these results strongly suggest that levels of AMPK gene expression in the rat hypothalamus determine the individual differences in the sensitivity to EA analgesia.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST-2012-0005755 and NRF-2013R1A1A1012403).
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