AMP-activated Protein Kinase mRNA Research Articles

AMPKα1 Deficiency in Astrocytes from a Rat Model of ALS Is Associated with an Altered Metabolic Resilience.

Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress.

Berberine Protects against Neurological Impairments and Blood-Brain Barrier Injury in Mouse Model of Intracerebral Hemorrhage

Background: Elevation of AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) signaling can suppress intracerebral hemorrhage (ICH)-induced neurological impairments. As an isoquinoline alkaloid, Berberine exerts neuroprotective effects in neurological disease models with activated AMPK/PGC1α signaling. Aim: We aim to study the effect of Berberine on ICH-induced brain injury and explore the potential molecular mechanism. Methods: ICH model was established in mice through intracerebral injection of autologous whole blood, followed by treatment with Berberine. Neurological impairments were assessed by the modified neurological severity score and behavioral assays. Brain edema and blood-brain barrier (BBB) integrity were assessed by water content in the brain, amount of extravasated Evans blue, and BBB tight junction components. Neuroinflammatory responses were assessed by inflammatory cytokine levels. AMPK/PGC1α signaling was examined by AMPK mRNA expression and phosphorylated AMPK and PGC1α protein levels. Results: Berberine (200 mg/kg) attenuated ICH-induced neurological deficits, motor and cognitive impairment, and BBB disruption. Berberine also suppressed ICH-induced inflammatory responses indicated by reduced production of inflammatory cytokines. Finally, Berberine drastically elevated AMPK/PGC1α signaling in the hemisphere of ICH mice. Conclusion: Our findings suggest that Berberine plays an important neuroprotective role against ICH-induced neurological impairments and BBB injury, probably by inhibition of inflammation and activation of AMPK/PGC1α signaling.

AICAR stimulates mitochondrial biogenesis and BCAA catabolic enzyme expression in C2C12 myotubes

Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism. Insulin resistant populations often exhibit reduced expression of genes governing mitochondrial metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Interestingly, PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, the consistently observed increased circulating levels of BCAA in diabetics may be partially explained by reduced PGC-1α expression. Conversely, PGC-1α upregulation appears to increase BCAA catabolism. PGC-1α activity is regulated by 5′-AMP-activated protein kinase (AMPK), however, only limited experimental data exists on the effect of AMPK activation in the regulation of BCAA catabolism. The present report examined the effects of the commonly used AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the metabolism and expression of several related targets (including BCAA catabolic enzymes) of cultured myotubes. C2C12 myotubes were treated with AICAR at 1 mM for up to 24 h. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via qRT-PCR and western blot, respectively. AICAR treatment significantly increased mitochondrial content and peak mitochondrial capacity. AICAR treatment also increased AMPK activation and mRNA expression of several regulators of mitochondrial biogenesis but reduced glycolytic metabolism and mRNA expression of several glycolytic enzymes. Interestingly, branched-chain alpha-keto acid dehydrogenase a (BCKDHa) protein was significantly increased following AICAR-treatment suggesting increased overall BCAA catabolic capacity in AICAR-treated cells. Together, these experiments demonstrate AICAR/AMPK activation can upregulate BCAA catabolic machinery in a model of skeletal muscle.

Effects of age on feeding response: Focus on the rostral C1 neuron and its glucoregulatory proteins.

Older people are likely to develop anorexia of aging. Rostral C1 (rC1) catecholaminergic neurons in rostral ventrolateral medulla (RVLM) are recently discovered its role in food intake control. It is well established that these neurons regulate cardiovascular function. This study aims to determine the effect of age on the function of rostral C1 (rC1) neurons in mediating feeding response. Male Sprague Dawley rats at 3-months (n = 22) and 24-months (n = 22) old were used and further divided into two subgroups; 1) treatment group with 2-deoxy-d-glucose (2DG) and 2) vehicle group. Feeding hormones such as cholecystokinin (CCK), ghrelin and leptin were analysed using enzyme-linked immunosorbent assay (ELISA). Rat brain was carefully dissected to obtain the brainstem RVLM region. Further analysis was carried out to determine the level of proteins and genes in RVLM that were associated with feeding pathway. Protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK) and neuropeptide Y Y5 receptor (NPY5R) were determined by western blot. Expression of TH, AMPK and NPY genes were determined by real-time PCR. This study showed that blood glucose level was elevated in young and old rats following 2DG administration. Plasma CCK-8 concentration was higher in the aged rats at basal and increased with 2DG administration in young rats, but the leptin and ghrelin showed no changes. Old rats showed higher TH and lower AMPK mRNA levels. Glucoprivation decreased AMPK mRNA level in young rats and decreased TH mRNA in old rats. Aged rC1 neurons showed higher NPY5R protein level. Following glucoprivation, rC1 neurons produced distinct molecular changes across age in which, in young rats, AMPK phosphorylation level was increased and in old rats, TH phosphorylation level was increased. These findings suggest that glucose-counterregulatory responses by rC1 neurons at least, contribute to the ability of young and old rats in coping glucoprivation. Age-induced molecular changes within rC1 neurons may attenuate the glucoprivic responses. This situation may explain the impairment of feeding response in the elderly.

Effect of electroacupuncture combined with treadmill exercise on body weight and expression of PGC-1α， Irisin and AMPK in skeletal muscle of diet-induced obesity rats

To investigate the effect of electroacupuncture (EA) plus treadmill exercise on the expression of peroxisome proliferator activated receptor γ coactivator 1α(PGC-1α), Irisin, AMP-activated protein kinase (AMPK) in skeletal muscle of diet-induced obesity (DIO) rats, so as to explore its mechanism underlying body reduction promotion. Forty-two male SD rats were divided into normal diet (control, n＝10), high fat diet (model), EA, treadmill exercise and EA plus treadmill exercise (combination) groups (n＝8 in each of the latter 4 groups). The obesity model was established by feeding the rats with high fat diet. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36) and "Tianshu" (ST25) for 30 min, 5 times per week for a total of 8 weeks. Rats of the treadmill exercise group were forced to perform exercise on a treadmill (16 m/min) for 30 min, 5 times per week for a total of 8 weeks. Rats in the combination group received the above-mentioned two methods. During the treatment, rats in the control group were fed with normal fodder, rats in other groups were fed with high fat fodder, and their body weight was measured once a week. The expression levels of PGC-1α， fibronectin type Ⅲ domain containing 5 (FNDC5), AMPK mRNA and protein of skeletal muscle were measured by quantitative real-time PCR and Western blot，respectively. After modeling, the body weight was significantly increased (P<0.05), and the expression levels of PGC-1α and FNDC5 mRNA and protein, AMPK mRNA and phosphorylated AMPK (p-AMPK) protein in the skeletal muscle were considerably decreased in the model group relevant to the control group (P<0.05). Following the treatment, the body weight was significantly down-regulated, while the expression levels of PGC-1α and FNDC5 mRNAs and proteins, AMPK mRNA and p-AMPK protein were obviously up-regulated in the EA, treadmill exercise and combination groups relevant to the model group (P<0.05). The therapeutic effect of EA plus treadmill exercise was significantly superior to those of both simple EA and simple treadmill exercise in down-regulating the body weight, as well as in up-regulating the expression of PGC-1α and FNDC5 mRNAs and proteins, AMPK mRNA, and p-AMPK protein (P<0.05). Both EA and treadmill exercise can significantly increase the expression of PGC-1α， FNDC5 and p-AMPK in skeletal muscle of DIO rats, suggesting their efficacy in restoring fatty acid oxidation in skeletal muscle cells and improving mitochondrial function, which may contribute to their function in body reduction. The therapeutic effect of EA plus treadmill exercise is better than that of simple EA and simple treadmill exercise.

Role of Phosphorylated AMP-Activated Protein Kinase (AMPK) in Myocardial Insulin Resistance After Myocardial Ischemia-Reperfusion During Cardiopulmonary Bypass Surgery in Dogs.

BackgroundThe aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in myocardial insulin resistance after myocardial ischemia-reperfusion during cardiopulmonary bypass surgery in dogs.Material/MethodsTwenty-four mongrel dogs were randomly assigned to 4 groups. The control group did not undergo aortic cross-clamping; the model group underwent 60 mins of aortic cross-clamping with 150 ml cardioplegic solution. The treatment group, the inhibition group respectively with 0.11mg/kg AICAR (AMPK agonist) in 150 ml cardioplegic solution and 0.11 mg/kg Compound C (AMPK inhibitor) in 150 ml cardioplegic solution. The blood flow was determined and left ventricular myocardial tissue were taken at pre-bypass, 15, 60, and 90 min after aorta declamping, respectively. Expression of AMPK mRNA, p-AMPK and GLUT-4 proteins was determined by RT-PCR, IHC and WB.ResultsCompared with the control group, receiving 60 min ischemia at 15 min after reperfusion, Myocardial Glucose Extraction Ratio were significantly decreased in the other 3 groups, it was significantly decreased from 20.0% to 1.2% at 60 min of reperfusion, and recovered to 6.1% after 90 min reperfusion in model group, while recovered to 4.1%, 12.0% after 90 min reperfusion respectively exposed to Compound C and AICAR. The expressions of p-AMPK, GLUT-4 protein and AMPK mRNA in myocardium were decreased in different experiment groups, but these changes occurred to a lesser extent in the treatment group.ConclusionsThe inability of GLUT-4 expression induced by the decreases in p-AMPK protein expression that may be one of the reasons for myocardial insulin resistance.

Expression of messenger RNA encoding two cellular metabolic regulators, AMP-activated protein kinase (AMPK) and O-GlcNAc transferase (OGT), in channel catfish: Their tissue distribution and relationship with changes in food intake

AMP-activated protein kinase (AMPK) is considered as the master cellular metabolism regulator that activates various proteins, including O-GlcNAc transferase (OGT). Physiological roles of AMPK and OGT, including the relationship between their mRNA expression and food intake, are poorly understood in channel catfish. This study examined the tissue distribution of AMPK and OGT mRNA and changes in their expression in response to changes in food intake in channel catfish. Expression of all AMPK subunit and OGT mRNA was detectable in the whole brain, liver, heart, spleen, white muscle, and kidney of channel catfish. The OGT mRNA was highly localized in the brain compared to other tissues. 28-day fasting increased hepatic expression of AMPK α1, β1, and OGT mRNA while refeeding fish for 14 days after the 14-day fast decreased their expression to the level similar to that of fish that were fed daily. No changes were noted in the expression of muscle and brain AMPK mRNA or OGT mRNA by fasting and refeeding. Hepatic AMPK α1, α2 and β1 mRNA decreased in response to increased feeding frequency, whereas no changes in the expression of AMPK or OGT mRNA were noted in the brain or the muscle. Results of the current study indicated that the hepatic expression of AMPK and OGT mRNA appeared to be more sensitive to changes in food intake in channel catfish. However, further studies are needed to clearly demonstrate if food intake influences the expression of AMPK and OGT mRNA in various tissues, including the hypothalamus.

The relationship between food exposure frequency and expression of AMP‐activated protein kinase (AMPK) mRNA in the brain, liver, and muscle of channel catfish

Obesity poses serious challenges to our healthcare system with serious consequences of associated diseases. Our laboratory has been examining how the expression of genes involved in food intake and tissue nutrient metabolism is associated with the development of obese‐like phenotypes in channel catfish. The objective of the study was to examine changes in expression of AMPK mRNA in response to changes in feeding frequency. Fish were fed every 12 (overfed), 24 (control), or 48 (underfed) hours (n= 4 tanks per treatment, 8 fish per tank) for 28 days. Brain, liver, and muscle tissue were collected at the end of the study. The expression of the AMPK mRNA was examined using quantitative RT‐PCR. The muscle expression of all AMPK subunit mRNA was unaffected by feeding frequency. In contrast, brain expression of alpha 1 and beta 1 subunit mRNA had a linear tendency for increase in response to increased feeding frequency. Hepatic expression of alpha 2 and beta 1 mRNA was lower in the overfeed group compared to those assigned as control or underfeed. Hepatic expression of alpha 1 mRNA decreased linearly in response to increased feeding frequency. Our results demonstrated that the hepatic and the brain expression of AMPK mRNA was influenced by increase in exposure to food in a transcript‐specific manner. Given that the AMPK serves as a regulator of tissue nutrient sensing and metabolism in mammals, it is possible that AMPK may share a similar function in the brain and liver but not in the muscle of channel catfish.Support or Funding InformationKansas IDeA Network of Biomedical Research Excellence Network (NIH/NIGMS grant number P20GM103418)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apparent Correlations Between AMPK Expression and Brain Inflammatory Response and Neurological Function Factors in Rats with Chronic Renal Failure.

To explore the correlations between AMP-activated protein kinase (AMPK) expression and brain inflammatory response and neurological function factors in rats with chronic renal failure. Chronic renal failure models in rats were established, and the healthy control group (normal group) was set. Chronic renal failure model rats were divided into model group (without any treatment), control group (intraperitoneal injection of normal saline), A-769662 group (intraperitoneal injection of AMPK specific activator), and compound C group (intraperitoneal injection of AMPK specific inhibitor). The results of HE staining showed renal tissue enlargement, and significant pathological changes. Compared with the normal group, AMPK level in peripheral blood and AMPK mRNA and protein expressions in brain tissue were significantly reduced, and AMPK pathway activation was significantly inhibited in other groups. Compared with the model group, rats in the A-769662 group had significantly decreased serum creatinine (Scr) and blood urea nitrogen (BUN) levels and γ-aminobutyric acid (γ-GABA) content, significantly increased brain-derived neurotrophic factor (BDNF) positive expressions and 5-hydroxytryptamine (5-HT) content, and decreased interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule 1 (ICAM-1) expressions (all P < 0.05), while it was just the opposite in compound C group (all P < 0.05). There is an apparent correlation between AMPK expression and brain inflammatory response in chronic renal failure rats. AMPK is expected to be an important pathway in the treatment of uremic encephalopathy.

Yiqi Huoxue prescription can prevent and treat post-MI myocardial remodeling through promoting the expression of AMPK signal pathway

ObjectiveTo investigate how Yiqi Huoxue (YQHX) prescription regulates mitochondrial biosynthesis and ATP synthesis via AMP-activated protein kinase (AMPK) and to reveal its molecular mechanism in preventing and treating post-MI myocardial remodeling. MethodsThe MI animal model of myocardial infarction were established by ligating Sprague Dawley (SD) rats' left anterior descending coronary arteries; the animals were randomly divided into MI group, YQHX prescription group and perindopril group, and a sham operation group was set at the same time. Related drug intervention was administered on the 2nd day after surgery, the YQHX group was given Astragalus, angelica, ginseng, Ligusticum wallichii and pseudoginseng, provided by the Dongzhimen Hospital of Beijing University of Chinese Medicine, once per day, at a dose of 21 g/kg body weight/day (the clinical equivalent dose based on a previous study), and the changes in relevant indicators were observed at 1 week and 4 weeks. Echocardiography (ECG) was used to observe the changes in rat cardiac structure and functions; the morphology-based technique was used to observe the changes in myocardial cells and mitochondria; the expression of AMPK signal pathway-related proteins and mRNA was detected using western blotting and real-time fluorescence quantification respectively, while fluorescence enzyme-labeled method was used for detecting ATP synthesis. ResultsCardiac structure and functions: Compared with the MI group at 1 week, the YQHX prescription group and the perindopril group exhibited increased LVEF and LVFS (Pall < .05); their LVEDs and LVEDd were reduced but suggested no statistically significant differences (F = 2.258, F = 0.3464, Pall > .05). At 4 weeks, both LVEF and LVFS were elevated in the YQHX prescription group (P = .008, .009) and the perindopril group (P = .279, .333), where differences in the later group indicated no statistical significance; the YQHX prescription group and the perindopril group were also featured by reduced LVEDs and LVEDd (Pall < .05). Morphology: Compared with the MI group at two time nodes, the YQHX prescription group and the perindopril group exhibited significant improvement in the pathological changes in myocardial cells and mitochondrial structure. Expression of AMPK signal pathway-related proteins and mRNA: The expression of both pLKB1 and pAMPK proteins followed a rising trend in the YQHX prescription group and the perindopril group at 1 week. The expression of LKB1mRNA and AMPKmRNA was elevated (Pall < .05). The increased expression of PGC-1α, NRF1 and mtTFA proteins demonstrated statistically significant differences (Pall < .05). The expression of mtDNA protein followed an increasing trend. At 4 weeks, the expression of both pLKB1 and pAMPK proteins was elevated (Pall < .05). Heightened expression was also reported in LKB1mRNA and AMPKmRNA (Pall < .05). The increased expression of PGC-1α, NRF1, mtTFA and mtDNA proteins demonstrated statistically significant differences (Pall < .05). ATP synthesis: ATP synthesis was increased in the YQHX prescription group and the perindopril group at both 1 week and 4 weeks (Pall < .001). ConclusionThe possible mechanism of YQHX prescription for preventing and treating post-MI myocardial remodeling may function through strengthening the activation AMPK signal pathway by LKB1, thus further increasing the expression of downstream transcription factor proteins and initiating mitochondrial replication and transcription; as a result, the YQHX prescription can improve the post-MI damage in mitochondrial morphological structure in the tissue at heart marginal zone as well as enhance mitochondrial biosynthesis and ATP synthesis.

Alpha-lipoic acid impairs body weight gain of young broiler chicks via modulating peripheral AMPK

In mammals, the AMP-activated protein kinase (AMPK) pathways in the central and peripheral tissues coordinately integrate inputs from multiple sources to regulate energy balance. The present study was aimed to explore the potential role of hepatic AMPK in the energy homeostasis of broiler chickens. Diets with 0, 0.05% or 0.1% alpha-lipoic acid (α-LA), a known AMPK inhibitor were provided to broiler chicks for 7days. As a result, α-LA supplementation decreased the relative growth rate of broiler chicks. Hepatic AMPKα2 mRNA levels were significantly upregulated by dietary α-LA, in concert with the increased phosphorylated AMPKα protein levels. In addition, hepatic FAS mRNA levels together with the malonyl-CoA to total CoA ester ratio were reduced by α-LA supplementation. Moreover, the hepatic phosphorylated glycogen synthase levels were increased resulting in a markedly decreased hepatic glycogen content. In conclusion, dietary α-LA supplementation decreased the in vivo hepatic glycogenesis and lipogenesis via stimulating hepatic AMPKα mRNA levels and the phosphorylated gene product. The stimulatory effect of α-LA on hepatic AMPK mRNA and pAMPKα protein levels together with our previous observations regarding its inhibitory effect on hypothalamic AMPK may have altered the energy balance and hence impaired body weight gain of broiler chicks.

Effects of Caloric Restriction and Aerobic Exercise on Adipose Tissue AMP-activated Protein Kinase Gene Expression in Obese Older Women

AMP-activated protein kinase (AMPK) plays a key role as a master regulator of cellular energy homeostasis. In adipose tissue, AMPK activation results in fatty acid oxidation and lowers chronic inflammation. PURPOSE: To investigate the effect of caloric restriction and caloric restriction plus aerobic exercise at different intensities on adipose tissue AMPK gene expression in obese older women. METHODS: Twenty nine overweight or obese, postmenopausal women were assigned to one of three 20-week intervention groups: caloric restriction (CR only, n=9), CR plus moderate-intensity aerobic exercise (CR+moderate-intensity, n=12), or CR plus vigorous-intensity aerobic exercise (CR+vigorous-intensity, n=8). Total calorie deficit was adjusted to approximately 2800 kcal/week, and the exercise involved treadmill walking at an intensity of 45-50% (moderate-intensity) or 70-75% (vigorous-intensity) of heart rate reserve. Subcutaneous abdominal adipose tissue samples were collected before and after the interventions. Adipose tissue AMPK mRNA levels were determined by real-time reverse transcription polymerase chain reaction. RESULTS: The average weight loss was 12.8±3.9 kg (14.6±5.2%) and it was not different among the three intervention groups. At baseline, adipose tissue AMPK mRNA levels did not differ among the three intervention groups. Compared to pre-intervention, CR increased adipose tissue AMPK mRNA levels (0.12±0.05 vs. 0.17±0.06, expressed as AMPK/β-actin mRNA ratio, P<0.05); however, CR+moderate-intensity and CR+vigorous-intensity did not alter adipose tissue AMPK mRNA levels (0.13±0.05 vs. 0.12±0.03 and 0.13±0.05 vs. 0.12±0.04, respectively). Moreover, change in AMPK mRNA levels in the CR group was significantly different from those in the CR+moderate-intensity and CR+vigorous-intensity groups (both P<0.05) CONCLUSION: Caloric restriction, but not caloric restriction plus aerobic exercise, increased abdominal adipose tissue AMPK gene expression in obese older women. The role of adipose tissue AMPK gene expression in regulating energy metabolism and inflammatory reactions during weight loss needs further investigation. (Supported by NIH R01-AG/DK20583, P30-AG21332, and M01-RR07122)

AMP-activated protein kinase is a biomarker of energetic status in freshwater mussels exposed to municipal effluents

Although biomarkers are frequently used to assess sublethal effects of contaminants, a lack of mechanistic linkages to higher-level effects limits the predictive power of biomarkers. Bioenergetics has been proposed as a framework for linking cellular effects to whole-animal effects. We investigated sublethal effects of exposure to wastewater treatment facility effluent in freshwater mussels in situ, thereby capturing ecologically relevant exposure conditions. Our study focused on the energetic biomarker AMP-activated protein kinase (AMPK), while also considering more traditional biomarkers like heat shock proteins (HSP70), and antioxidant enzymes (i.e., superoxide dismutase (SOD), glutathione-S-transferase (GST)). We examined biomarkers at mRNA and protein levels. Effluent exposure caused a reduction in total-AMPK protein abundance (p=0.05) and AMPK mRNA expression (p=0.02). Conversely, AMPK activity increased at downstream sites by 2.2-fold (p=0.05), indicating increased cellular energy consumption. HSP70 protein abundance was lower at downstream sites (p<0.05), while SOD and GST activity levels significantly increased. By using various biomarkers, we demonstrate that exposure to municipal effluent creates an energetically taxing situation. This is the first study to use AMPK to evaluate the effects of contamination in situ, and our results suggest that energetic biomarkers, like AMPK, complement traditional biomarkers and may help establish functional links between cellular and whole-animal effects.

Inhibition of AMPK expression in skeletal muscle by systemic inflammation in COPD rats.

BackgroundChronic obstructive pulmonary disease (COPD) is a disease characterized by airflow limitation and inflammation. Meanwhile, COPD also is associated with metabolic disorders, such as skeletal muscle weakness. Strikingly, activation of AMP-activated protein kinase (AMPK) exerts critical roles in energy metabolism. However, it remains unclear whether and how the expression levels of AMPK are affected in the COPD model rats which may lead to the dysfunction of the skeletal muscle in these rats.MethodsHere we developed a rat model of COPD, and we investigated the morphological changes of peripheral skeletal muscle and measured the levels of tumor necrosis factor -α (TNF-α) and AMPK in skeletal muscle by using approaches that include immunohistochemistry and polymerase chain reaction (PCR).ResultsWe found that the expression levels of both AMPK mRNA and protein in skeletal muscles were significantly reduced in the COPD model rats, in comparison to those from the control rats, the COPD model rats that received treatments with AICAR and resveratrol, whereas the expression levels of TNF-α were elevated in COPD rats.ConclusionSuch findings indicate that AMPK may serve as a target for therapeutic intervention in the treatment of muscle weakness in COPD patients.

발효울금 주정추출물부터 분리된 에틸아세테이트 분획물에 대한 3T3-L1 세포에서의 지방 형성 억제 효과

FCEE를 3T3-L1 전지방세포의 분화과정 중에 처리한 후, FCEE가 지방의 축적에 미치는 영향을 확인하였다. FCEE(200 μg/mL)를 세포에 처리한 후 Oil Red O 염색법과 AdipoRed 형광염색법을 이용한 지방 축적과 세포내 중성지방 함량을 측정한 결과, 무처리한 지방세포와 비교하여 각각 10.2%, 13.7% 유의하게 감소하는 것을 확인하였다. 이러한 결과에 대한 기작을 확인하고자 포도당 유입량과 유리 글리세롤 방출량을 측정하였다. FCEE(200 μg/mL) 처리 시 지방세포와 비교하여 포도당 유입량이 36.6% 유의적으로 감소하였으며, 유리 글리세롤 방출량도 8일째에 유의적으로 감소하였다. 이는 지방세포의 분화과정 중에 FCEE의 처리로 분화가 억제되어 지방구의 형성이 억제되고 배양액으로 배출되는 유리 글리세롤의 총량이 감소한 것으로 판단된다. 이러한 결과가 지방세포 분화의 억제에 의한 것인지 판단하기 위해 지방 형성 관련인자의 mRNA 발현량을 측정하였다. FCEE(200 μg/mL)의 처리로 AMPK mRNA 발현량은 지방세포와 비교하여 3배 증가하였으며, SREBP-1c, C/EBPα와 PPARγ mRNA 발현량은 각각 0.6, 0.6 및 0.8배 감소하는 것을 확인하였다. 이상의 결과들로부터 FCEE는 지방 합성을 억제하는 활성을 보유하고 있는 바, 향후 항비만 기능성 식품소재로 활용될 수 있을 것으로 사료된다.

Expression levels of the hypothalamic AMPK gene determines the responsiveness of the rats to electroacupuncture-induced analgesia.

BackgroundAlthough 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.MethodsSprague-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.ResultsThe 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).ConclusionsThe 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.

Globular adiponectin and its downstream target genes are up-regulated locally in human colorectal tumors: ex vivo and in vitro studies

ObjectiveLow plasma adiponectin levels are linked to obesity, insulin resistance, and the risk of several types of malignancy. Despite the decline in circulating adiponectin concentrations, the increase in the expression of adiponectin receptors AdipoR1 and AdipoR2 is greater in cancerous than in normal colonic tissue. The purpose of this study was to obtain new information regarding local adiponectin signaling in the pathogenesis of colorectal cancer (CRC). MethodsWe characterized the expressions of adiponectin and several of its downstream targets in paired samples of tumor tissue and adjacent noncancerous mucosa in 60 surgical patients with colorectal adenocarcinomas. ResultsAdiponectin was expressed in both colorectal tumors and the adjacent mucosa. The expressions of adiponectin mRNA and its globular protein variant (gAd), adiponectin receptor type 1 and 5′ AMP-activated protein kinase (AMPK) mRNA were significantly higher in colorectal tumors than in the adjacent mucosa. This finding was accompanied by increased mRNA expression of genes encoding proteins involved in fatty-acid trafficking and oxidation. The potential interference between adiponectin stimulation and AMPK activation through AMPK1 was examined in an in vitro model with the aid of silencing-RNA experiments. Furthermore, AMPK mRNA expression on tumors was positively correlated with a more advanced tumor stage in the patients. ConclusionWe propose that the globular adiponectin-AMPK pathway functions in an autocrine manner in colorectal tumors, explaining some of the beneficial changes in cellular oxidative capacity in tumors in favor of tumorigenesis.

Abstract 186: AMP-Activated Protein Kinase Mediates Cardioprotection in the Regenerative MRL Mouse Strain

Introduction: The Murphy Roths Large (MRL/MpJ) mouse model is well-known for its healing and regenerative properties. The actual mechanisms promoting regeneration are still unknown. AMP activated protein kinase (AMPK) has been lauded for its role as a cardioprotective sensor of cellular metabolic stress, cell growth, and ATP depletion. Based on our preliminary data, we hypothesize that increased levels of AMPK in the MRL hearts is primarily responsible for its altered mitochondrial biogenesis and metabolism, reduced oxidative stress, and altered cardiac function in the MRL mice. Methods: Adult C57BL/6 (control) and MRL mice (n=5/group) were studied pre- and post-7 days of exercise, and post-exercise with 3 days injection of either 5-amino -imidazole carboxamide riboside (AICAR, AMPK stimulator, 250ug/g, IP), or Compound C (AMPK inhibitor, 20ug/g, IP). Results: Compared to control at baseline, the MRL hearts had higher AMPK protein levels (9 fold increase, p&lt;0.001), equivalent AMPK mRNA levels by qRT-PCR, and higher AMP to ATP ratios by HPLC (3 fold increase, p&gt;0.05, n.s.). The MRL hearts also had increased mitochondria by electron microscopy (by 65 ± 4.5%, p&lt;0.05), reduced HW to BW ratio (48% decrease, p&lt;0.001), reduced cellular and mitochondrial reactive oxygen species (by DHE and Mitosox stains respectively), and increased glycolysis (Glut-4 sarcolemmal translocation). Hypertrophic response to exercise was greater in the MRL mice (HW/BW increased by 40% vs. 2% in control, p&lt;0.001). Mimicking injury, Compound C reduced active phosphorylated AMPK protein levels in control (by 26%, p&lt;0.05), while increased it in MRL mice (by 30%, p&lt;0.05). AICAR injection significantly increased pAMPK protein in both strains (p=0.008). Baseline MRL mice echocardiographic analysis (vs. control) indicated higher systolic function, but increased diastolic E/A and E/E’ ratios (p&lt;0.05). In the MRL hearts, Compound C treatment reduced %EF and %FS, while AICAR reduced post-exercise hypertrophy (p=0.02). Conclusions: This study highlights the cardioprotective role AMPK plays in the MRL mice, through its modulation of mitochondrial function and reduction of oxidative stress. This can in turn be used to develop new therapeutic treatment for heart failure.

AMPK Regulates Metabolic Actions of Glucocorticoids by Phosphorylating the Glucocorticoid Receptor through p38 MAPK

Glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, whereas AMP-activated protein kinase (AMPK) is the master regulator of energy homeostasis, sensing energy depletion and stimulating pathways of increasing fuel uptake and saving on peripheral supplies. We showed here that AMPK regulates glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. Transcriptomic analysis in the liver suggested marked overlaps between the AMPK and glucocorticoid signaling pathways directed mostly from AMPK to glucocorticoid actions. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive glucocorticoid-inducible leucine zipper protein, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Because increased action of glucocorticoids is associated with the development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacological interventions to these pathologies.

Impairment of PGC-1alpha expression, neuropathology and hepatic steatosis in a transgenic mouse model of Huntington's disease following chronic energy deprivation

We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARgamma coactivator-1alpha (PGC-1alpha) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1alpha, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRalpha mRNA and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA) reduced ATP and PCr levels and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1alpha, NRF1, Tfam and downstream genes in the striatum and cerebral cortex of wild-type (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1alpha and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1alpha, therefore, plays an important role in the behavioral phenotype, metabolic disturbances and pathology of HD, which suggests the possibility that agents that enhance PGC-1alpha function will exert therapeutic benefits in HD patients.