• The Korean Journal of Physiology and Pharmacology
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• v.17 no.4
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• pp.331-338
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• 2013

AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, is activated in response to cellular stress when intracellular levels of AMP increase. We investigated the neuroprotective effects of AMPK against scopolamine-induced memory impairment in vivo and glutamate-induced cytotoxicity in vitro. An adenovirus expressing AMPK wild type alpha subunit (WT) or a dominant negative form (DN) was injected into the hippocampus of rats using a stereotaxic apparatus. The AMPK WT-injected rats showed significant reversal of the scopolamine induced cognitive deficit as evaluated by escape latency in the Morris water maze. In addition, they showed enhanced acetylcholinesterase (AChE)-reactive neurons in the hippocampus, implying increased cholinergic activity in response to AMPK. We also studied the cellular mechanism by which AMPK protects against glutamate-induced cell death in primary cultured rat hippocampal neurons. We further demonstrated that AMPK WT-infected cells increased cell viability and reduced Annexin V positive hippocampal neurons. Western blot analysis indicated that AMPK WT-infected cells reduced the expression of Bax and had no effects on Bcl-2, which resulted in a decreased Bax/Bcl-2 ratio. These data suggest that AMPK is a useful cognitive impairment treatment target, and that its beneficial effects are mediated via the protective capacity of hippocampal neurons.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.4
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• pp.651-661
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• 2020

Objective: We hypothesized that Cr source can alter adipogenic-related transcriptional regulations and cell signaling. Therefore, the objective of the study was to evaluate the biological effects of chromium acetate (CrAc) on bovine intramuscular (IM) and subcutaneous (SC) adipose cells. Methods: Bovine preadipocytes isolated from two different adipose tissue depots; IM and SC were used to evaluate the effect of CrAc treatment during differentiation on adipogenic gene expression. Adipocytes were incubated with various doses of CrAc: 0 (differentiation media only, control), 0.1, 1, and 10 μM. Cells were harvested and then analyzed by real-time quantitative polymerase chain reaction in order to measure the quantity of adenosine monophosphate-activated protein kinase-α (AMPK-α), CCAAT enhancer binding protein-β (C/EBPβ), G protein-coupled receptor 41 (GPR41), GPR43, peroxisome proliferator-activated receptor-γ (PPARγ), and stearoyl CoA desaturase (SCD) mRNA relative to ribosomal protein subunit 9 (RPS9). The ratio of phosphorylated-AMPK (pAMPK) to AMPK was determined using a western blot technique in order to determine changing concentration. Results: The high dose (10 μM) of CrAc increased C/EBPβ, in both IM (p = 0.02) and SC (p = 0.02). Expression of PPARγ was upregulated by 10 μM of CrAc in IM but not in SC. Expression of SCD was also increased in both IM and SC with 10 μM of CrAc treatment. Addition of CrAc did not alter gene expression of glucose transporter 4, GPR41, or GPR43 in both IM and SC adipocytes. Addition of CrAc, resulted in a decreased pAMPKα to AMPKα ration (p<0.01) in IM. Conclusion: These data may indicate that Cr source may influence lipid filling in IM adipocytes via inhibitory action of AMPK phosphorylation and upregulating expression of adipogenic genes.

• The Korean Journal of Physiology
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• v.27 no.2
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• pp.175-183
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• 1993

There are many report suggesting that influx and intracellular calcium concentration $([Ca^{2+}]_i)$ are related to cell signalling in various cells. However, it has not been reported that calcium channel activation is affected by the substances involved in signal transduction pathways in the mouse eggs. In this study, the effects of isoprenaline (ISP) and cyclic AMP on calcium influx through calcium channels were investigated to show their relationship with the signal transduction process in unfertilized mouse eggs. Using whole cell voltage clamp techniques, calcium currents, elicited by the depolarizing pulses of 300 ms duration (from -50 mV to 50 mV in 10 mV increments) from a holding potential of -80 mV, were recorded. The current-voltage (I-V) relation of calcium currents was shown to be bell-shaped; the current began to activate at -50 mV and reached its maximum $(-1.33{\pm}0.16\;nA:\;mean{\pm}S.E.,\;n=7)$ at -10 mV, then decayed at around 50 mV. Calcium currents were fully activated within $7\;ms{\sim}20\;ms$ and completely inactivated 200 ms after onset of the step pulse. ISP within the concentration ranges of $10^{-8}\;M{\sim}10^{-4}\;M$ dose-dependently increased the amplitude calcium current. The permeable cyclic AMP analogue,8-bromocyclic AMP, also increased its maximal amplitude by 46ft at $10^{-5}\;M$, while protein kinase inhibitor (PKI), which is known to inhibit 0.02 phosphorylating units of cyclic AMP-dependent protein kinase (PKA) per microgram decreased calcium currents. Currents recorded in the presence of PKI were resistant to increase by the application of $10^{-5}\;M$. Also, PKI inhibited the calcium current increase elicited by ISP treatment. These results suggest that $\beta-adrenergic$ regulation of the calcium channel is mediated by the cAMP-dependent protein kinase. This signal transduction pathway might play a role in regulating $[Ca^{2+}]_i$, level due to the increase of calcium influx in mouse eggs.

• Korean Journal of Plant Resources
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• v.28 no.5
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• pp.582-590
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• 2015

In the present study, we investigated the effect of 70% EtOH extract from Hippophae Rhamnoides L. leaves (HRL) on the anti-obesity effect in 3T3-L1 cells. The effects of HRL on lipid accumulation in 3T3-L1 cells were examined using Oil Red O staining. In addition, we examined the gene expression levels by using RT-PCR and western blot. The results of this analysis showed that 100 ㎍/㎖ HRL significantly increased the inhibition of lipid accumulation by 82.25%; significantly decreased the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and fatty acid synthase (FAS) in 3T3-L1 cells as well as the stimulated protein expression of AMP-activated protein kinase (AMPK); and suppressed the expression level of PPARγ. These results suggest that HRL can prevent adipogenesis through activation of AMPKα and inhibition of adipogenesis transcription factors.

• Korean Journal of Veterinary Research
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• v.39 no.6
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• pp.1073-1080
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• 1999

Previous studies suggested that the inhibition of thyroxine ($T_4$) release by ${\alpha}_1$-adrenoceptor and muscarinic receptor stimulation results in activated protein kinase C (PKC) from mouse and guinea pig thyroids. In the present study, the effect of carbachol, methoxamine, phorbol myristate acetate (PMA), and R59022 on the release of $T_4$ from the mouse, rat, and guinea pig thyroids was compared to clarify the role of PKC in the regulation of the release of $T_4$. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for $T_4$ by EIA kits. Forskolin, an adenylate cyclase activator, chlorophenylthio-cAMP sodium, a membrane permeable analog of cAMP, and isobutyl-methylxanthine, a phosphodiesterase inhibitor, like TSH (thyroid stimulating hormone), enhaced the release of $T_4$ from the mouse, rat, and guinea pig thyroids. Methoxamine, an ${\alpha}_1$-adrenoceptor agonist, inhibited the TSH-stimulated release of $T_4$ in mouse, but not rat and guinea pig thyroids. In contrast, carbachol, a muscarinic receptor agonist, inhibited the release of $T_4$ in guinea pig, but not mouse and rat thyroids. These inhibition were reversed by prazosin, an ${\alpha}_1$-adrenoceptor antagonist or atropine, a muscarinic antagonist or $M_1$- and $M_3$-muscarinic antagonists, in mouse or guinea pig thyroids. In addition, staurosporine, a PKC inhibitor, reversed methoxamine or carbachol inhibition of TSH stimulation. Furthermore, PMA, a PKC activator, and R59022, a diacylglycerol (DAG) kinase inhibitor, inhibited the TSH-stimulated release of $T_4$ in mouse, rat, and guinea pig thyroids. These inhibition were blocked by staurosporine. These findings suggest that the activation of receptor or DAG inhibits TSH-stimulated $T_4$ release through a PKC-dependent mechanism in thyroid gland.

• Journal of Ginseng Research
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• v.46 no.4
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• pp.572-584
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• 2022

Background: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of trinucleotide CAG repeat in the Huntingtin (Htt) gene. The major pathogenic pathways underlying HD involve the impairment of cellular energy homeostasis and DNA damage in the brain. The protein kinase ataxia-telangiectasia mutated (ATM) is an important regulator of the DNA damage response. ATM is involved in the phosphorylation of AMP-activated protein kinase (AMPK), suggesting that AMPK plays a critical role in response to DNA damage. Herein, we demonstrated that expression of polyQ-expanded mutant Htt (mHtt) enhanced the phosphorylation of ATM. Ginsenoside is the main and most effective component of Panax ginseng. However, the protective effect of a ginsenoside (compound K, CK) in HD remains unclear and warrants further investigation. Methods: This study used the R6/2 transgenic mouse model of HD and performed behavioral tests, survival rate, histological analyses, and immunoblot assays. Results: The systematic administration of CK into R6/2 mice suppressed the activation of ATM/AMPK and reduced neuronal toxicity and mHTT aggregation. Most importantly, CK increased neuronal density and lifespan and improved motor dysfunction in R6/2 mice. Conversely, CK enhanced the expression of Bcl2 protected striatal cells from the toxicity induced by the overactivation of mHtt and AMPK. Conclusions: Thus, the oral administration of CK reduced the disease progression and markedly enhanced lifespan in the transgenic mouse model (R6/2) of HD.

• Journal of Veterinary Science
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• v.23 no.1
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• pp.4.1-4.15
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• 2022

Background: Flavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the antiadipogenic effects of flavonoids are unclear. Objectives: The present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells. Methods: The intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis. Results: The flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators. Conclusion: The overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

• Journal of Korean Medicine for Obesity Research
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• v.20 no.2
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• pp.109-121
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• 2020

Objectives: Tanshinone I is a bioactive constituent in Salvia miltiorrhiza. At present, the anti-obesity effect and mechanism of tanshinone I are not fully understood. Here we investigated the effect of tanshinone I on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Methods: Lipid accumulation and triglyceride (TG) content in 3T3-L1 cells were determined by Oil Red O staining and AdipoRed assay, respectively. The expression and phosphorylation levels of adipogenic/lipogenic proteins in 3T3-L1 cells were evaluated by Western blotting. The messenger RNA (mRNA) expression levels of adipogenic/lipogenic markers and leptin in 3T3-L1 cells were measured by reverse transcription polymerase chain reaction (RT-PCR). Lipid accumulation in zebrafish was assessed by LipidGreen2 staining. Results: Tanshinone I at 5 μM largely blocked lipid accumulation and reduced TG content in differentiating 3T3-L1 cells. Furthermore, tanshinone I decreased the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells. In addition, tanshinone I increased the phosphorylation of adenosine 3',5'-cyclic monophosphate (cAMP)-activated protein kinase (AMPK) while decreased the intracellular adenosine triphosphate (ATP) content with no change in the phosphorylation and expression of liver kinase-B1 in differentiating 3T3-L1 cells. Importantly, tanshinone I also reduced the extent of lipid deposit formation in developing zebrafish. Conclusions: These findings demonstrate that tanshinone I has strong anti-adipogenic effects on 3T3-L1 cells and reduces adiposity in zebrafish, and these anti-adipogenic effect in 3T3-L1 cells are mediated through control of C/EBP-α, PPAR-γ, STAT-3, FAS, ACC, perilipin A, and AMPK.

• Journal of Chest Surgery
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• v.25 no.4
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• pp.364-382
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• 1992

In order to investigate the effect of intracellular cyclic GMP on the calcium channel, whole cell patch clamp technique with internal perfusion method was used in the single ventricular myocytes of the rabbit. Cyclic GMP, cGMP analogues, cAMP, isopernaline and forskolin were perfused into cells and their effects on the calcium current were analysed by applying depolarizing step pulse of 10 mV in amplitude for 200 msec from holding potential of -40 mV. Calcium currents usually activated from -30 mV and then reached a peak at +10 mV. Amplitude of the calcium current was standardized with membrane capacitance, 50 pF. Peak amplitude at +10 mV in control was -0.15 nA/50pF. When 100 mM cAMP was applied from the pipette, peak amplitude of calcium current increased to -0.32 nA and addition of 1 mM isoprenaline further increased its amplitude. In the presence of cGMP it alone also produced an increase of the calcium current to -0.52 nA/50pF and addition of isoprenaline or forskolin increased its magnitude to -[0.55~0.95] nA/50pF. Simultaneous application of cGMP and cAMP increased the calcium current to -0.67 nA/50pF. Among the cGMP analogues, 8-Br-cGMP was the most potent stimulant for the calcium current activation. From the above results it could be concluded tlat cGMP increases the calcium current not through cAMP dependent protein kinase nor cAMP dependent phosphodiesterase pathway, but through independent phosphorylation pathway, possibly cGMP dependent protein kinase pathway.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.527-538
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• 2019

Background: Ginsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor (ER) via mitogen-activated protein kinase-mediated pathway. Our study aimed to delineate the mechanisms by which Rg1 activates the rapid ER signaling pathways. Methods: ER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidney HEK293 cells were treated with Rg1 ($10^{-12}M$, $10^{-8}M$), $17{\beta}$-estradiol ($10^{-8}M$), or vehicle. Immunoprecipitation was conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or their inhibitors were applied. Results: Rg1 rapidly induced $ER{\alpha}$ translocation to plasma membrane via caveolin-1 and the formation of signaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator of nongenomic activity of ER (MNAR), $ER{\alpha}$, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase (MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interfering RNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in these cells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor (EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). The increase in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could be abolished by G15. Conclusion: Ginsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containing signalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independently and exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogen receptor.