Mol. Cells 2016; 39(2): 163-168
Published online January 7, 2016
https://doi.org/10.14348/molcells.2016.2298
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: yshim@konkuk.ac.kr
Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner.
Keywords caffeine,
Caffeine is one of the most popular drugs in the world and its consumption had increased rapidly in modern society. Most of the caffeine consumed is from coffee. According to “caffeine informer” 2013 data, 2.6 kg coffee per capita (per person on average) is consumed in South Korea, making South Korea the 26th top coffee consuming country in the world. An optimal dose of caffeine has many beneficial effects, such increasing attention and focus, improving athletic performance, and improving many other aspects of health (Reviewed in Weinberg and Bealer, 2002). In contrast, caffeine is an addictive drug and has side effects, particularly during pregnancy (Kuczkowasaki, 2009). High caffeine consumption may increase the risk for high blood pressure, hypertension, insomnia, and panic disorder (Mesas et al., 2011; Vilarim et al., 2011). High caffeine consumption also has maternal effects during pregnancy, which are related with a greater risk for low birth weight infants (Chen et al, 2014). A lethal human caffeine dose is approximately 10 g (one cup of coffee contains ∼125 mg of caffeine) (Ekwall et al., 1998). Although high doses of caffeine can be lethal, the mode of action remains unclear.
In our previous study, we investigated effects of caffeine on
Maintenance and handling of
Appropriate amounts of caffeine (Sigma, USA) were included in the NGM before autoclaving to obtain final concentrations of 10-30 mM. After
Synchronized L1-stage worms were cultured in NGM containing 0 or 30 mM caffeine in a shaking incubator, and the total proteome was extracted and quantified as described previously (Ahn et al., 2006). After quantification, 2-D gel electrophoresis; that is, isoelectric focusing (p
Synchronized L1-stage worms were grown on NGM agar plates with or without caffeine for 24 h. Total RNA was extracted from the worms with Trizol reagent (Sigma), purified, and reverse transcribed with M-MLV reverse transcriptase (Gibco BRL, USA) using oligo-dT primers (Promega, USA) to synthesize the first-strand cDNA. Respective cDNA products were PCR-amplified using the following primers:
The RNAi analysis was performed using the “RNAi-by-soaking” method, as described previously (Min et al., 2015).
Synchronized L1-stage worms were grown on NGM agar plates at 20°C with or without caffeine for 24 h, and heat shock (30°C for 6 h) was used as a positive control. Worms were immobilized with levamisole before mounting on 2% agarose pads for microscopic observation with a Zeiss Axioskop 2 MOT fluorescence microscope (Carl Zeiss, Germany). Images were acquired under the same exposure conditions. Average pixel intensity values were calculated by sampling images from several animals. We calculated the mean and maximum pixel intensity for each animal from these images using Image J software (
Synchronized L1-stage worms were placed at the center of 0, 10, and 30 mM caffeine-containing NGM plates where a uniform
All experiments were repeated more than three times for statistical analysis. Student’s
Comparative proteomic analyses were carried out by comparing proteomes extracted from worms grown with or without caffeine to determine the
High-dose caffeine treatment is a developmental stressor because it inhibits the
Up-regulation of
To confirm induction of HSPs after caffeine treatment
Larval development was retarded when
The
Caffeine treatment has both positive and negative effects on physiological functions in a dose-dependent fashion. In general, a lower-dose results in positive effects and higher-doses result in negative effects. Therefore, understanding the mechanism of the lethal effects of high-dose caffeine treatment is important, as caffeine consumption is increasing worldwide. In this study, we performed a comparative proteomic analysis to identify caffeine-responsive proteins after treating
Many of the proteins that were up-regulated in response to caffeine have critical functions in the developmental processes (Supplementary Table S1). RNAi depletion of them causes either larval arrest or growth defects (
The balance between unfolded proteins and chaperones is regulated by the unfolded protein response (UPR) (Benedetti et al., 2006). In
In addition, HSP-4 alleviated food-avoidance behavior (aversion phenotype) in
Nevertheless, it is clear that expression of HSPs is critical. In summary, high-dose caffeine activated the UPR pathway and induced the expression of chaperone genes that protected worms from stress and alleviated the aversion phenotype.
. Differentially expressed heat shock proteins (HSPs) in worms grown with 30 mM caffeine
Spot ID | Gene name | Protein identified | Theoretical mass (kDa)/ p | Fold changed | % match |
---|---|---|---|---|---|
18471 | HSP 70 superfamily | 76.0./6.7 | +6.79 | 18 | |
18486 | HSP 70 superfamily | 72.3/5.0 | +2.72 | 23 | |
19229 | HSP 16 superfamily | 16.2/5.4 | +2.25 | 76 | |
694 | HSP 16 superfamily | 16.2/5.3 | +2.55 | 26 | |
18433 | HSP 70 superfamily | 73.0/5.0 | ? 4.88 | 27 |
Mol. Cells 2016; 39(2): 163-168
Published online February 29, 2016 https://doi.org/10.14348/molcells.2016.2298
Copyright © The Korean Society for Molecular and Cellular Biology.
Mohammad Al-Amin, Ichiro Kawasaki, Joomi Gong, and Yhong-Hee Shim*
Department of Bioscience and Biotechnology and Institute of KU Bio-technology, Konkuk University, Seoul 143-701, Korea
Correspondence to:*Correspondence: yshim@konkuk.ac.kr
Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner.
Keywords: caffeine,
Caffeine is one of the most popular drugs in the world and its consumption had increased rapidly in modern society. Most of the caffeine consumed is from coffee. According to “caffeine informer” 2013 data, 2.6 kg coffee per capita (per person on average) is consumed in South Korea, making South Korea the 26th top coffee consuming country in the world. An optimal dose of caffeine has many beneficial effects, such increasing attention and focus, improving athletic performance, and improving many other aspects of health (Reviewed in Weinberg and Bealer, 2002). In contrast, caffeine is an addictive drug and has side effects, particularly during pregnancy (Kuczkowasaki, 2009). High caffeine consumption may increase the risk for high blood pressure, hypertension, insomnia, and panic disorder (Mesas et al., 2011; Vilarim et al., 2011). High caffeine consumption also has maternal effects during pregnancy, which are related with a greater risk for low birth weight infants (Chen et al, 2014). A lethal human caffeine dose is approximately 10 g (one cup of coffee contains ∼125 mg of caffeine) (Ekwall et al., 1998). Although high doses of caffeine can be lethal, the mode of action remains unclear.
In our previous study, we investigated effects of caffeine on
Maintenance and handling of
Appropriate amounts of caffeine (Sigma, USA) were included in the NGM before autoclaving to obtain final concentrations of 10-30 mM. After
Synchronized L1-stage worms were cultured in NGM containing 0 or 30 mM caffeine in a shaking incubator, and the total proteome was extracted and quantified as described previously (Ahn et al., 2006). After quantification, 2-D gel electrophoresis; that is, isoelectric focusing (p
Synchronized L1-stage worms were grown on NGM agar plates with or without caffeine for 24 h. Total RNA was extracted from the worms with Trizol reagent (Sigma), purified, and reverse transcribed with M-MLV reverse transcriptase (Gibco BRL, USA) using oligo-dT primers (Promega, USA) to synthesize the first-strand cDNA. Respective cDNA products were PCR-amplified using the following primers:
The RNAi analysis was performed using the “RNAi-by-soaking” method, as described previously (Min et al., 2015).
Synchronized L1-stage worms were grown on NGM agar plates at 20°C with or without caffeine for 24 h, and heat shock (30°C for 6 h) was used as a positive control. Worms were immobilized with levamisole before mounting on 2% agarose pads for microscopic observation with a Zeiss Axioskop 2 MOT fluorescence microscope (Carl Zeiss, Germany). Images were acquired under the same exposure conditions. Average pixel intensity values were calculated by sampling images from several animals. We calculated the mean and maximum pixel intensity for each animal from these images using Image J software (
Synchronized L1-stage worms were placed at the center of 0, 10, and 30 mM caffeine-containing NGM plates where a uniform
All experiments were repeated more than three times for statistical analysis. Student’s
Comparative proteomic analyses were carried out by comparing proteomes extracted from worms grown with or without caffeine to determine the
High-dose caffeine treatment is a developmental stressor because it inhibits the
Up-regulation of
To confirm induction of HSPs after caffeine treatment
Larval development was retarded when
The
Caffeine treatment has both positive and negative effects on physiological functions in a dose-dependent fashion. In general, a lower-dose results in positive effects and higher-doses result in negative effects. Therefore, understanding the mechanism of the lethal effects of high-dose caffeine treatment is important, as caffeine consumption is increasing worldwide. In this study, we performed a comparative proteomic analysis to identify caffeine-responsive proteins after treating
Many of the proteins that were up-regulated in response to caffeine have critical functions in the developmental processes (Supplementary Table S1). RNAi depletion of them causes either larval arrest or growth defects (
The balance between unfolded proteins and chaperones is regulated by the unfolded protein response (UPR) (Benedetti et al., 2006). In
In addition, HSP-4 alleviated food-avoidance behavior (aversion phenotype) in
Nevertheless, it is clear that expression of HSPs is critical. In summary, high-dose caffeine activated the UPR pathway and induced the expression of chaperone genes that protected worms from stress and alleviated the aversion phenotype.
. Differentially expressed heat shock proteins (HSPs) in worms grown with 30 mM caffeine.
Spot ID | Gene name | Protein identified | Theoretical mass (kDa)/ p | Fold changed | % match |
---|---|---|---|---|---|
18471 | HSP 70 superfamily | 76.0./6.7 | +6.79 | 18 | |
18486 | HSP 70 superfamily | 72.3/5.0 | +2.72 | 23 | |
19229 | HSP 16 superfamily | 16.2/5.4 | +2.25 | 76 | |
694 | HSP 16 superfamily | 16.2/5.3 | +2.55 | 26 | |
18433 | HSP 70 superfamily | 73.0/5.0 | ? 4.88 | 27 |
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