JOURNAL OF MEDICINAL FOOD J Med Food 17 (9) 2014, 1003–1010 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.0175
Syzygium aromaticum L. (Clove) Extract Regulates Energy Metabolism in Myocytes Zheng Tu, Tijuana Moss-Pierce, Paul Ford, and T. Alan Jiang Technical Innovation Center, McCormick and Company, Inc., Hunt Valley, Maryland, USA. ABSTRACT The prevalence of metabolic syndrome and type 2 diabetes is increasing worldwide. Herbs and spices have been used for the treatment of diabetes for centuries in folk medicine. Syzygium aromaticum L. (Clove) extracts (SE) have been shown to perform comparably to insulin by significantly reducing blood glucose levels in animal models; however, the mechanisms are not well understood. We investigated the effects of clove on metabolism in C2C12 myocytes and demonstrated that SE significantly increases glucose consumption. The phosphorylation of AMP-activated protein kinase (AMPK), as well as its substrate, acetyl-CoA carboxylase (ACC) was increased by SE treatment. SE also transcriptionally regulates genes involved in metabolism, including sirtuin 1 (SIRT1) and PPARc coactivator 1a (PGC1a). Nicotinamide, an SIRT1 inhibitor, diminished SE’s effects on glucose consumption. Furthermore, treatment with SE dose-dependently increases muscle glycolysis and mitochondrial spare respiratory capacity. Overall, our study suggests that SE has the potential to increase muscle glycolysis and mitochondria function by activating both AMPK and SIRT1 pathways.
KEY WORDS: AMPK energy metabolism mitochondria PGC1a SIRT1 Syzygium aromaticum L. (Clove)
have been shown to significantly reduce serum glucose, triglyceride, and total cholesterol levels, and to repress genes encoding gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in both in vivo and in vitro models.12–14 The mechanisms, however, have yet to be elucidated. From earlier in vitro screenings on herbs and spices, we found that SE increases glucose consumption in muscle cells. In the present study, to understand the mechanisms by which SE regulates cellular metabolism, we investigated the effect of SE on critical metabolic pathways, including AMPK and SIRT1-PGC1a, in addition to glucose consumption and glycolysis in differentiated C2C12 cells. AMPK is a serine/threonine kinase that functions as an intracellular energy sensor, activated under conditions of low energy, such as elevated AMP/ATP ratio.15–17 The activation of AMPK switches off anabolic pathways that consume ATP, such as fatty acid, glycogen, and cholesterol synthesis, and switches on catabolic pathways which generate ATP, such as fatty acid oxidation and glycolysis.17 SIRT1 is an NAD + -sensing deacetylase. Induction of SIRT1 expression increases insulin sensitivity.18 PGC1a belongs to a family of transcription coactivators that regulate metabolic genes by activating a variety of nuclear receptors, including peroxisome proliferator-activated receptor (PPAR) a, b, c, estrogen receptor a (ERa), glucocorticoid receptor (GR), and liver X receptors (LXR).19 SIRT1-PGC1a pathway plays a critical role in the maintenance of mitochondrial function, thermogenesis, and energy homeostasis.20,21 Our data demonstrate that SE activates AMPK and SIRT1 pathways,
INTRODUCTION
T
he prevalence of obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM) has been increasing over the past decades due to the adoption of a sedentary lifestyle combined with excessive caloric intake. The number of people with diagnosed and undiagnosed diabetes in the United States reached 25.8 million in 2011, which represented 8.3% of the general population.1 A misbalance of energy homeostasis has been considered a main contributor to T2DM.2 Previous studies demonstrated that energy sensors, such as AMP-activated protein kinase (AMPK) and SIRT1, are vital links in a regulatory network for metabolic homeostasis.3–5 Understanding the mechanisms by which they act could help identify preventive and therapeutic strategies for metabolic diseases. Meanwhile, increasing evidence shows that a diet rich in phytochemicals and polyphenolic compounds may be related to a lower risk of T2DM and related metabolic disorders.6,7 Culinary herbs and spices have a long history of use in preventing and treating metabolic disorders, including T2DM.8–10 Syzygium aromaticum L. (Clove) is an important ingredient used in the preparation of meats, salad dressings, and desserts; it is popular in countries, including India, Sri Lanka, China, and Germany.11 S. aromaticum extracts (SE) Manuscript received 3 December 2013. Revision accepted 12 March 2014. Address correspondence to: Zheng Tu, MD, PhD, Technical Innovation Center, McCormick and Company, Inc., 204 Wight Avenue, Hunt Valley, MD 21031, USA, E-mail: [email protected]
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thereby possibly playing a role in regulating glucose and lipid metabolism in C2C12 myocytes. MATERIALS AND METHODS Reagents Metformin, resveratrol, insulin, nicotinamide, and antibody against b-actin were purchased from Sigma (St. Louis, MO, USA). Anti-phospho-AMPKa, anti-AMPKa, anti-phosphoACC, anti-ACC, and anti-SIRT1 were purchased from Cell Signaling (Danvers, MA, USA). Anti-PGC1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Preparation of S. aromaticum L. extracts Whole clove was ground, passed through a 40-mesh sieve, and extracted following protocols established by the National Cancer Institute.22 Briefly, the ground material was steeped overnight at room temperature in dichloromethane-methanol (CH2Cl2-MeOH, 1:1). The filtrate was collected under a slight vacuum followed by an MeOH extraction (30 min) on the remaining material. The MeOH eluent was combined with the other extract, and the solvent was removed from the combined extracts by rotary evaporation. Aliquots of SE were diluted in DMSO at 10 mg/mL and stored at - 20C. Cell culture, differentiation, and treatment C2C12 cells were purchased from ATCC (CRL-1772) and maintained in DMEM (Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS, penicillin/streptomycin (Invitrogen; 15140-122). For differentiation, C2C12 cells were plated on 6- or 96-well plates. Two days after plating, the cells reached confluence. Differentiation was then induced by switching the growth medium to differentiation medium (DMEM supplemented with 2% horse serum). The differentiation medium was changed every 24 h for 4 days. Then, cells were treated with or without different concentrations of SE or positive controls.
have been previously described.23 Briefly, C2C12 myocytes were treated with reagents indicated for 2 h (Fig. 3) or 16 h (Fig. 4). Cells were lysed with RIPA buffer supplied with 1 · Halt protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Scientific), separated by SDS-PAGE, and transferred to a polyvinylidene fluoride membrane, and endogenous proteins were detected by western blot using specific antibodies. RNA extraction and real-time PCR Total RNA was prepared from C2C12 myocytes using Trizol (Invitrogen) and RNeazy kit (Qiagen, Germantown, MD, USA) as previously described.24 The cDNA was generated from 2 lg of total RNA using cDNA Reverse Transcription kits (Applied Biosystems, Carlsbad, CA, USA). The samples were than analyzed using a real-time PCR system Via7 (Applied Biosystems). The primers and probes included mouse PGC1a (Mm01208835), SIRT1 (Mm00490758), and 18S rRNA (Hs99999901) as a reference gene. Glycolysis assay Glycolysis was determined by measuring the Extracellular Acidification Rate (ECAR) using a Seahorse XF96 analyzer (Seahorse Bioscience, North Billerica, MA, USA).25 C2C12 myocytes were treated with SE or metformin, resveratrol for 16 h. At that time, the medium was changed to unbuffered serum-free DMEM (Seahorse Bioscience) without glucose for 1 h. The measurement protocol used is as follows25: After four baseline measurements, 10 mM glucose and 100 mM 2-deoxyglucose (2-DG) were injected sequentially. For each injection, the ECAR value was monitored at four successive 4-min intervals, with a 2-min inter-measurement mixing. The experiment was performed thrice, and each condition had five replicates. Area under the curve (AUC) of ECAR was determined by plotting the acidification rate of the medium as a function of time, and normalized to baseline. Determination of NAD + /NADH ratio
Glucose consumption assay The glucose concentration in culture medium was measured using a glucose assay kit (Sigma) following the manufacturer’s instructions. Briefly, after treatment, 10 lL of cell culture medium were diluted to 100 lL with distilled H2O. Assay reagent (200 lL) was then added to each sample. The color reaction was stopped with 200 lL of 12 N H2SO4. OD was measured at 540 nm. Glucose consumption was calculated using the starting glucose concentration in culture medium (1 mg/mL) minus the glucose concentration measured at the end of the experiment. Glucose consumption was normalized to protein level of the cells. Cell protein level was quantitated with Coomassie Plus (Thermo Scientific, Waltham, MA, USA).
The ratio of NAD + /NADH was measured using an NAD + /NADH Quantification Colorimetric Kit (Biovision, Milpitas, CA, USA) following the manufacturer’s instructions. Briefly, cells were lysed with NADH/NAD Extraction Buffer by two freeze/thaw cycles followed by centrifugation. Fifty mL of extracted samples were loaded into a 96well plate to detect total NADt (NADH and NAD). For NADH, aliquot of the lysates was heated to 60C for 30 min to decompose NAD. Fifty microliter of NAD-decomposed samples were also loaded into the 96-well plate. OD was read at 450 nm. After measurement, the ratio of NAD + / NADH was calculated as (NADt–NADH)/NADH.
Western blotting
The measurement of mitochondria spare respiratory capacity
Cell processing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and membrane transfer
Mitochondria function was determined by measuring the Oxygen Consumption Rate (OCR) using a Seahorse XF96
1005
CLOVE ACTIVATES AMPK AND SIRT1 PATHWAYS *: p
Syzygium aromaticum L. (Clove) Extract Regulates Energy Metabolism in Myocytes Zheng Tu, Tijuana Moss-Pierce, Paul Ford, and T. Alan Jiang Technical Innovation Center, McCormick and Company, Inc., Hunt Valley, Maryland, USA. ABSTRACT The prevalence of metabolic syndrome and type 2 diabetes is increasing worldwide. Herbs and spices have been used for the treatment of diabetes for centuries in folk medicine. Syzygium aromaticum L. (Clove) extracts (SE) have been shown to perform comparably to insulin by significantly reducing blood glucose levels in animal models; however, the mechanisms are not well understood. We investigated the effects of clove on metabolism in C2C12 myocytes and demonstrated that SE significantly increases glucose consumption. The phosphorylation of AMP-activated protein kinase (AMPK), as well as its substrate, acetyl-CoA carboxylase (ACC) was increased by SE treatment. SE also transcriptionally regulates genes involved in metabolism, including sirtuin 1 (SIRT1) and PPARc coactivator 1a (PGC1a). Nicotinamide, an SIRT1 inhibitor, diminished SE’s effects on glucose consumption. Furthermore, treatment with SE dose-dependently increases muscle glycolysis and mitochondrial spare respiratory capacity. Overall, our study suggests that SE has the potential to increase muscle glycolysis and mitochondria function by activating both AMPK and SIRT1 pathways.
KEY WORDS: AMPK energy metabolism mitochondria PGC1a SIRT1 Syzygium aromaticum L. (Clove)
have been shown to significantly reduce serum glucose, triglyceride, and total cholesterol levels, and to repress genes encoding gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in both in vivo and in vitro models.12–14 The mechanisms, however, have yet to be elucidated. From earlier in vitro screenings on herbs and spices, we found that SE increases glucose consumption in muscle cells. In the present study, to understand the mechanisms by which SE regulates cellular metabolism, we investigated the effect of SE on critical metabolic pathways, including AMPK and SIRT1-PGC1a, in addition to glucose consumption and glycolysis in differentiated C2C12 cells. AMPK is a serine/threonine kinase that functions as an intracellular energy sensor, activated under conditions of low energy, such as elevated AMP/ATP ratio.15–17 The activation of AMPK switches off anabolic pathways that consume ATP, such as fatty acid, glycogen, and cholesterol synthesis, and switches on catabolic pathways which generate ATP, such as fatty acid oxidation and glycolysis.17 SIRT1 is an NAD + -sensing deacetylase. Induction of SIRT1 expression increases insulin sensitivity.18 PGC1a belongs to a family of transcription coactivators that regulate metabolic genes by activating a variety of nuclear receptors, including peroxisome proliferator-activated receptor (PPAR) a, b, c, estrogen receptor a (ERa), glucocorticoid receptor (GR), and liver X receptors (LXR).19 SIRT1-PGC1a pathway plays a critical role in the maintenance of mitochondrial function, thermogenesis, and energy homeostasis.20,21 Our data demonstrate that SE activates AMPK and SIRT1 pathways,
INTRODUCTION
T
he prevalence of obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM) has been increasing over the past decades due to the adoption of a sedentary lifestyle combined with excessive caloric intake. The number of people with diagnosed and undiagnosed diabetes in the United States reached 25.8 million in 2011, which represented 8.3% of the general population.1 A misbalance of energy homeostasis has been considered a main contributor to T2DM.2 Previous studies demonstrated that energy sensors, such as AMP-activated protein kinase (AMPK) and SIRT1, are vital links in a regulatory network for metabolic homeostasis.3–5 Understanding the mechanisms by which they act could help identify preventive and therapeutic strategies for metabolic diseases. Meanwhile, increasing evidence shows that a diet rich in phytochemicals and polyphenolic compounds may be related to a lower risk of T2DM and related metabolic disorders.6,7 Culinary herbs and spices have a long history of use in preventing and treating metabolic disorders, including T2DM.8–10 Syzygium aromaticum L. (Clove) is an important ingredient used in the preparation of meats, salad dressings, and desserts; it is popular in countries, including India, Sri Lanka, China, and Germany.11 S. aromaticum extracts (SE) Manuscript received 3 December 2013. Revision accepted 12 March 2014. Address correspondence to: Zheng Tu, MD, PhD, Technical Innovation Center, McCormick and Company, Inc., 204 Wight Avenue, Hunt Valley, MD 21031, USA, E-mail: [email protected]
1003
1004
TU ET AL.
thereby possibly playing a role in regulating glucose and lipid metabolism in C2C12 myocytes. MATERIALS AND METHODS Reagents Metformin, resveratrol, insulin, nicotinamide, and antibody against b-actin were purchased from Sigma (St. Louis, MO, USA). Anti-phospho-AMPKa, anti-AMPKa, anti-phosphoACC, anti-ACC, and anti-SIRT1 were purchased from Cell Signaling (Danvers, MA, USA). Anti-PGC1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Preparation of S. aromaticum L. extracts Whole clove was ground, passed through a 40-mesh sieve, and extracted following protocols established by the National Cancer Institute.22 Briefly, the ground material was steeped overnight at room temperature in dichloromethane-methanol (CH2Cl2-MeOH, 1:1). The filtrate was collected under a slight vacuum followed by an MeOH extraction (30 min) on the remaining material. The MeOH eluent was combined with the other extract, and the solvent was removed from the combined extracts by rotary evaporation. Aliquots of SE were diluted in DMSO at 10 mg/mL and stored at - 20C. Cell culture, differentiation, and treatment C2C12 cells were purchased from ATCC (CRL-1772) and maintained in DMEM (Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS, penicillin/streptomycin (Invitrogen; 15140-122). For differentiation, C2C12 cells were plated on 6- or 96-well plates. Two days after plating, the cells reached confluence. Differentiation was then induced by switching the growth medium to differentiation medium (DMEM supplemented with 2% horse serum). The differentiation medium was changed every 24 h for 4 days. Then, cells were treated with or without different concentrations of SE or positive controls.
have been previously described.23 Briefly, C2C12 myocytes were treated with reagents indicated for 2 h (Fig. 3) or 16 h (Fig. 4). Cells were lysed with RIPA buffer supplied with 1 · Halt protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Scientific), separated by SDS-PAGE, and transferred to a polyvinylidene fluoride membrane, and endogenous proteins were detected by western blot using specific antibodies. RNA extraction and real-time PCR Total RNA was prepared from C2C12 myocytes using Trizol (Invitrogen) and RNeazy kit (Qiagen, Germantown, MD, USA) as previously described.24 The cDNA was generated from 2 lg of total RNA using cDNA Reverse Transcription kits (Applied Biosystems, Carlsbad, CA, USA). The samples were than analyzed using a real-time PCR system Via7 (Applied Biosystems). The primers and probes included mouse PGC1a (Mm01208835), SIRT1 (Mm00490758), and 18S rRNA (Hs99999901) as a reference gene. Glycolysis assay Glycolysis was determined by measuring the Extracellular Acidification Rate (ECAR) using a Seahorse XF96 analyzer (Seahorse Bioscience, North Billerica, MA, USA).25 C2C12 myocytes were treated with SE or metformin, resveratrol for 16 h. At that time, the medium was changed to unbuffered serum-free DMEM (Seahorse Bioscience) without glucose for 1 h. The measurement protocol used is as follows25: After four baseline measurements, 10 mM glucose and 100 mM 2-deoxyglucose (2-DG) were injected sequentially. For each injection, the ECAR value was monitored at four successive 4-min intervals, with a 2-min inter-measurement mixing. The experiment was performed thrice, and each condition had five replicates. Area under the curve (AUC) of ECAR was determined by plotting the acidification rate of the medium as a function of time, and normalized to baseline. Determination of NAD + /NADH ratio
Glucose consumption assay The glucose concentration in culture medium was measured using a glucose assay kit (Sigma) following the manufacturer’s instructions. Briefly, after treatment, 10 lL of cell culture medium were diluted to 100 lL with distilled H2O. Assay reagent (200 lL) was then added to each sample. The color reaction was stopped with 200 lL of 12 N H2SO4. OD was measured at 540 nm. Glucose consumption was calculated using the starting glucose concentration in culture medium (1 mg/mL) minus the glucose concentration measured at the end of the experiment. Glucose consumption was normalized to protein level of the cells. Cell protein level was quantitated with Coomassie Plus (Thermo Scientific, Waltham, MA, USA).
The ratio of NAD + /NADH was measured using an NAD + /NADH Quantification Colorimetric Kit (Biovision, Milpitas, CA, USA) following the manufacturer’s instructions. Briefly, cells were lysed with NADH/NAD Extraction Buffer by two freeze/thaw cycles followed by centrifugation. Fifty mL of extracted samples were loaded into a 96well plate to detect total NADt (NADH and NAD). For NADH, aliquot of the lysates was heated to 60C for 30 min to decompose NAD. Fifty microliter of NAD-decomposed samples were also loaded into the 96-well plate. OD was read at 450 nm. After measurement, the ratio of NAD + / NADH was calculated as (NADt–NADH)/NADH.
Western blotting
The measurement of mitochondria spare respiratory capacity
Cell processing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and membrane transfer
Mitochondria function was determined by measuring the Oxygen Consumption Rate (OCR) using a Seahorse XF96
1005
CLOVE ACTIVATES AMPK AND SIRT1 PATHWAYS *: p
