Abstract
Women usually experience body weight gain with aging, which can put them at risk for many chronic diseases. Previous studies indicated that melatonin treatment attenuates body weight gain and abdominal fat deposition in several male animals. However, it is unclear whether melatonin affects female animals in the same way. This study investigated whether long-term melatonin treatment can attenuate body weight gain with aging and, if it does, what the mechanism is. Ten-week-old female ICR mice were given melatonin-containing water (100 μg/mL) or only water until 43 weeks. Melatonin treatment significantly attenuated body weight gain at 23 weeks (control; 57.2 ± 2.0 g vs melatonin; 44.4 ± 3.1 g), 33 weeks (control; 65.4 ± 2.6 g vs melatonin; 52.2 ± 4.2 g) and 43 weeks (control; 66.1 ± 3.2 g vs melatonin; 54.4 ± 2.5 g) without decreasing the amount of food intake. Micro-CT analyses showed that melatonin significantly decreased the deposition of visceral and s.c. fat. These results suggested that melatonin attenuates body weight gain by inhibiting abdominal fat deposition. Metabolome analysis of the liver revealed that melatonin treatment induced a drastic change in the metabolome with the downregulation of 149 metabolites, including the metabolites of glucose and amino acids. Citrate, which serves as a source of de novo lipogenesis, was one of the downregulated metabolites. These results show that long-term melatonin treatment induces drastic changes in metabolism and attenuates body weight gain and fat deposition with aging in female mice.
Introduction
Human obesity has become a global epidemic. Obesity increases the risk of many health problems, including diabetes mellitus, metabolic syndrome, cardiovascular diseases and cancer and hence leads to higher mortality (Flegal et al. 2013, Aune et al. 2016, Global et al. 2016). Therefore, obesity has been recognized as a public health problem. People usually experience body weight gain in middle age. This owes to the concurrence of different factors, such as inactivity, poor nutritional habits, and a decrease of basal metabolism, which increase abdominal fat deposition, particularly in visceral sites (Inelmen et al. 2003). Women especially experience weight gain around menopause (Guthrie et al. 1999). In fact, the prevalence of obesity in the elderly is higher in women than in men (Perissinotto et al. 2002). One of the likely mechanisms is the reduction in estrogen levels due to progressive loss of ovarian function (Mauvais-Jarvis et al. 2013).
Recently, obesity has been increasing not only in adults but also in children and adolescents worldwide (Wang & Lim 2012). Children and adolescents are likely to maintain their obese status into adulthood and are at high risk for developing chronic diseases in adulthood such as hypertension, dyslipidemia, diabetes mellitus, cardiovascular diseases and cancers (Wang & Lim 2012, Cote et al. 2013). Therefore, treatment and prevention of obesity should begin in childhood (Cunningham et al. 2014, Gortmaker & Taveras 2014). The prevalence of overweight and obesity in young adult women has dramatically increased in recent years (Wane et al. 2010). Compared with men, young adult women with obesity have risks associated with reproduction. Overweight and obesity in women are associated with infertility (Lainez & Coss 2019) and pregnancy complications, such as pre-eclampsia (Hurter et al. 2019), gestational diabetes mellitus (Chatzakis et al. 2019) and fetal macrosomia (Gaudet et al. 2014). Therefore, it is important to control body weight in women throughout life.
Melatonin (N-acetyl-5-methoxytryptamine) is a molecule secreted by the pineal gland in a circadian manner. In addition to regulating circadian rhythms, melatonin has a significant role in the regulation of metabolism, such as lipid metabolism and glucose metabolism (Tamura et al. 2008a, Cipolla-Neto et al. 2014, Szewczyk-Golec et al. 2015). Melatonin treatment reduced the body weight and abdominal fat deposition in male animals in several species (Prunet-Marcassus et al. 2003, Agil et al. 2011, Nduhirabandi et al. 2011, Favero et al. 2015). It is interesting that pineal melatonin secretion decreases with aging, which may contribute to weight gain with aging (Rasmussen et al. 2001, Cipolla-Neto et al. 2014). These observations suggested that melatonin could be a therapeutic reagent for the prevention of body weight gain with aging. However, studies of the effect of melatonin on body weight were done in male animals of several species. Thus, little is known about the inhibitory effect of melatonin on body weight gain in female animals. In addition, most studies on the effect of melatonin on weight were done on animals that were fed high-fat diets (Prunet-Marcassus et al. 2003, Rios-Lugo et al. 2010, Agil et al. 2011, Nduhirabandi et al. 2011). It remains unclear whether melatonin has similar effects in animals fed a normal diet. Furthermore, the mechanisms by which melatonin attenuates weight gain have not been fully clarified.
We have reported that melatonin works as an antioxidant within the ovarian follicle and protects oocytes and granulosa cells against oxidative stress, and that melatonin treatment improves fertilization and pregnancy rates in women with infertility (Tamura et al. 2008b, Tamura et al. 2009, 2012, 2013, 2014, 2020). In addition, we recently established a mouse model for examining the effects of long-term melatonin treatment on ovarian aging (Tamura et al. 2017). In this model, 10-week-old (corresponding to the adolescence of human) female mice were treated with melatonin until the age of 43 weeks (corresponding to the perimenopausal period of human). This long-term melatonin treatment delayed ovarian aging. Interestingly, in that study, we noticed that long-term melatonin treatment attenuated body weight gain with aging.
In this study, we examined the effect of long-term melatonin treatment on body weight gain with aging in normal female mice. We also analyzed the metabolome to examine how melatonin affects metabolisms and attenuates body weight gain.
Materials and methods
Animals
ICR mice were purchased from Japan SLC (Hamamatsu, Japan). The mice were housed at 24°C under controlled conditions (lights on from 5:00 to 19:00 h) under specific pathogen-free conditions and fed standard chow according to our published protocol (Tamura et al. 2017). All experimental protocols were approved by the Committee for Ethics on Animal Experimentation and performed under the guidelines for animal experiments at Yamaguchi University Graduate School of Medicine in accordance with Law No. 105 and Notification No. 6 of the Japanese government.
Experimental procedures
We first examined the effect of melatonin on body weight gain during aging as a pilot study. We performed the experiment according to the previous report (Nava et al. 2003). Ten female ICR mice (10 weeks old) were randomly allocated to two groups. Half were housed together and given only water to drink and the other half were given water supplemented with 100 μg/mL melatonin until 43 weeks of age. Melatonin was provided in the drinking water during the day as well as at night. The previous report showed that the 100 μg/mL of melatonin in drinking water increased serum melatonin levels up to 1~3 ng/mL (Nava et al. 2003). Body weights were measured at 10, 23, 33 and 43 weeks. To confirm reproducibility and to examine the possible mechanisms for the effect of melatonin on body weight gain, 16 female ICR mice (10 weeks old) were randomly allocated to two groups and were treated in the same way. The amount of food intake was checked every day per cage by subtracting the remaining food before feeding. The average daily food intake per one mouse was calculated in each group. At 43 weeks, these mice were subjected to the experiments described subsequently.
Quantification of abdominal fat deposition by micro-computed tomography imaging
Mice were anesthetized with 2% isoflurane (Wako Pure Chemical Industries), and CT images were acquired by 3D micro-CT (R_mCT2; Rigaku, Tokyo, Japan) as reported previously (Kina-Tanada et al. 2017). The CT images were visualized and analyzed using CTAtlas Metabolic Analysis Ver. 2.03 software (Rigaku). Abdominal fat was measured from the base of the ensiform cartilage to the pelvic floor and was divided into visceral and s.c. fat. Fat mass was expressed as a percentage of the body volume scanned.
Metabolome analysis
Mice were euthanized by cervical dislocation at 43 weeks. Liver tissues from five control and five melatonin-treated mice were used for metabolome analysis. Livers were harvested at the same time. Metabolome analysis was performed at Human Metabolome Technologies (Tsuruoka, Japan) as reported previously (Matsui et al. 2017). We analyzed 272 metabolites that were measurable in at least one out of ten mice. Statistical differences in single metabolites between two groups were evaluated with Welch’s two-sample t-test. Metabolites with a P-value less than 0.05 were considered as up- or downregulated metabolites by melatonin. The metabolomic data were subjected to hierarchical cluster analysis and principal component analysis using software (Human Metabolome Technologies). Data were also visualized on a metabolome-wide pathway map supported by VANTED software (https://immersive-analytics.infotech.monash.edu/vanted/). Pathways associated with metabolites that are downregulated by melatonin were analyzed with ConsensusPathDB (Kamburov et al. 2013). The 272 measurable metabolites were used as a background list. Pathways with a P-value less than 0.05 were considered as enriched pathways.
Statistical analysis
Statistics were analyzed with SPSS for Windows 13.0 (SPSS Inc.). Differences between the two groups were analyzed with an unpaired t-test. Differences were considered significant at P < 0.05.
Results
Effects of long-term melatonin treatment on body weight and fat deposition with aging
We first examined whether long-term melatonin treatment attenuates body weight gain during aging in female mice. Compared with the control, melatonin treatment significantly attenuated body weight gain at 23 weeks (control; 57.2 ± 2.0 g vs melatonin; 44.4 ± 3.1 g, P = 0.008), 33 weeks (control; 65.4 ± 2.6 g vs melatonin; 52.2 ± 4.2 g, P = 0.03) and 43 weeks (control; 66.1 ± 3.2 g vs melatonin; 54.4 ± 2.5 g, P = 0.003) (Fig. 1A). We then examined several parameters associated with body weight gain at 43 weeks. Final body weight at 43 weeks (control; 72.4 ± 2.8 g vs melatonin; 60.4 ± 3.1 g, P = 0.02) and total weight gain from 23 to 43 weeks were significantly lower in melatonin-treated mice than control mice (Fig. 1B). The amount of food intake was not different between the two groups while it was higher in melatonin-treated mice when corrected for final body weight (Fig. 1B). Micro-CT measurements showed that the depositions of visceral and s.c. fat were significantly lower in the melatonin-treated mice (Fig. 1C and D), suggesting that long-term melatonin treatment attenuates body weight gain with aging by inhibiting abdominal fat deposition.
Effects of long-term melatonin treatment on body weight and fat deposition with aging. (A) Effect of melatonin on body weight from 10 to 43 weeks. Five 10-weeks mice were given only water to drink as a control group, and five 10 weeks-mice were given water supplemented with 100 μg/mL melatonin as a melatonin-treated group until 43 weeks of age. Body weights were measured at 10, 23, 33 and 43 weeks of age. Values are mean ± s.e. Mean weight at each week; 10 weeks (control group; 33.4 g vs melatonin group; 33.1 g), 23 weeks (control group; 57.2 g vs melatonin group; 44.4 g), 33 weeks (control group; 65.4 g vs melatonin group; 52.2 g), 43 weeks (control group; 66.1 g vs melatonin group 54.4 g). *P < 0.05, **P < 0.01 vs control. (B) Control group mice (n = 8) and melatonin-treated group mice (n = 8) were treated in the same way until 43 weeks. Bodyweight at 43 weeks, total body weight gain from 10 to 43 weeks and amounts of food intake (g/day) were measured. Corrected food intake per final body weight is also shown. Values of each mouse are shown in dot plots. Mean ± s.e. is also presented. *P < 0.05, **P < 0.01 vs control. (C) Representative micro-CT images of abdominal fat (upper, transverse images; lower, coronal images) in control and melatonin-treated mice at 43 weeks. Abdominal fat was measured from the base of the ensiform cartilage to the pelvic floor and was divided into visceral fat (yellow) and s.c. fat (orange). (D) Quantification of fat deposition. Visceral fat deposition and s.c. fat deposition are expressed as a percentage of the scanned body volume. Values of each mouse are shown in dot plots. Mean ± s.e. is also presented. *P < 0.05 vs control.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Effects of long-term melatonin treatment on body weight and fat deposition with aging. (A) Effect of melatonin on body weight from 10 to 43 weeks. Five 10-weeks mice were given only water to drink as a control group, and five 10 weeks-mice were given water supplemented with 100 μg/mL melatonin as a melatonin-treated group until 43 weeks of age. Body weights were measured at 10, 23, 33 and 43 weeks of age. Values are mean ± s.e. Mean weight at each week; 10 weeks (control group; 33.4 g vs melatonin group; 33.1 g), 23 weeks (control group; 57.2 g vs melatonin group; 44.4 g), 33 weeks (control group; 65.4 g vs melatonin group; 52.2 g), 43 weeks (control group; 66.1 g vs melatonin group 54.4 g). *P < 0.05, **P < 0.01 vs control. (B) Control group mice (n = 8) and melatonin-treated group mice (n = 8) were treated in the same way until 43 weeks. Bodyweight at 43 weeks, total body weight gain from 10 to 43 weeks and amounts of food intake (g/day) were measured. Corrected food intake per final body weight is also shown. Values of each mouse are shown in dot plots. Mean ± s.e. is also presented. *P < 0.05, **P < 0.01 vs control. (C) Representative micro-CT images of abdominal fat (upper, transverse images; lower, coronal images) in control and melatonin-treated mice at 43 weeks. Abdominal fat was measured from the base of the ensiform cartilage to the pelvic floor and was divided into visceral fat (yellow) and s.c. fat (orange). (D) Quantification of fat deposition. Visceral fat deposition and s.c. fat deposition are expressed as a percentage of the scanned body volume. Values of each mouse are shown in dot plots. Mean ± s.e. is also presented. *P < 0.05 vs control.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Effects of long-term melatonin treatment on body weight and fat deposition with aging. (A) Effect of melatonin on body weight from 10 to 43 weeks. Five 10-weeks mice were given only water to drink as a control group, and five 10 weeks-mice were given water supplemented with 100 μg/mL melatonin as a melatonin-treated group until 43 weeks of age. Body weights were measured at 10, 23, 33 and 43 weeks of age. Values are mean ± s.e. Mean weight at each week; 10 weeks (control group; 33.4 g vs melatonin group; 33.1 g), 23 weeks (control group; 57.2 g vs melatonin group; 44.4 g), 33 weeks (control group; 65.4 g vs melatonin group; 52.2 g), 43 weeks (control group; 66.1 g vs melatonin group 54.4 g). *P < 0.05, **P < 0.01 vs control. (B) Control group mice (n = 8) and melatonin-treated group mice (n = 8) were treated in the same way until 43 weeks. Bodyweight at 43 weeks, total body weight gain from 10 to 43 weeks and amounts of food intake (g/day) were measured. Corrected food intake per final body weight is also shown. Values of each mouse are shown in dot plots. Mean ± s.e. is also presented. *P < 0.05, **P < 0.01 vs control. (C) Representative micro-CT images of abdominal fat (upper, transverse images; lower, coronal images) in control and melatonin-treated mice at 43 weeks. Abdominal fat was measured from the base of the ensiform cartilage to the pelvic floor and was divided into visceral fat (yellow) and s.c. fat (orange). (D) Quantification of fat deposition. Visceral fat deposition and s.c. fat deposition are expressed as a percentage of the scanned body volume. Values of each mouse are shown in dot plots. Mean ± s.e. is also presented. *P < 0.05 vs control.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Effects of long-term melatonin treatment on the metabolome profiles of liver
The attenuation of body weight gain and fat deposition by melatonin led us to hypothesize that long-term melatonin treatment alters metabolism in the body. Many of the nutrients from food, such as glucose and amino acids, are metabolized in the liver. Therefore, we considered that the liver metabolome would reveal the effects of melatonin on the body’s metabolism. Among the 272 metabolites that were measurable, 149 (54.7%) were significantly downregulated by melatonin treatment whereas only 5 (1.8%) were upregulated (Fig. 2A and Supplementary Table 1, see section on supplementary materials given at the end of this article). A hierarchical cluster analysis of the 272 metabolites clearly separated the two groups, and the melatonin group had a number of downregulated metabolites (Fig. 2B). A principal component analysis also showed distinct clustering of metabolomic changes by melatonin treatment (Fig. 2C). These results show that long-term melatonin treatment drastically changed the liver metabolisms in female mice.
Effects of long-term melatonin treatment on the metabolome profile of liver. (A) Pie chart showing the numbers of downregulated, updownregulated and no-changed metabolites by melatonin. (B) Heat map of hierarchical cluster analysis comparing the measurable 272 metabolites between the control and melatonin group. The x-axis shows the five control mice (C1~C5) and five melatonin-treated mice (M1~M5). The degrees of red color and of green color indicate higher and lower contents of the metabolites, respectively. (C) Principal component analysis comparing the measurable 272 metabolites between the control and melatonin group. Blue and red dots show five control mice (C1~C5) and five melatonin-treated mice (M1~M5), respectively. They are clearly separated into two groups. Blue box; control group. Red box; melatonin group.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Effects of long-term melatonin treatment on the metabolome profile of liver. (A) Pie chart showing the numbers of downregulated, updownregulated and no-changed metabolites by melatonin. (B) Heat map of hierarchical cluster analysis comparing the measurable 272 metabolites between the control and melatonin group. The x-axis shows the five control mice (C1~C5) and five melatonin-treated mice (M1~M5). The degrees of red color and of green color indicate higher and lower contents of the metabolites, respectively. (C) Principal component analysis comparing the measurable 272 metabolites between the control and melatonin group. Blue and red dots show five control mice (C1~C5) and five melatonin-treated mice (M1~M5), respectively. They are clearly separated into two groups. Blue box; control group. Red box; melatonin group.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Effects of long-term melatonin treatment on the metabolome profile of liver. (A) Pie chart showing the numbers of downregulated, updownregulated and no-changed metabolites by melatonin. (B) Heat map of hierarchical cluster analysis comparing the measurable 272 metabolites between the control and melatonin group. The x-axis shows the five control mice (C1~C5) and five melatonin-treated mice (M1~M5). The degrees of red color and of green color indicate higher and lower contents of the metabolites, respectively. (C) Principal component analysis comparing the measurable 272 metabolites between the control and melatonin group. Blue and red dots show five control mice (C1~C5) and five melatonin-treated mice (M1~M5), respectively. They are clearly separated into two groups. Blue box; control group. Red box; melatonin group.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Changes in liver metabolisms by long-term melatonin treatment
Figure 3 shows the pathway maps in the metabolome analysis with the expression levels of various metabolites. Figure 3A is the pathway map of glucose and its related metabolites. During the first step of glycolysis, the absorbed glucose is metabolized into glucose 6-phosphate (G6P) and then converted to fructose 6-bisphosphate (F6P). These intermediates in the glycolysis pathway were significantly downregulated by melatonin treatment, suggesting that melatonin treatment inhibited glucose utilization by the attenuation of glucose uptake or inactivation of glycolytic enzymes.
Changes in liver metabolisms by long-term melatonin treatment. Expression levels of control group (blue) and melatonin group (red) are expressed as mean ± s.d. *P < 0.05, **P < 0.01 vs control; N.D., not detected. (A) Pathway map of glycolysis pathway, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP). The metabolites described in the text are underlined in red. (B) Pathway map of non-essential amino acids (NEAAs) metabolism. They can be synthesized from the TCA cycle. NEAAs significantly downregulated by melatonin treatment are circled with red dashed line. (C) Pathway map of essential amino acids (EAAs) metabolism. All EAAs (circled with pink dashed line) are significantly downregulated by melatonin treatment. (D) Pathway map of ketone body metabolisms. 3-hydroxybutyric acid (3-HBA), one of representative ketone bodies, is underlined in red.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Changes in liver metabolisms by long-term melatonin treatment. Expression levels of control group (blue) and melatonin group (red) are expressed as mean ± s.d. *P < 0.05, **P < 0.01 vs control; N.D., not detected. (A) Pathway map of glycolysis pathway, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP). The metabolites described in the text are underlined in red. (B) Pathway map of non-essential amino acids (NEAAs) metabolism. They can be synthesized from the TCA cycle. NEAAs significantly downregulated by melatonin treatment are circled with red dashed line. (C) Pathway map of essential amino acids (EAAs) metabolism. All EAAs (circled with pink dashed line) are significantly downregulated by melatonin treatment. (D) Pathway map of ketone body metabolisms. 3-hydroxybutyric acid (3-HBA), one of representative ketone bodies, is underlined in red.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
Changes in liver metabolisms by long-term melatonin treatment. Expression levels of control group (blue) and melatonin group (red) are expressed as mean ± s.d. *P < 0.05, **P < 0.01 vs control; N.D., not detected. (A) Pathway map of glycolysis pathway, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP). The metabolites described in the text are underlined in red. (B) Pathway map of non-essential amino acids (NEAAs) metabolism. They can be synthesized from the TCA cycle. NEAAs significantly downregulated by melatonin treatment are circled with red dashed line. (C) Pathway map of essential amino acids (EAAs) metabolism. All EAAs (circled with pink dashed line) are significantly downregulated by melatonin treatment. (D) Pathway map of ketone body metabolisms. 3-hydroxybutyric acid (3-HBA), one of representative ketone bodies, is underlined in red.
Citation: Journal of Endocrinology 251, 1; 10.1530/JOE-20-0462
The tricarboxylic acid (TCA) cycle produces energy carriers (ATP and NADH) by catabolizing various metabolites (Akram 2014). Most of the intermediates in the TCA cycle and the energy carriers produced in this cycle were downregulated by melatonin treatment. Because the TCA cycle is downstream of the glycolysis pathway, we speculate that attenuation of glucose utilization by melatonin treatment would lead to the downregulation of intermediates in the TCA cycle. Citrate is one of the intermediates and is used as the source for de novo lipogenesis (Martinez-Reyes & Chandel 2020). Melatonin treatment significantly downregulated citrate, suggesting that de novo lipogenesis is inhibited by melatonin treatment through the downregulation of citrate.
The pentose phosphate pathway (PPP) produces energy carriers, such as NADPH, from G6P produced by glycolysis. PPP produces energy carriers, such as NADPH. It also produces the precursors for nucleotides synthesis, such as ribulose 5-phosphate (Ru5P) and ribose 5-phosphate (R5P). Melatonin downregulated the metabolites and energy carriers of the PPP, which in turn would have downregulated the metabolites in the nucleotide synthesis pathway (Supplementary Fig. 1). Although the attenuation of glucose utilization leads to the downregulation of nucleotides and energy carriers, how their downregulation is associated with the attenuation of body weight gain is unclear.
All non-essential amino acids (NEAAs) except cysteine (amino acids indicated by red dotted lines in Fig. 3B) and all essential amino acids (EAAs) (indicated by pink dotted lines in Fig. 3B) were significantly downregulated by melatonin treatment, and their downregulations were accompanied by decreases in a number of related metabolites. Because NEAAs are not only absorbed from the diet but are synthesized from the metabolites in the TCA cycle (Fig. 3B) (Akram 2014), it is speculated that the downregulation of NEAAs by melatonin treatment was due to the downregulation of the metabolites in the TCA cycle, possibly due to the inhibition of glucose utilization. In contrast to NEAAs, EAAs are not synthesized in the body but are absorbed from diet (Santana-Santos et al. 2008). Therefore, the downregulation of EAAs suggests that melatonin treatment inhibited the absorption of amino acids. Since amino acids are also catabolized and enter the TCA cycle, the decrease in the supply of amino acids by melatonin would also lead to the downregulation of intermediates in the TCA cycle including citrate.
Figure 3D shows the pathway map of ketone body metabolisms. 3-Hydroxybutyric acid (3-HBA) is one of the representative ketone bodies that increases under starvation (Cahill 2006). 3-HBA and its related metabolites were not upregulated by melatonin treatment, suggesting that melatonin treatment did not cause excessive energy deprivation or a disordering of energy utilization.
Pathway analysis of downregulated liver metabolites by long-term melatonin treatment
We analyzed the pathways associated with 149 metabolites that were downregulated by melatonin. Of these, 22 pathways were identified as enriched pathways for the downregulated metabolites by melatonin treatment (Table 1). Among them, seven terms were associated with the transport of glucose or amino acids (shown in bold), suggesting that melatonin treatment induced a massive decrease of metabolites by attenuating the absorption and transport of glucose and amino acids.
Pathways enriched in the downregulated metabolites by long-term melatonin treatment.
| Pathway | P value |
|---|---|
| Translation | 3.07E-04 |
| Transport of inorganic cations/anions and amino acids/oligopeptides | 1.95E-03 |
| Amino acid transport across the plasma membrane | 2.31E-03 |
| tRNA aminoacylation | 2.57E-03 |
| Mitochondrial tRNA aminoacylation | 2.57E-03 |
| Cytosolic tRNA aminoacylation | 2.57E-03 |
| Amino acid and oligopeptide SLC transporters | 3.95E-03 |
| Na+/Cl−-dependent neurotransmitter transporters | 7.25E-03 |
| Generic transcription pathway | 0.0105 |
| Gene expression (Transcription) | 0.0155 |
| Amine compound SLC transporters | 0.0155 |
| TP53 regulates metabolic genes | 0.0192 |
| Cellular responses to external stimuli | 0.0215 |
| Transcriptional regulation by TP53 | 0.0215 |
| Transport of bile salts and organic acids, metal ions and amine compounds | 0.0229 |
| RNA polymerase II transcription | 0.0299 |
| Interconversion of nucleotide di- and triphosphates | 0.0304 |
| Histidine, lysine, phenylalanine, tyrosine, proline and tryptophan catabolism | 0.0347 |
| Cellular responses to stress | 0.0426 |
| Metabolism of RNA | 0.0426 |
| SLC-mediated transmembrane transport | 0.0427 |
| Metabolism of proteins | 0.0461 |
Twenty-two pathways were identified as enriched in the metabolites downregulated by melatonin. Seven terms associated with the transport of amino acids or glucose are shown in bold.
Discussion
In the present study, we demonstrated that long-term melatonin treatment (from 10 weeks to 43 weeks of age) attenuated body weight gain during aging in female mice. We also revealed, using micro-CT, that melatonin treatment decreased the deposition of both visceral fat and s.c. fat. Furthermore, metabolome analysis revealed that melatonin treatment downregulated a number of metabolites involved in lipogenesis.
Melatonin regulates not only circadian rhythms but also metabolism (Tamura et al. 2008a, Cipolla-Neto et al. 2014, Szewczyk-Golec et al. 2015). Previous reports showed that melatonin treatment reduced the body weight and abdominal fat deposition in several male animals (Prunet-Marcassus et al. 2003, Agil et al. 2011, Nduhirabandi et al. 2011, Favero et al. 2015). However, it should be noted that there have been no reports demonstrating the inhibitory effects of melatonin on body weight gain and fat deposition in female mice. Women experience the risk of weight gain throughout life. During the perimenopausal period, they tend to be obese due to the decrease of circulating estrogen levels (Mauvais-Jarvis et al. 2013). Previous reports have shown the inhibitory effect of melatonin treatment on body weight gain in postmenopausal women (Walecka-Kapica et al. 2014, Chojnacki et al. 2015, Amstrup et al. 2016). However, women should avoid becoming obese not only after menopause but also starting at a young age. Therefore, there has been a need to develop a new therapy controlling women’s body weight throughout life. We recently showed that long-term melatonin treatment in mice from 10 to 43 weeks of age (corresponding to adolescence to the perimenopausal period in humans) delayed ovarian aging (Tamura et al. 2017). The present study clearly demonstrated that long-term melatonin treatment is also effective for controlling weight gain during aging. In addition, unlike most previous reports, this study used a normal diet to mimic naturally occurring body weight gain with aging. It is also interesting to note that that excessive light-at-night situation could also be a weight gain risk factor through the disruption of circadian rhythm (Park et al. 2019). Therefore, long-term melatonin treatment may be particularly effective in preventing weight gain in women who are under the excessive light-at-night situation, such as night-shift workers. Taken together, our results suggest that long-term melatonin treatment may be a promising way for women to control their body weight gain throughout life. It seems that melatonin does not decrease body weight but can inhibit weight gain in obesogenic situations. In fact, melatonin is effective in preventing weight gain not only in women experiencing postmenopausal weight gain (Amstrup et al. 2016) but also in women experiencing side effects of antipsychotics (Modabbernia et al. 2014).
Interestingly, the present results showed that long-term melatonin treatment decreased the depositions of both visceral fat and s.c. fat, which is consistent with previous reports showing that melatonin inhibits fat deposition in high-fat diet model animals (Jimenez-Aranda et al. 2013, de Farias et al. 2019). To elucidate the mechanisms, we performed a metabolome analysis and found that long-term melatonin treatment remarkably attenuated metabolism in the liver, and this was accompanied by a decrease in the number of metabolites, including metabolites of glucose and amino acids. Metabolites from the carbohydrates, proteins and lipids in our diet enter the TCA cycle where they are used to produce energy molecules, such as ATP (Akram 2014). The TCA cycle is a central pathway that connects almost all metabolic pathways. Citrate is an intermediate in the TCA cycle and is a source for de novo lipogenesis (Martinez-Reyes & Chandel 2020). When the nutrient supply is sufficient, surplus citrate is converted to triglycerides and transferred to fat tissue for storage, which increases fat mass (Alves-Bezerra & Cohen 2017). In this study, long-term melatonin treatment decreased the level of citrate in the liver. This may have contributed to the attenuation of de novo lipogenesis and fat deposition and the following weight gain. In fact, melatonin decreases plasma levels of triglycerides in rats with diet-induced obesity (Prunet-Marcassus et al. 2003, Rios-Lugo et al. 2010).
Several potential mechanisms by which melatonin attenuates body weight gain have been proposed (Tan et al. 2011, Cipolla-Neto et al. 2014, Genario et al. 2021). One is that melatonin inhibits appetite. Since the amount of food intake was not decreased by melatonin in this study, the effect of melatonin treatment is unlikely due to the loss of appetite. Considering that melatonin treatment attenuated weight gain independently of food intake, another possible mechanism is that melatonin treatment may have increased energy expenditure or energy loss, although we did not check them. Melatonin increases brown adipose tissue (BAT) in animals as well as humans (Tan et al. 2011, Halpern et al. 2019). BAT can convert extra energy into heat by oxidative phosphorylation in mitochondria (Porter 2017). Therefore, BAT can attenuate body weight gain by increasing energy expenditure via heat production. Therefore, it is possible that long-term melatonin treatment attenuated body weight gain through the increase of BAT, although we did not examine the effect of long-term melatonin treatment on the amount of BAT or thermogenesis. Further studies are needed to clarify the involvement of this mechanism. Another possible mechanism is that melatonin directly activated lipolysis of fat tissue. If lipolysis is activated with the increase of energy expenditure, body weight can be decreased. Under such a situation, ketogenesis should be generally activated and result in the increase of ketone bodies. However, as shown in Fig. 3D, ketone bodies were not increased by melatonin treatment. Therefore, the effect of melatonin treatment on body weight is unlikely due to the activation of lipolysis. As for energy loss, melatonin may have induced energy loss by increasing physical activities although this idea has been debated (Wolden-Hanson et al. 2000, Isobe et al. 2002, Raskind et al. 2007). We did not check the physical activities in this study. However, if physical activities were increased by melatonin treatment, energy-producing cycles, such as the TCA cycle, would be activated and result in the increase of ATP production in the liver. Because ATP was not increased by melatonin treatment in this study, it is unlikely that attenuation of body weight gain is due to the increase in physical activities.
An alternative possibility is that melatonin attenuated body weight gain by inhibiting the absorption of nutrients, such as amino acids and glucose. In our study, melatonin treatment decreased the level of not only NEAAs but also EAAs. Because EAAs are not synthesized in the body and are absorbed from the diet (Santana-Santos et al. 2008), the melatonin-induced decrease in the levels of EAAs suggests that melatonin treatment attenuated the absorption of amino acids. In addition, melatonin treatment downregulated glucose-related metabolites, suggesting that melatonin attenuated glucose uptake. This is supported by recent reports that melatonin has an inhibitory effect on lipid absorption in the intestine (Hong et al. 2020), and that melatonin decreases serum levels of several EAAs in colitis model mice (Liu et al. 2017). Furthermore, melatonin treatment in sarcopenic elderly patients decreases serum albumin levels while cotreatment with EAA recovers it (Rondanelli et al. 2018). Further studies are needed to test the hypothesis that the absorption of the nutrients is inhibited by melatonin treatment.
There are some limitations to this study. We did not measure energy expenditure or fasting serum levels of glucose, insulin or non-esterified fatty acids. We also did not confirm whether melatonin treatment actually increased the serum melatonin levels. Melatonin was contained in the drinking water with a dose of 100 µg/mL. This method can increase serum melatonin levels up to 1~3 ng/mL (Nava et al. 2003), which is the serum level similar to humans who were administered the general dose of melatonin (1 mg/day) (our unpublished data). The dose of 3 mg/day was found to be effective for improving oocyte quality in infertile women. Therefore, the dose of 100 µg/mL in the drinking water may have an effect almost similar to the oral administration of melatonin 1~3 mg/day in humans.
In this study, melatonin was administered during the day as well as at night, as reported previously (Favero et al. 2015, Tamura et al. 2017). It was reported that melatonin administration during the day could have detrimental effects on circadian rhythm in the body (Cipolla-Neto et al. 2014). Therefore, we cannot completely exclude such negative effects of melatonin treatment, although mice are nocturnal animals and drink less water during the day. In addition, because a non-standard light/darkness cycle (lights on from 5:00 to 19:00 h) was used in this study, the effects of melatonin may differ when a standard light/darkness cycle (lights on from 7:00 to 19:00 h) is applied.
Although changing life habits is the key to success in maintaining body weight in the long term (Greaves et al. 2017), several anti-obesity drugs are also available for the treatment of obesity at present (Franz et al. 2007). However, there are great concerns about their safety. Many drugs previously available in the market have been withdrawn due to the increased incidence of adverse effects, such as psychiatric and cardiac disorders (Li & Cheung 2011). On the other hand, few mild side effects of melatonin treatment, such as agitation, dizziness and sleepiness, have been reported in humans, even by long-term treatment (Andersen et al. 2016). Taken together, long-term melatonin treatment can be a novel effective and safe therapy for the prevention of body weight gain during aging. These encouraging findings in mice now need to be tested in a human trial.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-20-0462.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported in part by JSPS KAKENHI (Grant Number 17K11239, 18K09230, 18K09262, 19K09803, 20K09645, 20H03825 and 20K18191).
References
Agil A, Navarro-Alarcon M, Ruiz R, Abuhamadah S, El-Mir MY & Vazquez GF 2011 Beneficial effects of melatonin on obesity and lipid profile in young Zucker diabetic fatty rats. Journal of Pineal Research 50 207–212. (https://doi.org/10.1111/j.1600-079X.2010.00830.x)
Akram M 2014 Citric acid cycle and role of its intermediates in metabolism. Cell Biochemistry and Biophysics 68 475–478. (https://doi.org/10.1007/s12013-013-9750-1)
Alves-Bezerra M & Cohen DE 2017 Triglyceride metabolism in the liver. Comprehensive Physiology 8 1–8. (https://doi.org/10.1002/cphy.c170012)
Amstrup AK, Sikjaer T, Pedersen SB, Heickendorff L, Mosekilde L & Rejnmark L 2016 Reduced fat mass and increased lean mass in response to 1 year of melatonin treatment in postmenopausal women: a randomized placebo-controlled trial. Clinical Endocrinology 84 342–347. (https://doi.org/10.1111/cen.12942)
Andersen LP, Gogenur I, Rosenberg J & Reiter RJ 2016 The safety of melatonin in humans. Clinical Drug Investigation 36 169–175. (https://doi.org/10.1007/s40261-015-0368-5)
Aune D, Sen A, Prasad M, Norat T, Janszky I, Tonstad S, Romundstad P & Vatten LJ 2016 BMI and all cause mortality: systematic review and non-linear dose-response meta-analysis of 230 cohort studies with 3.74 million deaths among 30.3 million participants. BMJ 353 i2156. (https://doi.org/10.1136/bmj.i2156)
Cahill Jr GF 2006 Fuel metabolism in starvation. Annual Review of Nutrition 26 1–22. (https://doi.org/10.1146/annurev.nutr.26.061505.111258)
Chatzakis C, Goulis DG, Mareti E, Eleftheriades M, Zavlanos A, Dinas K & Sotiriadis A 2019 Prevention of gestational diabetes mellitus in overweight or obese pregnant women: a network meta-analysis. Diabetes Research and Clinical Practice 158 107924. (https://doi.org/10.1016/j.diabres.2019.107924)
Chojnacki C, Walecka-Kapica E, Klupinska G, Pawlowicz M, Blonska A & Chojnacki J 2015 Effects of fluoxetine and melatonin on mood, sleep quality and body mass index in postmenopausal women. Journal of Physiology and Pharmacology 66 665–671.
Cipolla-Neto J, Amaral FG, Afeche SC, Tan DX & Reiter RJ 2014 Melatonin, energy metabolism, and obesity: a review. Journal of Pineal Research 56 371–381. (https://doi.org/10.1111/jpi.12137)
Cote AT, Harris KC, Panagiotopoulos C, Sandor GG & Devlin AM 2013 Childhood obesity and cardiovascular dysfunction. Journal of the American College of Cardiology 62 1309–1319. (https://doi.org/10.1016/j.jacc.2013.07.042)
Cunningham SA, Kramer MR & Narayan KM 2014 Incidence of childhood obesity in the United States. New England Journal of Medicine 370 1660–1661. (https://doi.org/10.1056/NEJMc1402397)
de Farias TDSM, Cruz MM, de Sa RCDC, Severi I, Perugini J, Senzacqua M, Cerutti SM, Giordano A, Cinti S & Alonso-Vale MIC 2019 Melatonin supplementation decreases hypertrophic obesity and inflammation induced by high-fat diet in mice. Frontiers in Endocrinology 10 750. (https://doi.org/10.3389/fendo.2019.00750)
Favero G, Stacchiotti A, Castrezzati S, Bonomini F, Albanese M, Rezzani R & Rodella LF 2015 Melatonin reduces obesity and restores adipokine patterns and metabolism in obese (ob/ob) mice. Nutrition Research 35 891–900. (https://doi.org/10.1016/j.nutres.2015.07.001)
Flegal KM, Kit BK, Orpana H & Graubard BI 2013 Association of all-cause mortality with overweight and obesity using standard body mass index categories: a systematic review and meta-analysis. JAMA 309 71–82. (https://doi.org/10.1001/jama.2012.113905)
Franz MJ, VanWormer JJ, Crain AL, Boucher JL, Histon T, Caplan W, Bowman JD & Pronk NP 2007 Weight-loss outcomes: a systematic review and meta-analysis of weight-loss clinical trials with a minimum 1-year follow-up. Journal of the American Dietetic Association 107 1755–1767. (https://doi.org/10.1016/j.jada.2007.07.017).
Gaudet L, Ferraro ZM, Wen SW & Walker M 2014 Maternal obesity and occurrence of fetal macrosomia: a systematic review and meta-analysis. BioMed Research International 2014 640291. (https://doi.org/10.1155/2014/640291)
Genario R, Cipolla-Neto J, Bueno AA & Santos HO 2021 Melatonin supplementation in the management of obesity and obesity-associated disorders: a review of physiological mechanisms and clinical applications. Pharmacological Research 163 105254. (https://doi.org/10.1016/j.phrs.2020.105254)
Global B, Di Angelantonio E, Bhupathiraju ShN, Wormser D, Gao P, Kaptoge S, Berrington de Gonzalez A, Cairns BJ, Huxley R & Jackson ChL et al.2016 Body-mass index and all-cause mortality: individual-participant-data meta-analysis of 239 prospective studies in four continents. Lancet 388 776–786. (https://doi.org/10.1016/S0140-6736(1630175-1)
Gortmaker SL & Taveras EM 2014 Who becomes obese during childhood – clues to prevention. New England Journal of Medicine 370 475–476. (https://doi.org/10.1056/NEJMe1315169)
Greaves C, Poltawski L, Garside R & Briscoe S 2017 Understanding the challenge of weight loss maintenance: a systematic review and synthesis of qualitative research on weight loss maintenance. Health Psychology Review 11 145–163. (https://doi.org/10.1080/17437199.2017.1299583)
Guthrie JR, Dennerstein L & Dudley EC 1999 Weight gain and the menopause: a 5-year prospective study. Climacteric 2 205–211. (https://doi.org/10.3109/13697139909038063)
Halpern B, Mancini MC, Bueno C, Barcelos IP, de Melo ME, Lima MS, Carneiro CG, Sapienza MT, Buchpiguel CA, do Amaral FG, et al.2019 Melatonin increases brown adipose tissue volume and activity in patients with melatonin deficiency: a proof-of-concept study. Diabetes 68 947–952. (https://doi.org/10.2337/db18-0956)
Hong F, Pan S, Xu P, Xue T, Wang J, Guo Y, Jia L, Qiao X, Li L & Zhai Y 2020 Melatonin orchestrates lipid homeostasis through the hepatointestinal circadian clock and microbiota during constant light exposure. Cells 9 489. (https://doi.org/10.3390/cells9020489)
Hurter H, Vontelin van Breda S, Vokalova L, Brandl M, Baumann M, Hosli I, Huhn EA, De Geyter C, Rossi SW & Lapaire O 2019 Prevention of pre-eclampsia after infertility treatment: preconceptional minimalisation of risk factors. Best Practice and Research: Clinical Endocrinology and Metabolism 33 127–132. (https://doi.org/10.1016/j.beem.2019.05.001)
Inelmen EM, Sergi G, Coin A, Miotto F, Peruzza S & Enzi G 2003 Can obesity be a risk factor in elderly people? Obesity Reviews 4 147–155. (https://doi.org/10.1046/j.1467-789x.2003.00107.x)
Isobe Y, Torri T, Konishi E & Fujioi J 2002 Effects of melatonin injection on running-wheel activity and body temperature differ by the time of day. Pharmacology, Biochemistry, and Behavior 73 805–811. (https://doi.org/10.1016/s0091-3057(0200944-9)
Jimenez-Aranda A, Fernandez-Vazquez G, Campos D, Tassi M, Velasco-Perez L, Tan DX, Reiter RJ & Agil A 2013 Melatonin induces browning of inguinal white adipose tissue in Zucker diabetic fatty rats. Journal of Pineal Research 55 416–423. (https://doi.org/10.1111/jpi.12089)
Kamburov A, Stelzl U, Lehrach H & Herwig R 2013 The consensus path DB interaction database: 2013 update. Nucleic Acids Research 41 D793–D 800. (https://doi.org/10.1093/nar/gks1055)
Kina-Tanada M, Sakanashi M, Tanimoto A, Kaname T, Matsuzaki T, Noguchi K, Uchida T, Nakasone J, Kozuka C & Ishida M et al.2017 Long-term dietary nitrite and nitrate deficiency causes the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice. Diabetologia 60 1138–1151. (https://doi.org/10.1007/s00125-017-4259-6)
Lainez NM & Coss D 2019 Obesity, neuroinflammation, and reproductive function. Endocrinology 160 2719–2736. (https://doi.org/10.1210/en.2019-00487)
Li MF & Cheung BM 2011 Rise and fall of anti-obesity drugs. World Journal of Diabetes 2 19–23. (https://doi.org/10.4239/wjd.v2.i2.19)
Liu G, Jiang Q, Chen S, Fang J, Ren W, Yin J, Yao K & Yin Y 2017 Melatonin alters amino acid metabolism and inflammatory responses in colitis mice. Amino Acids 49 2065–2071. (https://doi.org/10.1007/s00726-017-2489-z)
Martinez-Reyes I & Chandel NS 2020 Mitochondrial TCA cycle metabolites control physiology and disease. Nature Communications 11 102. (https://doi.org/10.1038/s41467-019-13668-3)
Matsui T, Omuro H, Liu YF, Soya M, Shima T, McEwen BS & Soya H 2017 Astrocytic glycogen-derived lactate fuels the brain during exhaustive exercise to maintain endurance capacity. PNAS 114 6358–6363. (https://doi.org/10.1073/pnas.1702739114)
Mauvais-Jarvis F, Clegg DJ & Hevener AL 2013 The role of estrogens in control of energy balance and glucose homeostasis. Endocrine Reviews 34 309–338. (https://doi.org/10.1210/er.2012-1055)
Modabbernia A, Heidari P, Soleimani R, Sobhani A, Roshan ZA, Taslimi S, Ashrafi M & Modabbernia MJ 2014 Melatonin for prevention of metabolic side-effects of olanzapine in patients with first-episode schizophrenia: randomized double-blind placebo-controlled study. Journal of Psychiatric Research 53 133–140. (https://doi.org/10.1016/j.jpsychires.2014.02.013)
Nava M, Quiroz Y, Vaziri N & Rodriguez-Iturbe B 2003 Melatonin reduces renal interstitial inflammation and improves hypertension in spontaneously hypertensive rats. American Journal of Physiology: Renal Physiology 284 F447–F 454. (https://doi.org/10.1152/ajprenal.00264.2002)
Nduhirabandi F, Du Toit EF, Blackhurst D, Marais D & Lochner A 2011 Chronic melatonin consumption prevents obesity-related metabolic abnormalities and protects the heart against myocardial ischemia and reperfusion injury in a prediabetic model of diet-induced obesity. Journal of Pineal Research 50 171–182. (https://doi.org/10.1111/j.1600-079X.2010.00826.x).
Park YM, White AJ, Jackson CL, Weinberg CR & Sandler DP 2019 Association of exposure to artificial light at night while sleeping with risk of obesity in women. JAMA Internal Medicine 179 1061–1071. (https://doi.org/10.1001/jamainternmed.2019.0571)
Perissinotto E, Pisent C, Sergi G, Grigoletto F & ILSA Working Group (Italian Longitudinal Study on Ageing) 2002 Anthropometric measurements in the elderly: age and gender differences. British Journal of Nutrition 87 177–186. (https://doi.org/10.1079/bjn2001487)
Porter C 2017 Quantification of UCP1 function in human brown adipose tissue. Adipocyte 6 167–174. (https://doi.org/10.1080/21623945.2017.1319535)
Prunet-Marcassus B, Desbazeille M, Bros A, Louche K, Delagrange P, Renard P, Casteilla L & Penicaud L 2003 Melatonin reduces body weight gain in Sprague Dawley rats with diet-induced obesity. Endocrinology 144 5347–5352. (https://doi.org/10.1210/en.2003-0693)
Raskind MA, Burke BL, Crites NJ, Tapp AM & Rasmussen DD 2007 Olanzapine-induced weight gain and increased visceral adiposity is blocked by melatonin replacement therapy in rats. Neuropsychopharmacology 32 284–288. (https://doi.org/10.1038/sj.npp.1301093)
Rasmussen DD, Mitton DR, Larsen SA & Yellon SM 2001 Aging-dependent changes in the effect of daily melatonin supplementation on rat metabolic and behavioral responses. Journal of Pineal Research 31 89–94. (https://doi.org/10.1034/j.1600-079x.2001.310113.x)
Rios-Lugo MJ, Cano P, Jimenez-Ortega V, Fernandez-Mateos MP, Scacchi PA, Cardinali DP & Esquifino AI 2010 Melatonin effect on plasma adiponectin, leptin, insulin, glucose, triglycerides and cholesterol in normal and high fat-fed rats. Journal of Pineal Research 49 342–348. (https://doi.org/10.1111/j.1600-079X.2010.00798.x)
Rondanelli M, Peroni G, Gasparri C, Infantino V, Nichetti M, Cuzzoni G, Spadaccini D & Perna S 2018 Is a combination of melatonin and amino acids useful to sarcopenic elderly patients? A randomized trial. Geriatrics 4 4. (https://doi.org/10.3390/geriatrics4010004)
Santana-Santos L, Prosdocimi F & Ortega JM 2008 Essential amino acid usage and evolutionary nutrigenomics of eukaryotes – insights into the differential usage of amino acids in protein domains and extra-domains. Genetics and Molecular Research 7 839–852. (https://doi.org/10.4238/vol7-3x-meeting002)
Szewczyk-Golec K, Wozniak A & Reiter RJ 2015 Inter-relationships of the chronobiotic, melatonin, with leptin and adiponectin: implications for obesity. Journal of Pineal Research 59 277–291. (https://doi.org/10.1111/jpi.12257)
Tamura H, Nakamura Y, Narimatsu A, Yamagata Y, Takasaki A, Reiter RJ & Sugino N 2008a Melatonin treatment in peri- and postmenopausal women elevates serum high-density lipoprotein cholesterol levels without influencing total cholesterol levels. Journal of Pineal Research 45 101–105. (https://doi.org/10.1111/j.1600-079X.2008.00561.x)
Tamura H, Takasaki A, Miwa I, Taniguchi K, Maekawa R, Asada H, Taketani T, Matsuoka A, Yamagata Y, Shimamura K, et al.2008b Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. Journal of Pineal Research 44 280–287. (https://doi.org/10.1111/j.1600-079X.2007.00524.x)
Tamura H, Nakamura Y, Korkmaz A, Manchester LC, Tan DX, Sugino N & Reiter RJ 2009 Melatonin and the ovary: physiological and pathophysiological implications. Fertility and Sterility 92 328–343. (https://doi.org/10.1016/j.fertnstert.2008.05.016)
Tamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee L, Tamura I, Maekawa R, Aasada H, Yamagata Y, et al.2012 The role of melatonin as an antioxidant in the follicle. Journal of Ovarian Research 5 5. (https://doi.org/10.1186/1757-2215-5-5)
Tamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee L, Tamura I, Maekawa R, Asada H, Yamagata Y, et al.2013 Melatonin as a free radical scavenger in the ovarian follicle. Endocrine Journal 60 1–13. (https://doi.org/10.1507/endocrj.ej12-0263)
Tamura H, Takasaki A, Taketani T, Tanabe M, Lee L, Tamura I, Maekawa R, Aasada H, Yamagata Y & Sugino N 2014 Melatonin and female reproduction. Journal of Obstetrics and Gynaecology Research 40 1–11. (https://doi.org/10.1111/jog.12177)
Tamura H, Kawamoto M, Sato S, Tamura I, Maekawa R, Taketani T, Aasada H, Takaki E, Nakai A, Reiter RJ, et al.2017 Long-term melatonin treatment delays ovarian aging. Journal of Pineal Research 62. (https://doi.org/10.1111/jpi.12381)
Tamura H, Jozaki M, Tanabe M, Shirafuta Y, Mihara Y, Shinagawa M, Tamura I, Maekawa R, Sato S, Taketani T, et al.2020 Importance of melatonin in assisted reproductive technology and ovarian aging. International Journal of Molecular Sciences 21 1135. (https://doi.org/10.3390/ijms21031135)
Tan DX, Manchester LC, Fuentes-Broto L, Paredes SD & Reiter RJ 2011 Significance and application of melatonin in the regulation of brown adipose tissue metabolism: relation to human obesity. Obesity Reviews 12 167–188. (https://doi.org/10.1111/j.1467-789X.2010.00756.x)
Walecka-Kapica E, Klupinska G, Chojnacki J, Tomaszewska-Warda K, Blonska A & Chojnacki C 2014 The effect of melatonin supplementation on the quality of sleep and weight status in postmenopausal women. Przeglad Menopauzalny 13 334–338. (https://doi.org/10.5114/pm.2014.47986)
Wane S, van Uffelen JG & Brown W 2010 Determinants of weight gain in young women: a review of the literature. Journal of Women’s Health 19 1327–1340. (https://doi.org/10.1089/jwh.2009.1738)
Wang Y & Lim H 2012 The global childhood obesity epidemic and the association between socio-economic status and childhood obesity. International Review of Psychiatry 24 176–188. (https://doi.org/10.3109/09540261.2012.688195)
Wolden-Hanson T, Mitton DR, McCants RL, Yellon SM, Wilkinson CW, Matsumoto AM & Rasmussen DD 2000 Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of food intake and total body fat. Endocrinology 141 487–497. (https://doi.org/10.1210/endo.141.2.7311)
