Effects of Far Infrared Light on High-Fat Diet-Treated Mice

Obesity is a severe problem all over the world. One severe problem with obesity is the increased risk of severe diseases. To prevent obesity-related diseases, many people are undertaking prevention measures. Some people try to manage their body weight using supplement drinks, tablets, and health apparatuses. One type of health apparatus is based on the thermal effect. However, the detailed biological mechanisms of the effects of infrared light have not yet been elucidated. In this study, we attempted to clarify the beneficial effects of far infrared light using high-fat diet-treated mice. We exposed mice to far infrared light for 5 months. Body weights were not significantly different in high-fat diet-treated mice that were treated or not treated with far infrared light. However, body weights in far infrared-exposed control mice tended to increase compared to normal mice in the middle treatment term (from 7 to 15 weeks). In the Morris water maze, the goal time of far infrared light-treated control mice was nominally (but not significantly) shorter than the unexposed controls. Serum triglyceride concentrations were significantly increased in far infrared-exposed control mice compared to the unexposed controls. The levels of nerve growth factor and brain-derived neurotrophic factor protein were not significantly different except for nerve growth factor expression in the cerebellum of far infrared-exposed high-fat diet-treated mice. These results indicated that exposure to far infrared light did not provide clear beneficial effects in obese mice. However, the experimental schedule and methods may need to be modified to obtain clearer results. Received: January 13, 2021


Introduction
Obesity is a severe problem all over the world, and the percent of obese people is gradually increasing. One severe problem with obesity is an increased risk of secondary severe diseases such as type 2 diabetes mellitus, cardiovascular disease, and high blood pressure [1,2]. In particular, lipid metabolism including liver function is worse in obese people. The values of total cholesterol and triglyceride also significantly increase. On the other hand, calorie restriction extends the lifespan in rodents, C elegans, flies, and humans [3]. One mechanism of lifespan extension due to calorie restriction is oxygen metabolism. Treatment with tocotrienols, which are one type of vitamin E, showed significant anti-obesity effects in our previous studies [4,5]. Several lines of evidence have demonstrated that obesity produces oxidative stress in living tissues and enhances the risks of many diseases [6]. In addition, accumulation of oxidative damage is strongly related to many neurodegenerative disorders, such as Alzheimer's disease (AD) and dementia [7]. To prevent obesity-related diseases, many people undertake prevention measures such as walking, swimming, and food management. Some people try to manage their body weight using supplement drinks, tablets, and health apparatuses.
One health apparatus is based on the thermal effect and includes infrared (IR) saunas. However, the detailed biological mechanisms of the effect of IR light have not yet been elucidated.
Electromagnetic waves are common in our modern life. Sources include mobile phones, television systems, and medical imaging (X-ray imaging) [8]. Electromagnetic waves, which were discovered by Heinrich Rudolf Hertz in 1887, are classified by different wavelengths and include radio waves, microwaves, IR rays, visible light, ultraviolet, and X-rays. Far IR is a type of IR illumination corresponding to wavelengths ranging from 3 to 1000 μm. Far IR warms the body, but is not as harmful as ultraviolet, and is widely used in heaters, blankets, bedrock baths, etc. For far IR light, the absorbed IR rays stimulate molecules arranged in bonded lattices, resulting in vibration of molecules, and warming of substances [9]. Far IR light may dilate micro vessels, thereby activating blood circulation and increasing metabolism. We hypothesized that exposure to far IR light enhances blood flow and oxygen metabolism and plays a beneficial role against obesity. The blood flow volume gradually decreases during aging [10] and in AD [11,12]. Decreased blood flow may lead to decreased oxygen levels and malnutrition, accelerating the onset and progression of AD. Here we assessed the effects of far IR light in obese mice using a fabric that emits far IR light.

Animals and Reagents
All animal experiments were performed with the approval of the Animal Protection and Ethics Committee of the Shibaura Institute of Technology, Tokyo, Japan (Approval number 17006 and 19003). Wild-type C57BL/6 male mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) and were maintained until 8or 15 months old. All mice were maintained under conditions of controlled temperature (22 ± 2°C), and a 12-h light/dark cycle, and were provided free access to food and water. Following entry into the study, these mice were maintained for a 5-month period in the presence or absence of a fabric that generates far IR light (Control, n = 5; Control-Far IR, n = 5; high-fat diet (HFD), n = 5; HFD-Far IR, n = 5). HFD model mice were generated by feeding normal mice a diet (#D12492; Research Diets Inc., New Brunswick, NJ, USA) containing 5.24 kcal/g, with 60% of the calories from fat, 20% from protein, and 20% from carbohydrates, from 8 months of age until 12 months of age. Control diets (#D12450J; Research Diets Inc.) containing 3.85 kcal/g, with 10% of the calories from fat, 20% from protein, and 70% from carbohydrates were used for the control groups ( Table 1). The body weight and weight of consumed food (food intake) were measured once per week, and the relative body weights (normalized to the starting value for the respective animal) were calculated from the data. After the treatment period, cognition and motor function were assessed using trials, as described below.
Following assessments, blood (for serum) was collected from each mouse, mice were euthanized, and samples of brain (cerebral cortex, cerebellum, and hippocampus) were collected for analysis.
To assess survival, the mice were maintained until death from natural causes in the presence or absence of a fabric that generates far IR light (Control, n = 10; Control-Far IR, n = 10). The starting age  between the fabric and the animals, the volume of crumpled paper, which is typically used as a flooring material, was decreased from the usual amount. Control animal cages were supplied with the same fabric lacking the additive in the ores. In a pilot study, we frequently modified the experimental set-up regarding how to place the fabric in the cages. Each cage also included an enrichment tool.

Cognitive performance
Cognitive functions were assessed with the Morris water maze and Rota Rod tests, as described previously [13,14] with some

Blood Analysis
Prior to euthanasia, blood was collected without an anticoagulant. Following clotting (approximately 30 min at room temperature (RT)), the samples were centrifuged in a clinical centrifuge, and the resulting serum supernatants were transferred to fresh tubes, flash frozen, and stored at -80 °C until analysis. The samples were assessed for selected clinical chemistry parameters (including total protein, albumin, iron, alanine aminotransferase (ALT), lactic acid dehydrogenase, total cholesterol, and others) using an external inspection agency (Oriental Yeast Co., Ltd., Tokyo, Japan).

Western Blotting
All brain samples were homogenized in phosphate-buffered saline and used for western blotting as described previously [15],

in HFD-Treated Mice
To clarify the biological effects of far IR light, we measured the body weight of HFD-treated mice housed in the presence or absence of the fabric that generates far IR light. The relative body weight gain (normalized to baseline) of HFD-treated mice was not attenuated in animals housed with the fabric that generates far IR light (compared to those housed with control fabric) ( Figure 1). However, the ratio of body weight gain in far IR-exposed control mice nominally (but not significantly) increased in the middle treatment term (from 7 to 15 weeks) compared to the unexposed controls. Food intake weight of both HFD-treated groups significantly differed compared to both control groups ( Figure 2). However, calorie intake did not differ among any animal groups. Values were normalized to baseline (defined as 100%). Data are expressed as the mean ± SD (Control, n = 5; Control-Far IR, n = 5; HFD, n = 5; HFD-Far IR, n = 5). The timeline shows the treatment period. Statistical analysis was performed using two-way analysis of variance (ANOVA). No significant differences were found between the presence or absence of Far IR in the four groups.

Figure 2:
The daily average food and calorie intake of each mouse group. Food intake was measured once per week. These data show the daily average volume (g) for the entire 5-month period. Daily intake of calories was calculated according to food intake. Data re expressed as the mean + SD (Control, n = 5; Control-Far IR, n = 5; HFD, n = 5; HFD-Far IR, n = 5). The comparison was performed using the Tukey-Kramer test. (*, #, p < 0.01).

Measurement of Cognitive Function of HFD-Treated Mice in the Presence or Absence of Far IR Light
To clarify the effect of far IR light on the cognitive function of HFD-treated mice, 5 months after starting housing in a specific cage, we measured the learning ability using the Morris water maze task ( Figure 3). The mice were subjected to swim tests 4 times per day on 5 consecutive days. The time to achieve the goal did not differ significantly for the HFD-treated mice housed in the presence or absence of far IR light. However, the goal time in far IR light-

Changes in Serum Parameters of HFD-Treated Mice in the Presence or Absence of Far IR Exposure
Exposure to far IR light affects blood flow [16]. After the cognitive trials, the mice were bled, euthanized, and various regions of the brain were recovered. The resulting sera were tested for multiple clinical chemistry parameters (Table 2). Some parameters significantly changed in HFD-treated mice compared to the controls without far IR light. The level of triglyceride in far IR light-exposed controls was significantly increased compared to the unexposed controls. The level of ALT was significantly increase in far IR lightexposed HFD-treated mice compared to the unexposed HFD mice.
The level of triglyceride in far IR light-exposed HFD-treated mice showed a nominal (but not significant) decrease compared to the unexposed HFD mice.

Protein Levels in the Brains of HFD-Treated Mice
Neurotrophic factors such as NGF and BDNF play an important role in the maintenance of brain functions. Although the results of cognitive and motor functions were not different among groups, the levels of these factors and of their respective receptors (TrkA and TrkB) were assessed in the cerebral cortex, cerebellum, and hippocampus using western blotting ( Figure 5). The NGF protein level in the cerebellum was significantly higher in the far IR-exposed HFD mice than in the HFD controls. However, the other proteins did not differ significantly in any brain regions in the presence or absence of far IR light.

Mice
Finally, we measured longevity in the presence or absence of far IR light on control mice ( Figure 6). Longevity was not different according to treatment or no treatment with far IR light in the control mice. Figure 6: Survival rate of mice housed in the presence or absence of the fabric that generates far IR light. Data are expressed as the mean ± SD (Control (n = 10), Control-Far IR (n = 10)).

Far IR Light Exposure Inhibits Body Weight Gain in Mice
Far IR light is used in many places in everyday life, such as leg hyperthermia, saunas, cooking, and heat therapy [17]. These are all indispensable in our daily life, but the mechanisms of far IR light effects remain unclear. In the present study, to clarify the potential health benefits of far IR light, we exposed C57BL/6 mice to far IR light for a long-term period (5 months). Specifically, we assessed the effects of far IR light on HFD mice, given that far IR exposure may affect blood flow and change the oxygen concentration in small vessels in the brain. Lipid metabolism may be changed by obesity, and high levels of triglyceride and cholesterol may increase the risk of arteriosclerosis in small vessels. In fact, treatment of mice with the same HFD for 2 months significantly increased the total serum cholesterol level in our previous study [4]. However, contrary to

Far IR Exposure Does not Affect Cognitive Function and Brain Neurotrophic Factors
To clarify the effects of far IR light on spatial memory in HFDtreated mice, we measured learning ability using the Morris water maze task. This test measures the time taken by mice to reach a hidden acrylic platform [13,14]. The goal times did not differ significantly between the control and HFD-treated mice housed in the presence or absence of far IR light, although the goal times on the third and fourth days of testing were nominally (but not significantly) lower in the far IR-exposed control mice compared to the unexposed controls. we checked the protein expression of brain neurotrophic factors because we hypothesized that oxidation was promoted in the HFD-treated mouse brains. Accumulation of oxidative damage induces cognitive dysfunction and increases the risk of neurodegenerative disorders [19].
BDNF and NGF play important roles in the maintenance of brain function including cognition [20,21]. However, the protein expression of NGF, BDNF, and their receptors were not different among groups, except for NGF expression in the cerebellum of far IR-exposed HFD-treated mice. The cause of the disappointing result may be related to the difference in brain oxidation levels. We fed the mice a HFD for a relatively long period (5 months). However, our HFD does not contain high carbohydrates. Some researchers use a high-fat and high carbohydrate diet and then measure antioxidant effects such as vitamin E [22]. Although our diet provided excess nutrition, it may be insufficient for accelerating oxidation in the mouse's body. Another possible reason for this problem may be the animal species. Fernando et al. [23] reported that treatment of Sprague Dawley rats with a HFD for 2 months induces cognitive dysfunction via attenuation of hippocampal BDNF mRNA and protein expression, and that this dysfunction is improved by vitamin E treatment. Exposure of Wistar rats to a high oxygen concentration for 48 hours significantly increases brain lipid peroxidation levels and induces cognitive dysfunction as seen in the Morris water and radial arm mazes [13]. We suggest that mice have relatively strong resistance against oxidative stress compared to rats. Thus, modification of food components and the animal species may be needed for detection of a clear result following exposure to far IR light.
We found no significant difference in survival of control mice housed in the presence or absence of far IR light. The purpose of this study was to determine if far IR light suppresses body weight gain, affects lipid metabolism, and reduces oxidative stress production, all of which may extend the lifespan. In calorie restriction research, the lifespan is extended by changes in lipid metabolism and oxygen consumption [24]. We will assess animal survival using the obesity model soon.

Conclusion
In this study, we examined the possible biological effects of long-term exposure of HFD mice to far IR light. Notably, neither cognition nor motor function in HFD mice was improved after far IR exposure in our experimental models. However, body weight, some serum parameters, and brain neurotrophic factor protein expression changed by a small amount. Far IR light clearly plays a beneficial role in our everyday life including in the medical field. In the near future, we want to provide evidence to clarify the beneficial mechanism of far IR light.