The enhancement of fat oxidation during the active phase and suppression of body weight gain in glycerol-3-phosphate dehydrogenase 1 deficient mice

ABSTRACT

We investigated whether the deletion of glycerol-3-phosphate dehydrogenase (GPD) 1 would affect carbohydrate oxidation, fat oxidation, and body weight by using the GPD1 null mice (BALB/cHeA (HeA)). We found that fat oxidation in HeA mice was significantly high during the early active phase than in BALB/cBy (By) mice used as a control under ad libitum conditions. Metabolic tracer experiment revealed that fatty acid oxidation in the skeletal muscle of HeA mice tended to be high. The energy expenditure and fat oxidation in HeA mice under fasting conditions were significantly higher than that in the By mice. Moreover, we monitored body weight gain in HeA mice under ad libitum feeding and found lower body weight gain. These data indicate that GPD1 deficiency induces enhancement of fat oxidation with suppression of weight gain. We propose that GPD1 deletion contributes to the reduction of body weight gain via enhancement of fat oxidation.

GRAPHICAL ABSTRACT

(a) GPD1 deletion lowers cytosolic NAD⁺/NADH ratio and inhibits glycolysis. (b) GPD1 deletion reduces body weight gain due to enhancing energy expenditure and fat oxidation.

KEYWORDS:

• Glycerol-3-phosphate dehydrogenase
• glycerophosphate shuttle
• NAD⁺/NADH ratio
• energy expenditure
• fat oxidation

GPD1 encodes glycerol-3-phosphate dehydrogenase 1, which is an NADH-dependent cytosolic enzyme catalyzing the conversion of dihydroxyacetone phosphate derived from glucose to glycerol-3-phosphate (G3P). G3P is finally acylated to form phospholipids or triglycerides (TG). GPD1 makes an important link between the glycolytic pathway and triglyceride biosynthesis, and thus also between carbohydrate and fat metabolism. Furthermore, together with the mitochondrial isoform GPD2, GPD1 enables the transport of reducing equivalents from the cytosol to the mitochondria, as a glycerophosphate (GP) shuttle [1,2]. Transport of NADH to the mitochondria by the GP shuttle maintains the cytoplasmic NAD⁺/NADH ratio, which leads to the maintenance of proper carbohydrate metabolism. A previous paper reported that mice lacking GPD1 had a low lactate/pyruvate ratio, signifying a low cytosolic NAD⁺/NADH ratio in the skeletal muscle, and glycolysis was inhibited at the step catalyzed by GPD1 due to an impairment of GP shuttle [3]. Our previous study revealed that GPD1 deletion increases fat utilization to compensate for the decrease in carbohydrate utilization in the skeletal muscle during exercise [4]. Besides, GPD1 deficiency also affects amino acid metabolism in the liver and muscle during fasting [5]. These previous reports indicate that GPD1 may regulate the balance between carbohydrate metabolism and fat metabolism through maintaining the NAD⁺/NADH ratio. GPD1 has been reported to be one of the genes associated with obesity in human and rat studies [6–9]. Obesity results from a disruption of energy metabolism, inducing excessive accumulation of TG in the adipose tissue. As human adipocytes possess very low glycerol kinase activity, GPD1 is an essential enzyme for providing glycerol moiety required for TG synthesis in the adipose tissue [10]. One study which compared GPD1 enzymatic activity in lean and obese human subjects showed a positive correlation between the enzyme activity and body mass index [6]. Another human study reported that GPD1 gene expression in the skeletal muscle were increased with obesity and decreased by weight loss due to gastric bypass surgery [7]. Short-term high-fat diet intake increased GPD1 expression levels in the rat epididymal adipose tissue [8]. Based on these reports, GPD1 is a potential therapeutic target for obesity. However, whether GPD1 suppression prevents obesity has not been investigated.

GPD1 has been considered to be a key element that connects carbohydrate and fat metabolism and may influence weight gain. In this study, we used a GPD1 null model, BALB/cHeA (HeA) mice, to examine whether GPD1 deficiency and the resultant inhibition of the GP shuttle changed carbohydrate and fat metabolism and the effect of this change on body weight gain.

Materials and methods

Mice

BALB/cBy (By) mice were obtained from Japan CLEA (Tokyo, Japan). The origins of HeA mice and their breeding conditions have been described previously [11]. The mice were fed with a normal laboratory diet (MF diet, Oriental Yeast Co., Tokyo, Japan) for a week to stabilize their metabolism, and maintained under a 12-h light-dark cycle (light phase: 7:00–19:00, dark phase: 19:00–7:00) at constant temperature (22°C). The mice were cared for in accordance with the NIH Guide for the Care and Use of Laboratory Animals and our institutional guidelines. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the University of Shizuoka (No.135036). Body weight was measured once a week throughout the experimental period.

Measurement of oxygen (O₂) consumption and carbon dioxide (CO₂) production

Open-circuit indirect calorimetry was performed with an O₂/CO₂ metabolism measuring system ARCO-2000 for small animals (Arco System, Chiba, Japan). To habituate to the experimental condition, mice were placed in an individual chamber 2 h before the start of the measurement. Each mouse could freely access water and normal chow diet (ad libitum conditions) during the measurement of energy metabolism. During fasting experiments, mice could freely access water only. Energy expenditure (in calories per kilogram per minute), carbohydrate oxidation, fat oxidation (in milligrams per kilogram per minute), RQ and mice activity (in counts per minute) were measured every 10 min. The area under the curve (AUC) of each measurement were calculated by the trapezoidal rule. Measurements were performed during the dark period (from 19:00 to 7:00) and light period (from 7:00 to 17:00). Room temperature was constantly kept at 22°C while running light/dark cycles of 12 h. The rates of carbohydrate and fat oxidation were calculated using the Frayn’s equations [12]. The energy expenditure was calculated using the Lusk’s equation [13].

Palmitate oxidation in isolated muscle

To examine palmitate oxidation in muscles, gastrocnemius muscles were dissected and placed in a 20-mL glass reaction vial containing 2.5 mL of warmed (30°C), pre-gassed (95% O₂-5% CO₂, pH 7.4), modified Krebs-Henseleit buffer containing 4% FA-free BSA, 5 mM glucose, and 0.5 mM palmitate, giving a palmitate-to-BSA molar ratio of 1:1. After a 30-min preincubation period, muscle strips were transferred to vials containing 0.5 Ci/mL [1–¹⁴C] palmitate (PerkinElmer, Waltham, MA, USA) for 60 min. During this phase, exogenous palmitate oxidation was monitored by the production of ¹⁴CO₂. Gaseous ¹⁴CO₂ produced from the exogenous oxidation of [1–¹⁴C] palmitate during the incubation was measured by transferring 1 mL of the chase incubation medium to a 20-mL glass scintillation vial containing 1 mL of 1 M sulfuric acid and to a 0.5-mL microcentrifuge tube containing 1 M sodium hydroxide. Liberated ¹⁴CO₂ was trapped in the sodium hydroxide over 60 min, the microcentrifuge tube containing trapped ¹⁴CO₂ was placed in a scintillation vial, and radioactivity was counted.

Statistical analysis

Results are presented as mean ± SEM. Significant differences between each group were compared using the Student’s t-test (JMP 5.1.2; SAS, Cary, NC, USA). P < 0.05 (*) was considered as statistically significant. Other values were indicated as P < 0.01 (**) and P < 0.001 (***).

Results and discussion

We have previously revealed that the deletion of GPD1 induces an adaptation that enhances fat availability in the skeletal muscle during exercise [4]. MacDonald et al. reported that the low ratio of lactate/pyruvate via block in glycolysis in the skeletal muscle of GPD1 deficient mice under rest conditions, was consistent with a low cytosolic NAD⁺/NADH ratio [3]. Based on these reported studies, we hypothesized that GPD1 deficiency changes the balance of carbohydrate and fat utilization for energy production. To investigate the change of fuel utilization for energy production, we monitored energy expenditure, carbohydrate oxidation, and fat oxidation by an O₂/CO₂ metabolism measuring system for small animals and simultaneously measured locomotor activity during ad libitum feeding. In HeA mice, energy expenditure was higher than that in the control group at several time points during the active phase (19:00–7:00) and at the one time point in the rest phase (9:00) (Figure 1(a)). The amount of carbohydrate oxidation in the HeA mice was significantly low at two time points (21:00 and 2:00), while the amount of fat oxidation was significantly high during the early active phase (19:00, 21:00–1:00) (Figure 1(b) and (c)). There were no significant differences in the amount of carbohydrate and fat oxidation between the HeA mice and By mice during the rest phase (7:00 - 17:00). The AUC of energy expenditure, carbohydrate oxidation, and fat oxidation obtained from the measurement of all experimental periods were not significantly different. Respiratory quotients (RQ) in the HeA mice during the early active phase was significantly lower (time point at 19:00, 21:00–1:00) than that in the By mice (Figure 1(d)). Enhancement of anabolic reactions such as lipogenesis and TG synthesis induced by dietary intake increases RQ [14,15]. Glycolysis in HeA mice is inhibited due to an impaired GP shuttle [3]. Inhibition of glycolysis decrease the accumulation of visceral fat [16]. Moreover, since GPD1 is an enzyme that supplies glycerol moiety required for TG synthesis, the deletion of GPD1 may impair anabolic reaction included TG synthesis. Indeed, our previous study revealed that the deletion of GPD1 diminishes hepatic TG accumulation induced by alcohol administration [17]. The low RQ in HeA mice observed in the early active phase may be due to the reduction of anabolic reaction in peripheral tissue through GPD1 deletion. The average of RQ obtained from the measurement of all experimental periods were not significantly different. The activity of HeA mice was significantly lower at two time points (2:00 and 7:00), but there was no difference in the AUC (Figure 1(e)). It is widely perceived that physical activity is associated with increase in energy expenditure [18,19]. However, there was no difference in locomotor activity between the two strains. We considered that locomotor activity did not directly affect the difference in energy consumption and fat oxidation in HeA mice, observed at several time points. Feeding behavior has daily rhythm and nocturnal animals such as mice consume most of the daily food during the active phase [20,21]. Food intake promotes carbohydrate-dominant energy production in the peripheral organs, such as the skeletal muscle, while attenuating fat oxidation, thus food intake plays a role as a switch that dramatically changes the metabolic flux [22]. Since GPD1 deletion induces slower glycolysis resulting from a low NAD⁺/NADH ratio, GPD1 deficiency would prevent the switch to carbohydrate metabolism by feeding during the active phase. Our results indicate that GPD1 null mice tend to use fats during the active phase preferentially because of slower glycolysis induced by GPD1 deletion.

When the external energy supply is interrupted, e.g., during fasting, fat is the primary fuel for producing energy needed by the organs [23–25]. To investigate the contribution of GPD1 to energy metabolism during fasting conditions, we measured metabolic parameters such as energy expenditure using the same method as Figure 1. Under fasting conditions, energy expenditure and fat oxidation were significantly higher in HeA mice, regardless of the active or rest phase, and the AUC of these metabolic parameters in the HeA mice were significantly higher than that in the By mice (Figure 2(a) and (c)). We observed that carbohydrate oxidation in HeA mice at 21:00 was higher, but there was no significant difference in the AUC of carbohydrate oxidation (Figure 2(b)). There was no difference in the RQ between By and HeA mice at any time point. (Figure 2(d)). Since the amount of carbohydrate oxidation under the fasting condition decreased to approximately 10% of that under ad libitum feeding conditions, the contribution of carbohydrate oxidation to energy production was inconsiderable. Therefore, the RQ was extremely low and there was no significant difference between the By and HeA mice, even when fat oxidation in the HeA mice was significantly high. While we observed significant differences in activity at some points when compared between the By and HeA mice at each time point, the AUC obtained from all the time points was not significantly different (Figure 2(e)). These results indicate that GPD1 deficiency enhances fasting-induced fat oxidation, concomitant with increased energy expenditure. Our previous study has shown that GPD1 deficiency inhibits fasting-induced gluconeogenesis from glycerol and prompts an adaptation that enhances gluconeogenesis from glycogenic amino acids [5]. Besides, branched-chain amino acid (BCAA) levels in the muscle of HeA mice are decreased under fasting conditions. The increase of BCAA metabolic flux can enhance cellular respiration and fat oxidation via increases in TCA cycle activation in cultured muscle cells [26]. We speculate that further increases in fat oxidation in the HeA mice during fasting may be associated with changes in BCAA metabolism in the skeletal muscle.

As shown in Figures 1 and 2, we observed that fat oxidation in HeA mice was increased during the active phase, especially under fasting conditions. Skeletal muscle is responsible for the body’s energy expenditure, glucose and fatty acid uptake, and their utilization. Skeletal muscle comprises ~ 40% of the total body mass in mammals and accounts for ~30% of the resting metabolic rate in adult humans [27]. To investigate the rate of fatty acid oxidation in the skeletal muscle during the active phase, we measured the production of ¹⁴CO₂ from [¹⁴C] palmitate using isolated muscle obtained at active phase time period (21:00). Muscle fatty acid oxidation rate in the HeA mice tend to be higher (P = 0.054) compared to that in the By mice (Figure 3). This data suggests that the increase in fat oxidation during the active phase in the HeA mice observed in Figures 1 and 2 appeared to may be influenced by the enhancement of fatty acid utilization in the skeletal muscle. AMPK is activated by an increased AMP/ATP ratio associated with ATP consumption [28,29]. The role of AMPK in fatty acid oxidation was estimated with the use of an activator of AMPK, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) [30]. AICAR leads to an increase in uptake of fatty acids into the mitochondria via carnitine palmitoyltransferase 1, thereby stimulating fatty acid oxidation [30,31]. GPD1 deficient animals are unable to maintain normal ATP levels and show high AMP/ATP ratio in the skeletal muscle when forced to exercise vigorously [3]. Our previous studies have shown that GPD1 deficiency induces activation of AMPK and enhances fat availability in the skeletal muscle during exercise [4]. The enhancement of fat oxidation in the skeletal muscle of the mice lacking GPD1 may be involved in the change of AMPK activity due to lower ATP levels caused by inhibition of the GP shuttle.

Our metabolic measurement results showed that energy expenditure and fat utilization tended to be higher in GPD1 deficient mice in Figures 1 and 2. Increased fat oxidation is directly linked to the amount of energy expenditure, and influences weight gain simultaneously [32]. We hypothesized that metabolism flux changed by GPD1 deletion could also affect weight gain. To elucidate the contribution of higher energy expenditure induced by fat dominated metabolism to body weight gain in GPD1 deletion mice, we monitored body weight in HeA mice under normal chow feeding. We found no difference in body weight between the HeA and By mice at 6 weeks (Figure 4(a)). On the other hand, we observed that body weight of the HeA mice were significantly lower than that of the By mice at 10, 15, and 20 weeks. There was no difference in the amount of energy intake at 10, 15, and 20 weeks of age (Figure 4(b)). Reduced fat/carbohydrate oxidation ratio is a predictor of body weight gain. One human study reported that higher RQ, reflecting low rates of fat oxidation relative to carbohydrate oxidation, is associated with a higher rate of subsequent weight gain [33]. The result from Figure 1(d) showed that the RQ in HeA mice during the active phase were significantly low. Our study indicates that GPD1 deletion increases the fat/carbohydrate oxidation ratio and energy expenditure through metabolic flux change, and this metabolic change suppresses weight gain in GPD1 deficient mice.

In conclusion, the present study suggests that GPD1 deficiency increases fat oxidation and energy expenditure during the active phase, especially under fasting conditions. Our ex vivo experiment using the skeletal muscle showed that the utilization of fatty acid in the GPD1 deletion tended to be high under the active phase. Furthermore, GPD1 deficient mice had a smaller increase in body weight than that in the control mice under ad libitum normal chow feeding. We propose that GPD1 deletion contributes to the reduction of body weight gain via enhancing energy expenditure and fat oxidation. However, the HeA mice used in this study systemically lose GPD1 activity. Therefore, we cannot ignore the possibility that tissues other than the skeletal muscle contribute to the GPD1 deficient phenotype. In order to clarify the mechanism of fat-dominated metabolic changes by GPD1 deficiency observed in the present study, future studies will be needed to investigate the expression of genes and proteins involved in energy metabolism in the GPD1 null mice.

Figure 1. Energy metabolism measured by open-circuit indirect calorimetry under ad libitum feeding conditions. (a) Energy expenditure, (b) carbohydrate oxidation, (c) fat oxidation, (d) RQ, and (e) spontaneous physical activity in BALB/cBy (By) and BALB/cHeA (HeA) mice under ad libitum feeding conditions. The age of the mice used in the experiment was 14-16 weeks old. Oxygen consumption and carbon dioxide production were monitored using an O₂/CO₂ metabolism measuring system for small animals. Horizontal open bar, lights on; solid bar, lights off. Values are means ± SEM (n=12). *P < 0.05; **P < 0.01; ***P < 0.001 vs. the same time point in the By mice. The results have been summarized by determining the area under the curve (AUC).

Figure 2. Energy metabolism measured by open-circuit indirect calorimetry under fasting conditions. (a) Energy expenditure, (b) carbohydrate oxidation, (c) fat oxidation, (d) RQ, and (e) spontaneous physical activity in BALB/cBy (By) and BALB/cHeA (HeA) mice under fasting conditions. The age of the mice used in the experiment was 14-16 weeks old. Oxygen consumption and carbon dioxide production were monitored using an O₂/CO₂ metabolism measuring system for small animals. Horizontal open bar, lights on; solid bar, lights off. Values are means ± SEM (n=12). *P < 0.05; **P < 0.01; ***P < 0.001 vs. the same time point in the By mice. The results have been summarized by determining the area under the curve (AUC). ***P < 0.001 vs. the By mice.

Figure 3. Palmitate oxidation in isolated gastrocnemius muscle strips at the active phase. The age of the mice used in the experiment was 59 weeks old. Mice were fed normal chow diet in an ad libitum manner and gastrocnemius muscle was harvested at 21:00. Dissected muscles were immediately used for the measurement of palmitate oxidation. Values are means ± SEM (n=3 for By mice, n=4 for the HeA mice). P values compared to By mice are shown.

Figure 4. Changes of body weight and energy intake. (a) Body weight at 6, 10, 15, and 20 weeks. Male BALB/cBy (By) and BALB/cHeA (HeA) mice were fed normal chow diet under ad libitum conditions. Values are means ± SEM (n=9 for By mice, n=7 for the HeA mice). **P < 0.01 and ***P < 0.001 vs. the By mice. (b) Energy intake at 10, 15, and 20 weeks. Energy intake is shown as kcal/day which is total values of each cage divided by the number of mice in the same cage (n=7).

Author contribution

Tomoki Sato and Shinji Miura designed the experiments. Tomoki Sato, Neo Sayama and Mizuki Inoue performed mouse experiment. Tomoki Sato, Akihito Morita and Shinji Miura interpreted the results and analyzed the data. Tomoki Sato and Shinji Miura prepared figures and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments

We thank Dr. Nobuko Mori (Osaka Prefecture University, Japan) for the kind gift of the GPD1 null mice (BALB/cHeA mice). Grant-in-Aid for JSPS Research Fellow (KAKENHI, numbers 15J10165) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT, Tokyo), the Mishima Kaiun Memorial Foundation (Tokyo, Japan), and a University of Shizuoka Grant for Scientific and Educational Research. We thank the members of the Laboratory of Nutritional Biochemistry (Graduate School of Nutritional and Environmental Sciences, University of Shizuoka) for their technical assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by [Japan Society for the Promotion of Science] under Grant [KAKENHI, numbers 15J10165]; [Mishima Kaiun Memorial Foundation] under Grant [None]; [University of Shizuoka Grant for Scientific and Educational Research] under Grant [None].