
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72695
10.1038/s41598-024-72695-3
Article
Optimal fasting duration for mice as assessed by metabolic status
Fu Jian 1
Liu Sha 1
Li Mengyao 1
Guo Fangrui 1
Wu Xiaoran 1
Hu Jiahao 1
Wen Lixin 1
Wang Ji 1
Li Xiaowen lxw0630@csu.edu.cn
lxwwatermelon@163.com

123
1 https://ror.org/01dzed356 grid.257160.7 0000 0004 1761 0331 Hunan Engineering Research Center of Livestock and Poultry Health Care, Colleges of Veterinary Medicine, Hunan Agricultural University, Changsha, China
2 grid.452708.c 0000 0004 1803 0208 National Clinical Research Center for Metabolic Diseases, and Department of Metabolism and Endocrinology, Key Laboratory of Diabetes Immunology, Ministry of Education, and Metabolic Syndrome Research Center, The Second Xiangya Hospital of Central South University, Changsha, China
3 Animal Epidemic Prevention Department, Changsha Agriculture and Rural Affairs Bureau, Changsha, China
14 9 2024
14 9 2024
2024
14 215093 4 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
In the study of obesity and diabetes, mice are widely used for experimental research, and fasting is a common procedure used to reset metabolism in mouse models. The fasting duration for experimental mice varies greatly in nutritional and metabolic studies, ranging from 2 to 48 h. This study aims to assess the optimal fasting duration for mice fed low- and high-fat diets over a short period of time. C57BL/6J mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and fasted for 4, 6, 8, 10, 12, or 24 h. The effects of different conditions after fasting on the metabolic level of mice were explored, and the data were collected for analysis. Our data indicate that fasting has inconsistent effects on mice fed a low-fat or high-fat diet. To compare the metabolic differences between mice in different dietary levels and thereby secure better scientific data, mice should fast for 6 h in animal experiments. Fasting for 6 h is also recommended when comparing glucose tolerance with insulin tolerance.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72695-3.

Keywords

Metabolic research
Nutritional research
Fasting duration
Mouse model
High-fat diet
Subject terms

Animal physiology
Fat metabolism
Homeostasis
the National Key R&D Program of China2016YDF0501200 Wen Lixin the Horizontal topics of Hunan agricultural University2022xczx-414 Wang Ji issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

With increasing research on metabolic diseases, such as obesity and diabetes1, an increasing number of laboratory mice have been used to study the underlying mechanisms. For example, mouse models have been used to evaluate the physiological roles of potential targets in glucose homeostasis and diabetes development2. Fasting is commonly used to reset metabolism and reduce other physiological effects in mouse models3,4. However, the fasting duration for mice varies greatly between studies, ranging from 2 to 48 h5–8.

In general, the blood glucose of animals gradually drops after meals and remains in the lower range after fasting9. Studies have shown that significant weight loss is achieved after fasting for 2 h in mice10. Changes in observed insulin and glucagon levels have been observed after 12 h of fasting11. After 24 h of fasting, humans, rodents, and other mammals enter alternative metabolic phases, which rely less on glucose and more on ketone body-like carbon sources to regulate blood glucose homeostasis12. In most studies, 12 h of fasting is used to avoid the effect of either food in the GI tract or the effect of newly absorbed nutrients8. The standard approach for fasting mice prior to a GTT is the overnight fast13,14. This is likely a procedural remnant from GTTs performed in humans, typically conducted in overnight-fasted subjects15.

The length of time mice fast can affect both the research results and animal welfare16. In 2010, the Mouse Metabolic Phenotype Center (MMPC) consortium of the National Institutes of Health proposed to shorten standard fasting times to reduce metabolic stress in mice and suggested that fasting for 6 h should be the standard for glucose homeostasis metabolic testing in mice17. Some metabolic studies have experimented with shorter fasting periods (< 6 h)18,19. In addition, preoperative fasting is often performed to prevent pulmonary aspiration and improve intestinal preparation20. Interestingly, one study showed that fasting for as long as 24, 48, or 72 h did not cause gastric emptying in mice21. The welfare of animals should be considered continuously and abide by the 3R principle (replacement, reduction and refinement); therefore, the optimization of fasting procedures is worth exploring.

Physiological parameters including blood glucose levels21, related hormone levels22, and the status of blood glucose regulation pathways11 differ depending on feed composition23–25 and the duration of fasting25. Various studies have presented different requirements to obtain this series of changes. Mice-fed high-fat diets are commonly used for metabolic research. Therefore, the goal of this study was to fully understand the effects of fasting duration on blood glucose levels and to investigate the underlying mechanisms in mice. This information could greatly impact experimental design in animal model research and help optimize animal welfare conditions. In this study, mice were fed low- or high-fat diets to study the metabolic changes after fasting through a series of experiments. The overall goal was to determine the best fasting duration for mice fed a low- or high-fat diets and to provide a scientific basis for choosing appropriate fasting times in mice studies.

Results

Food intake and blood glucose levels of mice during feeding

The experimental design is shown in Fig. 1a. Mice did most of their feeding from 10:30 to 14:30 (Fig. 1b), while the highest and lowest blood glucose concentrations were found at 12:30 and 18:30, respectively (Fig. 1c). However, the variation in blood glucose during the daytime was generally small and there is no significant difference (p adjust ≥ 0.05). During the night, there were two feeding peaks for mice: from 22:30 to 02:30 and from 04:30 to 08:30 (Fig. 1d). The amount of food consumed at night was significantly higher than the amount consumed during the day. The blood glucose levels of the mice increased significantly during feeding (22:30 to 00:30), then began to decline significantly (p adjust = 0.0102, p adjust = 0.0099), and remained stable after 04:30 h (Fig. 1e). The blood glucose levels of mice fluctuated more at night than during the day. Our finding indicates that mice fed mostly during the night is consistent with the circadian rhythms that most mouse activity occurs at night.

Fig. 1 Food intake and blood glucose levels of mice during day and night. Schematic of the experimental design (a), the same animals were measured repeatedly every 2 h. Feed intake (b) and blood glucose levels (c) of mice during the day. Feed intake (d) and blood glucose levels (e) of mice during the night. Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and Benjamini, Krieger and Yekutieli multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p adjust < 0.05), and as long as there are the same letters indicate insignificant differences (p adjust ≥ 0.05) (n = 10/group).

Changes in feed intake and blood glucose levels in mice after fasting

The experimental design is shown in Fig. 2a. Mice were fed LFD or HFD for one week. After 12 h of fasting, the feed intake of LFD mice was concentrated in the first 3 h (Fig. 2b) and that of HFD mice was concentrated in the first 4 h (Fig. 2c). The blood glucose levels of LFD mice increased significantly 1 h after feeding (p adjust < 0.0001) and returned to stability after 3 h (Fig. 2d), whereas those of HFD mice did not change significantly after 6 h of feeding (p adjust ≥ 0.05; Fig. 2e). This indicated that prolonged fasting (12 h) had a greater effect on LFD mice.

Fig. 2 Feed intake and blood glucose levels of mice during 6 h feeding. Schematic of the experimental design (a), the same animals were measured repeatedly every 2 h. Feed intake in LFD mice (b) and HFD mice (c). Blood glucose levels in LFD mice (d) and HFD mice (e). Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and Benjamini, Krieger and Yekutieli multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p adjust < 0.05), and as long as there are the same letters indicate insignificant differences (p adjust ≥ 0.05) (n = 10/group).

Effects of time and duration of fasting on blood glucose and ketone levels in mice

The experimental design is shown in Fig. 3a, b. Mice were fed LFD or HFD for one week, we designed two experiments and the results were as follows. Through 12 h of fasting, the blood glucose levels of LFD mice decreased significantly after 2 h (p adjust = 0.0053) and remained stable from 4 h onward (Fig. 3c), whereas HFD mice had significantly decreased blood glucose levels after 2 h of fasting (p adjust < 0.0083) which then remained stable (Fig. 3d). This indicates that the blood glucose levels of HFD mice regained stability more quickly after fasting. Changes in blood ketone levels were consistent in both groups of mice: blood ketone levels significantly decreased after 2 h of fasting (p adjust < 0.0028, p adjust < 0.0442), remained stable from 2 to 4 h, and then increased after 6 h (Fig. 3e, f). These results suggest that the mice used pyruvate for gluconeogenesis as an energy supply after 2 h of fasting, whereas the increase in blood ketone levels after 6 h may be the result of lipolysis.

Fig. 3 Blood glucose and blood ketone levels in LFD and HFD mice. Schematic of the experimental design (a) fasting duration or (b) the time of fasting initiation to mice, the same animals were measured repeatedly every 2 h. Blood glucose levels in LFD mice (c) and HFD mice (d). Blood ketone levels in LFD mice (e) and HFD mice (f) (fasted for 12 h, fed for 1 h and then fasted for 12 h again). Blood glucose levels in LFD mice (g) and HFD mice (h) after fasting during the day (fasting from 8:30 a.m. to 00:30 a.m. the next day). Blood glucose levels in LFD mice (i) and HFD mice (j) after fasting during the night (fasting from 20:30 to 12:30 p.m. the next day). Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and Benjamini, Krieger and Yekutieli multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p adjust < 0.05), and as long as there are the same letters indicate insignificant differences (n = 10/group).

The experimental design for fasting at 8:30 am or 20:30 is shown in Fig. 3b. The fasting time from 8:30 to 00:30 of the next day, the blood glucose levels of LFD mice decreased significantly after fasting for 2 h (p adjust < 0.0001) and remained stable through 14 h of fasting (Fig. 3g). In HFD mice, blood glucose levels decreased significantly after fasting for 2 h (p adjust < 0.0001) and remained stable through 10 h of fasting (Fig. 3h). During the period of 20:30 to 12:30 the next day, blood glucose levels in both groups of mice decreased significantly after 2 h of fasting, remaining stable for 6 h in LFD mice (Fig. 3i, j). These results showed that fasting during the day and night had different effects on mice.

Effects of fasting duration on OGTT and ITT

The experimental design is shown in Fig. 4a. To determine the effect of fasting on glucose tolerance, LFD and HFD mice were fasted for 4, 6, and 8 h, and an oral glucose tolerance test (OGTT) was performed. During the OGTT, there was a significant increase in blood glucose levels in LFD vs. HFD mice only after 6 h of fasting (Fig. 4d). This was also illustrated with the AUC measurement (p = 0.0160; Fig. 4e). There were no significant differences at 4 and 8 h of fasting (p ≥ 0.05; Fig. 4b, c, f, g). In addition, to determine the effect of fasting on insulin tolerance, LFD and HFD mice fasted for 2, 4, and 6 h, and an insulin tolerance test (ITT) was performed. Interestingly, blood glucose levels during the ITT were also significantly increased following 6 h of fasting (p = 0.0459; Fig. 4l, m). There were no significant changes at 2 and 4 h of fasting (p ≥ 0.05; Fig. 4h-k).

Fig. 4 Effects of fasting duration on OGTT and ITT. Schematic of the experimental design (a), for different durations of fasting different groups of mice were measured. Blood glucose levels (b, d, f) in mice and the AUC of glucose (c, e, g) fasted for 4, 6, or 8 h during an OGTT. Blood glucose levels (h, j, l) in mice and the AUC of glucose (i, k, m) fasted for 2, 4, or 6 h during an ITT. Data are shown as mean ± SEM, and significance was determined using an unpaired t-test. *p < 0.05, ns p ≥ 0.05 (n = 10/group).

Effects of fasting on glucose metabolism

The experimental design is shown in Fig. 5a. To further investigate the effects of fasting on glucose metabolism in mice, three genes related to glucose metabolism were selected for RT-qPCR. Pdk4, Pygl, and Pck1 were selected for their roles in glucose metabolism, glycogenolysis, and gluconeogenesis. Compared with 0 h, Pdk4 expression in LFD mice and HFD mice was significantly downregulated after 8 h and 4 h of fasting, respectively (p adjust = 0.0399, p adjust = 0.0009; Fig. 5b, c), and glucose utilization was increased. The liver glycogen content of LFD mice tended to decrease after fasting, showing a significant difference at 4 h (p adjust < 0.0001), and remained stable after 8 h (Fig. 5d). HFD mice were significantly decreased at 8 and 12 h of fasting (p adjust = 0.0071, p adjust = 0.0050) and increased immediately after the decrease (Fig. 5e). The expression of Pygl in LFD mice and HFD mice were significantly upregulated at 4 h and 8 h of fasting, respectively (p adjust < 0.0001, p adjust = 0.0134; Fig. 5f, g). Serum pyruvate levels of LFD mice and HFD mice continued to decrease from 4 to 12 h of fasting, with significant differences observed at 12 h and 10 h, respectively (p adjust = 0.0143, p adjust = 0.0128; Fig. 5h, i). Muscle lactate levels in both groups decreased from 10 h of fasting onward and HFD mice were significantly lower at 24 h (p adjust = 0.0062; Fig. 5j, k). The expression of Pck1 was significantly upregulated at 10 h of fasting in two groups. (p adjust < 0.0028, p adjust < 0.0001; Fig. 5l, m).

Fig. 5 Changes of glucose metabolism in mice during fasting. Schematic of the experimental design (a). Relative Pdk4 expression in the liver of LFD mice (b) and HFD mice (c). Liver glycogen of LFD mice (d) and HFD mice (e). Relative Pygl expression in the liver of LFD mice (f) and HFD mice (g). Serum pyruvate of LFD mice (h) and HFD mice (i). Muscle lactic acid of LFD mice (j) and HFD mice (k). Relative Pck1 expression in the liver of LFD mice (l) and HFD mice (m). Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and Benjamini, Krieger and Yekutieli multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p adjust < 0.05), and as long as there are the same letters indicate insignificant differences (n = 8/period).

Effects of fasting on serum hormones

The serum insulin contents in LFD and HFD mice decreased significantly after 6 h and 10 h of fasting (p = 0.0431; p = 0.0315; Fig. 6a, b), respectively. The serum glucagon content in LFD mice increased significantly following 6 h of fasting (p = 0.0101; Fig. 6c) and remained fluctuating after decreasing at 8 h of fasting. The Serum glucagon content in LFD Mice were Substantially Stabilized During the Fasting Period (p ≥ 0.05; Fig. 6d), but the ratio of insulin to glucagon decreased significantly at 10 h (p = 0.0090; Fig. 6f). And the ratio in LFD mice decreased significantly after fasting for 6 h (p = 0.0193; Fig. 6e).

Fig. 6 Changes of serum hormones in mice during fasting. Serum insulin levels in LFD mice (a) and HFD mice (b). Serum glucagon levels in LFD mice (c) and HFD mice (d). The ratio of insulin to glucagon in LFD mice (e) and HFD mice (f). Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and least significance difference (LSD) multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p < 0.05), and as long as there are the same letters indicate insignificant differences (n = 8/period).

Effects of fasting on lipid

In parallel to glucose metabolism changes, there were profound changes in systemic lipid metabolism. A significant increase in serum NEFA levels was observed in LFD and HFD mice after 6 h of fasting, which then remained stable and continued to rise significantly again after 10 h of fasting (p adjust = 0.0011; p adjust = 0.0460; Fig. 7c, d). TG levels in the serum of LFD mice decreased significantly at 10 h of fasting, followed by a significant increase at 24 h (p adjust = 0.0284, p adjust < 0.0001; Fig. 7a). Serum TG in HFD mice were significantly increased at 6 h of fasting (p adjust = 0.0280), returned to initial levels at 10 h, and significantly increased again at 24 h (p adjust < 0.0001; Fig. 7b). The expression of Pparα in LFD mice and HFD mice were significantly upregulated at 4 h of fasting (p adjust = 0.0050, p adjust < 0.0001; Fig. 7e, f). The expression of Cpt1 in LFD mice and HFD mice were significantly upregulated at 8 h and 4 h of fasting, respectively (p adjust = 0.0152, p adjust = 0.0063; Fig. 7g, h), and a significant increase in serum pyruvate levels was observed at 24 h (p adjust < 0.0001; Fig. 5h, i).

Fig. 7 Changes of lipid metabolism in mice during fasting. Serum TG of LFD mice (a) and HFD mice (b). Serum NEFA of LFD mice (c) and HFD mice (d). Relative Pparα expression in the liver of LFD mice (e) and HFD mice (f). Relative Cpt1 expression in the liver of LFD mice (g) and HFD mice (h). Relative Hsl expression in epididymal adipose of LFD mice (i) and HFD mice (j). Relative Hsl expression in perirenal adipose of LFD mice (k) and HFD mice (l). Relative Hsl expression in subcutaneous adipose of LFD mice (m) and HFD mice (n). Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and Benjamini, Krieger and Yekutieli multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p adjust < 0.05), and as long as there are the same letters indicate insignificant differences (n = 8/period).

We then examined the changes in the expression of Hsl, which promotes triglyceride catabolism in subcutaneous, perirenal, and epididymal adipose tissues. Hsl expression levels in the epididymal adipose tissue of LFD mice were significantly upregulated at 4 h of fasting (p adjust = 0.0109; Fig. 7i). In the perirenal adipose tissue, Hsl expression was significantly upregulated after 6 h of fasting (p adjust = 0.0002; Fig. 7k). In the subcutaneous adipose tissue, Hsl expression was significantly downregulated at 4 h of fasting (p adjust = 0.0086), started to increase at 6 h, and was significantly upregulated at 8 h (p adjust = 0.0403; Fig. 7m). While Hsl expression levels in the epididymal and perirenal adipose tissues of HFD mice were significantly upregulated from 4 h of fasting onward (p adjust < 0.0001, p adjust = 0.0006; Fig. 7j, l), in subcutaneous adipose tissue were significantly upregulated only at 4, 10, and 24 h of fasting (p adjust < 0.0001, p adjust = 0.0003, 0.0329; Fig. 7n). These results indicate that lipolysis occurred in epididymal adipose tissue, perirenal adipose tissue, and subcutaneous adipose tissue of LFD mice after 4, 6, and 8 h of fasting, respectively. All three types of adipose mobilization began in HFD mice after 4 h of fasting, thus leading to an increase in transitional TG levels. However, this occurred mainly in epididymal and perirenal adipose catabolism, with occasional catabolism in the subcutaneous adipose tissue for energy supply.

Effects of fasting on protein metabolism

When fasting is prolonged after considerable glycogen and fat consumption, the body catabolizes protein for energy. Therefore, we evaluated the effect of fasting on protein metabolism in the skeletal muscle. Blood ammonia is derived from the catabolism of amino acids in various tissues and can be supplied by gluconeogenesis. Blood ammonia levels in LFD mice significantly decreased after 4 h of fasting (p adjust = 0.0022; Fig. 8a). There was a tendency for the blood ammonia level to rebound after 12 h of fasting, which was associated with a significant upregulation of the expression levels of the proteolytic genes Murf1 and Mafbx at 12 and 24 h of fasting (p adjust = 0.0032, p adjust < 0.0001, p adjust < 0.0001; Fig. 8c, e). Blood ammonia levels in HFD mice significantly decreased after 8 h of fasting and remained stable, while significantly increased at 24 h of fasting (p adjust = 0.0220, p adjust = 0.0005; Fig. 8b). Similarly, it was observed that a significant upregulation in the expression of the proteolytic genes Mafbx and Murf1 at 24 h of fasting (p adjust < 0.0001; Fig. 8d, f).

Fig. 8 Changes of protein metabolism in mice during fasting. Blood ammonia levels of LFD mice (a) and HFD mice (b). Relative Murf1 expression in skeletal muscle of LFD mice (c) and HFD mice (d). Relative Mafbx expression in skeletal muscle of LFD mice (e) and HFD mice (f). Each group was compared to the other groups separately. Data are shown as mean ± SEM, and significance was determined using one-way ANOVA and Benjamini, Krieger and Yekutieli multiple comparisons test. Different letters in the graphs indicate significant differences between different fasting durations with the same diets (p adjust < 0.05), and as long as there are the same letters indicate insignificant differences (n = 8/period).

Discussion

In mammals, energy intake and expenditure vary according to the sleep/wake and fasting/feeding periods, the timing of which depends on whether the species is diurnal or nocturnal26. Mice tend to sleep during the daytime with most of their eating, mating, and childbirth activities occurring at night. Research has shown that mice consume more food and water during scotophase than during photophase; approximately two-thirds of their total food and water intake is consumed during scotophase4. Consistent with the results of other studies, our results suggest that foraging in mice occurs primarily at night. In addition, most aspects of energy metabolism display clear variations during the day and night; these changes have been well documented. For example, plasma glucose concentration, glucose tolerance, and insulin sensitivity have been shown to vary throughout the day27. In the present study, we found that the blood glucose levels of the mice fluctuated at night.

Fasting is widely used in metabolic research to reduce the variability of physiological readouts, including blood glucose levels in mice. In recent years, the prevalence of many comorbidities of obesity, including type 2 diabetes and cardiovascular disease, has increased at an accelerated pace26. HFD mice are typically used to study obesity and related metabolic diseases. However, across metabolic experiments, a consistent duration of fasting for mice fed low-fat or high-fat diets has not been established. Therefore, we explored the differences in metabolism between mice fed low- or high-fat diets after different durations of fasting. Our data suggest that HFD mice regain glycemic stability more quickly than LFD mice after fasting and that prolonged fasting has a greater effect on LFD mice. More significant metabolic changes were seen when fasting was initiated at night than when fasting was initiated during the day21. We also observed that both LFD and HFD mice maintained stable blood glucose levels for longer periods during daytime fasting and that blood glucose levels were lower after nighttime fasting. Our data suggest that fasting induces greater changes in blood glucose levels if it begins at night.

OGTT and ITT are two classic experiments used to evaluate metabolic status. They evaluate glucose tolerance and insulin resistance, respectively, and are usually preceded by a fast to obtain baseline blood glucose levels. The duration of fasting before the experiment can have effects on the experimental results28. As discussed above, a shorter (6 h) fast is more physiological for mice. Therefore, three fasting times (4, 6, and 8 h)were tested in OGTT. Insulin administration in fasted animals carries the risk of inducing hypoglycaemia2, so we chose a shorter fasting time (2, 4, and 6 h) in ITT. We found that following 6 h of fasting there was a clear indication of glucose intolerance and insulin intolerance in the HFD mice compared with LFD mice. Therefore, for the sake of comparison of glucose tolerance and insulin tolerance in different dietary levels, we suggest that fasting for 6 h in OGTT and ITT. But this result only applied to a fasting period starting at the beginning of the photophase. After 3 h of fasting initiated in the scotophase, the plasma glucose concentrations reached a level similar to 18 h of fasting initiated in the photophase, a fasting period starting at the beginning of the scotophase may result in a possibly extended fasting period21. Therefore, if fasting was started with the beginning of scotophase, the results might have been different.

We further investigated the effects of fasting time on glucose, lipid, and protein metabolism in LFD and HFD mice. Our data showed that LFD and HFD mice had significantly increased glucose utilization after 8 h and 4 h of fasting, respectively. Serum insulin and glucagon levels changed significantly at 6 and 10 h after fasting to maintain blood glucose stability. This was paralleled by time-dependent depletion of hepatic glycogen in LFD mice reflecting an elevated rate of hepatic glycogen output during fasting to maintain steady blood glucose levels. In contrast, liver glycogen levels in HFD mice decreased significantly only at 8 h and 12 h. A previous study reported that mice fed on a HFD developed insulin resistance, displayed increased levels of blood glucose and insulin, as well as decreased glycogen synthesis29. This may explain the 3-fold difference in starting glycogen (0 h) between LFD and HFD. Prolonged fasting or starvation leads to the chronic activation of gluconeogenesis, thereby enabling enhanced glucose production in the liver to maintain glucose homeostasis30. Hepatic gluconeogenesis is an important mode of supplying fuel during fasting. Pyruvate is an important raw material for gluconeogenesis. We observed that the level of pyruvate continued to decrease after 4–12 h of fasting and lactate content began to decrease after 10 h. During energy deprivation, glucose is scarce. To decrease the utilization of glucose to preserve glucose to conserve glucose, pyruvate dehydrogenase complex (PDC) activity is suppressed by pyruvate dehydrogenase kinase (PDK)’s hyper-phosphorylation, limiting the conversion of pyruvate to acetyl-CoA31. Therefore, the expression of PDK4 was significantly up-regulated, and the content of pyruvate increased significantly after fasting for 24 h. PCK1 is a rate-limiting enzyme in the regulation of gluconeogenesis and is involved in the maintenance of blood glucose levels. We found that Pck1 expression was significantly upregulated in the livers of both groups at 10 h of fasting.

PPARα, a nuclear hormone receptor activated by fatty acid (FA), is central to the metabolic shift from glucose to fat utilization initiated by fasting32,33. Upon activation of PPARα, FA oxidation is stimulated and ketogenesis is enhanced34. Research has shown that after 6–8 h of fasting ketone production and LDL-C levels can be detected, while HDL-C levels increase, which represents a shift in the body from fat storage to fat use35. In this study, liver Ppara and Cpt1 expression levels in LFD and HFD mice were significantly upregulated from 4 h of fasting onward, reflecting a major metabolic switch in the liver to activate FA β-oxidation and ketogenesis33. The metabolic switch was also visible at the systemic level with rapid induction of whole-body lipolysis reflected by elevated circulating levels of NEFA in LFD and HFD mice after 4 and 6 h of fasting. Consistent with the increased Ppara and Cpt1 expression in the liver, pyruvic acid levels increased after 24 h of fasting. There were differences in whole-body lipolysis between the two groups of mice. After fasting, the epididymal adipose tissue of LFD mice was lipolyzed first, and the lipolysis of perirenal adipose tissue and subcutaneous adipose tissue were significantly increased after 6 h and 8 h of fasting, respectively. The three types of adipose tissue in HFD mice began to lipolyze simultaneously after 4 h of fasting, but epididymal and perirenal adipose tissue predominated, whereas subcutaneous adipose tissue was only occasionally lipolysis for energy supply. Thus, after fasting, the lipolysis of adipose tissue in HFD mice was earlier than in LFD mice. Earlier lipolysis may be responsible for the absence of significant changes in hepatic glycogen and faster activation of Pck1 in HFD mice. Meanwhile, as lipolysis continued to increase, the transitional TG content increased in both groups after 24 h of fasting.

Although fasting promotes a metabolic switch both in the liver and systemically, it inevitably induces skeletal muscle proteolysis. The loss of muscle proteins primarily results from the increased degradation of cell proteins36. Two major protein degradation pathways, the ubiquitin-proteasome and autophagy-lysosome systems, are activated during muscle atrophy and variably contribute to the loss of muscle mass37. Fasting for 12 and 24 h promotes the robust induction of Murf1 and Mafbx, two E3-ubiquitin ligases involved in muscle atrophy, in a variety of pathophysiological states38,39. Consistent with this finding, blood ammonia levels in mice in the current study rebounded after 24 h of fasting.

Conclusions

In conclusion, we have evaluated the metabolic changes in LFD and HFD mice after fasting. Our data indicate that fasting has inconsistent effects on mice fed a low-fat or high-fat diet. To minimize the discomfort of the animals, and hence increase animal welfare. To compare the metabolic differences between mice in different dietary levels and thereby secure better scientific data. In animal experiments, LFD and HFD mice should be fasted for 6 h. Fasting for 6 h is also recommended in OGTT and ITT. For practical reasons inherent to experimental research, fasting is often conducted during the day; however, our preliminary experimental data suggest that fasting at night has a greater effect on mouse metabolism. Therefore, if fasting is initiated at night, the fasting time should be reduced appropriately.

Methods

All six-week-old male C57BL/6 mice were obtained from the Hunan Slake Jingda Experimental Animals Co., Ltd. (Changsha, China). All experimental procedures complied with ARRIVE guidelines and the Animal Care and Use Guidelines of China and were approved by the Animal Care Committee of Hunan Agricultural University (No. 43322105, approval date: 7 November 2022).

1. Experiment 1: changes in blood glucose in mice during feeding

Six-week-old male C57BL/6J mice were housed at room temperature (22 ± 2℃) with a 12-hour-light/12-hour-dark cycle (the dark cycle going from 8:30 pm until 08:30 am). Mice were provided ad libitum access to water and food. After a week, twenty mice were randomly assigned to two experimental groups (n = 10 per group). Feed intake and blood glucose levels of one group were measured every 2 h for 12 h starting at 08:30, and the other group started at 20:30. The blood glucose levels were measured using an ACCU-CHEK glucose meter (Roche, Shanghai, China).

Experiment 2: effect of fasting duration on mice

Changes in feeding patterns and blood glucose after fasting

Twenty six-week-old C57BL/6J male mice were randomly divided into two groups (n = 10 per group) and fed a low-fat diet (LFD) or high-fat diet (HFD) for a week. Diet composition and calorie levels in mice are shown in Supplementary Tables S1 and S2. After fasting for 12 h, access to food was restored at 20:30. Feed intake and blood glucose were recorded at 0, 1, 2, 3, 4, 5, and 6 h after intake resumption.

Effect of fasting duration on blood glucose and ketone levels in mice

Twenty six-week-old male C57BL/6J male mice were randomly divided into two groups (n = 10 per group) and fed an LFD or HFD for a week. To ensure that the mice were fed before fasting, they were fasted for 12 h, fed for x h (where x was the time it took to reach maximum blood glucose levels after intake resumption in Sect. 2.1.), and then fasted for 12 h again. Blood glucose and ketone levels were measured every 2 h for 12 h from 08:30 am.

Effect of the time of fasting initiation on blood glucose levels in mice

Forty six-week-old male C57BL/6J mice were randomly divided into four groups with 10 mice per group. Two groups were fed LFD and two were fed HFD. One LFD group and one HFD group were fasted starting at 08:30 and blood glucose was measured every 2 h until 00:30 the next day. The other two groups were fasted from 20:30 and blood glucose was measured every 2 h until 12:30 the next day.

Effect of fasting duration on oral glucose tolerance test (OGTT) results in mice

Sixty six-week-old C57BL/6J male mice were randomly divided into two groups with 30 mice per group and fed an LFD or HFD. To ensure that the mice were fed before fasting, they were treated as described in Sect. 2.2. Each dietary group was split into sub-groups of ten mice each; each sub-group was fasted for 4, 6, or 8 h from 08:30 am. A 40% glucose solution was administered at a dose of 3 g/kg BW by oral gavage. Blood glucose was measured at 30, 60, 90, and 120 min after gavage and the area under the curve (AUC) was calculated for each mouse group.

Effect of fasting time on insulin tolerance test (ITT) results in mice

Sixty six-week-old C57BL/6J male mice were treated as in 2.4 and fasted for 2, 4, or 6 h from 08:30 am. Insulin 0.5 mU/g body weight (Novo Nordisk, Denmark) was injected intraperitoneally. Blood glucose was measured at 30, 60, 90, and 120 min after injection and the blood glucose AUC was calculated for each group of mice.

Experiment 3: The effect of fasting duration on metabolism and related regulatory pathways in mice

Experimental design

After a 3-day acclimation period (fed with an LFD), one hundred and twelve six-week-old C57BL/6J male mice were randomly divided into 2 groups and fed either an LFD or an HFD for one month. At the end of this month, 8 mice from each group were fasted for 0, 4, 6, 8, 10, 12, or 24 h then anesthetized via an intraperitoneal injection with 2.5% tribromoethanol at 0.01 mL/g body weight. The entire protocol started at 08:30 am and ended at 20:30 pm except for the 24 h fasting group, which was started to fast at 08:30 am the day before. Blood samples were taken from the orbital vein, and mice were euthanized by cervical dislocation.

Sample collection and preparation

Blood samples were collected overnight at 4 °C through the retro-orbital plexus of the mice. The sera were separated by centrifugation at 3500 g for 10 min at 4 °C and were then immediately stored at -80 °C until analyses. The liver, leg muscle, subcutaneous adipose, epididymal adipose and perirenal adipose tissues were collected. The tissues were washed with a physiologic saline solution and stored at -80 °C immediately until analyses.

Measurement of blood lipid and serum hormones

A biochemical analyzer (Mindray Bio-Medical Electronics Co., Shenzhen, China) was used to measure triglycerides (TG). Non-esterified fatty acid (NEFA) was measured using a non-esterified free fatty acids assay kit purchased from Nanjing Jian Cheng Biological Engineering Co., Ltd. (Nanjing, China). Insulin and glucagon levels were measured using ELISA kits purchased from Jingmei Biotechnology Co., Ltd. (Jiangsu, China) according to the manufacturer’s instructions.

Biochemical parameters of the blood, liver, and muscle

Serum pyruvate, serum ammonia, liver glycogen, muscle glycogen, and muscle lactic acid detection kits were purchased from Nanjing Jian Cheng Biological Engineering Co., Ltd. (Nanjing, China). The assays were performed according to the manufacturer’s instructions.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted using the TRIzol (Accurate Biology, Hunan, China) method according to the manufacturer’s instructions. RNA concentration was detected with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was then converted into complementary DNA (cDNA) using the Evo M-MLV Reverse Transcription Kit (Accurate Biology, Hunan, China). RT-qPCR was performed using the StepOne Real-Time PCR System (Applied Biosystems, USA) with the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701; Accurate Biology, Hunan, China), according to the protocols. Briefly, Thermal cycling conditions were as follows: one cycle at 95 °C for 10 min, 95 °C for 30 s, and 60 °C for 30 s. Primers of chosen genes are listed in Table S3. Relative CT amounts were normalized to β-actin expression, and the results were calculated using the 2−ΔΔCT method.

Statistical analysis

All data were analyzed using GraphPad Prism (version 9.0; GraphPad Software, San Diego, CA). Data were tested for normality using the Shapiro − Wilk test. Non-normally distributed data were analyzed using the Kruskal − Wallis rank-sum test. Normally distributed data were analyzed using one-way analysis of variance (ANOVA). The multiple comparison test was analyzed by using two-stage step-up method of Benjamini, Krieger and Yekutieli to control the false discovery rate (FDR). Statistical significance was set at p adjust < 0.05, while p adjust < 0.01 was considered highly significant.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

We would like to thank Editage for English language editing.

Author contributions

Conceptualization: J.W.; Methodology: J.F., S.L. and J.H.; Formal analysis: F.G. and X.W.; Writing – original draft: J.F., S.L. and M.L.; Writing – review & editing: L.W., J.W. and X.L.; Project administration: X.L. Funding acquisition: L.W.

Funding

The study was supported by the Horizontal topics of Hunan agricultural University (2022xczx-414) and the National Key R&D Program of China (Grant No. 2016YDF0501200).

Data availability

All data used in this study are available from Sha Liu (ls100585@stu.hunau.edu.cn) on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jian Fu and Sha Liu contributed equally to this work.
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