Time-restricted feeding improves metabolic syndrome by activating thermogenesis in brown adipose tissue and reducing inflammatory markers

A total of 56 male C57BL/6J mice, aged 6–8 weeks, were purchased from Jackson Laboratory (Bar Harbor, Maine) for this study. All mice were housed in a clean and ventilated environment and fed with standard diet (4–5% calories, SPFW01, SCBS, China) and high-fat diet (60% calories, D12492, research diet, USA). During the initial 2-week acclimation period, all animals were trained under controlled conditions: 12 hours of light and 12 hours of darkness, with ad libitum access to food and water. After two weeks, the animals were randomly assigned to one of two groups for 12 weeks: (i) a control group with free access to food and water during the 12-hour light and 12-hour dark cycles (Control), and (ii) a time-restricted feeding (TRF) group, where food was restricted to an 8-hour window (8:00 AM to 4:00 PM) under the same light/dark cycle. The feeding times were controlled by trained personnel. The experimental protocol was approved by the Ethics Committee of the First Affiliated Hospital of Gannan Medical University. After completing the experiments, tissue samples were collected and processed for biological analysis in an environmentally safe manner.

At 8:00 AM, glucose tolerance and insulin secretion were assessed in all mice after a 16-hour fasting period. For the glucose tolerance test, the mice received an intraperitoneal injection of 20% glucose (2 g/kg body weight). Blood glucose levels were measured at baseline and 15, 30, 45, 60, and 120 minutes post-injection. Plasma samples were collected at baseline and at 2.5, 5, and 15 minutes post-injection to assess insulin levels. For the insulin tolerance test, insulin sensitivity was assessed after a 6-hour fast at 10:00 PM. The mice were injected intraperitoneally with insulin (0.55 mU/g body weight), and blood glucose levels were measured at baseline and 15, 30, 45, 60, and 120 minutes post-injection.

Total RNA was extracted from freshly collected mouse liver and adipose tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany). For quantitative real-time PCR, mRNA was reverse-transcribed to complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA), and qRT-PCR was performed on a StepOnePlus machine (Applied Biosystems, Carlsbad, CA) under default thermal cycling conditions. The 2−ΔΔCT method was used for data analysis, and mRNA levels were normalized to β-actin expression for each sample. The primer sequences are presented in Table 1.

Blood glucose was measured using a Sinocare glucose meter (GA-3, China). Plasma insulin levels were determined using an ultrasensitive mouse insulin ELISA kit (ab277390, USA) according to the manufacturer’s instructions.

Mouse body temperature was measured using a small animal temperature analyzer. Specifically, temperature probes were placed near the oral cavity and interscapular brown adipose tissue (BAT), with readings taken for at least 10 seconds to allow stabilization. Two types of temperature conditions were used: first, room temperature (RT) conditions without any manipulation; and second, cold-induced conditions where mice were acclimated at 4°C for 6 hours before temperature measurement.

Inflammatory markers were measured from mouse serum samples. Plasma IL-6, IL-1β, and TNF-α levels were determined using enzyme-linked immunosorbent assays (ELISA) according to the manufacturer’s instructions.

Fresh tissues were fixed in 4% paraformaldehyde for 24 hours, then sectioned at 5 μm thickness. After hydration in distilled water, sections were stained with hematoxylin for a few minutes, differentiated in acid water and ammonia water, rinsed in running water for 1 hour, and dehydrated in 70% and 90% alcohol for 10 minutes each. Sections were then counterstained with eosin for 2-3 minutes, cleared in xylene, and mounted for microscopic observation. For Oil Red O staining, sections were washed in 60% isopropanol, stained with 0.5% Oil Red O solution for 10 seconds, differentiated again in 60% isopropanol, and mounted for microscopy.

Results are presented as mean ± SE. Statistical analyses were performed using GraphPad Prism v8.4 (San Diego, CA). Comparisons between two groups were made using two-tailed Student’s t-tests, while one-way or two-way ANOVA was used for comparisons among three or more groups. Unless otherwise specified, P < 0.05 was considered statistically significant.

To investigate the effects of time-restricted feeding (TRF) on metabolism and inflammation, we first used mice on a normal diet as the study subjects. The experimental group underwent a 16-hour fasting period with unrestricted access to water (8:00 AM to 12:00 AM) and an 8-hour feeding window, while the control group had free access to food and water. The observation period lasted 12 weeks (Figure 1A). Results showed that during the 12-week observation, there was no significant difference in body weight between the TRF group and the control group initially. However, by weeks 11 and 12, the TRF group showed a significantly lower body weight compared to the control group, with a reduction of approximately 15% (Figure 1B). This suggests that TRF (16:8) can reduce body weight in mice on a normal diet. To further evaluate the effects of TRF on glucose metabolism, we conducted glucose tolerance tests (GTT) and insulin sensitivity tests (ITT). The results revealed that TRF mice exhibited significantly improved glucose tolerance compared to the control group, as evidenced by a lower area under the glucose tolerance curve. Moreover, TRF enhanced insulin sensitivity, particularly at the 15- and 30-minute time points during the insulin test (Figures 2A–D). These findings indicate that TRF improves both glucose tolerance and insulin sensitivity in mice on a normal diet. The underlying mechanism for this improvement in glucose tolerance is unclear, but we hypothesize that TRF may activate pancreatic β-cells to release insulin rapidly upon glucose stimulation. To test this, we performed glucose-stimulated insulin secretion assays. The results showed that, 2.5 minutes after glucose injection, TRF mice exhibited a significantly higher insulin release compared to the control group. This enhanced insulin secretion persisted at 5 and 15 minutes post-injection (Figure 2F). This suggests that TRF positively regulates insulin secretion by pancreatic β-cells, possibly by increasing the number or function of β-cells. Additionally, to assess the impact of TRF on inflammation, we measured serum levels of IL-6, IL-1β, and TNF-α. The results showed that TRF significantly reduced serum IL-6 levels, while no significant changes were observed for IL-1β and TNF-α, which may be due to the relatively low baseline inflammation in mice on a normal diet (Figure 2E). These findings suggest that TRF (16:8) improves insulin sensitivity and mildly reduces serum inflammation in normal-diet mice.

Since TRF improved insulin sensitivity and reduced serum inflammation in normal-diet mice, we further explored its effects on metabolic syndrome in pathological states by using mice on a high-fat diet (HFD). We designed an experiment similar to that for the normal-diet mice, but replaced the standard diet with a high-fat diet (Figure 1A). Over the 12-week period, we found that TRF significantly slowed weight gain in HFD-fed mice compared to the control group, with the first significant difference appearing in week 9, and the difference becoming more pronounced by week 12 (Figures 3A, B). To assess the impact of TRF on glucose metabolism in HFD-fed mice, we performed GTT and ITT. TRF improved glucose tolerance, with the most significant effect observed at 30 minutes post-glucose injection, and a sustained effect through 120 minutes. Insulin sensitivity was also improved, with TRF mice showing significantly lower blood glucose levels 15 minutes after insulin injection compared to the control group, and this difference persisted through 120 minutes (Figures 3C–F). Additionally, glucose-stimulated insulin secretion assays showed that TRF enhanced insulin release in HFD-fed mice, with a significant increase in insulin secretion 2.5 minutes after glucose injection, and this effect persisted through 15 minutes (Figure 3G). These results suggest that TRF enhances the responsiveness of pancreatic β-cells to glucose, possibly by increasing their number or insulin secretion capacity. To investigate the effect of TRF on inflammation in HFD-fed mice, we measured serum levels of IL-6, IL-1β, and TNF-α. TRF significantly reduced serum levels of IL-6 and TNF-α, but no significant change was observed for IL-1β (Figure 3H). These findings suggest that TRF reduces inflammation in HFD-fed mice, alongside improving insulin sensitivity and attenuating obesity.

We further explored whether TRF improves hepatic lipid accumulation. Using Oil Red O staining, we examined lipid accumulation in the livers of HFD-fed mice. Results showed that sustained HFD feeding induced substantial lipid accumulation and vacuolar degeneration in the liver, while TRF significantly reduced both hepatic lipid accumulation and vacuolar degeneration, with a reduction of approximately 30% in lipid content compared to the control group (Figures 4A, B). These results suggest that TRF can reverse hepatic steatosis induced by a high-fat diet. To further understand the mechanism, we measured serum lipid levels, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and apolipoprotein A1 (ApoA-1). TRF significantly reduced serum TG, TC, and ApoA-1 levels but had no effect on LDL levels (Figure 4C), indicating that TRF lowers serum lipid levels, which may contribute to its protective effect against hepatic steatosis. To investigate whether TRF reduces hepatic lipid accumulation by downregulating lipid synthesis, we measured the expression of lipogenic genes (Fasn, Acc1, Srebp1c, and Chrebp). TRF significantly downregulated the expression of Fasn and Acc1, both of which are closely related to lipid synthesis, as well as Srebp1c, a key gene associated with non-alcoholic fatty liver disease (Figure 4D). These findings suggest that TRF ameliorates hepatic steatosis by reducing the expression of lipogenic genes.

Fasting has been found to improve glucose and lipid metabolism in mice fed a high-fat diet (HFD), particularly by reducing hepatic steatosis. Previous studies have shown that the effects of fasting on lipid accumulation are not limited to reducing lipid intake but also include increasing energy expenditure. Therefore, we further investigated the thermogenic effects of brown adipose tissue (BAT), which is a key organ involved in systemic energy metabolism.First, we performed histological staining of BAT from HFD-fed mice to observe changes in BAT size and the whitening effect of BAT under HFD challenge. The results showed that in the control group, BAT exhibited typical whitening under prolonged HFD feeding, characterized by the appearance of large lipid vacuoles, resembling those of white adipose tissue (Figure 5A). In contrast, fasting reversed this whitening phenomenon in the fasting group, where BAT remained denser, without large lipid vacuoles. This indicates that fasting has a protective effect against the whitening of BAT. However, the protective mechanism of fasting on BAT remains unclear.Literature suggests that BAT, as a thermogenic organ, has a heat-generating capacity closely related to its brown phenotype, and increased thermogenesis can result in elevated body temperature. To investigate further, we measured the oral and interscapular BAT temperatures in mice using a small animal temperature monitoring device. The results indicated that, in normal diet-fed mice, fasting had minimal impact on BAT thermogenesis under either room temperature (RT) or cold-induced (cold shock) conditions. However, in HFD-fed mice, fasting enhanced thermogenesis in the interscapular BAT region following cold shock, though no significant differences in thermogenesis were observed between the groups under RT conditions (Figures 5B, C).To verify these phenotypic observations, we measured the expression levels of key thermogenic genes and proteins (UCP1 and PGC-1α) in BAT. Our results demonstrated that time-restricted fasting increased the expression of both UCP1 and PGC-1α at the gene and protein levels in BAT (Figures 5D–F), which could explain the observed rise in BAT temperature in the fasting group. These findings suggest that fasting can regulate BAT thermogenesis, potentially contributing to its therapeutic effects on metabolic syndrome.Additionally, epididymal white adipose tissue (WAT), a representative of white fat, was used to assess fasting’s effects on lipid accumulation. We performed histological staining of epididymal WAT from HFD-fed mice and measured adipocyte diameter. The results showed that fasting reduced adipocyte diameter in epididymal WAT (Figure 5G). We hypothesize that this change may be due to a combination of reduced serum lipid levels and increased BAT thermogenesis induced by fasting.

These results provide insight into the mechanisms by which fasting influences fat thermogenesis and lipid accumulation, offering a potential explanation for its beneficial effects in the treatment of metabolic syndrome (Figure 6).