Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet

Abstract

While diet-induced obesity has been exclusively attributed to increased caloric intake from fat, animals fed a high-fat diet (HFD) ad libitum (ad lib) eat frequently throughout day and night, disrupting the normal feeding cycle. To test whether obesity and metabolic diseases result from HFD or disruption of metabolic cycles, we subjected mice to either ad lib or time-restricted feeding (tRF) of a HFD for 8 hr per day. Mice under tRF consume equivalent calories from HFD as those with ad lib access yet are protected against obesity, hyperinsulinemia, hepatic steatosis, and inflammation and have improved motor coordination. The tRF regimen improved CREB, mTOR, and AMPK pathway function and oscillations of the circadian clock and their target genes' expression. These changes in catabolic and anabolic pathways altered liver metabolome and improved nutrient utilization and energy expenditure. We demonstrate in mice that tRF regimen is a nonpharmacological strategy against obesity and associated diseases.

Figure 1. Time of feeding shapes diurnal pattern of whole-body metabolism and influences body weight gain

(A) Schematic outline of four feeding regimens used in this study. Time restricted fed mice were allowed access to food from ZT13 through ZT21. Food availability is indicated by light beige boxes. (B) Food ingested, (C) respiratory exchange ratio (CO₂ exhaled/O₂ inhaled), (D) average activity (+ SEM, n = 4 mice) and (E) whole body energy expenditure as measured by volume of O₂ consumed in 2 h bins plotted against time. Since the high fat (HF) diet is energy rich (5.51 Kcal/g), the mice on HF diet consume equivalent amount of energy as the mice on normal chow (NC) (3.36 Kcal/g). (F) Area under the curve analyses of energy expenditure (from Figure 1E) (+ SEM, n = 4, *p < 0.05). Given the differences in body composition, metabolic activities in different organs and heterogeneity of substrate uses in different groups of mice, both food intake and energy expenditures were expressed relative to individual animal or unit body weight. (G) Cumulative average energy intake or (H) average daily energy intake (+ SEM, n = 24 over 17 weeks) by individual mice on NC or HF diet is not significantly different (n.s., p > 0.05) under ad lib or tRF paradigm. The near equivalent average energy intake from NC and HF diet is not different from several published studies. Voluntary energy intake is often independent of dietary fat content both in rodents and human twins (examples include but are not limited to (Bray et al., 2010; Fujisaka et al., 2011; Hosooka et al., 2008; Kennedy et al., 2007; Lin et al., 2000; Saltzman et al., 1997; Samuel et al., 2004)), and these studies also show irrespective of caloric intake, voluntary ingestion of high fat diet predisposes to obesity, diabetes and related diseases. The higher proportion of energy intake from fat is usually considered the cause for diet-induced obesity in these studies. (I) Average energy intake (+ SEM) normalized to unit body weight shows no difference at the beginning of the experiment. In the subsequent weeks with gradual increase in body weight, this value progressively declines. By the end of 16–18 weeks, mice on tRF consume more energy/unit body weight than the ad lib counterparts. (J) Representative FT mice were remarkably leaner than the FA mice. (K) Average body weight (+ SEM, n = 20 – 32 mice). Also see Figure S1.

Figure 2. Time restricted feeding improves diurnal rhythms in metabolic regulators and the circadian oscillator

Representative immuno blots and densitometry quantification of the immuno blots (average (+ SEM, n = 3) during feeding and fasting in the tRF mice which coincide with night and day, respectively) for (A) transcriptionally active phospho-Ser133-CREB and (B) phospho-S6 in the mouse liver. (C) Whisker plot showing the AMP level in the liver of FT mice is significantly higher than that in FA mice. (D) Proportion of phospho-ACC (pACC) relative to total ACC from mouse liver (Also see Figure S2). (E) Double-plotted average (+ SEM, n = 4) mRNA levels of circadian oscillator components Per2, Bmal1,Rev-erbα, Cry1 and additional clock components (Figure S2D) in liver at different times of the day. Transcript levels were measured by qRT-PCR and normalized to Gapdh RNA levels. Broken line separates double plotted data. Also see Figure S2.

Figure 3. Time restricted feeding alters glucose metabolism and restores normal glucose tolerance

qRT-PCR estimates of mRNA levels of gluconeogenic CREB targets (A) Pcx and (B) G6pc in the liver show reduced expression during feeding in the liver of FT mice. This is accompanied by (C) increased levels of G6-P and TCA cycle metabolites, malate, fumarate and citrate. (D) Elevated mRNA level of G6pdx in the livers of FT mice correlated with (E) a rise in pentose phosphate cycle (PPC) metabolites ribulose-5-P, ribose 5-P, fructose 6-P and sedoheptulose 7-P and higher levels of reduced glutathione. tRF regimen also improved oscillation of (F) Umps and (G) Tk1 mRNA in liver and resultant improvement in (H) nucleotide metabolites (also see Figures S3 and S4A) which are produced from PPC intermediates. (I) IPGTT shows normal glucose tolerance in FT mice. Average blood glucose levels (+ SEM, n = 6 mice) in overnight fasted and after glucose infusion are shown. (J) Levels of blood insulin after overnight fasting or 1 h after glucose infusion. (K) Time spent (average + SEM, n = 6, *p < 0.05) on an accelerating rotarod.

Figure 4. Time restricted feeding alters fatty acid metabolism and prevents obesity and liver pathology

(A) Reduced hepatic mRNA levels of REV-ERBα target gene Fasn in the livers of FT mice contributes to (B) the reduction in free fatty acids myristate and palmitate. Reduced mRNA levels of (C) Pparγ, (D) Scd1 and (E) Elovl5 in the liver of FT mice (also see Figure S3) accompanied reduced levels of several unsaturated fatty acids including (F) oleate, palmitoleate, eicosenoate and vaccinate (also see Figure S4B). Reduced malonyl-carnitine, increased BHBA (see also Figure S4B) and (G) increased Lipc mRNA are indicative of increased β-oxidation. (H) Body composition analyses by MRI illustrates tRF prevents excessive whole-body fat accumulation in mice fed HF diet. Average weights of fat, lean and remaining body mass are shown. (I) Increased levels of leptin after overnight fasting (−) and after glucose administration (+) indicative of increased adipose tissue in FA mice are absent in the FT mice. (J) tRF also prevents enlarged liver and (K) liver damage indicated by increased serum ALT.

Figure 5. Time restricted feeding prevents liver diseases

(A) Representative histopathology (upper panel, H&E, Scale bars 200 μm) and Sirius red (lower panel, Scale bars 200 μm) of the liver. Steatohepatitis was scored by a histopathologist who was blinded to the source condition of each sample using a semi-quantitative method derived from Brunt et al. (Brunt et al., 2004) measuring the degree of steatosis (0–3), ballooning degeneration (0–2), lobular (0–3) and portal (0–2) inflammation and fibrosis (0–4). (B) Average Steatosis score (+ SEM, n = 4 mice) of liver sections. (C) Representative scanning electron microscope image of a liver from FA mice shows large lipid droplets and vacuoles that are reduced in FT liver. Scale bar = 1 μm. (D) Measurement of several volume fractions show FT regimen prevents the decline in density of mitochondria and ER under ad lib HF diet (FA). Measurements are from volume rendering obtained from serial block-face images of the liver. See also Table S2. (E) Gene expression signature of diet-induced obesity in the mouse liver is attenuated by tRF. Average expression (+ SEM) from 8 different time points (Figure S2A) are shown in bar-graphs. Significant differences between FA and FT (*p < 0.05) were found. Temporal expression profiles of these genes are shown in Figure S5.

Figure 6. Time restricted feeding modulates bile acid metabolism, energy expenditure and inflammation

tRF regimen (A) suppressed expression of Hmgcs2 and (B) increased expression of Cyp7a1 in the liver, which accompanied (C) a reduction in serum cholesterol and (D) increase in hepatic bile acids. Relative levels of representative bile acids are shown (also see Figure S6). (E) qRT-PCR measurements of relative mRNA levels of Bmal1, Dbp and Pparα show robust circadian oscillation in the BAT of FT mice (also see Figure S3 for NA and NT gene expression). Increased expression of UCP1-3 during the late night correlates with increased energy expenditure in FT mice (Figure 1). H&E stained sections of (F) BAT or (G) WAT show adipocyte hypertrophy in FA mice is prevented in the FT mice. Scale bar = 50 μm. Arrows indicate infiltrating cells that are most likely macrophages. Reduction in infiltrating macrophages also correlates with a reduction in (H) the mRNA levels of several pro-inflammatory cytokines in the WAT of FT mice. Average (+ SEM, n = 8, *p < 0.05) mRNA levels of pro-inflammatory cytokines TNFα, CXCL2, IL6 and IL1 in the WAT are reduced under tRF paradigm.

Figure 7. Time of feeding exerts a larger effect on liver metabolites in mice on a high fat diet

(A) Heat-map rendering of normalized levels of 324 liver metabolites at eight different time points in the liver of NA, NT, FA and FT groups of mice. The metabolites were clustered by hierarchical clustering. (B) Summary of metabolite changes in the liver highlights the larger effect of temporal feeding pattern when animals were fed high fat diet. For statistical analyses, all eight time-points for each feeding regimen were treated as replicates. Number of metabolites that changed between any two of the six different contrasts is shown in the bottom panel. (C) Heatmap rendering of a subset of metabolites of a cluster enriched for fatty acids and (D) another cluster enriched in intermediates of energy- and anabolic- metabolism. Tissues were harvested at ZT14, 17, 20, 23, 2, 5, 8 and 11. The time of food access is indicated in yellow boxes. Red = high, Green = low. Steady state levels of several of the metabolites at 8 different time points representing one full day from (C) and (D) marked with underlined text are shown in (E). Normalized values presented in Table S1 are plotted against time.