Autophagy failure is associated with metabolic insufficiency. Although caloric restriction (CR) extends healthspan, its adherence in humans is poor. We established an isocaloric twice-a-day (ITAD) feeding model wherein ITAD-fed mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. We hypothesized that ITAD feeding will provide two intervals of intermeal fasting per circadian period and induce autophagy. We show that ITAD feeding modifies circadian autophagy and glucose/lipid metabolism that correlate with feeding-driven changes in circulating insulin. ITAD feeding decreases adiposity and, unlike CR, enhances muscle mass. ITAD feeding drives energy expenditure, lowers lipid levels, suppresses gluconeogenesis, and prevents age/obesity-associated metabolic defects. Using liver-, adipose-, myogenic-, and proopiomelanocortin neuron-specific autophagy-null mice, we mapped the contribution of tissue-specific autophagy to system-wide benefits of ITAD feeding. Our studies suggest that consuming two meals a day without CR could prevent the metabolic syndrome.

Because fasting activates autophagy, we established an isocaloric twice-a-day (ITAD) feeding model wherein test mice eat the same amount of food as ad libitum (Ad-lib) controls (Con), albeit they eat their food at two 2 hr windows early and late in the diurnal cycle. We hypothesized that adopting the ITAD feeding strategy will eliminate scattered feeding, and provide two windows of intermeal fasting in each circadian period, which in principle will sustain autophagy without the need to restrict calories or alter the type of food consumed. Here we show that ITAD feeding promotes system-wide benefits including reduction of body fat and increased lean mass that accompany significant tissue remodeling. ITAD feeding sustains autophagy levels in aged mice, and prevents age-associated energy imbalance, dyslipidemia, and glucose intolerance. Using liver-, adipose-, myogenic-, and hypothalamic proopiomelanocortin (POMC) neuron-specific Atg7 KO mice, we identified the contribution of cell-specific autophagy to system-wide benefits of ITAD feeding.

Caloric restriction (CR) extends healthspan and lifespan in multiple organisms (). Despite its remarkable benefits, humans adhere poorly to CR (), which has motivated the search for sustainable approaches to extend healthspan. Alternate feeding strategies, including intermittent fasting (), fasting-mimicking intervention (), and time-restricted feeding () each mimic the effects of CR. Since fasting activates autophagy, it is conceivable that dietary interventions mediate their benefits, in part, through autophagy. The integrative physiology of autophagy and its ability to promote metabolic correction in a dietary intervention model remains unexplored.

Decreased quality control and accumulation of damaged organelles are factors contributing to chronic diseases including the metabolic syndrome. Autophagy, a lysosomal quality control pathway critical for cellular cleanliness, is compromised with age, setting the basis for chronic diseases (). In fact, mice knocked out (KO) for the autophagy gene Atg7 or lacking Beclin function display early lethality () and metabolic defects including fat accumulation (), muscle loss (), and glucose intolerance ().

Given the effect of ITAD feeding on WAT browning, we investigated the role of autophagy in ITAD feeding-induced iWAT browning. Our studies revealed that ITAD feeding led to induction of UCP1 ( Figure 3 C), and Eva1, Zic1, and Fbxo31 in iWAT, indicating adipose browning ( Figure 3 D). Notably, loss of Atg7 blocked ITAD-driven WAT browning, as indicated by reduced expression of Eva1 and Zic1 ( Figures 7 E and 7F). Consistent with these changes, Atg7KOmice failed to decrease their adipocyte size ( Figure S7 C) or increase their VO2, VCO2, and EE rates in response to ITAD feeding ( Figures 7 G–7I). Failure to increase their EE was not due to reduced locomotion since all groups displayed equivalent activity ( Figure 7 J). Although ITAD feeding reduced eWAT mass ( Figure 7 C) and lowered serum leptin levels to varying degrees in Con and Atg7KOmice ( Figure 7 K, left), ITAD-fed KO mice remained modestly glucose intolerant ( Figure S7 D) and insulin insensitive, as indicated by elevated serum insulin levels ( Figure 7 K, right) compared with Con. Most surprisingly, impaired glucose clearance in ITAD-fed Atg7KOmice occurred in part from failure to suppress gluconeogenesis when subjected to PTT ( Figure 7 L) suggesting that adipose autophagy contributes to ITAD feeding-driven lipid/glucose homeostasis by modulating iWAT browning and hepatic glucose production.

Since ITAD feeding reduced fat mass, we next tested whether autophagy is required for the fat-intrinsic benefits of ITAD feeding. Loss of Atg7 in adipose tissue using the aP2-Cre line revealed eWAT browning and reduced adiposity (); however, aP2 is expressed in several non-adipogenic tissues (). Consequently, we used the adiponectin-Cre line () to delete Atg7 in WAT to identify the benefits of ITAD feeding that are lost in adipose-specific Atg7KO mice (Atg7KO). Immunoblots revealed loss of ATG7 and accumulation of LC3-I in eWAT and iWAT, validating loss of autophagy ( Figure S7 A). Loss of Atg7 did not affect adipocyte differentiation, as indicated by equivalent expression of markers of differentiated fat: Pparγ, aP2, C/EBPα, C/EBPβ, FAS, and PLIN1 ( Figure S7 B). Under basal Ad-lib-fed condition, 5- to 6-month-old RD-fed Atg7KOmice showed no differences in body wt compared with Con (25.6 ± 0.9 versus 28.3 ± 0.8, p = 0.09, n = 6), and no differences in fat pad wt (data not shown). After 4 months of ITAD feeding on HFD, while ITAD-fed Con mice reduced their body wt by ∼20%, ITAD-fed Atg7KOmice lost only ∼7% of their wt ( Figure 7 A). These data suggest that autophagy is required in adipose tissue ( Figure 7 A) and POMC neurons ( Figure 6 A) for the body wt-reducing effect of ITAD feeding. Accordingly, qNMR analyses revealed that Atg7KOmice failed to significantly lower their body fat content when subjected to 4 months of ITAD feeding ( Figure 7 B). Intriguingly, while eWAT from ITAD-fed Con and Atg7KOmice each lost ∼50% of their mass ( Figure 7 C), iWAT from Atg7KOmice completely resisted losing its mass following ITAD feeding ( Figure 7 D), demonstrating that autophagy is required for reduction of iWAT mass, but not eWAT mass, in response to ITAD feeding. Since ITAD feeding increases OCR in iWAT ( Figure 3 G), and not eWAT ( Figure 3 H), ITAD feeding-driven increase in OCR/EE is likely coupled to loss of iWAT mass.

Because Myf5+ progenitors give rise to muscle, and since ITAD feeding induced Myf5 expression and autophagy in muscle ( Figures 3 N and 2 G), we explored whether Myf5 progenitor cell-specific autophagy is required for muscle-specific benefits of ITAD feeding. Consistent with immunofluorescence in Figure 3 P, GA from 6 month ITAD-fed Con mice displayed an increase in type IIB MyHC protein levels, while TA revealed an increase in embryonic (e)MyHC protein levels without affecting those of MyHC IIA and MyHC I ( Figures 6 I and 6J). ITAD-fed Con mice also increased their expression of glycolytic genes in GA, hexokinase 2 (Hk2), phosphofructokinase (Pfk), and pyruvate kinase (Pk) ( Figure S6 D). By contrast, ITAD-fed mice lacking Atg7 in Myf5+ progenitors (Atg7KO) failed to induce MyHC type IIB and eMyHC protein levels ( Figures 6 I and 6J) or induce glycolytic gene expression to levels observed in Con ( Figure S6 D), demonstrating the requirement of autophagy in Myf5+ progenitors for glycolytic type IIB fiber expansion in the context of ITAD feeding. Consistent with these changes, RD-fed Atg7KOmice remained modestly glucose intolerant despite ITAD feeding ( Figure 6 K). Impaired glucose intolerance in KO mice likely occurred from muscle-intrinsic defects, and not from increased glucose production in liver, since Atg7KOmice displayed reduced basal gluconeogenesis compared with Con in PTT ( Figure S6 E). In sum, autophagy failure in myogenic progenitors may explain age-associated loss of type IIB fibers that is reversible in part by ITAD feeding.

Surprisingly, depleting Atg7 in liver suggests multiple roles of autophagy in decreasing liver TG in ITAD-fed mice. As noted earlier, ITAD feeding suppressed the expression of lipogenic genes, e.g., Fas ( Figure 4 L), and increased expression of Pgc1α, Pparα, and Pparα target Fgf21 ( Figures 6 H and 4 I), which drive fat oxidation. Intriguingly, depleting Atg7 in liver reversed ITAD feeding-driven suppression of Fas expression, suggesting that autophagy is required to suppress de novo lipogenesis in ITAD-fed mice ( Figure 6 H). Further, ATG7-depleted livers failed to induce Pgc1α and Fgf21 expression in ITAD-fed mice ( Figure 6 H), supporting the notion that autophagy coordinates lipohomeostatic responses during ITAD feeding via time-restricted changes in lipophagy and lipogenesis ( Figure 4 P).

Because POMCergic autophagy drives hepatic lipophagy in a cell-nonautonomous manner (), we investigated the role of POMCergic autophagy in hepatic lysosomal degradation of LD in ITAD mice at 11 a.m. Consistent with increased autophagy flux and OCR at 11 a.m. ( Figures 2 A and 2H), lysosomal inhibition with i.p. leupeptin (plan in Figure S6 C, left) for 2 hr in ITAD-fed Con mice led to ∼3-fold increase in liver TGs, indicating lysosomal turnover of TGs at 11 a.m. ( Figure S6 C, right). By contrast, livers from ITAD-fed Atg7KOmice displayed higher basal TG levels, which failed to accumulate upon lysosomal inhibition ( Figure S6 C), indicating that POMCergic autophagy is required for lipophagy of liver TG in ITAD-fed mice at 11 a.m. In fact, acutely depleting liver Atg7 by injecting Cre-expressing adenoviruses in Atg7mice or denervating the liver via vagotomy to uncouple the liver from CNS each blocked ITAD feeding-driven increases in liver OCR at 11 a.m. ( Figure 6 G), demonstrating that POMCergic and liver autophagy act in concert to mobilize lipid in ITAD mice.

Since ITAD feeding activates autophagy in liver, MBH, WAT, and muscle at 11 a.m., we sought to map the contribution of autophagy in each tissue system to metabolic benefits of ITAD feeding. POMCergic autophagy plays crucial roles in regulation of body wt () and fat utilization in peripheral tissues (). Consequently, we asked to what extent is POMCergic autophagy required for benefits of ITAD feeding. To that end, body wt analyses revealed that, while Con mice lost ∼20% of their body wt after 4 months of ITAD feeding on HFD, ITAD-fed mice lacking Atg7 in POMC neurons (Atg7KO) resisted losing their body wt ( Figure 6 A). Further, while ITAD-fed Con mice on HFD decreased their eWAT wt by ∼40%, ITAD-fed KO mice maintained their eWAT mass ( Figure 6 B). In fact, ITAD-fed KO mice failed to induce their iWAT OCR to levels observed in ITAD-fed Con ( Figure 6 C), possibly due to the reported loss of WAT sympathetic tone in Atg7KOmice (). ITAD-fed Atg7KOmice also resisted lowering their liver and serum TG levels compared with ITAD-fed Con mice ( Figures 6 D and 6E). To exclude that loss of Atg7 in POMC neurons from birth led to developmental defects in the hypothalamus, which, in turn, reduced the benefits of ITAD feeding, we generated Atg7KOmice wherein Atg7 was deleted during adulthood via tamoxifen (Tmx)-driven expression of Cre in POMC neurons (). As anticipated, 6 weeks of ITAD feeding on HFD significantly decreased serum TG levels in Con and Atg7KOmice prior to administration of Tmx (i.e., day 0) ( Figure 6 F). Con and Atg7KOmice were then subjected to Tmx injections (day 1) and serum TG levels were analyzed on day 15. While Tmx-injected control mice maintained reduced serum TG levels, Tmx-injected Atg7KOmice lost their ability to lower their serum TG levels in response to ITAD feeding ( Figure 6 F). Despite these defects in lipid metabolism in Atg7KOmice, ITAD-fed Con and Atg7KOmice each reduced their glucose production to similar levels in intraperitoneal (i.p.) PTT ( Figure S6 A), and, accordingly, each displayed equivalent improvements in glucose clearance when subjected to i.p. GTT ( Figure S6 B). These data show that POMCergic autophagy is required to mediate the effects of ITAD feeding on lipohomeostasis in liver and iWAT, but not glucose homeostasis.

Since ITAD feeding increases EE, we tested its ability to restore EE in aged mice. Indeed, ITAD feeding prevented age-associated loss of VO2, VCO2, and EE rates ( Figures 5 H–5J), without changing locomotor activity ( Figure 5 K). Seahorse respirometry ( Figure 5 L) revealed patterns of increased liver OCR in ITAD-fed aged mice, which were supported by increases in VO2 ( Figure 5 H). Prevention of age-associated loss of OCR was associated with increased expression of mitochondrial genes Cox4 and Cpt2, and induction of Pgc1α, in ITAD-fed aged mice ( Figure 5 M). ITAD feeding also prevented age-associated reduction in expression of Atg genes and lysosomal Lamp1 ( Figure 5 N), and increased LC3-II flux in aged livers at 11 a.m. ( Figure 5 O). Finally, ITAD feeding improved glucose clearance in aged or obese mice subjected to glucose tolerance tests (GTTs) ( Figures 5 P and 5Q), validating its effectiveness in preventing age/obesity-associated metabolic compromise.

To determine whether ITAD feeding prevents age/obesity-associated metabolic compromise, we subjected 4- and 18-month-old mice to Ad-lib or ITAD feeding on HFD for 6 months ( Figure 5 A). ITAD feeding significantly reduced body wt in 10- (data not shown) and 24-month-old mice ( Figure 1 H), and reduced fat mass and increased lean mass in 10-month-old mice ( Figure 5 B), while similar statistically insignificant trends were observed in 24-month-old mice ( Figure 5 B). ITAD feeding significantly decreased liver wt in young and aged mice ( Figure 5 C), and reduced liver and serum TG in 10-month-old mice ( Figures 5 D and 5E), while a trend for decreased liver TG was noted in 24-month-old mice ( Figure 5 D). Consistent with qNMR data ( Figure 5 B), ITAD feeding significantly increased GA-sol wt in 10-month-old mice, while modestly increasing GA-sol wt in 24-month-old mice ( Figure 5 F). ITAD feeding also reversed hypertriglyceridemia by ∼50% when mice fed HFD Ad-lib for 8 months were switched to ITAD feeding for 4 months ( Figure 5 G), indicating that ITAD feeding can reduce hyperlipidemia and potentially lower cardiovascular disease risk.

(H–O) VO2, VCO2, EE rates, and z axis movements in young/aged male mice fed Ad-lib on HFD or ITAD-fed on HFD for 6 months (H–K) (n = 3–4). Liver OCR and AUC for OCR (L) (n = 3–4), liver qPCR analyses for mitochondrial and autophagy-related genes (M and N) (n = 8), and net LC3-II flux in liver explants cultured in presence (+) or absence (−) of Lys Inh in aged male mice (O) (n = 8).

Restricted feeding uncouples peripheral clocks from the light-entrained central clock (), suggesting that changes in peripheral clocks may contribute to the phenotype of ITAD-fed mice. In fact, analyses of oscillations of core clock genes in livers from ITAD-fed mice revealed a modest, albeit statistically insignificant, increase in expression of circadian driver Bmal1 ( Figure S4 C). We also noted changes in oscillations of clock repressors Per1, Per2, and Per3, with shifts in phase in expression of Per1 and Per3 ( Figures S4 C–S4H). Given the role of circadian proteins in metabolic regulation (), it is likely that diurnal ITAD feeding, and resulting changes in expression of clock genes, shapes the phenotype of ITAD-fed mice. To explore this possibility, we compared changes in body wt, eWAT wt, iWAT browning, and iWAT OCR in mice fed ITAD or ITAN (fed at 8–10 p.m. and 5–7 a.m.) on RD ( Figure S5 A). After 4 months, we noted no difference in body wt between both groups ( Figure S5 B), although ITAN-fed mice displayed significantly increased eWAT wt (Ad-lib versus ITAN eWAT wt/body wt; 14.0 ± 0.9 versus 19.2 ± 0.8; p < 0.05, t test), while ITAD-fed mice showed reduced eWAT wt when compared with Con (Ad-lib versus ITAD eWAT wt/body wt; 16.1 ± 1.1 versus 14.1 ± 0.4; p < 0.05, t test). ITAD-fed mice also showed an increase in expression of brown fat marker Eva1 in iWAT ( Figure S5 C) that was associated with increased OCR ( Figure S5 D). ITAN-fed mice displayed similar trends for Eva1 expression and iWAT OCR, although these values did not acquire statistical significance ( Figures S5 C and S5D). Strikingly, improvement in glucose clearance occurred earlier in ITAN-fed mice than ITAD-fed mice after 4 months on RD ( Figure S5 E), suggesting that ITAD or ITAN feeding of young (8 months old) RD-fed mice each leads to distinct metabolic benefits.

Liver TG analyses revealed significantly decreased lipid levels in ITAD-fed mice ( Figure 4 F), while serum TGs were only modestly lower in ITAD-fed mice on RD ( Figure 4 G). Increases in autophagy flux and OCR in liver at 11 a.m. ( Figures 2 A and 2H) correlated with ∼3-fold increase in Pparα expression at 11 a.m. ( Figure 4 H), a key driver of autophagy (), suggesting a role for lipophagy in ITAD feeding-driven liver TG depletion ( Figure 4 F). At 11 a.m., we also noted increased expression of Pparα target Fgf21, and a trend toward increased FGF21 secretion ( Figures 4 I and 4J) (), which may have contributed to liver fat loss. Quite surprisingly, qPCR analyses for Srebp1c, the master regulator of lipogenesis, and its targets Fas, Elovl6, Acsl5, and Gpat1, indicated maximal suppression of lipogenesis in ITAD mice at 7 p.m. ( Figures 4 K–4O). Since autophagy flux and OCR were suppressed from 3 to 11 p.m. ( Figures 2 A, 2I, and 2J), decreased liver TG after 7 p.m. in ITAD-fed mice ( Figure 4 F) may have resulted from suppressed lipogenesis despite the surge in serum insulin, a key driver of TG synthesis. These results suggest that induction of lipophagy (11 a.m.) and suppression of lipogenesis (7 p.m.) act in concert to limit hepatic TG accumulation in ITAD-fed mice ( Figure 4 P).

To explore the effect of ITAD feeding on glucose/lipid metabolism, we characterized circulating insulin/glucose levels, liver/serum triglycerides (TGs), and expression of glucose/lipid metabolism genes in livers from Ad-lib and ITAD-fed mice across 24 hr. Surprisingly, ITAD-fed mice displayed a surge in serum insulin levels that correlated with 5–7 p.m. feeding ( Figure 4 A), after which insulin levels dropped to levels lower than those in Ad-lib mice. Increased serum insulin at 7 p.m. in ITAD-fed mice was associated with reduced blood glucose levels from 7 p.m. to 3 a.m. ( Figure 4 B), suggesting that 7 p.m. insulin release possibly increased tissue glucose uptake and/or suppressed gluconeogenesis. Supporting the latter, livers from ITAD-fed mice displayed varying degrees of reduction in expression of gluconeogenic genes G6pc, Pck1, and Fbp1 at 7 p.m. compared with Con ( Figures 4 C, S4 A, and S4B). Pyruvate tolerance tests (PTTs) initiated at 6 p.m. in mice food deprived from 10 a.m. onward and fed for 10 min at 5 p.m. ( Figure 4 D) displayed reduced blood glucose levels in ITAD mice ( Figure 4 E), confirming decreased gluconeogenesis. Although we cannot explain the reason for increased serum insulin at 7 p.m. (and not after the first feeding window), it is likely that insulin's ability to suppress autophagy inhibited autophagy flux at 7 p.m. in ITAD mice ( Figures 2 A and 2I).

(A–O) Serum insulin (A) (n = 8), blood glucose levels (B) (n = 14), hepatic gluconeogenic gene Pck1 at indicated time points (C) (n = 8), pyruvate tolerance test (PTT) at 6 p.m. (D and E) (n = 4), liver and serum TG (F and G) (n = 8), qPCR for hepatic Pparα and Fgf21 genes (H and I) (n = 8), serum FGF21 levels (J) (n = 3), and qPCR for lipogenic genes at indicated time points in male mice subjected to Ad-lib or ITAD feeding on RD for 10 months (K–O) (n = 8).

Since aging is associated with preferential loss of type IIB glycolytic fibers (), we investigated the effect of ITAD feeding on type IIB fiber content. Staining for myosin heavy chain (MyHC) glycolytic type IIB and oxidative type I fibers in GA from 10-month-old ITAD-fed mice (analyzed at 14 months of age) revealed a remarkable increase in type IIB fibers without changes in type I fiber content, indicating glycolytic fiber expansion ( Figures 3 P and 3Q). Since increased glycolytic fiber number is associated with reduced endurance, we tested the effect of ITAD feeding on exercise capacity. During 3 days of acclimatization on a treadmill-based exercise regime ( Figures S3 F and S3G) (), we failed to observe significant differences in exercise capacities by both groups. During the test ( Figure S3 H), when treadmill speed was increased by 1 m/min every min, consistent with increased type IIB fiber content, ITAD-fed mice fatigued earlier at 28 m/min speed, indicated by increased shocks required to stay on the treadmill. Nevertheless, ITAD feeding leads to retention of key attributes of skeletal muscle that are typically lost with age: mass and type IIB fiber content.

To understand how ITAD feeding increases muscle mass ( Figures 1 K and S3 D), we examined the effect of ITAD feeding on myocyte proliferation and fiber-type changes. H&E-stained GA from ITAD-fed mice for 9–10 months revealed myocytes that were ∼25% smaller in size than Con mice ( Figures 3 K and S3 E). In fact, we noted an abundance of myocytes with cross-sectional area 5,000–15,000 pixelon a scale from 0 to 40,000 pixel Figure 3 L). ITAD feeding also increased the number of cells with centralized nuclei ( Figures 3 K and 3M). Small myocytes with centralized nuclei reflect proliferating and regenerating muscle (). Accordingly, we noted an ∼30%–40% increase in expression of myogenic factors Myf5, Myf6, and Myog without changes in Myod1 and Ckm expression ( Figure 3 N), and an ∼1.6-fold increase in expression of proliferation marker Cyclin D1 ( Figure 3 O), indicating active myogenesis in ITAD-fed mice.

Surprisingly, ITAD feeding increased F4/80 positivity in WAT, indicating macrophage infiltration ( Figure S3 C). Because alternatively activated M2 macrophages are anti-inflammatory in nature (), we tested whether increased F4/80 positivity reflected an increase in M2 macrophage content. Indeed, qPCR analyses in eWAT, a fat depot prone to inflammation, revealed remarkably increased anti-inflammatory M2 macrophage markers Arg1 (∼4-fold), Ym1 (∼2-fold), and IL-10 (∼2-fold) in ITAD-fed mice, while only a modest increase in pro-inflammatory IL-6 expression was observed ( Figure 3 J). By contrast, expression of anti- or pro-inflammatory cytokine genes remained unremarkable in iWAT ( Figure 3 J).

Since ITAD feeding decreases fat mass, we characterized the effect of ITAD feeding on WAT. Consistent with reduced fat mass, ITAD-fed mice displayed reduced serum leptin levels, with values displaying statistical significance at 7 a.m. and 11 p.m., indicating improved leptin sensitivity ( Figure S3 A). Hematoxylin and eosin (H&E) stains of WAT revealed decreased adipocyte size in ITAD-fed mice ( Figure 3 A), which in conjunction with increased EE ( Figures 1 L and 1M) suggested increased fat utilization. Indeed, iWAT from ITAD mice displayed pockets of uncoupling protein 1 (UCP1)-positive brown adipocytes displaying multiloculated lipid droplets (LDs) ( Figures 3 A–3C and S3 B). Increased expression of brown genes Zic1, Eva1, and Fbxo31 in iWAT, and no changes in expression of beige genes Tmem26, Klhl13, and Tbx1 (), supported iWAT browning in ITAD-fed mice ( Figure 3 D). We also noted an ∼3-fold increase in expression of adipogenic precursor Ebf2 () and Pdgfrα () in iWAT from ITAD mice ( Figure 3 E). Since Ebf2 determines brown adipocyte identity, we suspect that Ebf2 orchestrates iWAT browning in ITAD mice. Although we detected a statistically insignificant increase in expression of myogenic factor Myf5 in eWAT from ITAD mice ( Figure 3 E), the significance of this increase is unclear. In keeping with increased mitochondrial mass in brown adipocytes, we noted significant increases in expression of mitochondrial markers Cpt1b and Cox4 and of Pgc1α, a driver of mitochondrial biogenesis, in iWAT, and ∼1.5- to 2-fold increase in expression of adipogenic factor Pparγ in iWAT and eWAT ( Figure 3 F). Consistent with these data, respirometry revealed an ∼2-fold increase in OCRs in iWAT (p = 0.07) ( Figure 3 G), but not in eWAT ( Figure 3 H). Further, ITAD feeding improved the ability to respond to cold (4°C for 1 hr), indicated by increased expression of brown fat genes, but not beige genes, in iWAT ( Figure 3 I).

Because lipophagy () drives fat utilization and oxygen consumption rates (OCRs) (), autophagy activation at 11 a.m. in ITAD-fed mice was associated with ∼2-fold increase in hepatic OCRs ( Figure 2 H), while suppression of LC3-II flux at 7 p.m. ( Figures 2 I and 2A) was associated with normalization of OCRs to basal rates ( Figure 2 J). Our studies do not reveal the mechanism for time-dependent modulation of autophagy in ITAD-fed mice; however, it is likely that complex interplay between AMPK, a regulator of the circadian clock () and autophagy (); mTOR; and possibly a subset of core circadian proteins differentially regulates autophagy at distinct time points during ITAD feeding.

Since mechanistic target of rapamycin (mTOR) and AMP-regulated kinase (AMPK) () regulate autophagy, we examined how their activities correlated with changes in LC3-II flux across 24 hr. Immunoblotting of liver lysates revealed that increases in LC3-II flux at 11 a.m. from ITAD mice were associated with significantly increased phosphorylated (P)-AMPK levels ( Figures S2 C and S2D). AMPK is induced by starvation, yet, surprisingly, we noted increased P-AMPK levels in the 8–10 a.m. feeding window in ITAD mice, suggesting that autophagy induction is perhaps AMPK driven. ITAD feeding also increased P-S6 levels in both feeding windows reflecting nutrient-driven mTOR complex 1 activity ( Figures S2 E and S2F). Indeed, recent work has shown that availability of nutrients concurrently activates AMPK and mTOR (). Since mTOR suppresses autophagy, and because autophagy is active between 7 and 11 a.m. in ITAD mice ( Figure 2 A), mTOR signaling at 11 a.m. is likely uncoupled from autophagy as demonstrated in secretory cells ().

Because ITAD feeding provides two periods of intermeal fasting, which activates autophagy, we tested if and when ITAD feeding stimulates autophagy. Our initial qPCR analyses at six time points across 24 hr (7 a.m., 11 a.m., 3 p.m., 7 p.m., 11 p.m., and 3 a.m.) revealed modest increases in expression of autophagy-related genes Lc3 (light chain 3) and Beclin1 during the first feeding window in ITAD-fed mice (data not shown). Consequently, we comprehensively tested the effect of ITAD feeding on autophagy activity across 24 hr via LC3-II flux analyses in livers exposed or not to lysosomal inhibitors at each of the six time points. LC3-II flux analyses from distinct pools of mice subjected to ITAD feeding for 8–10 months revealed progressive increases in autophagy from 7 to 11 a.m. and maintenance of flux until 2 p.m., following which autophagy flux steadily declined until 7 p.m. to levels lower than those in Ad-lib mice ( Figures 2 A and S2 A). After 7 p.m., LC3-II flux gradually increased to reach its zenith at 3 a.m. in IT AD-fed mice ( Figure 2 A). Upon comparing the oscillations of LC3-II flux in both groups ( Figure 2 A), we noted a clear shift in phase of autophagy flux in ITAD-fed mice characterized by 8–10 a.m. feeding-associated induction of autophagy, a clear departure from the typical increase in autophagy during starvation. Consistent with maximal autophagy flux at 11 a.m., Atg gene expression was increased in GA ( Figure 2 B), iWAT ( Figure 2 C), and mediobasal hypothalamus (MBH) ( Figure S2 B) at 11 a.m. after as early as 4 months of ITAD feeding. Beclin1 protein levels were also increased to varying degrees at 11 a.m. in several tissues from 4 month ITAD-fed mice ( Figure 2 D). Tissue-wide autophagy flux analyses revealed ∼2.5-fold increase in flux of autophagy cargo p62 in MBH, ∼3-fold increase in LC3-II flux in brown adipose tissue, and ∼2-fold increase in LC3-II flux in GA at 11 a.m. from mice subjected to ITAD feeding for 8 months ( Figures 2 E–2G).

(H–J) Oxygen consumption rates (OCRs) in livers at 11 a.m. (H), IB for LC3 in livers at 7 p.m. and treated (+) or not (−) with Lys Inh for 2 hr (I), and OCR in livers at 7 p.m. from RD-fed male mice on Ad-lib or ITAD for 10 months (J) (n = 3). Quantification for net LC3-II flux and steady-state LC3-II are shown.

(B–D) qPCR for indicated autophagy and lysosomal genes in gastrocnemius (GA) and iWAT (n = 8) (B and C) and immunoblots (IB) for Beclin1 and ATG12-ATG5 conjugate in the indicated tissues harvested at 11 a.m. from RD-fed male (n = 4) and female mice (n = 4) on Ad-lib or ITAD feeding for 4 months (total n = 8) (D). Densitometry values in (D) are shown (right).

To determine whether ITAD feeding increased muscle mass, we subjected mice to X-ray computed tomography (CT). CT reconstructions confirmed that ITAD feeding on RD for 12 months reduced total fat mass and decreased subcutaneous WAT (sWAT) mass in abdominal (Abd) and scapular (Sca) planes ( Figure 1 I). Further, CT revealed a trend of reduced eWAT mass in the Abd plane ( Figure 1 I). CT also showed a significant increase in lean mass in the Sca plane and a trend for the same in the Abd plane of ITAD-fed mice compared with Con ( Figure 1 J). Consistent with increased lean mass, gastrocnemius/soleus (GA-sol) muscles from 12-month-old ITAD mice weighed modestly more than those in Con ( Figure 1 K). Reduction of fat mass in ITAD mice was associated with increased oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure (EE) ( Figures 1 L, 1M, S1 G, and S1H), which did not result from increased locomotion ( Figures S1 I and S1J). Thus, ITAD feeding normalizes age- and diet-associated energy imbalance.

Monthly body weight (wt) analyses for 12 months, and at 16 months of ITAD feeding, revealed no differences between RD-fed Ad-lib and ITAD mice, supporting similar caloric intake by both groups ( Figures 1 B and 1C). However, quantitative nuclear magnetic resonance (qNMR) analyses revealed progressive loss of body fat and proportionate increase in lean mass as early as 3 months of ITAD feeding ( Figures 1 D and 1E), indicating that partitioning calories into two meals is sufficient to alter body composition. In fact, analysis of tissue wt from RD-fed mice subjected to ITAD feeding for 4 months showed significantly decreased liver and epididymal white adipose tissue (eWAT) wt ( Figure 1 F) in absence of changes in body wt ( Figures 1 B and 1C). However, after 16 months of ITAD feeding, decreases in liver and eWAT wt did not acquire statistical significance ( Figure 1 F). By contrast, 3-month-old ( Figure 1 G) and 18-month-old ( Figure 1 H) mice subjected to ITAD feeding on HFD for 8 and 6 months, respectively, resisted wt gain compared with Con mice.

To develop a feeding strategy that incorporates periods of fasting between feeding windows, we randomized 4-month-old C57BL/6J male mice into Ad-lib Con and ITAD groups. ITAD mice were fed between 8 and 10 a.m. (feeding window 1) and between 5 and 7 p.m. (feeding window 2), such that food consumed at these two diurnal windows equals the food consumed by Ad-lib mice in 24 hr ( Figure 1 A). Analyses of food consumed per cage revealed that test mice (5 mice per cage) acclimatized to ITAD feeding by day 6, indicated by progressive increases in cumulative chow intake in the two windows ( Figure S1 A, lower panel). Thereafter, we noted that each cage of five mice consumed the same amount of food per day (Ad-lib versus ITAD; 15.1 ± 0.7 g/cage/day versus 14.9 ± 0.8 g/cage/day) ( Figures S1 A and S1B). After 16 months, both groups had consumed similar amounts of regular chow diet (RD) (Ad-lib cage versus ITAD cage; 7,109 versus 6,975 g) ( Figure S1 C). Similarly, test mice (5 mice per cage) fed high-fat diet (HFD; 60% calories in fat) acclimatized to ITAD feeding by day 3 (Ad-lib versus ITAD; 8.95 ± 0.6 g/cage/day versus 8.77 ± 0.6 g/cage/day) ( Figures S1 D and S1E). After 8 months, Con and ITAD groups had consumed similar amounts of HFD (Ad-lib cage versus ITAD cage; 2,077 versus 2,035 g) ( Figure S1 F). Our goal was to establish diurnal ITAD feeding and nocturnal isocaloric twice-a-night (ITAN) feeding strategies and compare their abilities to induce autophagy and prevent metabolic syndrome. Since ITAD and ITAN feeding led to similar effects on body weight in RD-fed mice, we pursued our long-term feeding studies in ITAD-fed mice.

(A–F) The isocaloric twice-a-day feeding (ITAD) strategy wherein test mice feed between 8 and 10 a.m. and between 5 and 7 p.m. the same amount of food that ad libitum (Ad-lib)-fed controls (Con) eat in 24 hr (A). Body weight (wt) (B and C), body composition (D and E), and tissue wt (F) at indicated intervals in regular chow diet (RD)-fed male mice subjected to Ad-lib or ITAD feeding for indicated duration (n = 5).

Discussion

Lee et al., 2014 Lee J.M.

Wagner M.

Xiao R.

Kim K.H.

Feng D.

Lazar M.A.

Moore D.D. Nutrient-sensing nuclear receptors coordinate autophagy. Martinez-Lopez et al., 2016 Martinez-Lopez N.

Garcia-Macia M.

Sahu S.

Athonvarangkul D.

Liebling E.

Merlo P.

Cecconi F.

Schwartz G.J.

Singh R. Autophagy in the CNS and periphery coordinate lipophagy and lipolysis in the brown adipose tissue and liver. Here we show that ITAD feeding/intermeal fasting in absence of CR promotes metabolic flexibility and prevents age/obesity-associated metabolic defects. Consolidating the system-wide metabolic benefits of ITAD feeding ( Figure 7 M), we have found that ITAD feeding/intermeal fasting activates autophagy in liver, adipose tissue, muscle, and MBH at 11 a.m. LC3-II flux analyses in liver at each of the six time points revealed that autophagy is modified in a time-dependent manner in ITAD-fed mice. This time-restricted change in autophagy is characterized by: (1) maximal activation at 11 a.m. in response to feeding between 8 and 10 a.m. and its suppression at 7 p.m. immediately after the second feeding window, and (2) a complete shift in phase of LC3-II flux compared with Con. Induction of autophagy at 11 a.m. led to expression of key drivers of fat utilization, Pparα, Fgf21, and Pgc1α, since acutely depleting Atg7 in liver blocked ITAD feeding-driven expression of these genes. Since PPARα signaling induces autophagy (), it is possible that feedforward autophagy-PPARα-lipophagy regulatory loops help maximize fat utilization during ITAD feeding. Interestingly, although autophagy flux decreased at 7 p.m. in ITAD mice, KO of Atg7 via Cre injections reversed the suppression of lipogenesis between 3 and 11 p.m., indicating a role of autophagy in suppression of lipogenesis in ITAD-fed mice. However, how autophagy activity is modified in ITAD-fed mice, and how this impacts de novo lipogenesis, is unknown and will remain the subject of future studies. Our data allow us to speculate that AMPK and mTOR and their opposing influences on autophagy activator protein ULK1/ATG1 could potentially reorganize autophagy in response to changes in nutrient availability; however, validation of this notion will require future studies. In sum, activation of autophagy and increased fat utilization during the first feeding window, and suppression of lipogenesis at 7 p.m., act in concert to decrease liver TG in ITAD-fed mice ( Figure 4 P). In accordance with findings that cold-induced lipophagy in liver is governed by functional autophagy in POMC neurons (), we propose that POMCergic autophagy is required for ITAD feeding-driven fat utilization in liver and iWAT, solidifying the integrative physiology of CNS to peripheral autophagy in energy balance.

ITAD feeding led to significant brown fat-like remodeling of iWAT and an abundance of markers of anti-inflammatory M2 macrophage in eWAT. Brown fat-like remodeling of iWAT was autophagy dependent, since iWAT from Atg7KOAdipoq mice displayed reduced browning and decreased EE rates. However, we were most surprised to find that, while ITAD feeding reduced eWAT mass in both Con and Atg7KOAdipoq, Atg7KOAdipoq mice failed to decrease their iWAT mass in response to ITAD feeding. While we are unable to explain these results, it is possible that different origins or innervation patterns of distinct fat depots is the reason why autophagy is required in iWAT, and not eWAT, for the benefits of ITAD feeding.

POMC and Con mice each displayed similar improvements in glucose clearance, which excluded the requirement of POMCergic autophagy for glucose homeostasis in the context of ITAD feeding. By contrast, ITAD-fed Atg7KOAdipoq and Atg7KOMyf5 mice each failed to completely improve glucose clearance rates, indicating that autophagy is required in these tissue systems for ITAD feeding-driven control of glucose homeostasis (Myf5 mice failed to increase their glycolytic type IIB fibers or increase expression of glycolytic genes compared with Con. ITAD feeding suppressed gluconeogenesis to similar levels in Atg7KOMyf5 and Con mice, excluding the role of hepatic gluconeogenesis in altered glucose homeostasis in Atg7KOMyf5 mice. Surprisingly, ITAD-fed Atg7KOAdipoq mice remained pyruvate intolerant, indicating that adipose autophagy is required to suppress hepatic gluconeogenesis in ITAD-fed mice, although the inter-organ crosstalk linking adipose autophagy to hepatic gluconeogenesis remains unknown. A major benefit of ITAD feeding is improved glucose tolerance. ITAD-fed Atg7KOand Con mice each displayed similar improvements in glucose clearance, which excluded the requirement of POMCergic autophagy for glucose homeostasis in the context of ITAD feeding. By contrast, ITAD-fed Atg7KOand Atg7KOmice each failed to completely improve glucose clearance rates, indicating that autophagy is required in these tissue systems for ITAD feeding-driven control of glucose homeostasis ( Figure 7 M). Since improved glucose clearance in ITAD mice was associated with increased glycolytic type IIB fiber number and increased expression of glycolytic genes in GA, it is possible that ITAD feeding enhances the efficiency of skeletal muscles to take up glucose in an autophagy-dependent manner. Supporting this contention, ITAD-fed Atg7KOmice failed to increase their glycolytic type IIB fibers or increase expression of glycolytic genes compared with Con. ITAD feeding suppressed gluconeogenesis to similar levels in Atg7KOand Con mice, excluding the role of hepatic gluconeogenesis in altered glucose homeostasis in Atg7KOmice. Surprisingly, ITAD-fed Atg7KOmice remained pyruvate intolerant, indicating that adipose autophagy is required to suppress hepatic gluconeogenesis in ITAD-fed mice, although the inter-organ crosstalk linking adipose autophagy to hepatic gluconeogenesis remains unknown.