While activation of beige thermogenesis is a promising approach for treatment of obesity-associated diseases, there are currently no known pharmacological means of inducing beiging in humans. Intermittent fasting is an effective and natural strategy for weight control, but the mechanism for its efficacy is poorly understood. Here, we show that an every-other-day fasting (EODF) regimen selectively stimulates beige fat development within white adipose tissue and dramatically ameliorates obesity, insulin resistance, and hepatic steatosis. EODF treatment results in a shift in the gut microbiota composition leading to elevation of the fermentation products acetate and lactate and to the selective upregulation of monocarboxylate transporter 1 expression in beige cells. Microbiota-depleted mice are resistance to EODF-induced beiging, while transplantation of the microbiota from EODF-treated mice to microbiota-depleted mice activates beiging and improves metabolic homeostasis. These findings provide a new gut-microbiota-driven mechanism for activating adipose tissue browning and treating metabolic diseases.

Recently, intermittent fasting was demonstrated to optimize energy metabolism and promote health (). However, the mechanism for these benefits is unclear. Notably, one study found that time-restricted feeding can counteract obesity without reducing energy intake (). Although perturbation of circadian rhythm was considered as a significant contributor to the increased energy expenditure (), the possibility exists that white adipose browning would be a more direct mechanism. Therefore, in the current study, mice were placed on an every-other-day fasting (EODF) regimen to explore its effect on white adipose beiging and metabolic disorders. Evidence suggests that EODF selectively activates beige fat thermogenesis and ameliorates obesity-related metabolic diseases, probably via a microbiota-beige fat axis.

Obesity and related metabolic disorders are growing health challenges for Western countries; they mainly result from an imbalance between energy intake and energy expenditure (). Emerging evidence suggests that non-shivering thermogenesis can re-establish energy balance and therefore counter the effects of elevated energy intake (). This process is mediated primarily by the thermogenic activity of uncoupling protein 1 (UCP1), mainly in brown and beige fat cells (). In this context, activating brown adipose tissue (BAT) or browning of white adipose tissue (WAT) could be a promising therapy for obesity and related metabolic diseases. Given current thinking that adult humans do not have active BAT (), conversion of white fat to beige fat rather than BAT activation would hold more therapeutic potential. While many browning strategies have been described, mainly in rodent models, only a very limited number of them have so far supported selective browning of WAT (). Thus, the therapeutic potential associated with these approaches in a clinical setting is not yet clear.

Gut microbiota play a critical role in energy metabolism and lipid homeostasis, and germfree or microbiota-depleted rodents have decreased susceptibility to diet-induced obesity and metabolic syndrome (). Based on the above findings, EODF treatment could alter the microbiota compositions and prevent HFD-induced obesity and metabolic disorders. To further clarify the role of gut microbiota in mediating the beneficial effects of EODF regimen on metabolic diseases, the effect of EODF in control and microbiota-depleted DIO mice was compared. EODF treatment significantly reduced obesity and hepatic steatosis and improved insulin sensitivity in control mice, but not in microbiota-depleted mice ( Figures 6 A–6G ), indicating that the effects of EODF depend on gut microbiota. To examine whether gut microbiota are sufficient to replicate the effects of EODF, microbiota-depleted DIO mice were transplanted with AL microbiota and EODF microbiota, respectively. Compared with the AL microbiota-transplanted group, EODF microbiota transplantation did mimic all the beneficial effects of EODF treatment on metabolic dysfunctions ( Figures 6 A–6H). Moreover, in line with the above results from lean mice, EODF-induced inguinal WAT beiging occurred in control DIO mice, but not in their microbiota-depleted counterparts ( Figure 7 A ). EODF microbiota transplantation to DIO mice promoted inguinal WAT Ucp1 mRNA and UCP1 protein expression, an increase in multilobular adipocytes, and enhanced energy expenditure with lower RER ( Figures 7 B–7F). Accordingly, the EODF-induced elevated levels of serum acetate, serum lactate, and inguinal WAT Mct1 mRNA were abolished by microbiota depletion but restored by EODF microbiota transplantation ( Figures 7 A, 7B, 7G, and 7H). These findings underscore an important role for gut microbiota in the beneficial effects of EODF on WAT beiging and the subsequent improvements in metabolic diseases.

Data are presented as mean ± SEM. Different lowercase letters indicate statistical significance by two-way ANOVA with Sidak multiple comparisons (A) or two-tailed unpaired t test (B and E–H): a, p < 0.05; c, p < 0.005; and d, p < 0.001. Black letters show the effects of EODF or EODF/MT (EODF versus AL or EODF/MT versus AL/MT drunk with the same water), and red letters show the effects of microbiota depletion (AB versus CV within the same feeding regimen).

(G and H) Serum acetate (G) and lactate (H) of DIO mice after EODF or MT treatment. Before EODF or MT treatment, all mice were given access to CV or AB water for 4 weeks. n = 6–8 mice/group.

(C) Representative H&E staining of inguinal WAT from mice transplanted with EODF microbiota (EODF/MT, right) and AL microbiota (AL/MT, left). Scale bar: 100 μm.

(A and B) mRNA expression of thermogenic genes in inguinal WAT in the fed state. All mice were given access to control vehicle (CV) water or water supplemented with an antibiotics cocktail (AB) for 4 weeks and were then treated with AL or EODF (A) or microbiota transplantation (MT, B). n = 6–8 mice/group.

Data are presented as mean ± SEM. Different lowercase letters indicate statistical significance by two-tailed unpaired t test: a, p < 0.05; b, p < 0.01; c, p < 0.005; and d, p < 0.001. Black letters show the effects of EODF or EODF/TM (EODF versus AL, or EODF/MT versus AL/MT with access to the same water).

(A) Body weight. Before EODF or microbiota transplantation (MT) treatment, all mice were given access to control vehicle (CV) water or water supplemented with an antibiotics cocktail (AB) for 4 weeks. n = 6–8 mice/group.

BAT and inguinal WAT gene expression in DIO mice, in response to the EODF regimen, displayed a similar trend to that found in lean mice. Ucp1 and Pgc1a mRNA upregulation occurred in the inguinal WAT, but not in the BAT, of EODF mice ( Figures 5 D and 5H). The mRNAs encoding β-AR and FGF21 signaling-related receptors were not altered ( Figure 5 D). These results hint that EODF-induced WAT beiging might be involved in the beneficial effects of EODF on obesity-associated metabolic syndrome in DIO mice.

Given that activation of beige cells can suppress obesity and metabolic disease (), the effect of an EODF regimen on diet-induced obesity (DIO) mice was explored. Mice were fed a HFD for 3 months and then subjected to either the AL or EODF regimens (15 cycles). EODF mice displayed pronounced weight loss, in contrast to their AL counterparts, without any difference in their cumulative food intake ( Figures 5 A and 5B ). EODF mice had dramatically lower body weights that correlated with the number of EODF cycles ( Figure 5 C). Consistent with the above data that EODF upregulated Glut4 in lean mice ( Figure S3 ), EODF also increased inguinal WAT Glut4 mRNA expression and thus improved insulin sensitivity in DIO mice ( Figures 5 D and 5E). Moreover, liver steatosis and injury markers in EODF mice were obviously ameliorated ( Figure 5 F). EODF resulted in a more significant reduction of inguinal fat mass than of epididymal fat mass ( Figure 5 G). Thus, EODF could differentially impact visceral and subcutaneous fat depots.

Data are presented as mean ± SEM; n = 6 mice/group. Different lowercase letters indicate different statistical significance by two-tailed unpaired t test: a, p < 0.05; b, p < 0.01; c, p < 0.005; and d, p < 0.001 versus AL.

(F) Liver function. Upper left, representative image of livers from AL mouse (left) and EODF mouse (right). Scale bar: 5 mm. Middle left, liver triglycerides; lower left, serum ALT. Right, representative H&E staining of liver sections from AL (upper) and EODF(lower) mice. Scale bar: 100 μm.

To further determine the potential mechanism by which gut microbiota influence EODF-induced beiging,H NMR-based metabolomics was carried out on cecal contents obtained from long-term and short-term EODF-treated mice. In order to maximize the discrimination between AL and EODF mice, pairwise orthogonal projection to latent structure-discriminant analysis (OPLS-DA) was performed on normalized NMR data obtained from the cecal contents. Many metabolites changed after EODF treatment, including acetate, lactate, formate, bile acids, propionate, succinate, cytidine monophosphate (CMP), and xanthine, with noted lower levels of trimethylamine (TMA), uracil, and some amino acids, including branch chain amino acids (BCAAs), glycine, tyrosine, and phenylalanine ( Figures 4 A and 4B ). Among these molecules, both acetate and lactate were increased after both long-term and short-term EODF ( Figures 4 C and 4D). Consistently, EODF treatment also elevated serum acetate and lactate levels ( Figures 4 C and 4D). Shotgun metagenomics sequencing analysis also showed that EODF upregulated the pathway of “pyruvate fermentation to acetate and lactate by Lactobacillus reuteri” and “Pyruvate fermentation to acetate and lactate by unclassified bacteria” ( Figure S7 ). Intriguingly, recent studies indicate that both acetate and lactate are beiging inducers (). In addition, both BAT and WAT express proton-linked monocarboxylate transporter 1 (MCT1) encoded by Mct1, which drives acetate and lactate transport across the plasma membrane of adipocytes (). Given recent evidence that the expression of Mct1 is controlled by physiological stimuli of beiging (), the selective upregulation of Mct1 was noted in inguinal WAT, but not in the BAT of either long-term or short-term EODF-treated mice ( Figures 4 E–4G). The induction of Mct1, together with changes in serum acetate and lactate levels, was abolished in microbiota-depleted mice and restored in mice transplanted with EODF microbiota ( Figure S6 H). Taken together, these data revealed that EODF primarily alters the gut microbiota composition to promote the generation of acetate and lactate and, subsequently, to induce inguinal WAT beiging. Moreover, neither the thermoneutral condition nor the deficiency of PPARα abolished the effect of EODF on promoting the increase of Mct1 mRNA expression in inguinal WAT and of serum acetate and lactate levels ( Figures 2 A and 2I–2K), supporting the conclusion that the effects of EODF are independent of β-AR or PPARα signaling, but associated with gut-microbiota-derived metabolites.

Data are presented as mean ± SEM. Different lowercase letters indicate statistical significance by two-tailed unpaired t test: a, p < 0.05; b, p < 0.01; and d, p < 0.001 versus AL.

(E and F) mRNA expression of Mct1 in inguinal WAT (E) and BAT (F) from mice after long-term EODF treatment in the fed state; n = 7–8 mice/group.

(C and D) Cecum and serum acetate (C) and lactate (D) levels from mice after long-term and short-term EODF treatment; n = 7–10 mice/group.

(B) OPLS-DA scores (left) and correlation-coefficient-coded loadings plots for the models (right) from NMR spectra of cecal content aqueous extracts from mice after short-term EODF treatment; n = 10 mice/group.

(A) Orthogonal projection to latent structure-discriminant analysis (OPLS-DA) scores (left) and correlation-coefficient-coded loadings plots for the models (right) from NMR spectra of cecal content aqueous extracts from mice after long-term EODF treatment; n = 7–8 mice/group.

To investigate whether the EODF-induced microbial shift directly contributes to WAT beiging, microbiota from EODF and AL mice—referred as EODF microbiota and AL microbiota, respectively—were transplanted to microbiota-depleted mice. Transplantation of EODF microbiota significantly upregulated inguinal WAT Ucp1 mRNA and increased small intestine length when compared to the mice transplanted with AL microbiota ( Figures 3 A and 3F). Notably, both the intestine length and Ucp1 mRNA expression in mice transplanted with AL microbiota also increased compared to those of AL mice ( Figures 3 A and 3F), which is consistent with a previous report that microbiota depletion promotes WAT beiging (). To explore whether gut microbiota were necessary for EODF-induced beiging, EODF was performed on the microbiota-depleted mice. Indeed, EODF activated beiging only in control mice, but not in microbiota-depleted mice, although the microbiota depletion itself also induced WAT beiging ( Figure S6 A). Accordingly, the beneficial effects of EODF on several indices of metabolic function were also decreased when the gut microbiota were depleted ( Figures S6 B–S6G). Therefore, these findings suggest that the effects of EODF on inguinal WAT beiging might be mediated by the gut microbiota.

Recently, correlative evidence revealed a metabolic interaction between the gut microbial communities and the host, and gut bacteria have an important role in the regulation of brown and beige adipose tissues (). Previous studies revealed that fasting and feeding rhythms significantly alter the gut microbiota () and that major changes in microbiota composition can directly promote WAT beiging (). Therefore, it is reasonable to speculate that EODF might induce beiging by altering gut microbiota composition. To test this hypothesis, cecum microbiota communities were profiled by 16S rRNA gene amplicon sequencing. EODF increased the length of the small intestine ( Figure 3 A ) and caused clear alterations in the microbiota content, as indicated by the generalized UniFrac distances ( Figure 3 B). Hierarchical clustering of individual species confirmed an effect of EODF on the gut microbiome ( Figure 3 C). According to a previous study (), Firmicutes and Bacteroidetes are the most abundant phyla identified in healthy mice; these were present in both EODF and AL groups. However, EODF significantly altered their relative abundance ( Figure 3 D). Moreover, significant differences were observed in Operational Taxonomic Unit (OTU) abundance at the phylum level in Firmicutes, Bacteroidetes, Actinobacteria, and Tenericutes ( Figure 3 E). The EODF regimen increased the OTU abundance of Firmicutes while decreasing most other phyla ( Figure 3 E). Notably, the ratio of Firmicutes:Bacteroidetes increased from 3.4 in AL mice to 8.9 in EODF mice. Intriguingly, similar shifts in the ratio of Firmicutes:Bacteroidetes in mice were reported to be associated with increased glucose uptake in inguinal WAT, but not in interscapular BAT (). These data are consistent with the present results that EODF selectively induces beiging of inguinal WAT.

Data are presented as mean ± SEM. Different lowercase letters indicate different statistical significance by two-tailed unpaired t test (E) or one-way ANOVA with Bonferroni posttest (A and F): a, p < 0.05; b, p < 0.01; c, p < 0.005; and d, p < 0.001. Black letters are versus AL, and green letters are versus AL/MT.

In addition to β-AR signaling, FGF21 is another classical signaling hormone known to regulate WAT browning () and the adaptive fasting response (). However, its expression or its correspondent receptor or obligate co-receptor expression levels in WAT were not altered after the EODF regimen ( Figure S3 ). Liver-derived FGF21 is the most important contributor to circulating FGF21 levels, and PPARα is a key regulator of hepatic FGF21 (). In addition, PPARα is known to mediate acute fasting-induced upregulation of both hepatic Fgf21 mRNA and circulating FGF21 protein levels (). Therefore, Pparaand Pparamice were used to examine the role of hepatic FGF21 in the beiging phenotype induced by the EODF regimen. Contrary to an acute fasting model, EODF in wild-type Pparamice was associated with a modest decrease, not increase, in FGF21 expression ( Figures 2 F and 2G). Pparamice on AL had lower basal Fgf21 mRNA and circulating FGF21 protein levels ( Figures 2 F and 2G). As expected, the EODF regimen in Pparamice had no impact on either hepatic Fgf21 mRNA expression or circulating FGF21 levels ( Figures 2 F and 2G). EODF did, however, result in a corresponding increase in inguinal WAT Ucp1 mRNA when compared to PparaAL animals, indicating a comparable beiging phenotype ( Figure 2 H); this increase was independent of changes in FGF21. Interestingly, WAT from PparaAL mice also exhibited elevated basal Ucp1 expression when compared to Pparamice ( Figure 2 H). This suggests that loss of PPARα signaling promotes a modest increase in beiging in the absence of EODF. Taken together, these data indicate that EODF-induced inguinal WAT beiging does not appear to require FGF21.

The discrepancy of activation between brown and beige cells after EODF conditioning might be due to restricted β-AR-independent thermogenesis in subcutaneous WAT (). The present data suggest that the EODF-induced browning could be independent of β-AR signaling, as EODF treatment significantly downregulated expression of Adrb3 in both BAT and inguinal WAT under the fed state, and the trend was the same in fasted BAT and WAT (Figures S2 D and S3 ). To further exclude the role of β-AR signaling, mice were acclimatized to thermoneutrality (30°C) for 1 day and then treated with either AL or EODF over the entire period at 30°C. The EODF at 30°C still reduced body weight and fat mass and induced pronounced inguinal WAT beiging, as indicated by increased expression of Ucp1 mRNA, UCP1 protein, other beige-fat-thermogenic-associated markers, and evident beige morphology ( Figures 2 A–2E ).

Data are presented as mean ± SEM. Different lowercase letters indicate statistical significance by two-tailed unpaired t test: a, p < 0.05; c, p < 0.005; and d, p < 0.001. Black letters show the effects of EODF (EODF versus AL within the same strain), and red letters show the effects of Ppara-null (Pparα −/− versus Ppara +/+ mice within the same treatment). SCFAs, short-chain fatty acids.

(J) mRNA expression of Mct1 in inguinal WAT of Ppara +/+ and Ppara −/− mice with or without EODF treatment in the fed state; n = 5 mice/group.

(H) mRNA expression of Ucp1 in inguinal WAT of Ppara +/+ and Ppara −/− mice with or without EODF treatment in the fed state; n = 5 mice/group.

(F and G) Liver Fgf21 mRNA expression (F) and serum FGF21 levels (G) in Ppara wild-type (Ppara +/+ ) and Ppara-null (Ppara −/− ) mice with or without EODF treatment in the fed state; n = 5 mice/group.

To examine the chronological relationship between WAT beiging and increased energy expenditure induced by EODF, the mice were exposed to short-term EODF. Following 3 cycles of EODF treatment, evident beiging in inguinal WAT was observed as indicated by a striking increase in Ucp1 mRNA expression ( Figure S5 A), while body weight and energy expenditure were not obviously changed ( Figures S5 B–S5D). These results suggest that the effects of EODF on energy expenditure and weight loss are subsequent to its effect on WAT beiging.

Intriguingly, and in contrast, EODF induced an obvious browning in subcutaneous (inguinal) WAT, as indicated by tissue color ( Figure 1 G) and UCP1 expression ( Figures 1 H and 1I). Histological analysis of inguinal WAT in EODF mice also clearly revealed a significant increase in multilobular adipocytes ( Figure 1 I), a typical characteristic of beige adipocytes. Although the fold change of Ucp1 mRNA in the fasted state was relatively lower than in the fed state, the browning phenotype was still pronounced ( Figure S3 ). To determine the washout period for the EODF effect, the 15-cycle EODF-treated mice were placed back on AL feeding. The elevated Ucp1 mRNA levels in inguinal WAT were significantly diminished on the 7day after returning to AL feeding and fell to normal levels on the 15day ( Figure S4 A). However, the effect of EODF on suppressing weight gain was sustained, as the body weight gain was still significantly lower than the AL group on the 15day ( Figure S4 B).

Non-shivering thermogenesis is mainly mediated by activating brown and/or beige adipocytes (). Although the above data indicate a mild mass increase in the interscapular BAT in EODF mice ( Figure 1 C), Ucp1 expression was significantly suppressed ( Figure S2 A), and other thermogenic genes, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a) and type II iodothyronine deiodinase (Dio2), were not upregulated in either the fed or fasted states of mice treated with EODF ( Figures S2 B and S2C). The mechanism by which EODF induces higher energy expenditure might differ from that of cold exposure; cold exposure is closely tied to β-AR signaling (), while fasting is known to suppress norepinephrine turnover () and norepinephrine-induced thermogenesis in BAT (). In line with these data, EODF did not affect expression of the β-adrenergic receptor (Adrb3) mRNA in BAT in the fasted state and suppressed it in the fed state ( Figure S2 D).

To determine the adaptive metabolic changes in energy homeostasis induced by intermittent fasting, the effect of 15-cycle EODF on body weight was first analyzed in mice fed a chow diet. When compared with the ad libitum (AL) group, EODF did not affect cumulative food intake ( Figure 1 A ), but reduced body mass gain ( Figure 1 B), indicative of lower metabolic efficiency and/or higher energy expenditure in EODF mice. To determine the fate of additional energy unaccounted for by body mass gain, the masses of various body depots were analyzed. EODF significantly reduced fat mass—more specifically, visceral epididymal WAT; however, it did not change lean mass and even increased the BAT mass ( Figure 1 C). These data indicate that EODF treatment might promote adaptive non-shivering thermogenesis and the burning of fat. The circadian core body temperature measured rectally in EODF mice was higher than in the AL group ( Figure 1 D), as was total energy expenditure ( Figures 1 E and S1 ), which suggests that the additional energy intake in EODF mice is released as heat. To determine the primary fuel utilized during this process, whole-body respiratory exchange ratios (RER) were assayed in one cycle of EODF. On the first day (the “fasted” day) of the RER assay, the RER of EODF mice was significantly lower than that of the AL mice, and nearly equal to 0.7 ( Figures 1 F and S1 B), indicating that lipid utilization was a major energy fuel. Although on the second day (the “fed” day), when all mice were feeding, the EODF mice had higher RER, their average RER in a whole EODF cycle was relatively lower than that of their AL counterparts ( Figures 1 F and S1 B). These results indicate that increased utilization of lipid, rather than carbohydrate, is responsible for the increased energy expenditure in EODF mice. Therefore, the EODF treatment appears to favor fat burning.

Data are presented as mean ± SEM. Different lowercase letters indicate different statistical significance by two-tailed unpaired t test: a, p < 0.05; b, p < 0.01; c, p < 0.005; and d, p < 0.001 versus AL.

(E and F) Daily total energy expenditure (E) and respiratory exchange ratio (F) during one cycle of EODF. On day 1 (“fasted”), the EODF mice were fasted while the AL mice were fed a chow diet. On day 2 (“fed”), both groups had ad libitum access to chow. Data marked as “1 cycle” show the average value of the 2 days (“fasted” plus “fed”). n = 4 mice/group.

Discussion

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et al. Genetic and functional characterization of clonally derived adult human brown adipocytes. A striking finding from this study is that EODF selectively activates beige, but not brown, adipocytes. Cold acclimation was originally found to increase the number of brown adipocytes in the parametrical fat pad in mice (). Since that finding, more than a hundred alternate treatments have been shown to activate both brown and beige adipose (). Brown adipocyte activation by cold exposure or by β-AR agonists is clinically non-feasible, and adult human BAT is largely composed of beige-like adipocytes (). Therefore, understanding the underlying mechanisms driving the selective beiging of certain white depots during EODF could reveal new therapeutic targets for the prevention and treatment of metabolic disease, which might provide alternative pharmaceutical options for those who cannot sustain intermittent fasting for long periods of time.

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et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Another important finding from the present study is that EODF causes dramatic weight loss and attenuates metabolic dysfunction in mice by directly activating beige cells through shaping the gut microbiota. A recent report revealed that restriction of feeding periods prevents HFD-induced weight gain and metabolic syndrome without reducing total caloric intake; these effects were attributed to alterations in circadian rhythms (). The present work revealed that EODF treatment also does not affect cumulative food intake, thus demonstrating that weight loss may result from increased energy expenditure. Conversely, EODF treatment did not appear to affect circadian rhythms, as rectal temperature profiles and metabolic curves in EODF animals paralleled those of the AL control groups. Therefore, the mechanism for EODF-induced weight loss does not appear to be the result of circadian perturbation. On the other hand, the prominent function of activated beige cells is to promote thermogenesis and suppresses obesity (). The present study reveals pronounced beiging of inguinal WAT, and the mechanism for this EODF-induced improvement in metabolic syndrome could be, at least in part, attributed to increased thermogenesis as a result of WAT beiging. Moreover, consistent with an important role for gut microbiota in inducing beiging, EODF microbiota transplantation can reproduce the effects of an EODF regimen, and EODF fails to further improve obesity-related diseases in microbiota-depleted mice.

In summary, the present work uncovered novel perspectives on beige-fat development in the inguinal WAT. EODF was shown to selectively activate beige fat, probably by re-shaping the gut microbiota, which led to increases in the beiging stimuli acetate and lactate. EODF also dramatically ameliorated metabolic syndrome in a mouse model of obesity. This alternative beige fat activation by EODF offers new insights into the microbiota-beige fat axis and provides a novel therapeutic approach for the treatment of obesity-related metabolic disorders.