All animal experiments were conducted in adherence to the animal research methods outlined in the “Guidelines for the Care and Use of Laboratory Animals” approved by the ethics committee of Shinshu University School of Medicine. Male 6-week-old mice on a C57BL/6J genetic background were purchased from CLEA Japan, Inc. (Tokyo, Japan) and maintained in a controlled and clean environment (25°C, 12-h light/dark cycle). The control diet was purchased from ORIENTAL YEAST Co., Ltd (Tokyo, Japan). The HFD (D12492M) containing 60% kcal of fat was purchased from Research Diet (New Brunswick, NJ). The ingredients of these diets were shown in Supplementary Tables 1, 2, respectively. Acidic activated charcoal was provided from Sumi Plus Lab Co., Ltd. (Yokohama, Kanagawa, Japan), with dextrin (Pinedex#2, Matsutani Chemical Industry Co., Ltd., Itami, Hyogo, Japan) used as a vehicle. The acidic charcoal was prepared by carbonizing plants and trees at constant atmospheric pressure and temperature above 700°C after soaking acidic deep seawater (pH 5.5). The activated charcoal powder was completely mixed with HFD and then administered to mice. Inulin (Fuji FF, Fuji Nihon Seito Corporation, Tokyo, Japan) and raffinose (Nitten Raffinose FP, Nippon Beet Sugar Manufacturing Co., Ltd., Tokyo, Japan) were added to prevent charcoal-induced constipation. In the first experiment, mice (n = 21) weighing 22–26 grams were randomly divided into three groups after acclimatization for at least 2 weeks into (1) a control diet group (Con, n = 5), (2) a HFD with 5% dextrin and 5% inulin raffinose group (HFD + Veh, n = 8), and (3) a HFD with 5% acidic activated charcoal and 5% inulin raffinose group (HFD + 5%C, n = 8). As an additional dose-dependency experiment, mice (n = 28) weighing 23–28 grams were randomly divided into five groups fed (1) a control diet (Con, n = 4), (2) a HFD with 5% dextrin and 5% inulin raffinose (HFD + Veh, n = 6), (3) a HFD with 1.5% acidic activated charcoal and 1.5% inulin raffinose (HFD + 1.5%C, n = 6), (4) a HFD with 3% acidic activated charcoal and 3% inulin raffinose (HFD + 3%C, n = 6), and (5) a HFD with 4.5% acidic activated charcoal and 4.5% inulin raffinose (HFD + 4.5%C, n = 6). During the experiments, the animals’ body weight (BW) and food intake were measured weekly. At 12 weeks of treatment, the mice were sacrificed by CO₂ asphyxiation after 6-h fasting for collecting blood and tissues. Blood samples were centrifuged twice at 3,000 rpm for 15 min to obtain serum and stored at –80°C until use. The organs were harvested, washed with 0.9% sterile saline, and weighed, and the length of the small intestine was measured. The tissues were preserved either by immediate fixation in 10% neutral formalin or immediate freezing with dry ice and storage at –80°C. The feces during 6-h fasting before killing were collected manually, weighted, and stored at –80°C until use.

For the glucose tolerance test (GTT), the mice were injected intraperitoneally with 1 g/kg BW of glucose dissolved in 0.9% sterile saline using a 26-gauge needle after 18-h fasting. For the insulin tolerance test (ITT), the mice were injected intraperitoneally with 0.6 unit/kg BW of insulin (Humulin R; Eli Lilly, Indianapolis, IN) in the same manner after 6-h fasting. Blood glucose concentrations were determined at 0, 15, 30, 60, 90, and 120 min after injection using a FreeStyle Freedom Lite glucose meter (NIPRO, Osaka, Japan).

Serum levels of aspartate aminotransferase (AST) (#431-30901), alanine aminotransferase (ALT) (#431-30901), triglycerides (TGs) (#632-50991), total cholesterol (T-Chol) (#294-65801), non-esterified fatty acids (NEFAs) (#279-75401), phospholipids (PLs) (#433-36201), total bile acid (TBA) (#431-15001), and glucose (#298-65701) were measured with commercially available enzyme assay kits (Wako Pure Chemical Industries Co., Ltd., Osaka, Japan). Serum insulin, leptin, and high-molecular-weight adiponectin were determined by ELISA kits (AKRIN-011T, AKRLP-011, and AKMAN-011, respectively, FUJIFILM Wako Shibayagi, Gunma, Japan). Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using the equations published by Matthews et al. (7). Serum thyroid-stimulating hormone (TSH) was determined by ELISA kits (KT-29922, KAMIYA BIOMEDICAL COMPANY, Seattle, WA) (8).

Total liver lipids and fecal lipids were extracted according to the hexane:isopropanol method (9) with a slight modification and quantified using the abovementioned kit (Wako Pure Chemical Industries Co., Ltd.). For extracting liver lipids, approximately 50 mg of liver tissues were disrupted and sonicated in 5–10 volumes of sodium phosphate buffer (NaPi, 50 mM). The lysate (50 μL) was drawn into a new tube, and 900 μL of hexane/isopropanol (3:2, vol/vol) (HIP) was added. The tube was vigorously vortexed for 1 min and centrifuged at 2,500 rpm for 5 min at 4°C, and the upper layer was transferred to a new tube and centrifuged under vacuum at 40–50°C. After evaporation, the lipid extracts were dissolved in 100 μL of HIP containing 1% (wt/vol) Triton X-100 and evaporated again by vacuum centrifuge. Then, 100 μL of distilled water was added to the sample, incubated at 37°C for 30 min, and vortexed. Finally, the lipid extracts were solubilized in 1% Triton X-100/water as described previously (10), and 10 μL of the lipid extracts (corresponding to 0.25 mg of livers) were used for measuring TG, PL, T-Chol, and NEFAs (11).

For extracting fecal lipids, 20–30 mg of dry feces were collected and placed in 19 volumes of 50 mM NaPi. The tube was kept on ice for 30–60 min until the feces become swollen, and then sonicated on ice until the feces were completely dispersed in the solution. The total lipids in feces were extracted in a similar manner as those in the liver and finally solubilized in 1% Triton X-100/water at a concentration of 0.025 mg feces eq./μL. Ten μL of the lipid extracts (corresponding to 0.25 mg of feces) were used for measuring TBA, TG, T-Chol, PL, and NEFAs.

Formalin-fixed liver tissues were embedded in paraffin, cut into 3 μm sections, and stained with hematoxylin and eosin (12). To quantify the size of adipocytes and the relative area of islets, 10 independent sections of epididymal white adipose tissue (eWAT) and pancreas were photomicrographed (×200 magnification) in each group, and the size of each adipocyte and the area of each islet were quantified using the 1.52V version of Image J (National Institutes of Health, Bethesda, MD). Values were expressed as the percentage of adipocytes of the corresponding size. The total islet area was also expressed as the percentage of the total area of pancreatic parenchyma in the sections.

Total RNA was isolated from frozen liver tissues using a NucleoSpin RNA Plus Kit (QiagenMACHEREY-NAGEL Gmbh & Co., KG, Neumann, Germany). Total RNA from the stomach, upper small intestine, lower small intestine, large intestine, brown adipose tissue (BAT), and eWAT were extracted using TRI reagent (MOR Molecular Research Center, Inc., Cincinnati, OH). Tissues (50 mg) were disrupted and sonicated in 500 μL of TRI reagent. The homogenate was incubated at room temperature for 5 min, 100 μL of chloroform was added, and the homogenate was shaken vigorously for 15 sec. The homogenate was then incubated at room temperature for 2–3 min and centrifuged at 12,000 × g for 15 min at 4°C. The upper layer was transferred to a new tube and 1 volume of 70% ethanol was added and vortexed. The mixture was transferred to a column with a silica membrane and centrifuged at 12,000 × g for 1 min at room temperature. The silica membrane was washed and dried, and RNA was eluted and collected using RNase-free water. The RNA samples were reverse transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd., Osaka, Japan). All mRNA levels were determined by the real-time quantitative polymerase chain reaction (qPCR) using a SYBR qPCR mix (Toyobo Co., Ltd.) on a Thermo Fisher QuantStudio 3 Real-Time PCR Instrument (Thermo Fisher Scientific, Waltham, MA). The mRNA levels were normalized to 18S ribosomal RNA (18S rRNA) levels and expressed as fold changes relative to those of C57BL/6J mice fed the control diet. The primer sequences were listed in Supplementary Table 3.

The detailed method of metabolomic analysis is described in Supplementary Information.

Results were expressed as the mean ± standard error of the mean (SEM). Two-tailed Student’s t-tests and a one-way analysis of variance (ANOVA) with the Bonferroni’s correction were conducted using SPSS statistics version 22 (IBM, Armonk, NY). A P-value of less than 0.05 was considered statistically significant.

In the first experiment, test mice were given the three different diets for 12 weeks. In the HFD + 5%C group, the limbs, fur and tail turned black from the charcoal (Supplementary Figure 1A). Weight gain in the HFD + 5%C group was significantly suppressed as compared with the HFD + Veh group and was similar to the Con group (Figure 1A and Supplementary Table 4). There were no significant differences in food intake between the HFD + Veh and HFD + 5%C groups (Figure 1B), nor were remarkable differences seen for the gross appearance of the liver, lungs, or subscapular BAT among the three groups (Figures 1C,D). We observed that eWAT became larger, the cecum became smaller, and the small intestine became shorter by HFD treatment, which were all mitigated by the charcoal-containing HFD (Figures 1C,D and Supplementary Table 4). Since thyroid dysfunction can lead to darker fur and weight loss, we measured serum levels of TSH, a sensitive indicator of primary hypothyroidism. The TSH levels were not changed by charcoal treatment (Supplementary Figure 1B). These results demonstrated that acidic activated charcoal prevented HFD-induced obesity.

Since obesity has been found to impair glucose metabolism, the GTT was conducted in the tenth week of HFD treatment. The blood glucose levels of the HFD + 5%C group were consistently lower than those of the HFD + Veh group (Figure 2A). Accordingly, the area under the receiver operating characteristic curve (AUC) was significantly smaller in the HFD + 5%C group than in the HFD + Veh group (Figure 2A).

To assess whether the improved glucose intolerance stemmed from enhanced insulin sensitivity, the ITT was performed in the twelfth week of HFD treatment. After insulin injection, glucose levels in the HFD + 5%C group fell faster than in the HFD + Veh group (Figure 2B). The blood glucose initial ratio in the HFD + 5%C group was consistently lower than in the HFD + Veh group. Consequently, the AUC was also significantly smaller in the HFD + 5%C group (Figure 2B). These results indicated that acidic activated charcoal intake could reverse HFD-induced insulin resistance.

In agreement with the GTT/ITT results, serum insulin levels and HOMA-IR were significantly lower in the HFD + 5%C group than in the HFD + Veh group, while serum leptin and high-molecular-weight adiponectin were comparable between the HFD + Veh and HFD + 5%C groups (Figure 2C). To exclude the possibility that decreased serum insulin levels were due to impaired pancreatic function, we assessed the histology of pancreas. There were no significant abnormalities, such as atrophy, inflammation, and fibrosis, in pancreatic parenchyma of charcoal-containing HFD-fed mice. Quantification of the relative islet area and pancreatic expression levels of Ins2 (insulin 2) revealed no significant differences between the groups (Supplementary Figures 2A,B).

In the determination of serum lipid profiles, while serum TBA levels were significantly decreased in the HFD + 5%C group, no significant differences in the other parameters were observed between the HFD + Veh and HFD + 5%C groups (Figure 2C).

In both the HFD + Veh and HFD + 5%C groups, the serum levels of AST and ALT, which are conventional indices of liver damage, were within the reference values, and no abnormal morphologies were found in the liver tissue (Figure 2C and Supplementary Figure 3A). In the latter group, no charcoal deposition was observed in the liver. Quantification of mRNA in the liver demonstrated no significant differences in gene expression related to fatty acid (FA)/TG metabolism, inflammation, or fibrosis (Supplementary Figures 3B–F). Quantitative analysis of hepatic lipids showed that TGs in the HFD + 5%C group were increased, while PL, T-Chol, and NEFA levels were comparable among the groups (Supplementary Figure 3G). Taken together, it appeared that the liver was not a primary target of acidic activated charcoal for preventing HFD-induced BW gain and insulin resistance.

The finding that acidic activated charcoal prevented eWAT weight gain prompted us to assess the phenotypic changes in white adipocytes. Microscopic examination of eWAT revealed marked adipocyte hypertrophy and inflammatory cell infiltration around adipocytes in the HFD + Veh group, and the proportion of larger adipocytes (4,001–12,000 μm²) in this group was significantly increased. These alterations were prevented by 5% charcoal intake (Figures 3A,D).

We next evaluated whether eWAT mRNA levels were associated with adipocyte enlargement and inflammation. Compared with the HFD + Veh group, the expression levels of the genes related to inflammation and fibrosis, including interleukin 1 beta (Il1b), chemokine (C-C motif) ligand 2 (Ccl2), transforming growth factor beta 1 (Tgfb1), and actin alpha 2 (Acta2), were significantly decreased in the HFD + 5%C group (Figures 3B,C). The mRNA levels of other inflammation-related genes, such as cluster of differentiation 68 (Cd68) and tumor necrosis factor alpha (Tnf), were decreased as well but did not reach statistical significance (Figures 3B,C).

The improvement of adipocyte hypertrophy in the HFD + 5%C group may have been associated with increased FA catabolism, browning of white adipocytes, and decreased FA uptake and TG synthesis. Among several genes related to FA/TG metabolism, the mRNA levels of the cluster of differentiation 36 (Cd36) and FA-binding protein 4 (Fabp4) genes, both responsible for FA uptake into adipocytes, tended to be decreased in the HFD + 5%C group (Supplementary Figure 4). No browning of white adipocytes was detected in the HFD + 5%C group (Supplementary Figure 4).

BAT plays an important role in the maintenance of whole-body energy/fuel metabolism through the promotion of fat burning. To evaluate whether the anti-obesity effect of charcoal stemmed from enhanced fat burning in BAT, we assessed for phenotypic changes in this tissue. Histological analysis revealed that the HFD + Veh group had increased unilocular lipid droplets and white adipocytes (i.e., whitening), with slight inflammatory cell infiltration around adipocytes, while such changes were absent in the HFD + 5%C group (Supplementary Figure 5A). In mRNA quantifications of BAT, compared with the HFD + Veh group, the expression levels of the inflammation-related genes Cd68 and Ccl2 tended to be decreased in the HFD + 5%C group. The mRNA levels associated with fat burning, including uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), type 2 iodothyronine deiodinase (Dio2), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Ppargc1a), and cytochrome c oxidase subunit 5B (Cox5b) and 8B (Cox8b), were not increased by the co-administration of charcoal (Supplementary Figure 5B). Cell death-inducing DNA fragmentation factor alpha-like effector C (Cidec) as a gene related to lipid storage was decreased but did not reach statistical significance. The above results indicated that although activated charcoal could improve whitening in BAT, it was not due to enhanced fat oxidation.

Since acidic activated charcoal powder was not digested or absorbed into the body and charcoal was excreted into the feces and mouse bedding in the cage turned black by the charcoal treatment (Supplementary Figure 6A), we next examined for possible pulmonary and gastrointestinal toxicity. To assess the presence of inflammation in the lungs and gastrointestinal tract, we determined mRNA levels in the lung (Supplementary Figure 6B), stomach (Supplementary Figure 6C), upper small intestine (Supplementary Figure 6D), lower small intestine (Supplementary Figure 6E), and large intestine (Supplementary Figure 6F). The mRNA levels of genes related to inflammation [Il1b, Ccl2, and nitric oxide synthase 2 (Nos2)], fibrosis (Tgfb1), and neutrophil infiltration [myeloperoxidase (Mpo)] were not significantly different between the HFD + 5%C and HFD + Veh groups. The possibility that co-treatment with acidic activated charcoal could injure/irritate the respiratory and gastrointestinal mucosa was therefore considered low.

Since shortening of the small intestine and a reduction in cecal contents were prevented by treatment with acidic activated charcoal, we tested the possibility that the intestinal environment was altered by such treatment by the metabolomic analysis of cecal contents. A volcano plot identified 42 metabolites that were significantly increased in the HFD + 5%C group as compared with the HFD + Veh group (Figure 4). The 20 most significantly increased metabolites are listed in Supplementary Table 5. Nine metabolites were neutral lipids, FAs, or BAs such as docosapentaenoate, deoxycholic acid (DCA), and hyodeoxycholic acid (Figure 4). To validate these metabolomic results, fecal lipids and BAs were quantified, revealing that the amounts of TGs, PLs, and TBA were significantly increased in the feces of all mice in the HFD + 5%C group (Figure 5A).

BA metabolism, including DCA, lithocholic acid, and taurocholic acid, is controlled by farnesoid X receptor (FXR). Analysis of the FXR signaling pathway in the lower small intestine and liver revealed that the mRNA levels of Fxr and its target genes, including small heterodimer partner (Shp), fibroblast growth factor 15 (Fgf15), apical sodium BA transporter (Asbt), and FA-binding protein 6 (Fabp6), were significantly down-regulated in the HFD + 5%C group vs. the HFD + Veh group in the intestine (Figure 5B) while those of Fxr-regulated genes were unchanged in the liver, indicating that intestinal FXR was specifically down-regulated by charcoal co-administration. BAs have also been shown to effectively activate Takeda G protein-coupled receptor 5 (TGR5), although the mRNA levels of Tgr5 in the lower small intestine were not significantly increased in the HFD + 5%C group (Figure 5B).

The hepatic mRNA expression of cholesterol 7 alpha-hydroxylase (Cyp7a1), a rate-limiting enzyme of BA synthesis, as well as those of hydroxymethylglutaryl-CoA synthase 1 (Hmgcs1) and reductase (Hmgcr), both rate-limiting enzymes of de novo cholesterol synthesis, were significantly increased in the HFD + 5%C group as compared with the HFD + Veh group, while those of ATP-binding cassette sub-family B member 11 (Abcb11) and Fxr were comparable to the HFD + Veh group (Figure 5C). These changes were likely due to an adaptive response to decreased BA reabsorption in the intestine, i.e., increased BA excretion into the feces, by acidic activated charcoal.

To evaluate the reproducibility and dose dependency of the anti-obesity effect of acidic activated charcoal, a second mouse cohort was given a HFD supplemented with different charcoal contents for 12 weeks. In the 1.5, 3, and 4.5% charcoal-containing HFD groups (HFD + 1.5%C, HFD + 3%C, and HFD + 4.5%C, respectively), weight gain was significantly suppressed vs. the HFD + Veh group, which was enhanced with charcoal dosage (Figure 6A and Supplementary Table 6). There were no significant differences in the gross appearance of the liver or the liver/body weight ratio among the five groups (Figures 6B,C). As in the first experiment, eWAT became larger and the cecum became smaller in the HFD + Veh group over the Con group. Acidic activated charcoal improved these changes in a dose-dependent manner without affecting thyroid function (Figures 6B,C, Supplementary Figure 1B and Supplementary Table 6).

The GTT was conducted in the ninth week of HFD treatment. The blood glucose levels of all HFD + C (1.5%C, 3%C, and 4.5%C) groups were consistently lower than that of the HFD + Veh group, with the HFD + 4.5%C group exhibiting the lowest blood sugar level (Figure 7A). The AUC became smaller with increasing doses in the HFD + C groups vs. the HFD + Veh group (Figure 7A).

The ITT in the eleventh week of treatment showed that the glucose levels in all HFD + C groups fell faster than in the HFD + Veh group. The decrease in the HFD + 4.5%C group was especially larger than in the Con group (Figure 7B), which was reflected in the AUC results (Figure 7B). Serum insulin levels and HOMA-IR significantly decreased in the HFD + C groups with rising charcoal doses (Figure 7C). These findings indicated that 4.5%C exerted the strongest therapeutic effect on HFD-induced insulin resistance. Similarly to the results of the first experiment, there were no significant differences in serum and hepatic lipid profile parameters between the HFD + Veh and HFD + C groups (Figure 7D and Supplementary Figure 7A).

Microscopic examination of eWAT revealed that the adipocyte hypertrophy and inflammatory cell infiltration around adipocytes in the HFD + Veh group were dose-dependently corrected by charcoal intake; as the charcoal doses increased, the proportion of larger adipocytes decreased significantly (Figures 8A,B). Compared with the HFD + Veh group, the expression levels of inflammation- and fibrosis-related genes, such as Cd68, Il1b, Ccl2, Tgfb1, Acta2, and Col1a1, were decreased in the HFD + C groups with rising charcoal doses (Figure 9A). Histological analysis of BAT revealed that the HFD-induced unilateral lipid droplets and slight inflammatory cell infiltration around adipocytes were ameliorated with increasing charcoal doses (Figure 8A). Overall, our results indicated that the improvement of HFD-induced eWAT hypertrophy and inflammation by acidic activated charcoal, as well as BAT whitening, were dose-dependent.

The amounts of TBA, TGs, T-Chol, PLs, and NEFAs all significantly increased with higher charcoal doses in feces samples collected from the HFD + C groups (Figure 9B). However, Fxr and its related genes (Shp, Fgf15, Asbt, and Fabp6) showed no remarkable differences between the HFD + Veh group and the HFD + C groups. Interestingly, the mRNA level of Tgr5 increased with greater doses of charcoal, which was significant in the HFD + 3%C groups (Figure 9C).

The finding that the hepatic mRNA expressions of Hmgcr and Cyp7a1 were significantly increased in the HFD + 4.5%C group indicated enhanced synthesis of cholesterol and BAs in the liver (Figure 9D), likely reflecting dose-dependent increases in cholesterol/BA excretion into the feces.