Branched-chain amino acid supplements may cause insulin resistance and trigger inflammation through the INFGR1/JAK1/STAT1 signaling pathway

Total 40 C57BL/6 J male mice (22 ± 2 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd, they were randomly divided into 3 groups. STC group (n = 20), HFD (n = 10), High BCAA group (n = 10). STC group mice were fed with standard chow for 16 weeks. HFD group mice were fed with a high-fat diet for 16 weeks. High BCAA group was fed with a high BCAA diet (L-Amino Acid Rodent Diet Based on Teklad 7002 Rodent Chow With 150% Added BCAA, Research Diet Inc, catalog #A12030801). The housing conditions for the animals were maintained under specific pathogen-free circumstances, with a controlled temperature of 22 ± 2 °C and a relative humidity of 55% (ranging from 45 to 70%), and under a 12-h light–dark cycle. Mice were sacrificed by CO2 asphyxiation after 16 weeks of experimental period, all procedures involving the animals were performed following China's national regulations on the use of experimental animals and were in line with the standards set by the Laboratory Animal Welfare and Ethics Committee of Jinhua Municipal Central Hospital, which reviewed and approved the guidelines (AL-JHYY202415).

The collected subcutaneous white adipose tissue (sWAT) was fixed by inflation with a 4% buffered paraformaldehyde solution (dissolve in water, v/v). The tissues were then embedded in paraffin, after dehydration, cut into sections (5 μm) and stained using Hematoxylin & Eosin (H&E), anti-TNF-α, anti-MCP-1 and anti-IL-1β antibody. These stained sections were examined under a light microscope and analyzed using the Image J software. The antibodies used for TNF-α staining were sourced from Abcam (Catalog #ab220210) at a dilution of 1/200, for IL-1β staining from Abcam (Catalog #ab205924) at a dilution of 1/200, and for MCP-1 staining from Abcam (Catalog #ab214819) at a dilution of 1/200.

Adipose tissue samples were collected and rapidly frozen in liquid nitrogen. Upon experimentation, the tissues were homogenized in 500 µL of 0.2 M sodium acetate solution (pH 4.5) using a Precellys tissue homogenizer. The homogenate was centrifuged at 800g for 10 min at room temperature to remove debris, and the resulting supernatant was used to measure TNF-α (Catalog# DY410, R&D Systems), IL-1β (Catalog# DY401, R&D Systems), and MCP-1 (Catalog# DY479, R&D Systems) by using commercial ELISA kits.

For immunofluorescence staining, antigen retrieval was performed on the sections by boiling them in sodium citrate buffer (0.1 mol/L sodium citrate, 0.1% Tween 20, pH 6.0) for 20 min. This was followed by blocking with 5% FBS in PBS for 1 h at room temperature. The sections were then incubated overnight at 4 °C with mouse anti-F4/80 (Abcam, Cat#ab6640, 1/500 dilution) and anti-iNOS (Abcam, Cat#ab178945, 1/500 dilution) antibodies. The next day, fluorescent dye-conjugated secondary antibodies (anti-rabbit 594 nm and anti-mouse 488 nm) were applied at room temperature for 1 h. Finally, the sections were mounted using ProLong™ Glass Antifade Mountant (Catalog# P36980↗, Invitrogen). The distribution of fluorescence in sWAT was observed using a laser scanning confocal microscope (Leica TCS SP8 CARS, Germany).

Mice were fasted for 12 h under glucose tolerance test in 15 weeks. After measuring the baseline blood glucose concentration from a tail cut by a Glucometer test strip, mice were injected intraperitoneally with 20% glucose at 1.5 mg/g body weight. Blood glucose concentrations were measured at 15, 30, 45, 60, 90, and 120 min after glucose injection. Mice were fasted for 6 h under insulin tolerance test in 16 weeks. After measuring the baseline blood glucose concentration, mice were injected intraperitoneally with recombinant human insulin at 1.2 mU/g body weight (Catalog# 91,077 C, Sigma–Aldrich). Blood glucose concentrations were then measured at 15, 30, 45, 60, 90, and 120 min after insulin administration.

To procure visceral adipose tissue, we meticulously excised any apparent blood vessels and connective tissue using sterilized forceps and scissors. We then measured out two grams of this tissue, rinsed it thoroughly with 20 mL of Dulbecco's Phosphate-Buffered Saline (DPBS), and finely chopped it into pieces ranging from 1 to 2 mm in size. Following this, we subjected the chopped tissue to centrifugation at 550g in DPBS at a temperature of 4 °C for 10 min, which served to eliminate any red blood cells. Afterward, we introduced a digestion solution to the tissue and maintained the mixture at 37 °C for an hour to facilitate digestion. We then filtered the digested material through a 70 µm strainer (Catalog #CLS431751, Merck), performed another round of centrifugation, and collected the resultant pellet. This pellet was further processed to extract macrophages, utilizing CD14 + magnetic beads (Invitrogen, Catalog# 11149D) in alignment with the guidelines provided by the bead manufacturer. Once isolated, we quantified the macrophages, evaluated their viability, and prepared them for plating, setting the stage for further experimental investigations.

Isolated adipose tissue macrophage was analyzed using FACS assays on an LSRFortessa instrument (BD Biosciences). Subsequent statistical analysis of the data was performed using FlowJo software. For staining, cells were incubated with fluorochrome-conjugated antibodies, specifically anti-F4/80 (Abcam, Catalog #ab6640, at a dilution of 1/500), anti-206 (Abcam, Catalog #ab64693, at a dilution of 1/500), anti-CD86 (Abcam, Catalog #ab239765, at a dilution of 1/500), anti-11b (Abcam, Catalog #ab133357, at a dilution of 1/500).

Total RNA was isolated using TRIzol reagent (Catalog# 15,596,026, Invitrogen), and reverse transcription of 1 μg of this RNA was conducted utilizing the GoScript™ Reverse Transcription Mix (Catalog# A2801, Promega). Subsequent real-time quantitative PCR (QPCR) analyses were carried out on an Applied Biosystems ViiA 7 Real-Time PCR System, employing QuantiNova SYBR® Green PCR Kit (Catalog# 208,056, QIAGEN) alongside specific primers for various genes as listed in Supplementary Table 1.

Total RNA was isolated using the RNeasy mini kit (Qiagen). Strand-specific RNA-seq libraries were prepared with the TruSeq stranded total RNA sample preparation kit (Illumina), quantified by the Qubit 2.0 Fluorometer (Life Technologies), and assessed for insert size with the 2100 bioanalyzer (Agilent). Clusters were generated on the cBot at a concentration of 10 pM and sequenced on the NovaSeq 6000 system (Illumina). Library preparation and sequencing were carried out by the Meiji Biotechnology Corporation.

20 C57B/L mice (8 weeks around 22 g) male mice were randomly divided into 2 groups. The STC group was fed a standard chow diet, the HFD group was fed a high-fat diet, and the experiment continued for 16 weeks. Every 2 weeks, the body weight and food intake were recorded. After 16 weeks, we sacrificed the mice from the 2 groups and isolated the adipose tissue macrophage for analysis. The body weight of the HFD group was significantly higher (14 weeks P = 0.0001 and 16 weeks P < 0.0001) than the STC group (Fig. 1A), which suggested the HFD-induced obesity mice model has been established successfully. There was higher food intake in the HFD group than the other group too (Fig. 1B). Then the adipose tissue macrophage was isolated and the cellular BCAA and BCKA were extracted for LC/MS/MS measurement, the results showed that in HFD-induced obesity mice, there were the accumulation of both BCAA (Leucine, Isoleucine and Valine) and BCKA (KIC, KMV and KIV) in adipose tissue macrophage (Fig. 1C, D). We have detected the mRNA expression of BCAA catabolic enzymes, the QPCR results indicated that the mRNA levels of BCAT1, BDK, and P-BCKDHA were decreased significantly, and the mRNA expression of PP2CM was significantly increased (Fig. 1E). The results from immunoblotting experiment were consistent with QPCR results (Figs. 1F), the expression of BCAT1, BCAT2,BDK and phosphorylated BCKDH-A were decreased after BCAA supplementation and the expression of PP2CM was increased (Fig. 1G). These results showed that BCAA catabolism in adipose tissue macrophages was disrupted in HFD-induced obesity mice.

20 C57B/L mice (8 weeks around 22 g) male mice were randomly divided into 2 groups. The STC group was fed with normal diet, High BCAA group was fed with BCAA supplementation diet for 16 weeks. The body weight of each group was recorded every 2 weeks. In 14 weeks (P = 0.0395) and 16 weeks (P = 0.0307), we can see significantly increased body weight in the high BCAA group (Fig. 2A). There was a higher food intake of the High BCAA group than STC group (Fig. 2B). And in 15 and 16 weeks, we performed glucose tolerance experiment and insulin sensitivity experiment respectively, the results showed that high BCAA group has poor glucose tolerance compared to the STC group (Fig. 2C). And high BCAA supplementation caused lower insulin sensitivity compared to the STC group (Fig. 2D), which implied that high BCAA supplementation would cause insulin resistance in mice.

Following a 16-week study period, the mice were euthanized, and their subcutaneous white adipose tissue (sWAT) was collected for immunohistochemical analysis. The staining results revealed that the group receiving high levels of branched-chain amino acid (BCAA) supplementation exhibited a more pronounced presence of pro-inflammatory markers IL-1β, TNF-α, and MCP-1 compared to the standard treatment control (STC) group, as depicted in Fig. 3A. Quantitative analysis confirmed a significant upregulation of these pro-inflammatory cytokines in the high BCAA group (P < 0.0001), shown in Figs. 3 B-D. Additionally, ELISA assays conducted on the adipose tissue confirmed the elevated expression of IL-1β, TNF-α, and MCP-1 in the high BCAA-supplemented mice (P < 0.0001), as illustrated in Figs. 3E–G. These findings suggest that BCAA supplementation may induce increased inflammation in adipose tissue.

Since adipose tissue macrophage is the mainly source of pro-inflammatory cytokine, then we examined the macrophage polarization in different groups. sWAT was used to do immunofluorescence staining, we co-stained the DAPI (nucleus marker), F4/80 (macrophage marker) and iNOS (pro-inflammatory macrophage marker). The IF staining results showed that F4/80 signal was no different in the two groups, but the signal of iNOS was significantly increased in the high BCAA group (Fig. 4A), which indicated that BCAA supplementation can promote adipose tissue macrophage pro-inflammatory polarization. To verify our finding, we also performed a flow cytometry experiment, after adipose tissue macrophage isolation, we co-stained the cell with F4/80, CD11b (macrophage marker), CD206 (anti-inflammatory macrophage marker) and CD86 (pro-inflammatory macrophage marker) (Fig. 4B). The results were consistent with IF staining, the population of total macrophage (F480 + CD11b +) showed no change between the two groups (Fig. 4C), but CD206 + macrophage decreased (from 42.2 to 25.0%) and CD86 + macrophage increased (from 24.3 to 43.3%) in the high BCAA group (Fig. 4D, E). These results indicated that BCAA supplementation can promote adipose tissue macrophage from an anti-inflammatory phenotype to a pro-inflammatory phenotype, contributing to the increased levels of pro-inflammatory cytokines observed in adipose tissue after BCAA supplementation.

To investigate the molecular signal pathway activated by BCAA supplementation, adipose tissue macrophages were isolated from mice in both the STC and BCAA supplementation groups. Then RNA-seq was performed to reveal changes in the mRNA profile. The transcriptomic analysis identified 393 differentially expressed genes (DEGs), with 80 genes upregulated and 313 genes downregulated (Fig. 5A, B). Then we used these DEGs to perform KEGG enrichment analysis. The results showed most DEGs enriched on immune system signaling by interferons, Interferon signaling, IL-22 soluble receptor signaling pathway, PI3K class IB pathway, interferon-γ signaling pathway, Integrin beta-5 pathway, EGF/EGFR signaling pathway, STAT3 pathway and BDNF signaling pathway (Fig. 5C). Gene Set Enrichment Analysis (GSEA) indicated that BCAA supplementation activated the IFN-γ and TNF-α pathways (Fig. 5D, E). Additionally, the protein–protein interaction (PPI) network provided a platform to identify disease-related genes based on their functional relationships (Fig. 5F).

The RNA-seq data has suggested that BCAA supplementation may contribute to regulating the interferon-γ signaling pathway (Fig. 5D). To verify whether BCAA supplementation can activate this pathway, further experiments were conducted. Adipose tissue macrophages were isolated from the two groups, and the protein expression of iNOS indicated that BCAA supplementation can induce pro-inflammatory macrophage polarization (Fig. 6A, B), consistent with previous results (Fig. 4A). Next, protein expression in the IFN-γ signaling pathway was examined. INF-γ recognizes cell membrane receptors IFNGR1 and/or IFNGR2. Western blot analysis showed that BCAA supplementation increased the protein expression of IFNGR1 but not IFNGR2 (Fig. 6A, C and D). Additionally, the expression of JAK1, P-JAK1, STAT1, and P-STAT1 proteins was assessed. The results demonstrated that BCAA supplementation enhanced the P-JAK1/JAK1 and P-STAT1/STAT1 ratios (Fig. 6E, F). All above results indicated that BCAA supplementation can activate IFNGR1/JAK1/STAT1 signal pathway and regulate adipose tissue macrophage polarization.

To verify the IFNGR1/JAK1/STAT1 signal pathway is the mainly pathway of BCAA supplantation’s function, adipose tissue macrophages were isolated, and IFNGR1 was silenced using siRNA. Immunoblotting results indicated that the knockdown efficiency of siRNA achieved approximately 60% reduction in IFNGR1 expression (Fig. 7A, B). Subsequently, pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1) in the culture medium were measured. ELISA results showed that IFNGR1 knockdown inhibited the secretion of these pro-inflammatory cytokines following high BCAA stimulation (Fig. 7C–E). And IFNGR1 knockdown also inhibited the pro-inflammatory macrophage polarization cause by BCAA supplementation (Fig. 7F), there was no change of F4/80 + CD11b + (total macrophage), but significantly inhibited the CD86 + cell (pro-inflammatory macrophage) from 41.96 to 33.11% (Fig. 7G, B). The above results suggested that IFNGR1 Silence can reverse the effect of BCAA supplementation-induced adipose tissue inflammation and pro-inflammatory macrophage polarization. Thus, IFNGR1 serves as a critical target for the high BCAA stimulus.

All procedures involving the animals were performed in accordance with China's national regulations on the use of experimental animals and were in line with the standards set by the Laboratory Animal Welfare and Ethics Committee of Jinhua Municipal Central Hospital, which re-viewed and approved the guidelines (AL-JHYY202415).

Not applicable.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The RNA-seq raw data are available upon request to interested researchers.