Indole-3-carbinol ameliorates ER stress-mediated hyperleptinemia in western diet-fed apoE-/- mice

TL;DR

Indole-3-carbinol reduces ER stress and high leptin levels in obese mice on a high-fat diet, potentially preventing atherosclerosis.

Contribution

This study demonstrates that I3C alleviates ER stress-induced hyperleptinemia in western diet-fed apoE-/- mice.

Findings

I3C reduced adipose tissue weight and plasma leptin levels in apoE-/- mice.

I3C decreased ER stress markers and increased genes related to cholesterol efflux and fatty acid β-oxidation.

I3C reduced aortic ER stress markers in atherosclerotic lesions.

Abstract

Endoplasmic reticulum (ER) stress during overnutrition causes leptin resistance in obese animals and humans. ER stress induces the activation of the unfolded protein response, which disrupts the leptin signaling pathway, accelerating atherosclerosis development and its complications.

Indole-3-carbinol (I3C) improves metabolic dysfunction in diet-induced obesity; however, its role in protecting against ER stress-induced hyperleptinemia remains unclear. Herein, we explored whether dietary I3C alleviates ER stress in apolipoprotein E-deficient (apoE-/-) mice fed a western diet (WD).

ApoE-/- mice were fed either WD (60 kcal from fat, n = 10) or WD supplemented with 0.05% I3C (w/w, n = 10) for 12 weeks.

I3C supplementation (0.05%) resulted in reduced adipose tissue weight and plasma leptin levels compared with those in WD-fed apoE-/- mice after 12 weeks. I3C also significantly decreased the protein expression of ER stress markers, whereas increased the mRNA expression of genes related to cholesterol efflux and fatty acid β-oxidation in the liver, despite no changes in plasma cholesterol and triglyceride levels. Immunohistochemistry revealed reduced aortic localization of glucose-related protein 78 compared with the WD group, suggesting that I3C partially alleviated ER stress in atherosclerotic lesions of WD-fed apoE-/- mice.

I3C may serve as a feasible compound for preventing atherosclerosis and its associated complications.

Figures

Biosynthesis of I3C. Glucobrassicin from cruciferous vegetables is hydrolyzed by myrosinase to form isothiocyante, which decomposes to 3-methylene-3H-indolium under neutral pH. Under acidic conditions, such as in the stomach, it is converted to I3C, which then undergoes condensation reactions.

Effects of I3C on hepatic mRNA expression levels in ApoE-/- mice fed a WD. Results are presented as the mean ± standard error (n = 5 per group). *P < 0.05, P < 0.01 versus WD.

Effects of I3C on endoplasmic reticulum stress in the liver of ApoE-/- mice fed a WD. Representative protein levels of p-eIF2α, eIF2α, XBP1, CHOP, and GRP78 as measured by western blotting. Results are presented as the mean ± standard error (n = 4–5 per group). *P < 0.05, P < 0.01 versus WD. p-eIF2α, phospho-eukaryotic initiation factor 2 subunit alpha; XBP1, X-box binding protein 1; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein.

Effects of I3C on histology and immunohistochemistry in the aortic root of ApoE-/- mice fed a WD. Representative hematoxylin & eosin (a, b) staining (scale bar, 20 µm) and GRP78 immunostaining (c, d) of aortic root sections from ApoE-/- mice fed a WD. Original magnification: 200 × I indicates intima; M, media; and L, lumen.

A proposed mechanism of I3C supplementation in ApoE-/- mice fed a WD. Accumulation of free cholesterol or ox-LDL in endothelial cells due to hypercholesterolemia or hyperleptinemia triggers ER stress-mediated apoptosis in endothelial or liver cells, all of which were reversed by I3C supplementation in ApoE-/- mice. The figure was created in BioRender.com and modified by Geng et al. (49).

Tables

Table 1. Biochemical parameters

Group	ApoE-/-
	WD	0.05% I3C
Body weight gain (g/day)	0.18 ± 0.01	0.17 ± 0.01NS
Food intake (g/day)	3.05 ± 0.05	2.80 ± 0.10NS
Food efficacy ratio (%)	5.95 ± 0.35	5.98 ± 0.41NS
Liver weight (%)	4.51 ± 0.30	4.75 ± 0.21NS
Abdomial fat weight (g)	1.69 ± 0.06	1.38 ± 0.09*
Epididymal fat weight (g)	3.98 ± 0.18	3.33 ± 0.21*
Brown fat weight (g)	0.49 ± 0.05	0.36 ± 0.03*
Leptin (pg/mL)	27477.00 ± 3880.45	14497.00 ± 1517.00**
Plasma Total cholesterol (mg/dL)	695.66 ± 28.61	642.23 ± 39.39NS
Plasma Triglyceride (mg/dL)	94.14 ± 9.96	104.19 ± 6.20NS
Plasma CETP (ρmol/μL/h)	28.00 ± 0.40	25.64 ± 0.71NS
Fasting glucose (mg/dL)	164.58 ± 11.09	182.72 ± 12.24NS
IL-6 (pg/mL)	4.37 ± 0.37	6.11 ± 0.73NS
TNF-α (pg/mL)	36.52 ± 2.38	44.30 ± 3.42NS
Plasma TBARS (nmol/mL)	15.34 ± 1.20	9.30 ± 0.40*

Full text

Popular scientific summary

Hyperleptinemia caused by overnutrition triggers ER stress and activates the UPR, contributing to atherogenesis development and complications.I3C supplementation reversed the increase in fat weight and hyperleptinemia as well as the WD-induced activation of ER stress in apoE^-/-^ mice.Controlling hyperleptinemia-induced ER stress may offer a potent strategy against atherosclerosis and its complications.

Atherosclerosis is a chronic and complex disorder characterized by the deposition of lipid-laden foam cells in the arterial wall provoked by proatherogenic stimuli such as hypercholesterolemia, inflammatory mediators, elevated plasma leptin levels, hyperglycemia, and oxidative stress (1, 2). Accumulation of free cholesterol (FC) in macrophage foam cell triggers endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR), contributing to all stages of atherosclerotic lesion development and its complications (3). The accumulation of unfolded proteins from excess fat induces ER stress pathways, such as the phosphorylation of eukaryotic initiation factor 2α (eIF2α) and expression of X-box binding protein (XBP)1 and C/EBP homologous protein (CHOP), which consequently induce proapoptotic signaling pathways (4, 5). Although ER stress is associated with all stages of atherosclerosis, the UPR is considered one of the mechanisms determining cell survival or death (6).

Leptin is an adipokine that serves as the main messenger transmitting signals to maintain energy homeostasis (7). Although leptin resistance is a major hallmark of obesity that facilitates atherosclerosis and vascular dysfunction, its function in atherosclerotic lesion development and vascular calcification remains controversial (8, 9). ER stress blocks leptin signaling through multiple mechanisms, including impaired pro-opiomelanocortine processing and inflammation induction (10). Chemical chaperones and leptin sensitizer, which improve ER function, enhance leptin sensitivity and reduce leptin resistance (11, 12).

Therefore, exploring potential pharmacological candidates derived from natural plants containing phytochemicals has gained global attention as a strategy against hyperleptinemia associated with increased adiposity in atherosclerosis and related metabolic complications.

The Brassicaceae family includes broccoli, cabbage, and cauliflower, which are widely produced worldwide (13). The recommended daily intake of vegetables for adults in the USA ranges at 150–450 g, with the daily intake of Brassicaceae vegetables estimated to be approximately 85.0 g/day, indicating them as a major diet constituent (14). In addition, consumption of cruciferous vegetables provides 20–120 mg of indole-3-carbinol (I3C), which is produced via bacterial myrosinase activity or glucobrassicin hydrolysis (15). I3C demonstrates anticancer, antioxidant, antiobesity (16, 17), antiatherosclerotic (18) and antiinflammatory effects (19). Accumulating data have indicated that I3C exerts antiobesity effects by downregulating the expression of genes related to adipogenesis and lipogenesis in cells and obese animal models (17, 20?–22). In particular, I3C has received considerable attention owing to its beneficial effects against various types of cancer by inducing autophagy and inhibiting cell proliferation via the mTOR pathway (23, 24). Nevertheless, the protective effects of I3C against the development of hyperleptinemia induced by a western diet (WD) in apolipoprotein E-deficient (apoE^-/-^) mice are poorly understood. Therefore, we examined whether I3C supplementation ameliorates hyperleptinemia-induced ER stress. Our results revealed that I3C mitigates hyperleptinemia-induced ER stress markers, accompanied by a reduction in adiposity and leptin resistance in apolipoprotein E-deficient (apoE^-/-^) mice fed a WD.

Materials and methods

Materials

I3C was purchased by Sigma-Aldrich (St. Louis, MO, USA). I3C is a naturally occurring compound in cruciferous vegetables, produced by myrosinase during food preparation or digestion (13, Fig. 1). Notably, I3C is highly unstable and undergoes self-condensation, with its primary derivative, 3,3′-diindolymethane (DIM), exhibiting enhanced stability and bioavailability (25). Although I3C and DIM have demonstrated various beneficial effects in metabolic diseases, they have also been associated with side effects such as liver toxicity, immunotoxicity, reduced vitamin A levels, and telomerase inhibition (26). Most of these toxic effects occur at high doses rather than at at therapeutic doses.

Biosynthesis of I3C. Glucobrassicin from cruciferous vegetables is hydrolyzed by myrosinase to form isothiocyante, which decomposes to 3-methylene-3H-indolium under neutral pH. Under acidic conditions, such as in the stomach, it is converted to I3C, which then undergoes condensation reactions.

Animals and diets

The animal study was conducted in a blinded manner, approved by the Experimental Ethics Committee, and performed in accordance with the ARRIVE guidelines. Six-week-old male apoE^-/-^ mice (18–23 g) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and were acclimated for 1 week in a temperature- and humidity-controlled room. Twenty apoE^-/-^ mice were divided into two main diet groups: 1) WD (60% kcal from fat, Research Diet, New Brunswick, NJ, USA) and 2) WD supplemented with 0.05% I3C, based on a single dose of 400–1,200 mg (27, Table S1). Body weight and food intake were monitored weekly, and every alternate day thereafter. At the end of the experiment, the mice were fasted for 18 h and euthanized by CO_2_ asphyxiation. Blood was collected from the abdominal aorta and centrifuged at 1,200 × g for 10 min at 4°C. The aorta and liver were resected and stored at −80°C. The aortic arch was immersed in 10% formalin for pathophysiological observation.

Measurement of biochemical parameters

Plasma glucose (AM201), total cholesterol (TC; AM202-K), and triglyceride (TG; AM157S-K) levels were measured using kits from Asan Pharm Co., Ltd. (Seoul, South Korea). CETP activity (MAK106-1KT) was determined using a kit from Sigma-Aldrich (St. Louis, MO, USA). IL-6 (04-BI-IL6), TNF-α (ab46105), and leptin (22-LEPMS-E01) levels were measured using commercial ELISA kits from Abcam (Cambridge, UK) and ALPCO Diagnostics (Salem, NH, USA). Plasma thiobarbituric acid reactive substance (TBARS) levels were measured using a previously described method (28), with minor modifications.

Quantitative reverse transcription-PCR

Total hepatic RNA was extracted using the Total RNA Isolation Kit (RC101; Vazyme Biotech Co., Ltd., Ladkrabang, Bangkok, Thailand). Real-time PCR (Polymerase Chain Reaction) was performed as previously described (29), using the primers listed in Table S2. Primers were designed and supplied by BIONEER (Daejeon, Korea). Data were normalized to β-actin levels.

Western blot analysis

Western blotting was conducted as previously described (30). Protein concentration was measured using a BCA kit (Pierce, Rockford, IL, USA). Subsequently, equal amounts of protein (20 µg) were subjected to 4–20% SDS-PAGE and transferred onto 0.2-µm PVDF (Polyvinylidene fluoride) membranes. The membranes were incubated with primary antibodies against p-eIF2α (#3398), eIF2α (#2103), XBP1 (#27901), CHOP (#2895) (1:1,000; Cell Signaling Technology; Danvers, MA, USA), GRP78 (ab21685, Abcam Inc., Waltham, MA, USA) and β-actin (A5441, 1:10,000; Sigma-Aldrich). The membranes were washed three times, incubated with secondary anti-mouse or anti-rabbit IgG antibodies, and visualized using enhanced chemiluminescence (SYNGENE, Frederick, MD, USA). Band densities were quantified using the ImageJ software. Data were normalized to β-actin levels.

Histology and immunohistochemistry

The aortic arch was fixed with 10% formalin for hematoxylin and eosin (H&E) staining and GRP78 immunohistochemistry, and stained according to a previously published method (31). For immunohistochemistry, paraffin sections (4 µm) of formalin-fixed aorta tissues were deparaffinized with xylene, and antigens were unmasked using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a pressure cooker (high power) for 10 min, and then cooled down to 25°C. After rinsing once with PBS, the sections were treated with 1% H_2_O_2_ for 10 min and blocked with donkey serum, followed by incubation with avidin D and biotin-blocking solution (Vector Laboratories, Burlingame, CA, USA). The sections were incubated with mouse anti-GRP 78 (ab21685, Abcam Inc.) at 4°C overnight. The slides were rinsed with PBS, incubated with mouse biotinylated IgG, and subsequently treated with an avidin-biotin complex (ABC kit, Vector Laboratories). Immunoreactivity was detected by incubation with 3,3′-diaminobenzidine (DAB kit, Vector Laboratories). Sections incubated with 10% nonimmune mouse serum instead of primary antiserum were used as negative controls.

Statistical analysis

Data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 10.0 software (La Jolla, CA, USA). mRNA and protein levels are presented relative to those in WD mice. Data were analyzed using the t-test, and statistical significance was set at P < 0.05.

Results

Effects of I3C on biochemical parameters

Table 1 shows the biochemical parameters of apoE^-/-^ mice fed a WD supplemented with 0.05% I3C for 12 weeks. We did not detect any changes in body weight gain, food intake, food efficacy ratio, and liver weight between the WD- and I3C-supplemented groups after 12 weeks (Table 1). I3C supplementation (0.05%) significantly reduced abdominal, epididymal, and brown fat mass by 18, 16, and 27%, respectively, compared with those in the WD group (P < 0.05). Plasma leptin levels were markedly decreased by 47% in the I3C-supplemented group compared with those in the WD group (P < 0.01), concurrent with reduced fat weight in I3C-supplemented apoE^-/-^ mice. These results suggested that I3C regulates WD-induced leptin signaling in apoE^-/-^ mice. Although I3C supplementation significantly attenuated the increase in fat weight, we did not find any significant differences in the plasma levels of TC (Total cholesterol), TG (Triglyceride), CETP (Cholesteryl ester transfer protein), fasting glucose, and cytokines between the two groups. The increase in plasma TBARS levels in the WD group was significantly reduced by 39% in the I3C-supplemented group (P < 0.05), indicating that I3C may exert its antioxidant activity against oxidative- or ER stress under hypercholesterolemic conditions.

Effects of I3C on hepatic mRNA expression levels

I3C supplementation (0.05%) resulted in a significant increase in the mRNA expression of ABCA1 and PPAR-α by 4.8- and 1.6-fold, respectively, compared with that in the WD group. However, we did not observe any changes in the mRNA expression levels of PPAR-γ, SR-B1, and SREBP-1c following I3C supplementation (Fig. 2). These results suggested that I3C partially affects cholesterol efflux and fatty acid β-oxidation in the liver of WD-fed apoE^-/-^ mice.

*Effects of I3C on hepatic mRNA expression levels in ApoE-/- mice fed a WD. Results are presented as the mean ± standard error (n = 5 per group). *P < 0.05, *P < 0.01 versus WD.

Effects of I3C on hepatic ER stress markers

I3C supplementation led to a significant reduction in the WD-induced increase in protein expression of the ER stress-related markers, p-eIF2α, XBP-1, CHOP, and GRP78 in the liver of apoE^-/-^ mice by 40, 20, 50, and 58%, respectively (Fig. 3, P < 0.05). This reduction was accompanied by a decrease in fat weight, indicating that hyperleptinemia caused by an increase in fat evoked ER stress in the liver of WD-fed apoE^-/-^ mice; conversely, 0.05% I3C supplementation attenuated these changes.

*Effects of I3C on endoplasmic reticulum stress in the liver of ApoE-/- mice fed a WD. Representative protein levels of p-eIF2α, eIF2α, XBP1, CHOP, and GRP78 as measured by western blotting. Results are presented as the mean ± standard error (n = 4–5 per group). *P < 0.05, *P < 0.01 versus WD. p-eIF2α, phospho-eukaryotic initiation factor 2 subunit alpha; XBP1, X-box binding protein 1; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein.

Effects of I3C on aortic histopathology and immunohistochemistry

Images of the aortic roots from the WD group revealed marked plaques and cell proliferation, which were reduced by 0.05% I3C supplementation (Fig. 4a, b). GRP78 was noticeably expressed in the aortic root in the WD group (Fig. 4c, d). However, we observed a significant reduction in GRP78 localization in the aortic arch of mice in the I3C-supplemented group compared with that in WD-fed apoE^-/-^ mice, indicating that hypercholesterolemia-induced atherosclerotic lesions were accompanied by GRP78 overexpression, which was reversed by I3C treatment.

Effects of I3C on histology and immunohistochemistry in the aortic root of ApoE-/- mice fed a WD. Representative hematoxylin & eosin (a, b) staining (scale bar, 20 µm) and GRP78 immunostaining (c, d) of aortic root sections from ApoE-/- mice fed a WD. Original magnification: 200 × I indicates intima; M, media; and L, lumen.

Discussion

This study demonstrated that I3C supplementation partially improved metabolic abnormalities and suppressed atherosclerotic ER stress in WD-fed apoE^-/-^ mice. In particular, I3C supplementation reversed the increase in fat weight and hyperleptinemia as well as the WD-induced activation of ER stress in the liver and aorta of apoE^-/-^ mice.

Leptin is a hormone derived from adipocytes, and is directly involved in energy homeostasis (32). ER stress is closely associated with leptin resistance, and activation of the UPR results in disruption of the leptin signaling pathway (11, 33). In this study, 0.05% I3C supplementation restrained hyperleptinemia and increased adipose tissue weight without altering serum lipid and glucose levels, indicating that I3C may mitigate hyperleptinemia by reducing adipose tissue weight and hepatic ER stress markers in WD-fed apoE^-/-^ mice. In addition, 3,3′-diindolylmethane (DIM), a derivative of I3C, improved the dysregulation of glucose metabolism and enhanced insulin sensitivity by upregulating glucose uptake but not I3C (34), suggesting that I3C does not affect glucose metabolism or the insulin signaling pathway in diet-induced obese mice. DIM has demonstrated better biological effects than I3C (35). Sulforaphane, an isothiocyanate from cruciferous vegetables, was demonstrated to ameliorate increased weight gain and visceral adiposity, as well as leptin-induced vascular cell proliferation in diet-induced obese animal models (36). Celastrol, a leptin sensitizer and powerful antiobesity agent, not only increased leptin sensitivity by activating leptin receptor-STAT3 pathway but also reduced ER stress by reducing PERK (Protein kinase R-like ER kinase) phosphorylation in the hypothalamus of diet-induced obese mice (12). Therefore, I3C may modulate the leptin signaling pathway under hyperleptinemic conditions induced by a high-fat diet in this study, and the underlying molecular mechanism need to be clarified in future studies.

The ER plays a pivotal role in lipid synthesis, assembly, and droplet formation; accordingly, ER dysfunction due to the accumulation of excess lipids causes ER stress (37?–39). FC buildup in macrophages within atherosclerotic lesions induces ER stress-CHOP-mediated apoptosis (40). Oxidized lipids such as 7-ketocholesterol and 4-hydroxynonenal act as ER stress inducers and activate the IRE1α-eIF2α pathway, which can be restored by the action of ER stress inhibitors or antioxidants (5, 41). In this study, WD-induced hypercholesterolemia evoked oxidative damage in plasma and the UPR response in the liver and aorta of apoE^-/-^ mice, which was reversed by I3C supplementation. These results are in agreement with studies indicating that oxidized LDL or its byproducts causes apoptosis of vascular and macrophage cells through the induction of the ER stress-mediated CHOP pathway in the atherosclerotic lesions of apoE^-/-^ mice (3, 5, 41, 42). In addition, these results indicated that dietary supplementation with I3C can ameliorate leptin resistance, which is accompanied by a reduction in the levels of hepatic and aortic ER stress markers in WD-fed apoE^-/-^ mice, implying that I3C may serve as a potent phytochemical agent for preventing diet-induced hyperleptinemia and its complications by reducing ER stress. However, further research is needed to elucidate the effect of I3C in ER stress-mediated leptin signaling pathway. Interestingly, leptin-induced GRP78 expression in neuronal cells, which is dependent on the PI3K-mTOR pathway, may contribute to protection against ER stress (43). Thus, oxidized lipoproteins or their byproducts induce ER stress and upregulate the expression of UPR markers, such as GRP78, in the atherosclerotic lesions of apoE^-/-^ mice and in patients with atherosclerosis and comorbid metabolic syndrome (3, 44).

Several studies have attempted to clarify the preventive roles of I3C on diet-induced metabolic dysfunction. I3C upregulated the expression of sirtuin (SIRT)1, PPAR-α, and PPAR-γ, which participate in fat tissue thermogenesis, lipid accumulation, and inflammation (16, 17) and downregulated the ER stress pathway (20). In addition, I3C supplementation (40 mg/kg BW) attenuated oxidative stress, inflammatory response, and apoptosis via the gut-liver-adipose tissue axis in alcoholic fatty liver (45). These results indicated that the preventive effect of I3C against obesity is mediated, at least partially, via the upregulation of the SIRT-AMP-activated protein kinase (AMPK) signaling pathway. Furthermore, I3C may prevent atherosclerotic lesion formation by remodeling the gut bacterial composition and reducing lipid accumulation; however, I3C (15 mg/kg) only modestly affected the serum lipid profile in high-choline-fed apoE^-/-^ mice (15, 46).

Several studies have demonstrated that I3C supplementation protects against hepatic steatosis by stimulating important hepatic signaling systems in diet-induced obese mice. These findings are in agreement with those of other studies that have described a positive correlation between ER stress and lipogenesis. The reduction in lipid accumulation in HepG2 cells following I3C treatment was associated with the downregulation of lipid synthesis and its downstream genes, FASN (Fatty acid synthase) and ACAT2 (Acetyl-CoA acetyltransferase 2), representing one possible mechanism for reducing apoB1 production and secretion, suggesting that I3C directly affects the SREBP-1c pathway (22). In our study, I3C significantly increased hepatic ABCA1 and PPARα mRNA levels, implying that cholesterol efflux and fatty acid β-oxidation was associated with hepatic ER stress dysfunction. ER stress induced by excess fat leads to dysregulation of lipid metabolism via the lipogenesis pathway and promotes hepatic steatosis progression through inflammatory pathways (20). Our findings suggested that the I3C-mediated reduction in hyperleptinemia may directly be explained by the observed reduction in adipose tissue mass and inhibition of ER stress markers. In addition, a lack of hepatic XBP-1 and eIF2α has been associated with fatty liver improvement in obese animals (47, 48). Overall, our results revealed that a reduction in the levels of hepatic ER stress markers through I3C supplementation partially improved hepatic lipid efflux and catabolism by upregulating ABCA1 and PPAR-α mRNA levels, despite not altering plasma lipid concentration.

I3C readily converts to DIM under acidic conditions in the stomach. Although the WD of apoE^-/-^ mice was supplemented with 0.05% I3C in this study, DIM has been investigated to play a more potent role than I3C in high fat diet-induced obese animals. Therefore, we aim to investigate the metabolic effects of I3C compared with those of DIM in the adipose tissue of this model in an upcoming study. This study suggested that the increase in FC or oxidative products caused by hypercholesterolemia or hyperleptinemia triggers ER stress, adiposity, and leptin resistance, all of which were reversed by I3C supplementation in WD-fed apoE^-/-^ mice (Fig. 5). Overall, our results revealed that controlling hyperleptinemia-induced ER stress may offer a potent strategy against atherosclerosis and its complications.

A proposed mechanism of I3C supplementation in ApoE-/- mice fed a WD. Accumulation of free cholesterol or ox-LDL in endothelial cells due to hypercholesterolemia or hyperleptinemia triggers ER stress-mediated apoptosis in endothelial or liver cells, all of which were reversed by I3C supplementation in ApoE-/- mice. The figure was created in BioRender.com and modified by Geng et al. (49).