(-)-Epicatechin mitigates high-fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress

Abstract

We investigated the capacity of dietary (-)-epicatechin (EC) to mitigate insulin resistance through the modulation of redox-regulated mechanisms in a rat model of metabolic syndrome. Adolescent rats were fed a regular chow diet without or with high fructose (HFr; 10% w/v) in drinking water for 8 weeks, and a group of HFr-fed rats was supplemented with EC in the diet. HFr-fed rats developed insulin resistance, which was mitigated by EC supplementation. Accordingly, the activation of components of the insulin signaling cascade (insulin receptor, IRS1, Akt, and ERK1/2) was impaired, whereas negative regulators (PKC, IKK, JNK, and PTP1B) were upregulated in the liver and adipose tissue of HFr rats. These alterations were partially or totally prevented by EC supplementation. In addition, EC inhibited events that contribute to insulin resistance: HFr-associated increased expression and activity of NADPH oxidase, activation of redox-sensitive signals, expression of NF-κB-regulated proinflammatory cytokines and chemokines, and some sub-arms of endoplasmic reticulum stress signaling. Collectively, these findings indicate that EC supplementation can mitigate HFr-induced insulin resistance and are relevant for defining interventions that can prevent/mitigate MetS-associated insulin resistance.

Keywords: Endoplasmic reticulum stress; Epicatechin and flavonoids; Free radicals; Insulin resistance; Metabolic syndrome; NADPH oxidase; Redox signaling.

Figure 1. Effects of EC supplementation on metabolic parameters in HFr-fed rats

A- (-)-Epicatechin chemical structure, B- ITT, C-GTT, D- Area under the curve from ITT and GTT performed on week 7 on the respective treatments, and E-plasma insulin concentration during GTT in rats fed control diet and regular drinking water (empty circles and empty bars), a control diet and drinking water supplemented with 10% (w/v) fructose (black circles and black bars), or a diet supplemented with 20 mg EC/kg body weight and fructose-containing drinking water (grey circles and grey bars). Results are shown as means ± SE and are the average of 8 animals/group. D- Values having different symbols (*,#) are significantly different; E- *are significantly different from the other groups at the corresponding time points; (p < 0.05, one way ANOVA).

Figure 2. EC supplementation enhances insulin signaling in epididymal adipose and liver tissues in HFr-fed rats

After 8 weeks on the corresponding diets, rats were fasted overnight then injected with saline or insulin (10 mU/g body weight) and then sacrificed after 10 minutes. Phosphorylation of IR, IRS1, ERK1/2, and Akt are shown for A- epididymal adipose tissue and B-liver. Bands were quantified and results for the HFr and HFr + EC were referred to control group values (C). Results are expressed as the ratio of phosphorylated/total protein level. Results are shown as mean ± SEM of 5 animals/treatment. *, # are significantly different between them and from the insulin untreated groups, ^&are significantly different from the insulin untreated control and HFr groups, and ** are significantly different from all other groups (p<0.05, one way ANOVA test).

Figure 3. Effects of EC supplementation on epididymal adipose and liver tissue insulin signaling in HFr-fed rats: inhibitory signaling

A,B- Phosphorylation of IKKα/β (Ser178/180, JNK (Thr183, Tyr185) and PKCδ (Thr505), and PTP-1B protein levels in epididymal adipose tissue (A) and liver (B) after 8 weeks on the corresponding diets. Bands were quantified and results for the HFr (HF) and HFr + EC (EC) were referred to control group values (C). Results are expressed referred to either total protein or β-tubulin levels. Results are shown as mean ± SEM of 8 animals/treatment. *, # are significantly different from all other groups (p<0.05, one way ANOVA test);

Figure 4. Effects of EC supplementation on epididymal adipose and liver tissue NOX upregulation

A,B: Protein levels of NOX subunits (NOX2, NOX4, p47) were measured by Western blot in A- adipose tissue and B- liver, and bands were quantified. B- NOX activity was measured in liver as described in methods. Results for the HFr (HF) and HFr + EC (EC) were referred to control group values (C). C- NOX2 and NOX4 mRNA were measured by quantitative real-time PCR, normalized against TATA-Box binding protein (TBP) and were referred to control group values (C). D- Protein carbonyls were measured as described in methods. Results are shown as mean ± SEM of 4-8 animals/treatment. *Significantly different from other groups (p<0.05, one way ANOVA test).

Figure 5. Effects of EC supplementation on epididymal adipose and liver tissue activation of the pro-inflammatory NF-κB signaling pathway

Different steps in the NF-κB pathway were evaluated in rat epididymal adipose tissue and liver after 8 weeks on the corresponding diets, measuring: A- phosphorylation (Ser32) and total levels of IκBα, and phosphorylation of p65 (Ser536); B- TNFα and MCP-1 (NF-κB target genes) protein levels. Bands were quantified and results for the HFr (HF) and HFr + EC (EC) were referred to control group values (C). Results are shown as mean ± SEM of 5 animals/treatment. *,^# are significantly different from the untreated controls, and are significantly different among them. (p<0.05, one way ANOVA test).

Figure 6. Effects of EC supplementation on parameters of ER stress in epididymal adipose tissue and liver

The three branches of the UPR response were evaluated by Western blot measuring PERK (Tyr980), eIF2α (Ser51), and IRE1α (Ser724) phosphorylation, sXBP-1 and cleaved ATF6 (cATF6) in A- adipose tissue and B- liver. Bands were quantified and results for the HFr (HF) and HFr + EC (EC) were referred to control group values (C). Phosphorylated/total ratios were calculated for PERK, eIFα and IRE1α; sXBP-1 and cATF6 were normalized to tubulin content. Results are shown as mean ± SEM of 5-8 animals/treatment. *Significantly different from other groups (p<0.05, one way ANOVA test).