Insights into the molecular mechanisms of the anti-atherogenic actions of flavonoids in normal and obese mice

Abstract

Obesity is a major and independent risk factor for cardiovascular disease and it is strongly associated with the development of dyslipidemia, insulin resistance and type 2 diabetes. Flavonoids, a diverse group of polyphenol compounds of plant origin widely distributed in human diet, have been reported to have numerous health benefits, although the mechanisms underlying these effects have remained obscure. We analyzed the effects of chronic dietary supplementation with flavonoids extracted from cranberry (FLS) in normal and obese C57/BL6 mice compared to mice maintained on the same diets lacking FLS. Obese mice supplemented with flavonoids showed an amelioration of insulin resistance and plasma lipid profile, and a reduction of visceral fat mass. We provide evidence that the adiponectin-AMPK pathway is the main mediator of the improvement of these metabolic disorders. In contrast, the reduced plasma atherogenic cholesterol observed in normal mice under FLS seems to be due to a downregulation of the hepatic cholesterol synthesis pathway. Overall, we demonstrate for the first time that the molecular mechanisms underlying the beneficial effects of flavonoids are determined by the metabolic state.

Figure 1. Body weight changes and Glucose Tolerance Test (GTT) in mice on different dietary regimens.

A, mouse body weight was measured weekly from 6 to 24 weeks of age. n = 12 mice in each group at 6–14 wks of age; n = 8 mice in each group at 14–24 wks of age. B, GTT in mice at 24 wks of age. Glucose (1.5 g/ kg body weight) was injected intraperitoneally after overnight fasting (16 hours). Plasma glucose was measured using OneTouch Ultra Glucose Meter (LifeScan) at 0, 15, 30, 60, and 90 min after injection. Data are expressed as means±SE. ∧P = 0.06 HFDC vs. HFD, n = 6–8 mice per group.

Figure 2. FLS increases adiponectin levels and restores multimers' adiponectin profile altered by high-fat feeding.

A, quantitative analysis of adiponectin mRNA levels in visceral adipose tissue by real-time RT-PCR and adiponectin protein levels in serum by EIA or western blot (WB). B–D, representative western blots of serum samples. 0.5 µl (B) or 1 µl (C, D) of serum was subjected to 15% (B) or 8–15% (C, D) SDS-PAGE under reducing, heat-denaturing (B) or non-reducing, non-heat-denaturing (C, D) conditions at different voltage: 20–25 V (C), 40–45 V (D). Adiponectin was detected using antibodies specific for both globular (g) and full-length (f) adiponectin [(g+f), B–D)] or full length [(f), B)]. Albumin was detected by Ponceau staining (B) and used as a loading control for reducing conditions. E, quantitative analysis of adiponectin multimers in serum. Each serum sample was analyzed 2–4 times on different western blot membranes. HMW, high molecular weight; MMW, medium molecular weight; LMW, low molecular weight multimers. LFD group was used as a reference for quantification. Data are expressed as mean±SE. *P<0.05, **P<0.01, ^#P<0.001 LFDC vs. LFD or HFDC vs. HFD; n = 7−8 in each group. §C, adopted from (21).

Figure 3. FLS downregulates expression of genes involved in hepatic cholesterol synthesis and hepatic LDL uptake in non-obese mice in fasting state.

A, real-time RT-PCR analysis of genes involved in cholesterol synthesis and uptake in liver. B, representative western blot for low density lipoprotein receptor (LDLR). 70 µg of liver protein was subjected to 8% SDS-PAGE under reducing, heat-denaturing conditions. Tubulin was used as a loading control. Each sample was analyzed 2–4 times on different western blot membranes. LFD group was used as a reference for quantification. C, real-time RT-PCR analysis (mRNA) and quantification of western blot analysis (WB) for LDLR. Data are expressed as mean±SE. *P<0.05, **P<0.01, ^#P<0.001 LFDC vs. LFD or HFDC vs. HFD; n = 7−8 in each group. Sterol-regulatory element binding protein 2 (SREBP2); 3-hydroxy-3-methylglutaryl-Co-A reductase (HMG-R); 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-S); farnesyl diphosphate synthase (FDS); squalene synthase (SS).

Figure 4. FLS upregulates expression of LPL and genes involved in FA utilization in muscle in obese mice.

Real-time RT-PCR analysis of lipoprotein lipase (LPL) and very low density lipoprotein receptor (VLDLR) (A), genes involved in FA utilization in muscle: transcriptional regulation (B), free FA (FFA) uptake and activation (C), and FA oxidation (D). E, representative western blot for TIM23 (E). 50 µg of muscle protein was subjected to 15% SDS-PAGE under reducing, heat-denaturing conditions. Tubulin was used as a loading control. F, quantitative analysis of TIM23 in muscle. LFD group was used as a reference for quantification. Data are expressed as mean±SE. *P<0.05, **P<0.01, HFDC vs. HFD n = 8 in each group. Peroxisome proliferator-activated receptor α (PPARα); PPARγ coactivator-1α (PGC-1α); cluster of differentiation (CD36); fatty acid transport proteins 1 and 4 (FATP1 and FATP4); carnitine palmitoyltransferases 1β and 2(CPT1β and CPT2); uncoupling protein 2 (UCP2); acyl-coenzyme A oxidase (AOX), translocase of the inner mitochondrial membrane (TIM23).

Figure 5. FLS increases AMPK phosphorylation in liver in non-obese mice and in muscle in obese mice.

A and B, representative western blots of muscle (A) and liver (B) samples. 70 µg of total protein was subjected to 12.5% SDS-PAGE under reducing, heat-denaturing conditions. C, quantitative analysis of western blots, pAMPK/AMPK ratio. Samples were repeated on different western blot membranes. LFD group was used as a reference for quantification. Data are expressed as mean±SE. *P<0.05, **P<0.01, LFDC vs. LFD or HFDC vs. HFD; n = 3−6 (liver) and n = 8 (muscle) in each group.

Figure 6. FLS inhibits lipid accumulation in visceral adipose tissue in obese mice.

Real-time RT-PCR analysis of genes involved in lipid accumulation: lipolysis (LPL), FFA uptake (CD36) and lipoprotein uptake (LRP). LFD group was used as a reference for quantification. Data are expressed as mean±SE. *P<0.05, HFDC vs. HFD. Lipoprotein related protein (LRP).

Figure 7. Molecular mechanisms underlying the anti-atherogenic effects of flavonoids in normal and obese mice.

A, activation of AMPK pathway in muscle of HFDC mice. Adiponectin/AdipR1 signaling in muscle activates the AMPK pathway, resulting in elevated transcriptional activity of PGC-1α and PPARα, and consequently increased expression of their target genes. B, molecular mechanisms underlying the anti-atherogenic effects of flavonoids are different in normal and obese mice.