Antidiabetic effects of chamomile flowers extract in obese mice through transcriptional stimulation of nutrient sensors of the peroxisome proliferator-activated receptor (PPAR) family

Abstract

Given the significant increases in the incidence of metabolic diseases, efficient strategies for preventing and treating of these common disorders are urgently needed. This includes the development of phytopharmaceutical products or functional foods to prevent or cure metabolic diseases. Plant extracts from edible biomaterial provide a potential resource of structurally diverse molecules that can synergistically interfere with complex disorders. In this study we describe the safe application of ethanolic chamomile (Matricaria recutita) flowers extract (CFE) for the treatment and prevention of type 2 diabetes and associated disorders. We show in vitro that this extract activates in particular nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) and its isotypes. In a cellular context, in human primary adipocytes CFE administration (300 µg/ml) led to specific expression of target genes of PPARγ, whereas in human hepatocytes CFE-induced we detected expression changes of genes that were regulated by PPARα. In vivo treatment of insulin-resistant high-fat diet (HFD)-fed C57BL/6 mice with CFE (200 mg/kg/d) for 6 weeks considerably reduced insulin resistance, glucose intolerance, plasma triacylglycerol, non-esterified fatty acids (NEFA) and LDL/VLDL cholesterol. Co-feeding of lean C57BL/6 mice a HFD with 200 mg/kg/d CFE for 20 weeks showed effective prevention of fatty liver formation and hepatic inflammation, indicating additionally hepatoprotective effects of the extract. Moreover, CFE treatment did not reveal side effects, which have otherwise been associated with strong synthetic PPAR-targeting molecules, such as weight gain, liver disorders, hemodilution or bone cell turnover. Taken together, modulation of PPARs and other factors by chamomile flowers extract has the potential to prevent or treat type 2 diabetes and related disorders.

Figure 1. Binding and transcriptional activation of PPARs by natural-products contained in camomile flowers extract (CFE).

(A, B) PPARγ bound and activated by CFE (µg/ml) or rosiglitazone (nM). (C, D) PPARα bound and activated by CFE (µg/ml) or GW7647 (nM). (E,F) PPARβ/δ bound by CFE (µg/ml) or GW0742 (nM). Binding of compounds was measured in a competitive time-resolved fluorescence resonance energy transfer assay. Transcriptional activation was determined in a reporter gene assay and is represented relative to the reference compound. Data are expressed as mean ± SD (n=3-4).

Figure 2. Gene expression profile of camomile flowers extract (CFE) in human adipocytes and hepatocytes.

(A) Human primary adipocytes were treated with either 10 µM RGZ, 300 µg/ml CFE or vehicle only for 24 h. Gene expression was analyzed by qPCR. (B) Human primary adipocytes were transfected with PPARγ siRNA (hatched bars) or control siRNA (unhatched bars) and were treated with either 10 µM RGZ, 300 µg/ml CFE or vehicle only for 24 h. (C) Human HepG2 hepatocytes were treated with either 10 µM GW7647, 300 µg/ml CFE or vehicle only for 24 h and gene expression was analyzed by qPCR. (D) Human HepG2 hepatocytes were transfected with PPARα siRNA (hatched bars) or control siRNA (unhatched bars) and were treated with either 10 µM GW7647, 300 µg/ml CFE or vehicle only for 24 h. Data are expressed as mean ± SEM (n=2-4/group). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. vehicle. RGZ, rosiglitazone ; CFE, camomile flowers extract; GW, GW7647.

Figure 3. Antidiabetic effects of camomile flowers extract in insulin-resistent DIO mice.

(A) Fasting blood glucose of untreated HFD-fed mice or mice treated for 2 weeks with RGZ or CFE. (B) Fasting plasma insulin after 2 weeks of treatment. (C) Effect of treatment for 2 weeks on insulin resistance determined by homeostatic model assessment of insulin resistance (HOMA-IR). (D, E) Glucose and insulin concentrations during oral glucose tolerance test (OGTT) after 2 weeks of treatment with vehicle, RGZ or CFE. AUC, area under the curve. Data are expressed as mean ± SEM. *p≤0.05, **p≤0.01 vs. vehicle-treated HFD-fed mice. HFD, high-fat diet; VEH, vehicle (n=13-14); RGZ, rosiglitazone (n=8-14) ; CFE, camomile flowers extract (n=12-14).

Figure 4. Camomile flowers extract improves dyslipidemia in obese DIO mice.

(A) Fasting plasma NEFA after 6 weeks of treatment. (B) Fasting plasma triacylglycerol after 6 weeks of treatment. (C) Fasting plasma total, HDL and LDL/VLDL cholesterol in DIO mice after 6 weeks of treatment. Data are expressed as mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, n.s. not significant vs. vehicle-treated HFD-fed mice. LFD, low-fat diet; HFD, high-fat diet; VEH, vehicle (n=13-14); RGZ, rosiglitazone (n=8-14) ; CFE, camomile flowers extract (n=12-14).

Figure 5. Prevention of insulin resistance and dyslipidemia by camomile flowers extract during HFD-feeding of healthy C57BL/6 mice.

(A) Fasting plasma glucose of LFD-fed, untreated HFD-fed or HFD-fed mice treated for 20 weeks with CFE (n=14/group). (B) Fasting plasma insulin after 20 weeks of intervention (LFD n=7, HFD n=14, HFD+CFE n=14). (C) Effect of 20 weeks preventive feeding on homeostatic model assessment of insulin resistance (HOMA-IR) index (LFD n=7, HFD n=13, HFD+CFE n=12). AUC, area under the curve. (D) Fasting plasma NEFA after 20 weeks of treatment (LFD n=8, HFD n=10, HFD+CFE n=9). (E) Fasting plasma triacylglycerol after 20 weeks of treatment (LFD n=8, HFD n=14, HFD+CFE n=14). Data are expressed as mean ± SEM. n.s. not significant, *p≤0.05, **p≤0.01 vs. untreated HFD-fed mice. LFD, low-fat diet; HFD, high-fat diet; CFE, camomile flowers extract.

Figure 6. Prevention of non-alcoholic fatty liver disease (NAFLD) by camomile flowers extract during HFD-feeding of healthy C57BL/6 mice.

(A) Plasma levels of liver alanine transaminase (ALT) after 20 weeks of treatment (n=12/group). (B) Effect treatment over 20 weeks on liver morphology. (C, D) Liver concentrations of triacylglycerol and NEFA after 20 weeks of treatment (n=10-12/group). (E) TNFα protein concentrations in liver (n=9/group). (F) Liver qPCR analysis of genes involved in lipid metabolism (n=4-6 pools, 2 mice/pool). (G) Liver qPCR analysis of genes involved in inflammation and macrophage infiltration (n=4-6 pools, 2 mice/pool). Data are expressed as mean ± SEM. n.s. not significant, *p≤0.05, **p≤0.01 vs. untreated HFD-fed mice. LFD, low-fat diet; HFD, high-fat diet; CFE, camomile flowers extract.

Figure 7. Camomile flowers extract (CFE) does not induce adverse effects commonly linked with PPAR agonists.

(A) Effect of CFE on cellular viability in human HepG2 cells after treatment for 24 h. Data are expressed as mean ± SD (n=3/group). (B, C) Mouse body weight during treatment of DIO mice for 6 weeks with CFE or HFD alone (B) and during the preventive study by 20 weeks feeding of healthy C57BL/6 mice with LFD, HFD alone or HFD with CFE (C). Data are expressed as mean ± SEM (n=14/group). Data are shown as mean ± SEM. (D, E) Food intake during treatment of DIO mice for 6 weeks with CFE or HFD alone (D) and during the preventive study by 20 weeks feeding of healthy C57BL/6 mice with LFD, HFD alone or HFD with CFE (E). Data are expressed as mean ± SEM (n=14/group). (F) Hematocrit of treated DIO mice after 6 weeks (mean ± SEM, n=14/group). (G) Effect of CFE on plasma osteocalcin levels after treatment of DIO mice for 6 weeks (mean ± SEM, n=14/group). *p≤0.05, n.s. not significant vs. untreated HFD-fed mice. LFD, low-fat diet; HFD, high-fat diet; CFE, camomile flowers extract.

Figure 8. Concept of PPAR polypharmacology.

Ethanolic extracts of chamomile flowers represent a complex mixture of plenty diverse compounds, particularly small molecules. In dependance of their bioavailability, a set of small molecules bind to peroxisome proliferator-activated receptors (PPARs) and potentially other yet unidentified targets. Ligand-binding then induces conformational changes in PPARs that lead to their transcriptional activation/modulation and thus to beneficial regulation of glucose and lipid metabolism. In summary, the polypharmacological mechanism is driven by the composition of structurally diverse small molecules in chamomile flowers extracts and by the large binding pocket of PPARs leading to promiscuous ligand-binding properties. The proportion of activation between the different PPAR subtypes is determined by the cellular context (e.g. cell type) and by the definite chemical composition of the plant extract.