Anti-diabetic and Anti-obesity effects of flavonoids in Premature citrus extract

Abstract

Abstract 1: Flavonoids are natural polyphenols widely found in citrus fruits and peel and known to possess anti-adipogenic effects. However, the detailed mechanisms for their anti-adipogenic effects remain to be fully elucidated. In the present study, we observed the anti-adipogenic effects of five major citrus flavonoids including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN) associated with AMPK activation in palmitate (PA)-treated HepG2 cells. Intracellular lipid accumulation and triglyceride (TG) content were quantified using Oil-Red O staining and a TG assay, respectively. Glucose uptake was assessed using a 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The phosphorylation levels of AMPK, ACC, and GSK3β, as well as the expression levels of SREBP-2 and HMGCR were analyzed using Western blot analysis. The potential interactions between the flavonoids and the γ-subunit of AMPK were investigated using molecular docking. Flavonoid treatment significantly reduced intracellular lipid accumulation and TG content, increased glucose uptake in an insulin-independent manner, increased the phosphorylation levels of AMPK, ACC, and GSK3β, and decreased the expression levels of SREBP-2 and HMGCR in PA-treated HepG2 cells. Molecular docking analysis revealed that the flavonoids bind to CBS domains of the regulatory γ-subunit of AMPK with high binding affinities, and may act as potential AMPK activators. Overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK.

Abstract 2: The present study examined the relationship between the anti-diabetic effect of hesperidin (HES) and differential gene expression in HES treated high fat diet (HFD)-induced obese mice. Based on a 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) uptake assay, treatment with HES restored glucose uptake to control levels in an insulin-independent manner in palmitate (PA)-treated HepG2 cells. Western blot analysis confirmed that HES increased the insulin-stimulated phosphorylation of Akt and GSK3β in insulin-resistant PA-treated HepG2 cells. High fat diet (HFD)-induced obese mice treated with HES showed reduced serum insulin, blood glucose, and homeostatic model assessment for insulin resistance (HOMA-IR) values. In addition, both glucose tolerance and insulin tolerance were significantly improved to normal level by HES in HFD-induced obese mice. RNA sequencing disclosed that the expression levels of twelve up-regulated and six down-regulated genes related to insulin signaling and glucose metabolism were restored to normal level by HES in the liver of HFD-induced obese mice. A protein-protein interaction (PPI) network was constructed via search tool for the retrieval of interacting genes/proteins (STRING) analysis, and Eno1, Pik3cd, Hk2, Trib3, Myc, Nos3, Ppargc1a, and Igf2 were located in the functional hubs of the PPI network of glucose metabolism. Furthermore, Western blot analysis confirmed that HES improved insulin sensitivity and glucose homeostasis by normalizing the expression levels of hexokinase-II, enolase-1, and PI3 kinase p110δ to normal level. Overall, these results suggest that HES exerts a potential anti-diabetic effect by normalizing the expression levels of genes related to the insulin signaling and glucose metabolism, which were perturbed in the liver of HFD-induced obese mice.

Abstract 3: The present study was conducted to examine the effect of premature citrus extract (PCE) on the serum parameters of beagle dogs. The body weight did not significantly differ between the groups supplemented with PCE and control group throughout the experiment. The groups supplemented with PCE showed significantly decreased serum triglyceride levels compared with the control group at week 12. Serum cholesterol analysis confirmed that the PCE-supplemented group had significantly decreased serum levels of low-density lipoprotein-cholesterol and increased serum levels of the beneficial high-density lipoprotein-cholesterol compared to those in the control group at week 12. In addition, the group supplemented with PCE for 12 weeks showed significantly decreased serum glucose levels compared with those in the control group at week 0. Furthermore, serological tests revealed that PCE did not alter functional parameters of liver, kidney, and pancreas in the serum of beagle dogs over 12 weeks. These results suggest that supplementation of PCE in the diet decreased triglyceride, low-density lipoprotein-cholesterol, and glucose levels and increased the high-density lipoprotein-cholesterol level without affecting the functional parameters of liver, kidney, and pancreas in the serum of beagle dogs.