Dietary Eriodictyol Alleviates Adiposity, Hepatic Steatosis, Insulin Resistance, and Inflammation in Diet-Induced Obese Mice

The present study aimed to investigate the molecular mechanisms underlying the anti-obesity effect of flavonoid eriodictyol (ED) supplementation in mice fed with a high-fat diet (HFD). C57BL/6N mice were fed with normal diet (ND), HFD (40 kcal% fat), or HFD + 0.005% (w/w) ED for 16 weeks. In HFD-induced obese mice, dietary ED supplementation significantly alleviated dyslipidemia and adiposity by downregulating the expression of lipogenesis-related genes in white adipose tissue (WAT), while enhancing fecal lipid excretion. ED additionally improved hepatic steatosis and decreased the production of pro-inflammatory cytokines by downregulating the expression of hepatic enzymes and the genes involved in lipogenesis and upregulating the expression of hepatic fatty acid oxidation-related enzymes and genes. In addition, ED improved insulin resistance (IR) by suppressing hepatic gluconeogenesis, enhancing glucose utilization, and modulating the production and release of two incretin hormones, namely gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Taken together, the current findings indicated that ED can protect against diet-induced obesity and related metabolic disturbances, including dyslipidemia, inflammation, fatty liver disease, and IR in diet-induced obese mice.


Introduction
High-fat food products are highly consumed, particularly in the United States, leading to high rates of obesity, which represents one of the most severe burdens on healthcare systems. Obesity has been closely linked to metabolic syndrome and is often accompanied by increased adiposity, dyslipidemia, chronic inflammation, insulin resistance (IR), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) [1]. Although the mechanisms underlying abdominal obesity and metabolic syndrome are not fully understood, the dysregulation of lipid metabolism in liver and white adipose tissue (WAT) is considered a significant factor that contributes to adiposity and obesity-related complications [2].

Eriodictyol Supplement Improved Plasma and Hepatic Lipid Levels, and Modulated the Expression of Hepatic Lipid-Regulating Enzymes and Genes
The results revealed that ED supplementation in HFD-fed mice significantly reduced plasma total-cholesterol (TC), triglyceride (TG), and free fatty acid (FFA) levels, as well as atherogenic index (AI) and apolipoprotein B (ApoB) levels. In addition, ED-supplemented HFD-fed mice showed markedly higher HDL-cholesterol-to-TC ratio (HTR) and ApoA-I/ApoB ratio ( Figure 2).

Eriodictyol Supplement Improved Plasma and Hepatic Lipid Levels, and Modulated the Expression of Hepatic Lipid-Regulating Enzymes and Genes
The results revealed that ED supplementation in HFD-fed mice significantly reduced plasma total-cholesterol (TC), triglyceride (TG), and free fatty acid (FFA) levels, as well as atherogenic index (AI) and apolipoprotein B (ApoB) levels. In addition, ED-supplemented HFD-fed mice showed markedly higher HDL-cholesterol-to-TC ratio (HTR) and ApoA-I/ApoB ratio ( Figure 2).

Eriodictyol Supplementation Ameliorated Insulin Resistance, Glucose Tolerance, and Adipokine/Cytokine Dysregulation
ED-supplemented mice showed significantly reduced plasma glucose and insulin levels relative to mice in the HFD group ( Figure 4A,B). Furthermore, ED-supplemented HFD mice showed improved insulin resistance based on the results of an intraperitoneal glucose tolerance test (IPGTT) and a homeostasis model assessment of insulin resistance (HOMA-IR) index relative to mice in the HFD group ( Figure 4C,D). Moreover, the ED supplementation significantly decreased the circulating levels of the gastric inhibitory polypeptide (GIP), which stimulates insulin secretion; however, ED supplementation increased the plasma levels of glucagon-like peptide-1 (GLP-1), which increases insulin sensitivity and activity ( Figure 4E). In addition, the expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) enzymes and their encoding genes (PCK and G6PC) were significantly down-regulated, whereas hepatic glucokinase (GK) activity and insulin receptor substrate 2 (IRS2) gene expression level was dramatically increased in the ED-supplemented group compared to those in the HFD group ( Figure 4F,G).

Eriodictyol Supplementation Ameliorated Insulin Resistance, Glucose Tolerance, and Adipokine/Cytokine Dysregulation
ED-supplemented mice showed significantly reduced plasma glucose and insulin levels relative to mice in the HFD group ( Figure 4A, B). Furthermore, ED-supplemented HFD mice showed improved insulin resistance based on the results of an intraperitoneal glucose tolerance test (IPGTT) and a homeostasis model assessment of insulin resistance (HOMA-IR) index relative to mice in the HFD group ( Figure 4C, D). Moreover, the ED supplementation significantly decreased the circulating levels of the gastric inhibitory polypeptide (GIP), which stimulates insulin secretion; however, ED supplementation increased the plasma levels of glucagon-like peptide-1 (GLP-1), which increases insulin sensitivity and activity ( Figure 4E). In addition, the expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) enzymes and their encoding genes (PCK and G6PC) were significantly down-regulated, whereas hepatic glucokinase (GK) activity and insulin receptor substrate 2 (IRS2) gene expression level was dramatically increased in the ED-supplemented group compared to those in the HFD group ( Figure  4F, G).

Discussion
HFD is known to cause obesity, which is linked with a higher risk of metabolic complications, such as dyslipidemia, adiposity, hepatic steatosis, chronic inflammation, and IR, through multiple mechanisms. In the present study, long-term ED supplementation significantly attenuated the increase in HFD-induced WAT weight without altering the food intake and body weight in HFD-fed mice. Similar results were reported in a previous study [9], in which mice fed with a high-fat and high-sucrose (HF/HS) with ED diet for 8 weeks showed no significant differences in body weight but showed a moderate reduction in the weights of perirenal and epididymal fat relative to the HF/HS group. Furthermore, ED supplementation significantly downregulated the expression of the adipocyte genes involved in FA uptake (CD36 and LPL) and lipogenesis (SREBP1, FAS, and SCD), which could be associated with reduced adiposity observed in ED-fed obese mice.
Multiple epidemiologic studies have investigated the effects of HFD on NAFLD and obesity [10,11]. Furthermore, the metabolic profiles of the majority of patients with obesity and metabolic syndrome (MS), which includes NAFLD and dyslipidemia, were found to be characterized by high TC, LDL-C, TG, and FFA, and low HDL-C levels [12]. In this respect, dyslipidemia is closely linked to obesity, NAFLD, and MS. NAFLD is characterized by increased circulating concentrations of FFAs, excess lipid accumulation in the liver, hepatocyte apoptosis, and progressive inflammation. Inflammation significantly contributes to fibrogenesis, leading to NASH, a severe form of NAFLD. In the present study, ED supplementation was found to reduce the plasma levels of TC, TG, and FFA and decrease the atherogenic index, indicating improved dyslipidemia. Notably, ED supplementation significantly increased fecal excretion of cholesterol and FA, which appeared to

Discussion
HFD is known to cause obesity, which is linked with a higher risk of metabolic complications, such as dyslipidemia, adiposity, hepatic steatosis, chronic inflammation, and IR, through multiple mechanisms. In the present study, long-term ED supplementation significantly attenuated the increase in HFD-induced WAT weight without altering the food intake and body weight in HFD-fed mice. Similar results were reported in a previous study [9], in which mice fed with a high-fat and high-sucrose (HF/HS) with ED diet for 8 weeks showed no significant differences in body weight but showed a moderate reduction in the weights of perirenal and epididymal fat relative to the HF/HS group. Furthermore, ED supplementation significantly downregulated the expression of the adipocyte genes involved in FA uptake (CD36 and LPL) and lipogenesis (SREBP1, FAS, and SCD), which could be associated with reduced adiposity observed in ED-fed obese mice.
Multiple epidemiologic studies have investigated the effects of HFD on NAFLD and obesity [10,11]. Furthermore, the metabolic profiles of the majority of patients with obesity and metabolic syndrome (MS), which includes NAFLD and dyslipidemia, were found to be characterized by high TC, LDL-C, TG, and FFA, and low HDL-C levels [12]. In this respect, dyslipidemia is closely linked to obesity, NAFLD, and MS. NAFLD is characterized by increased circulating concentrations of FFAs, excess lipid accumulation in the liver, hepatocyte apoptosis, and progressive inflammation. Inflammation significantly contributes to fibrogenesis, leading to NASH, a severe form of NAFLD. In the present study, ED supplementation was found to reduce the plasma levels of TC, TG, and FFA and decrease the atherogenic index, indicating improved dyslipidemia. Notably, ED supplementation significantly increased fecal excretion of cholesterol and FA, which appeared to promote the beneficial effects of ED on dyslipidemia by suppressing the absorption of exogenous fats. In addition to improving dyslipidemia, ED markedly reduced hepatic lipid droplet accumulation and lipid content (TG and FA). Higher FFA plasma levels were found to induce FFA accumulation in hepatocytes by increasing hepatic fatty acid uptake, which interferes with lipid metabolism and enhances lipogenesis, thereby leading to the progression of NAFLD and lipotoxic hepatocellular injury [13]. In the present study, ED not only significantly reduced hepatic lipogenesis by decreasing the activities of ME, FAS, and PAP and downregulating the expression of the SREBP1, ACC, and FAS genes, but also markedly increased the expression of enzymes (CPT and β-oxidation) and the PPARα gene, which regulates fatty acid oxidation, in the liver of HFD-fed mice. The above findings suggested that the coordinated action of increased fecal excretion of FA and cholesterol, attenuated dyslipidemia, decreased hepatic lipogenesis, and enhanced hepatic fatty acid oxidation by ED supplementation appeared to attenuate hepatic steatosis. Furthermore, ED decreased the levels of plasma pro-inflammatory cytokines, including PAI-1, IFN-γ, and IL-1β, and increased the anti-inflammatory cytokine IL-10 level in the plasma, suggesting that ED exerts a protective role against the development of NASH/NAFLD. NAFLD with adiposity and dyslipidemia are strongly associated with IR [14]. Moreover, IR is one of the multiple factors that influences disease progression from NAFLD to NASH [15]. In the present study, we also observed that ED decreased the plasma glucose and insulin levels, thereby improving IR based on IPGTT and the reduced HOMA-IR measurements. In addition, ED-supplemented HFD mice showed dramatically reduced circulating GIP levels and higher plasma levels of GLP-1 relative to the HFD mice. Between the two incretin hormones GIP and GLP-1, GIP is involved in lipid metabolism, and elevated plasma GIP level promotes hyperinsulinemia and obesity-induced IR development [16,17]. By contrast, GLP-1 exerts anti-inflammatory effects against NAFLD, and the increase in GLP-1 secretion ameliorates IR in HFD-induced obese mice [16]. In the fasting state, glucose production via gluconeogenesis is primarily regulated by the liver, and fasting hyperglycemia occurs as a result of dysregulated expression of hepatic gluconeogenic enzymes and genes, such as PEPCK and G6Pase [18]. Thus, the current findings indicated that the suppression of gluconeogenesis via downregulation of the expression of hepatic PEPCK and G6Pase enzymes and their genes is associated with improved insulin sensitivity and activity in ED-supplemented obese mice.
Moreover, ED supplementation significantly enhanced glucose utilization by increasing the activity of hepatic GK, which is involved in glycolysis, and by upregulating hepatic IRS2 mRNA expression. A previous study using IRS2 −/− mice suggested that IRS2-deficient mice developed diabetes as a result of severe IR paired with β-cell failure [19] and showed higher lipogenic enzyme FAS and hepatic lipid levels [20]. Therefore, the above findings suggested that attenuation of NAFLD by ED supplementation is partially linked with glucose homeostasis and leads to the prevention of insulin insensitivity and IR.
Adiposity is linked with the production and release of several inflammatory mediators, which can cause chronic low-grade inflammation and IR [21,22]. Leptin is an adipokine that is primarily expressed in adipose tissue and plays a major role in body weight regulation. Leptin can modulate the production of pro-inflammatory adipokines/cytokines [23]. Obesity and its associated complications, such as IR, are associated with increased plasma leptin concentrations [24][25][26]. The above findings indicated that leptin acts as a potential link between inflammation and IR. ED treatment significantly lowered plasma levels of leptin and markedly decreased the levels of plasma pro-inflammatory cytokines, thereby improving inflammation coupled with adiposity, which in turn prevents IR.

Experimental Animals and Diet
C57BL/6N mice (4 weeks old, male) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were individually housed under controlled temperature conditions (24 • C) with the 12 h light-dark cycle and fed with a normal chow diet for 1 week of acclimation. The mice were then randomly assigned (n = 10 mice/group) to one of the following experimental diets for 16 weeks: ND (AIN-76 purified diet), HFD (20% fat based on AIN-76 diet plus 1% cholesterol), or HFD with 0.005% (w/w) of ED. The HFD contained 40 kcal% fat, 17 kcal% protein and 43 kcal% carbohydrate with the fat sources consisting of lard (85% (w/w) of total fat) and corn oil (15% (w/w) of total fat). At the end of the experimental period, the mice were anesthetized with isoflurane (5 mg/kg body weight; Baxter, Deerfield, IL, USA) after 12 h of fasting. After blood collection to determine the plasma lipid, adipokine, and hormone concentrations, liver and WAT tissues were removed, and samples were rinsed with physiological saline, weighed, snap-frozen in liquid nitrogen, and stored at −70 • C until enzymatic activity determination and/or RNA analysis. The animal study protocols were approved by Kyungpook National University Ethics Committee (Approval No. KNU 2016-0040, April 18, 2016).

Blood Analysis
The plasma TC, HDL-C, TG, and glucose concentrations were determined using commercial kits (Asan Pharm Co., Seoul, South Korea). The plasma FFA concentration was determined using a commercial kit (Wako Chemicals, Osaka, Japan). The ApoA-I and ApoB100 levels were additionally measured using enzymatic kits (Eiken Chemical Co., Tokyo, Japan). The non HDL-C, HTR, and AI values were calculated as follows: HOMA-IR was calculated as (fasting glucose (mmol/L) × fasting insulin (µIU/mL))/22.5. For the glucose tolerance test, mice were fasted for 12 h at 15 weeks after the start of the diet experiments and then injected intraperitoneally with glucose (0.5 g/kg body weight). Blood glucose levels were measured from the tail vein with a glucose analyzer (One Touch Ultra, Wayne, PA, USA) at 0, 30, 60, and 120 min after glucose injection.

Hepatic and Fecal Lipid Contents
Hepatic and fecal lipids were extracted following the method of Folch et al. [27]. Fecal samples from each group were collected daily for 1 week and subjected to lipid extraction. The dried hepatic lipid residues were dissolved in 1 mL of ethanol for TG, cholesterol, and FA assays. Triton X-100 and a sodium cholate solution in distilled water were added to 200 µL of the dissolved lipid solution for emulsification. The fecal samples were dried and extracted in ice-cold chloroform and methanol (2:1, v/v) for 24 h at 4 • C. After centrifugation at 900 g for 10 min, the supernatant was collected, dried at 50 • C and dissolved in ethanol. Hepatic and fecal TG, cholesterol, and FA concentrations were analyzed using the same enzymatic kits used for the plasma analyses.

Activities of Hepatic Lipid-and Glucose-Regulating Enzymes
Hepatic mitochondrial, cytosolic, and microsomal fractions were prepared as previously described [28] with slight modifications. Protein concentrations were determined by the Bradford method [29]. Cytosolic FAS activity was measured by monitoring the malonyl coenzyme A-dependent oxidation of NADPH at 340 nm [30]. Cytosolic ME activity was measured according to the method described by Ochoa et al. [31] by monitoring the cytosolic NADPH production at 340 nm. The G6PD activity was determined in the cytosol following the method of Pitkanen et al. [32] based on the reduction of 1 µmol of NADP per minute at 25 • C measured at 340 nm using a spectrophotometer. Mitochondrial fatty acid β-oxidation was measured by monitoring the reduction of NAD+ to NADH at 340 nm [33]. Mitochondrial CPT activity was assayed using the method described by Markwell et al. [34]. Microsomal PAP activity was determined following a spectrophotometric method [35]. GK activity in the cytosol was measured using a spectrophotometric assay as described by Davidson and Arion [36], in which the formation of glucose-6-phosphate at 37 • C was coupled to its oxidation by glucose-6-phosphate dehydrogenase and NAD+. PEPCK activity was measured relative to the rate of oxaloacetate synthesis and the reduction of NADH to NAD following the spectrophotometric assay developed by Bentle and Lardy [37]. G6Pase activity in the microsomes was determined following the method described by Alegre et al. [38] with slight modifications.

RNA Isolation and Gene Expression Analysis
The liver and epididymal WAT were homogenized in TRIzol reagent (Invitrogen, Grand Island, NY, USA). Total RNA was isolated according to the manufacturer's instructions. DNase digestion was performed to remove contaminating DNA. RNA purity and integrity were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Total RNA (1 µg) was reverse-transcribed into cDNA using the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany). mRNA expression levels were quantified by real-time RT-PCR using the SYBR green PCR kit (Qiagen) and the CFX96TM real-time system (Bio-Rad, Hercules, CA, USA). The gene-specific primers used for real-time RT-PCR are presented in Table 1. The following amplification profile was used: 35 cycles of 10 min at 90 • C, 15 s at 95 • C, and 60 s at 60 • C. Cycle thresholds were determined based on SYBR green emission intensities during the exponential phase. The Ct data were normalized using GAPDH, and the relative gene expression levels were calculated using the 2 ∆∆Ct method. Table 1. Primer sequences used for RT-qPCR.

Morphological Examination of Liver and Fat Tissues
The liver and epididymal WAT were removed from the mice, fixed in 10% (v/v) paraformaldehyde/phosphate-buffered saline, and embedded in paraffin for H&E staining. The stained areas were viewed under an optical microscope (Nikon, Tokyo, Japan) at 200× magnification.

Statistical Analysis
All data were presented as the mean ± standard error of the mean. Statistical analyses were performed using the statistical package for the social sciences software program SPSS (SPSS Inc., Chicago, IL, USA). Statistically, differences between groups (HFD and ND; and ED and HFD) were determined using Student's t-test.

Conclusions
In conclusion, our current findings demonstrated that dietary ED attenuated lipogenesis in WAT and increases fecal lipid excretion, thereby reducing adiposity and dyslipidemia. In addition, ED improves NAFLD partly by decreasing hepatic lipogenesis and increasing hepatic FA oxidation. In addition, ED supplementation improves hyperglycemia, hyperinsulinemia, and IR by enhancing glucose utilization and decreasing hepatic gluconeogenesis, as well as improving inflammation. Taken together, these findings indicated that 16 weeks of ED supplementation could be helpful in preventing obesity and metabolic disorders, such as adiposity, dyslipidemia, NAFLD, and IR in HFD-induced obese mice.