Bitter melon extract attenuating hepatic steatosis may be mediated by FGF21 and AMPK/Sirt1 signaling in mice

We sought to evaluate the effects of Momordica charantia (bitter melon, BM) extract on insulin sensitivity, NAFLD, hepatic FGF21 and AMPK signaling in mice fed a high-fat diet. Male C57/B6 mice were randomly divided into HFD and HFD supplementation with BM for 12 week. Body weight, plasma glucose, FGF21 and insulin levels, hepatic FGF21 and AMPK signaling proteins were measured. The results showed that plasma FGF21 and insulin concentrations were significantly decreased and hepatic FGF21 content was significantly down-regulated, while FGF receptors 1, 3 and 4 (FGFR1, FGFR3 and FGFR4) were greatly up-regulated in BM group compared to the HFD group (P < 0.05 and P < 0.01). BM also significantly increased hepatic AMPK p, AMPK α1 AMPK α2 and Sirt1 content compared to the HFD mice. We, for the first time, demonstrated that BM extract attenuated hepatic steatosis in mice by enhancing hepatic FGF21 and AMPK/Sirt1 signaling.

plasma apoB-100 and apoB-48 in HFD-fed mice, and modulate the phosphorylation of IR, IRS-1, and its downstream signaling molecules [20][21][22] . Bioactive compounds of BM, cucurbitane triterpenoids, stimulate GLUT4 translocation to the cell membrane by activation of the AMPK pathway in both L6 myotubes and 3T3-L1 adipocytes 23 . Supplementation of BM to rats fed a high-fructose diet during gestation and lactation ameliorates fructose-induced dyslipidemia and hepatic oxidative stress in male offspring 1 . Our previous study shows that BM extract enhances insulin signaling, increases GLUT4 abundance and modulates acylcarnitine content in the skeletal muscle of HFD fed mice 24 . The precise mechanism by which BM extract improves glucose metabolism is largely unknown, and the effects of BM extracts on FGF21 signaling in obesity and metabolic syndrome have not been studied. Based on the above rationale, and given the role of FGF21 in lipid metabolism, we postulated that FGF21 may mediate the hypoglycemic action of BM. To test this hypothesis, we evaluated the effects of a BM extract on glucose and lipid metabolism, liver fat content, insulin sensitivity, and FGF21 signaling in mice fed a HFD.

Results
Effects of BM extracts on body weight, food intake, body composition and Food convert ratio (FCR) in the HFD fed mice. No significant difference was found in the body weight, food intake, Fat mass (FM), or fat-free mass (FFM) (Fig. 1A-D) between the HFD and BM-V groups at baseline (week 0). Energy intake at week 2 and week 3 was significantly lower in the BM-V animals when compared with HFD animals, and there were no significant difference between HFD and BM groups after week 3 of initiate treatment. From weeks 5 to 12 of initiated intervention, body weight was significantly lower in the BM-V group than in HFD animals. The FM was significantly increased and FFM were significantly decreased in the HFD group during 12-week study, but these parameters between week 0 and week 12 were not significantly altered in BM-V mice. FM was significantly higher at week 12 than week 0 in HFD group (P , 0.01). FM was significantly lower and FFM were significantly higher in BM-V mice in compared with HFD mice after the 12-week intervention ( Fig. 1C and D). Actual food intake was no difference between HFD and BM-V groups (Fig. 1 E). Feed efficiency data in these animals show that FCR was much higher in the BM-V group than in the HFD from week 6 to week 12 (P , 0.05, P , 0.01 and P , 0.001, Fig. 1F).
BM extract significantly improved glucose metabolism and enhanced insulin sensitivity in mice. No differences in plasma glucose concentrations were observed between HFD and BM-V groups at baseline. However, glucose levels significantly increased in the HFD groups from week 6 to week 12 (P , 0.05, Fig. 2A) when compared with week 0, but no significant changes were observed in BM-V group relative to its week 0. Plasma glucose concentrations were significantly lower in BM-V mice in comparison to HFD mice at week 6 and week 12 (P , 0.05, Fig. 2A). Plasma insulin concentration was significantly decreased, and insulin sensitivity (assessed by HOMA-IR) was significantly improved in BM-V group when compared with HFD group (P , 0.05 and P , 0.01, Fig. 2 B, C). IPGTT data show that glucose concentrations were significantly Figure 1 | Effects of BM extract on body weight, food intake, body composition and FCR in mice fed a HFD. Two groups of mice were fed a HFD with or without BM-V for 12 weeks. Body weight and food intake are shown in panels (A) and (B), respectively. Body composition was measured as described in the methods. (C) Fat mass (FM) (% of body weight), (D) free fat mass (FFM) (% of body weight). (E) FCR results which were calculated as mass of the food eaten divided by the body mass gain over one week. The actual food intakes were presented as mean food intake/g/mouse/week (Fig. 1 F). Mean 6 SEM (n 5 10/group). Asterisk symbol stands for statistical comparison of BM-V group vs. HFD group; * P , 0.05, ** P , 0.01 and *** P , 0.001. # P , 0.05 and ## P , 0.01, Wk 0 vs. Wk12 in the HFD group. BM-V extract altered liver FGF 21 signaling in mice. Fasting plasma FGF21 levels and hepatic FGF21 content were significantly reduced in the BM-V group in comparison with HFD group (P , 0.05, Fig. 3A-B). Moreover, BM-V supplementation significantly increased hepatic FGFR1 (57%), FGFR3 (49%), FGFR4 (82%) and PGC-1a (55%) content (P , 0.001, P , 0.01, P , 0.001 and P , 0.01, respectively), slightly increased bKlotho (5%) and PPARa (7%) content when compared with HFD animals (Fig. 3C).
Morphological features of liver tissues were evaluated by H&E staining. The typical macrovesicular steatosis, hepatocellular ballooning, portal and lobular inflammatory cell infiltrations were observed in the HFD animals after 12 week of feeding ( Figure 4A). Hepatic FGF21 content determined by Immunofluorescence microscopy was higher in HFD than in the BM-V group ( Figure 4B). Supplementation with BM-V was shown to significantly reduce liver TG content in comparison with the HFD group (BM-V vs. HFD, P , 0.05, Figure 4C).
Plasma lipid profile analysis showed that fasting plasma TG, cholesterol and LDL-cholesterol concentrations were lower in the BM-V animals than in the HFD animals, but only plasma TG in the BM-V group was significantly lower (P , 0.05). There was no significant difference in plasma HDL-cholesterol levels between groups (Table 1).

Discussion
In this study we assessed the effects of a BM extract on insulin sensitivity, liver fat content, and FGF21 and AMPK/Sirt-1 signaling in mice fed a high-fat diet. We observed that the BM-V extract significantly reduced body weight, fasting plasma glucose, insulin, and FGF21 concentration as well as liver FGF21 and TG content in mice fed a HFD. Furthermore, the extract improved plasma lipid profiles and enhanced insulin sensitivity when compared with the HFD group without significantly affecting food intake 25 . BM extract reduced the palatability of the diet as shown by the food intake at first three weeks of initiate intervention, which was significant lower in the BM-V group than in HFD group. However, these animals were able to adapt BM mixed diet quickly and consumed the same amount of food when compared with HFD after 3 weeks (Fig. 1B) and actual food intake (g/mouse/week) was slightly higher in BM mice than in HFD mice (Fig. 1E). Compared with regular 5%-10% dietary fiber supplementation experiments and caloric restriction studies i.e. 20%-30% lower food intake, HFD mixed with 1.2% BM extract seemed to not significantly affect the energy density of the diet in comparison with pure HFD. Therefore, the higher FCR in the BM-V group may contribute to either increased energy expenditure or reduced caloric absorption from the gut or both of them. Although the oxygen consumption rate in these mice were not measured, our data indicate that decreased adiposity in BM-supplemented rats may result from lower metabolic efficiency, a consequence of increased lipid oxidation and mitochondrial uncoupling 26 .
A major contributor to NAFLD may be insulin resistance, and a major contributor to insulin resistance is obesity, especially abdominal obesity 4 . In an in vitro study, BM extract reduced lipid accumulation during differentiation from pre-adipocyte to adipocyte, with a reduction in overall triglyceride of 32.4% after 72 hours compared with untreated control cells 27 . Recent study shows that aqueous extract of Momordica charantia seeds (MCSE) primarily regulated the insulin signaling pathway in muscles and adipose tissues with targeting insulin receptor (IR) 28 . Here, for the first time illustrated that the novel effects of BM-V on attenuated or reversed fatty accumulated in the liver of mouse on a HFD by modulating FGF21 signaling. FGF21 has potential insulin mimetic effects on lowering plasma glucose and liver lipid levels, suggesting that FGF21 may play a role in the pathogenesis of liver and whole-body insulin resistance in T2DM 17 . FGF21 injection in the brain increased energy expenditure and insulin sensitivity in obese rats 29 . On the other hand, clinical studies showed that plasma FGF21 levels were significantly higher in both insulin resistant obese and T2DM patients than in healthy subjects 7,29,30 . We observed that FGF21 resistance develops in mice fed a HFD (Plasma FGF21 levels in the HFD group were 4.7-fold higher than low-fat diet fed group, P , 0.001, unpublished data). Increase of FGF21 in the HFD mice may indicate that there is a compensative mechanism of the liver to secrete more FGF21 protein in the conditions of impaired FGF21 signaling. More recent studies reported that FGF21 levels were increased in association with obesity and NAFLD 31 . Thus, obesity is also linked to a FGF21-resistant condition 32 . In this study, we demonstrated that BM-V reduced plasma FGF21 levels and hepatic FGF21 content, and attenuated HFD-induced liver steatosis, while BM-V also significantly enhanced peripheral insulin sensitivity. The effects of BM extracts on reducing FGF21 levels may be contributed to enhance FGF21 signaling, and therefore, BM works more like as FGF21 ''sensitizer'' instead of agonist or antagonist of FGF21. Hyperinsulinemia, elevated FFA and glucose have also been noted to induce FGF21 expression in human studies 9,33,34 . BM extract robustly reduced hepatic FGF21 content in HFD-fed mice, but it remains to evaluate whether BM extract directly suppresses liver FGF21 expression without affecting PPARa transcription or indirectly inhibits FGF21 expression by blocking the effect of PPARa on FGF21 regulation. In vitro studies suggest that FGF21 initiates its action by activating a unique dual receptor complex consisting of a co-receptor bklotho and the tyrosine kinase FGFR 35 . bKlotho binds FGF21 and facilitates the activation of other FGFRs 36 . It is well documented that FGF21 is selective for FGFR1 isoform 1c, with varying reports of using isoforms 2c or 3c 37,38 . FGFR4 is dominant in mature hepatocytes and involved in the control of hepatic bile acid and lipid metabolism 39 . Recent study shows that FGF21 binds FGFR1 with much higher affinity than FGFR4 in presence of bKlotho; while FGF19 binds both FGFR1 and FGFR4 in presence of bKlotho with comparable affinity 40 . The increase of FGFR1, FGFR3 and FGFR4 protein abundance may reflect a negative feed-back regulatory mechanism of FGF21 on its receptors. BM supplementation resulted in reduction of liver FGF21expression and induction of its receptor expression in the HFD fed mice, indicating that BM extract enhances FGF21 signaling, by which attenuates HFD-induced insulin resistance and hepatic steatosis.
Skeletal muscle and adipose tissues secrete FGF21, but hepatocytes contribute greatly to FGF21 levels in response to free fatty acid (FFA) stimulation of a PPARa/RXR dimeric complex 37,38 . Other studies have reported that there was a positive correlation between FGF21 concentration, insulin levels, and body mass index (BMI) in human clinical and animal studies 5,31 . A study in Chinese subjects found a positive association of plasma FGF21 with circulating triglycerides, total cholesterol and gamma-glutamyltransferase, but not insulin sensitivity 41 . FFAs also increased circulating FGF-21, while insulin had little effect under physiological conditions. These observations may help explain the apparent paradox of increased FGF21 levels in obesity, insulin resistance, and starvation 38 . Consistent with Tan's report that BM extracts increased activity of AMPK, a key pathway mediating glucose uptake and fatty acid oxidation 23 , we observed that BM-V significantly increased AMPK phosphorylation, AMPKa1 and AMPKa2 protein abundance when compared with the HFD mice. Furthermore, BM-V significantly increased hepatic Sirt1 protein abundance in comparison with the HFD. The findings that BM extract enhanced FGF21 and AMPK-Sirt1 signaling pathways support the notion that FGF21 regulates mitochondrial activity and enhances oxidative capacity through an AMPK-SIRT1-PGC1alphadependent mechanism in adipocytes 12 .
Taken together, BM extract supplementation greatly enhances insulin sensitivity, reverses HFD-induced liver damage, and significantly reduces plasma FGF21 levels and body fat mass in mice fed a HFD. This study suggests that the favorable effects of a BM extract on increasing insulin sensitivity and attenuating hepatic steatosis may be mediated by enhanced FGF21 and AMPK-Sirt1 signaling. Therefore, a BM extract presents a potential botanical target for the development of new therapeutic alternatives to treat NAFLD, obesity, and diabetes.   All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Pennington Biomedical Research Center. Thirty 5-week-old male C57BL/6J mice were ordered from Charles River Laboratories, Inc (Wilmington, MA) and maintained at constant temperature and humidity (21 6 2uC with humidity 65-75%) with a 12512-h light-dark cycle. Mice were housed two/cage, labeled with ear punch, and allowed access to water and food ad libitum.

Methods
Mice were randomly divided into two groups; high-fat diet control (HFD) and HFD supplementation with bitter melon extract (BM-V). Animals were fed a HFD containing 58% of energy from fat (D-12331) purchased from Research Diets Inc. (New Brunswick, NJ). BM was administered by incorporating the extract into the high-fat diet at a dose of 1.2% (W/W). Briefly, 500 g of high-fat diet and 6 g of BM extract powder were mixed with a food processor (Cuisinart DLC-2014, Sears, Hoffman Estates, IL); food dye was added to monitor HFD completely mixed with BM extract. Then the mixed diet was stored in 220uC for the feeding experiments. Chosen 1.2% BM extract was based on our previous study 24 . Food intake and body weight were recorded weekly. Fasting glucose was measured at weeks 0, 6, and 12 of the study respectively. Body composition was determined (described in the methods) at weeks 0 and 11. Fasting plasma insulin, FGF21 levels, and lipid profiles were measured at week 12. At the end of the study, the mice were euthanized. The liver and other tissues were dissected and snap frozen in the liquid nitrogen and stored at 280uC for future measurements.
Blood chemistry and hormone analysis. After 4 hours of fasting, blood samples were collected by tail stick. Plasma glucose levels were measured by a colorimetric hexokinase glucose assay (Sigma Diagnostics, St Louis, MO). Plasma insulin levels were determined by mouse insulin enzyme-linked immunosorbent assay (ELISA) kits (Millipore Co. Billerica, MA). Homeostasis model assessment -insulin resistance (HOMA-IR) was calculated using the following formula: HOMA-IR 5 [I 0 (mU/ml) 3 G 0 (mmol/liter)]/22.5 42 . Plasma FGF21 was measured by mouse FGF-21 ELISA Kits according to the manufacturer's instructions (R & D Systems, Minneapolis, MN). Intra-assay and inter-assay CVs of FGF21 were 4.5% and 6.1%, respectively. FGF21 quality control result was 278 pg/ml (range 191-319 pg/ml).
Body composition measurement. Body composition for all animals was measured using a Minispec TD-NMR Spectrometer (Bruker Optics, TX) 43 . Total fat mass (FM) and fat free mass (FFM) were recorded.
Feed conversion ratio (FCR). FCR is a measure of an animal's efficiency in converting feed mass into increased body mass. It was calculated at weeks 2, 4, 6, 89 and 12 as the ratio of feed intake to gain in body weight 44,45 .
Intraperitoneal glucose tolerance testing (IPGTT) and intraperitoneal insulin tolerance testing (IPITT) were measured at weeks 10 and 11 respectively. IPGTT was performed after overnight fast, before and after mice were intraperitoneal injected glucose at dose 1 gram/kg body weight, blood glucose concentrations were measured at 0, 15, 30, 60 and 120 min using glucose strips described as below. For IPITT, after 4 h fast mice were IP injected insulin at 0.75 U/kg body weight 43 . Blood glucose concentrations were measured from the tail vein at time 0 (baseline) or 15, 30, 60 and 120 minutes after insulin injection using the Freestyle blood glucose monitoring system (Thera Sense, Phoenix, AZ).
Plasma lipid profile analysis. Fasting plasma TG concentrations were assessed using a TG reagent kit from Eagle Diagnostics Inc (DeSoto, TX). Plasma cholesterol levels were measured with a cholesterol assay kit from BioVision Inc (Mountain View, CA). HDL-cholesterol level was determined by phosphotungstic acid and magnesium chloride precipitation method 46 .
Liver FGF21 content assessment. Liver tissues (,25 mg) were added to ten volumes of homogenization buffer (w/v), minced with scissors in Eppendorf microcentrifuge tubes, and homogenized by micro-homogenizer 43 . Tubes were centrifuged at 8000 3 g for 5 min. 50 ml of supernatant was assayed using a FGF21 ELISA kit as described above.
Liver lipid extract for TG measurement. Liver lipid extracts were prepared by the Folch procedure 47 . Briefly, liver tissues (about 25 mg) were added to five volumes of PBS (w/v), minced with scissors in Eppendorf microcentrifuge tube, and homogenized by sonication. The liver lysates were added to ten volumes of an extract solvent containing chloroform and methanol in the ratio of 251 (v/v). After vortexing, the tubes were centrifuged at 5000 3 g for 10 min. Aliquots of 100 ml were removed from the bottom of the tube, transferred to a new tube and dried under nitrogen gas. After adding 100 ml of PBS to the tube, 10 ml of mixture was taken to measure TG content using a TG assay kit (DeSoto, TX). The results were normalized by protein concentration.
Histological studies in the liver. The sections of liver tissues from the center of the largest liver lobes were fixed in 10% buffered formaldehyde, and then embedded in paraffin. A 5 mm-thick section cut from a paraffin-embedded block was stained with hematoxylin and eosin (HE staining). All specimens were observed and photomicrographed using an Olympus 1X71 inverted microscope and Olympus PP72 camera, Olympus America Inc (Melville, NY).
Immunofluorescence microscopy. Immunofluorescence for FGF21 was performed on liver slides. Briefly, the sections of liver tissues from the center of the largest liver lobes were fixed in 10% buffered formaldehyde, and then embedded in paraffin. A 5 mm-thick section cut was taken from the paraffin-embedded block. Slides were dewaxed using xylene, rehydrated with an alcohol gradient, and then incubated with a 1% Citrate buffer in 0.1% Triton X100, wash with PBS solution for 8 minutes to unmask antigen. A blocking solution containing 10% normal goat serum was incubated with the sample overnight at 4uC. Samples were incubated with a monoclonal anti-FGF21 (15250) for 60 min at room temperature, and with Alexa Fluor 594-conjugated goat anti-mouse IgG (15300 dilution; Invitrogen, Carlsbad, CA) for 60 min at room temperature. After sealing the slides, images were obtained on a Zeiss 510 Axiophot microscope equipped with a Nikon digital camera and processed using Metamorph imaging software, version 6.1 (Universal Imaging).
Western blotting analysis. Liver lysates were prepared by homogenization in buffer A (25 mM HEPES, pH 7.4, 1% Nonidet P-40 (NP-40), 137 mM NaCl, 1 mM PMSF, 10 mg/ml aprotinin, 1 mg/ml pepstatin, 5 mg/ml leupeptin) using a PRO 200 homogenizer (PRO scientific, Oxford, CT). The samples were centrifuged at 14,000 g for 20 minutes at 4uC and protein concentrations of the supernatants were determined by Bio-Rad protein assay kit (Bio-Rad laboratories, Inc. Hercules, CA). Supernatants (50 mg) were resolved by 8% or 12% SDS-PAGE and subjected to immunoblotting. The protein abundance was detected with antibodies against FGF21 (R & D Systems, Minneapolis, MN), FGFR1, AMPK p (Thr172) , AMPK a1, AMPKa2, PPARa, and PGC-1a (Millipore, Billerica, MA), FGFR3 (Bioworld Technology, Inc, Louis Park, MN), bklotho and FGFR4 (Santa Cruz, CA), and b-actin (Affinity Bioreagents, Golden, CO) using Chemiluminescence Reagent Plus (PerkinElmer Life Science, Boston, MA), and quantified via densitometer. All the proteins were normalized to b-actin. Spectra TM multicolor broad range protein ladder (10-260 kDa, Pierce Biotech, Rockford, IL) was used as reference for the molecular weight of interesting protein. www.nature.com/scientificreports Statistical analysis. SAS univariate procedure with the normal option and the QQ plot statement was conducted to test normality of the data. Results were expressed as mean 6 SEM. Comparisons between groups were determined by Student t-test. P values , 0.05 were considered statistically significant.