Chlorogenic Acid Improves High Fat Diet-Induced Hepatic Steatosis and Insulin Resistance in Mice
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- Ma, Y., Gao, M. & Liu, D. Pharm Res (2015) 32: 1200. doi:10.1007/s11095-014-1526-9
Chlorogenic acid (CGA), the most abundant component in coffee, has exhibited many biological activities. The objective of this study is to assess preventive and therapeutic effects of CGA on obesity and obesity-related liver steatosis and insulin resistance.
Two sets of experiments were conducted. In set 1, 6-week old C57BL/6 mice were fed a regular chow or high-fat diet (HFD) for 15 weeks with twice intra-peritoneal (IP) injection of CGA (100 mg/kg) or DMSO (carrier solution) per week. In set 2, obese mice (average 50 g) were treated by CGA (100 mg/kg, IP, twice weekly) or DMSO for 6 weeks. Body weight, body composition and food intake were monitored. Blood glucose, insulin and lipid levels were measured at end of the study. Hepatic lipid accumulation and glucose homeostasis were evaluated. Additionally, genes involved in lipid metabolism and inflammation were analyzed by real time PCR.
CGA significantly blocked the development of diet-induced obesity but did not affect body weight in obese mice. CGA treatment curbed HFD-induced hepatic steatosis and insulin resistance. Quantitative PCR analysis shows that CGA treatment suppressed hepatic expression of Pparγ, Cd36, Fabp4, and Mgat1 gene. CGA treatment also attenuated inflammation in the liver and white adipose tissue accompanied by a decrease in mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and Tnfα, Mcp-1 and Ccr2 encoding inflammatory proteins.
Our study provides direct evidence in support of CGA as a potent compound in preventing diet-induced obesity and obesity-related metabolic syndrome. Our results suggest that drinking coffee is beneficial in maintaining metabolic homeostasis when on a high fat diet.
KEY WORDSChlorogenic acid (CGA) Hepatic steatosis Inflammation Insulin resistance Obesity
Prevalence of obesity and overweight is progressively expanding worldwide and now affects more than 60% of adults in the US (1,2). Obesity has been identified as the major cause of type 2 diabetes, cardiovascular diseases, various cancers, and other health problems, which lead to further morbidity and mortality. Emergence of metabolic syndrome incidence has resulted in an increased demand for new strategies that are safe and effective in preventing and treating obesity and obesity-associated diseases.
Coffee is one of the most widely consumed beverages, and an epidemiological study has shown coffee is inversely related to the risk of developing chronic diseases, such as type 2 diabetes mellitus, cardiovascular diseases and cancer (3). Among the components in coffee, chlorogenic acid (CGA), an ester of caffeic acid and quinic acid (4), is the most abundant form of phenolic acid and major bioactive compound, which is also widely available in other plants, fruits and vegetables such as apples, pears, tomatoes, blueberries, and many others (5). In addition to its strong antioxidative property, the potential role of CGA in glucose and lipid metabolism has been explored in recent years. CGA has been reported to inhibit glucose-6-phosphate translocase 1, resulting in the reduction of glucose transport in the intestine (6). In the meanwhile, CGA enhances glucose uptake in isolated skeletal muscle cells and L6 muscle cell line (7,8) and improves glucose tolerance in db/db mice (9), indicating its potential antidiabetic activity. In addition, CGA significantly decreased plasma and liver lipid levels in (\( fa \)/\( fa \)) Zucke rats and rats fed a high-cholesterol diet (10,11). Using a fat enriched diet containing 37% fat calorie and 0.02% of CGA, Cho and colleagues (12) showed a reduced weight gain of ICR mice by 16% (40.05 g vs 47.7 g for high fat diet control without CGA). These studies suggest that CGA may exert a beneficial influence on metabolic diseases. The current study investigates the preventive and therapeutic activity of CGA in mice fed a HFD. Special attention was paid to the CGA effect on obesity-related liver steatosis and insulin resistance and the underlying molecular mechanism. Our results demonstrate that CGA significantly blocked diet-induced weight gain but did not affect body weight or fat mass in obese mice. In both prevention and treatment studies, CGA suppressed hepatic steatosis, suppressed obesity-related inflammation and improved glucose tolerance and insulin sensitivity. Our results provide critical information regarding the beneficial effects of CGA in managing obesity and obesity-associated metabolic disorders.
MATERIALS AND METHODS
Chlorogenic acid (CGA) was purchased from Cayman Chemical (Ann Arbor, Michigan). The TRIZOL reagent and the SuperScript® III First-Strand Synthesis System are from Life Technologies (Grand Island, NY). The RNeasy Lipid Tissue Mini Kit was from Qiagen (Valencia, CA). PerfeCTa® SYBR® Green FastMix was acquired from Quanta BioSciences (Gaithersburg, MD). The Oil Red O solution was obtained from Electron Microscopy Science (Hatfield, PA). Infinity™ Triglycerides kit was purchased from Fisher Diagnostics (Middletown, VA). Total cholesterol assay kit was from Genzyme Diagnostics (Charlottetown, PE Canada) and NEFA-HR assay kits for free fatty acid was from Wako Bioproducts (Richmond, VA). The Mercodia Insulin ELISA kit was purchased from Mercodia Developing Diagnostics (Winston Salem, NC). A TUREtrack glucometer and test strips were purchased from Nipro Diagnostics, Inc. (Fort Lauderdale, FL). High-fat diet (F3282, 60% kJ/fat) was purchased from Bio-serv (Frenchtown, NJ). C57BL/6 mice were purchased from Charles River (Wilmington, MA).
Animals and Treatment
All procedures performed on mice were approved by the Institutional Animal Care and Use Committee at the University of Georgia, Athens, Georgia. Two sets of experiments were carried out. In the first set, 6-week-old male C57BL/6 mice fed a HFD received two injections of CGA (100 mg/kg, intra-peritoneal) per week or carrier solution [dimethyl sulfoxide (DMSO)] for 15 weeks. In the second, obese mice (average body weight 50 g) were administrated CGA (100 mg/kg, intra-peritoneal) or DMSO twice per week for 6 weeks. Body weight and food intake were monitored weekly and animal body composition was determined at the end of the experiment using EchoMRI-100™ from Echo Medical Systems (Houston, TX).
After mice were sacrificed, the liver and epididymal white adipose tissue (eWAT) and brown adipose tissues (BAT) were collected, fixed in 10% formalin, embedded in paraffin, and sectioned at a thickness of 6 μm. Hematoxylin and eosin (H&E) staining was performed. Frozen sections (8 μm) were stained with 0.2% Oil Red O in 60% of isopropanol for 15 min and washed three times with phosphate buffered saline. A microscopic examination was performed and photographs were taken under a regular light microscope.
Lipid Extraction and Analysis
Hepatic lipids were extracted following an established procedure (13). Briefly, liver tissues were homogenized in phosphate buffered saline. Total lipids in 300 μl of homogenate were extracted by addition of 5 ml of chloroform-methanol (2:1, vol/vol) mixture. An aliquot of the organic phase was evaporated to dry and disolved in 1% Triton X-100. Hepatic cholesterol and triglyceride assays were performed according to the manufacturer’s instructions.
Determination of Blood Lipid and Insulin Level
Blood samples were collected from fasted mice. Cholesterol, triglyceride, free fatty acid and insulin levels in the plasma were measured using commercial assay kits according to the manufacturer’s instructions.
Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)
For GTT, mice were injected intraperitoneally with glucose at 2 g/kg body weight after fasting overnight. A small cut at the tip of a mouse tail was made at a selected time to give a small drop of blood which was directed absorbed into a test strip for determination of glucose level using a glucometer. For ITT, mice fasted for 4 h and blood glucose levels were measured after an intraperitoneal injection of insulin (0.75 U/kg) from Eli Lilly (Indianapolis, IN).
Gene Expression Analysis by Real Time PCR
Primer Sets for Real Time PCR Analysis of Gene Expression
All data were analyzed using GraphPad Prism 6 (GraphPad Software, San Diego, CA). One-way ANOVA was performed to evaluate the difference following Tukey post hoc test. All data are reported as mean ± standard deviation (SD) with statistical significance set at p < 0.05.
CGA Prevented Animals from Development of High-Fat Diet-Induced Obesity and Macrophage Infiltration in eWAT
CGA Suppressed PPARγ-Associated Development of Fatty Liver in HFD-fed Mice
CGA Suppressed Diet-Induced Hyperinsulinemia and Hyperglycemia in HFD-Fed Mice
CGA Treatment did not Affect Body Weight but Improved Insulin Sensitivity in Obese Mice
CGA Treatment Reduced Hepatic Lipid Accumulation and Obesity-Related Chronic Inflammation in Obese Mice
Consistent with improved hepatic lipid level, expression of macrophage marker genes such as chemoattractant Mcp-1 and its receptor C-C chemokine receptor type 2 (Ccr2), F4/80, Cd68 as well as Tnfα were also lower in CGA-treated animals (Fig. 8c). Similarly, mRNA levels of the same set of genes in eWAT also significantly reduced by CGA treatment (Fig. 8d). Taken together, these data suggest that CGA treatment of obese mice reduces HFD-induced chronic inflammation and improved hepatic steatosis.
Previous studies have demonstrated that CGA is capable of effecting glucose and lipid metabolism as well as weight gain induced by HFD feeding (6,9, 10, 11, 12). The current study comprehensively assesses both preventive and therapeutic activities of CGA on diet-induced obesity and obesity-associated metabolic syndromes, demonstrates the effect of CGA on obesity-related liver steatosis and insulin resistance, and explores underlying molecular mechanism. Utilizing C57BL/6 mice fed a HFD with 60% calorie from fats as an animal model and intraperitoneal injection as a route of drug administration, we examined the CGA activity in blocking HFD-induced weight gain and in reducing the body weight of obese mice. We demonstrate that CGA was effective in preventing HFD-induced weight gain (Fig. 1), inhibiting development of liver steatosis (Fig. 3), and blocking HFD-induced insulin resistance (Fig. 5). CGA treatment of obese mice did not yield weight loss, but improved insulin sensitivity (Fig. 6) and reduced lipid accumulation in the liver (Fig. 7). The beneficial effects were associated with CGA activity in blocking HFD-induced inflammation (Fig. 2), inhibiting diet-induced expression of Pparγ and its target genes in the liver, and increasing expression of genes responsible for lipid metabolism (Figs. 4 and 8).
The protective activity of CGA against HFD-induced obesity can be attributed to its antioxidant activity. Accumulating evidence suggests that chronic inflammation is closely associated with diet-induced obesity (15). HFD elevates production of reactive oxygen species in adipose tissue and boosts macrophage infiltration (16,17). We have previously shown that macrophage elimination by clodronate liposomes blocks HFD-induced obesity (18). We also recently demonstrated that HFD-induced obesity could be blocked by overexpression of superoxide dismutase 3 gene as a means to maintain redox homeostasis (19). CGA is a well-known anti-oxidant and capable of suppressing inflammation by inhibiting NF-κB and JNK/AP-1 activation and inhibition of the toll-like receptor 4 signaling pathway (20,21) Consistent with these results, we observed that CGA treatment greatly reduced the mRNA level of macrophage marker genes in eWAT including F4/80, Cd68, Cd11b, Cd11c, Tnfa and Mcp-1 (Fig. 2), indicating a lack of diet-induced macrophage infiltration. Mechanistically, twice injection of CGA into mice per week provides sufficient antioxidants for animals to remove reactive oxygen species generated by consumption of lipids-enriched diet, thereby suppressing the expression of the pro-inflammatory cytokine gene induced by HFD, and consequently blocking fat accumulation and weight gain. Once the development of obesity is blocked, other metabolic parameters such glucose and insulin sensitivity maintain normal (Fig. 5). Results shown in Fig. 6a suggest that blockade of inflammation is not sufficient in reducing body weight in fats already accumulated in adipose tissue, indicating that antioxidants would not be effective in reducing body weight.
Results in Figs. 4 and 8 clearly show that CGA prevented and improved liver steatosis by inhibiting the PPARγ pathway. PPARγ, especially PPARγ2, is expressed highly in adipose tissue (22,23), promoting fatty acid uptake into adipocytes and adipocyte differentiation. Overexpression of hepatic Pparγ resulted in exacerbated liver steatosis (24). Conversely, liver and hepatocyte specific Pparγ knockout mice were protected against hepatic lipid accumulation (25, 26, 27). These studies suggest that PPARγ plays an important role in the development of hepatic steatosis. Our results show that HFD stimulated both Pparγ1 and Pparγ2 gene expression in the liver, concordant with HFD-induced hepatic lipid accumulation. Hepatic fatty acid transporter CD36 is a common target of PPARγ (28) and increased hepatic CD36 activity is critical for the development of steatosis in obesity (29, 30, 31). Consistent with previous reports (13,32, 33, 34), HFD significantly induced hepatic Cd36 expression, facilitating transport of long-chain fatty acids into liver. In both prevention and treatment studies, CGA dramatically reduced hepatic Pparγ mRNA level and inhibited expression of fatty acid transporter Cd36 and Fabp4 genes (Figs. 4a and 8a). In addition to blockade of fatty acid uptake, CGA also dramatically inhibited PPARγ-regulated Mgat1 expression. Mgat1 is involved in incorporation of fatty acids into triglyceride (35) and knockdown of this enzyme in the liver markedly reduced hepatic steatosis in diet-induced and genetic obese model (36). Therefore, by targeting the hepatic PPARγ pathway, CGA exerts its protective function against obesity-induced hepatic steatosis. Here, we quantify the change of PPARγ and its target genes by real time PCR. It should be noticed that the gene expression level is not necessarily equal to the protein level. Measurement in protein level will further confirm the effect of CGA on this pathway. In addition to the effect on Pparγ, previous studies have shown that CGA could enhance PPARα level in the liver and stimulate lipid utilization (11,12). In consistent with these earlier reports, our data also show that CGA promoted expression of Pparα and its target genes Acox1 and Fgf21 (Figs. 4b and 8b).
In summary, results presented in this study demonstrate CGA prevents mice from diet-induced obesity and obesity-related metabolic syndrome. CGA also improves liver steatosis, insulin sensitivity and reduces chronic inflammation in obese mice. These findings provide direct evidence in support of the potential health benefits of CGA in managing obesity and obesity-associated metabolic disorders. The next step for CGA research may be the development of improved CGA formulation to reach an effective CGA concentration in humans without toxicity and inconvenience.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported in part by the National Institute of Health (RO1EB007357 and RO1HL098295). We thank Ms. Ryan Fugett for proof-reading and English editing.