articles nature publishing group Intervention and Prevention Viscous Dietary Fiber Reduces Adiposity and Plasma Leptin and Increases Muscle Expression of Fat Oxidation Genes in Rats Ajmila Islam1, Anthony E. Civitarese2, Robert L. Hesslink3 and Daniel D. Gallaher1 Dietary interventions that reduce accumulation of body fat are of great interest. Consumption of viscous dietary fibers cause well-known positive metabolic effects, such as reductions in the postprandial glucose and insulin concentrations. However, their effect on body composition and fuel utilization has not been previously studied. To examine this, rats were fed a viscous nonfermentable dietary fiber, hydroxypropyl methylcellulose (HPMC), for 6 weeks. Body composition was measured by dual-energy X-ray absorptiometry (DXA) and fat pad weight. Plasma adipokines, AMP kinase activation, and enzyme and mRNA analysis of key regulators of energetics in liver and soleus muscle were measured. The HPMC diet significantly lowered percent body fat mass and increased percent lean body mass, compared to a cellulose-containing diet (no viscosity). Fasting leptin was reduced 42% and resistin 28% in the HPMC group compared to the cellulose group. Rats fed HPMC had greater activation of AMP kinase in liver and muscle and lower phosphoenolpyruvate carboxykinase (PEPCK) expression in liver. mRNA expression in skeletal muscle was significantly increased for carnitine palmitoyltransferase 1B (CPT-1B), PPARγ coactivator 1α, PPARδ and uncoupling protein 3 (UCP3), as was citrate synthase (CS) activity, in the HPMC group relative to the cellulose group. These results indicate that viscous dietary fiber preserves lean body mass and reduces adiposity, possibly by increasing mitochondrial biogenesis and fatty acid oxidation in skeletal muscle, and thus represents a metabolic effect of viscous fiber not previously described. Thus, viscous dietary fiber may be a useful dietary component to assist in reduction of body fat. Obesity (2011) 20, 349–355. doi:10.1038/oby.2011.341 Introduction The prevalence of obesity in the United States has reached alarming levels. As of 2008, the Centers for Disease Control reported that 32 states had a prevalence ≥25% (1), with the prevalence of overweight and obesity at 66.3% of adults in the United States in 2003–2004 (2). The causes of being overweight or obese are complex, but certainly involve several factors such as reduced physical activity and over consumption of energydense, highly palatable foods (3). The primary concern regarding obesity is the increased risk for a number of comorbidities, including insulin resistance, hypertension, type 2 diabetes, and coronary heart disease. Given the importance of reducing adiposity in overweight or obese individuals, there is great interest in developing drugs that facilitate this reduction. Considerable effort has focused on the development of incretin-based drugs in metabolic disease, such as GLP-1, GIP agonists, and dipeptidyl peptidase-4 inhibitors. These drugs work in part by slowing the rate of gastric emptying, reducing food intake, improving satiety, and minimizing postprandial glucose excursions by insulinotropic responses. A nonpharmacological approach, with potential improved tolerability, is the use of foods or food components that result in a slower rate of absorption of glucose from a meal. A number of studies indicate that a diet leading to a reduced glycemic response reduces adiposity relative to a diet producing a greater glycemic response. Rats fed a high amylose-based diet for 7 weeks, which leads to a low glycemic response, had a significantly lower epididymal fat pad weight and a lower concentration of plasma leptin, when compared to rats fed a waxy corn starch-based diet, which has a high glycemic response (4). Similarly, rats fed mung-bean starch, which has a low glycemic response, for 5 weeks had a strong trend for a smaller epididymal fat pad, fewer adipocytes, and smaller adipocyte volume compared to rats fed a high glycemic response wheat starch-based diet (5). Viscous polysaccharides are a category of dietary fiber that is well established to blunt the postprandial plasma glucose and insulin response to a meal. Therefore, viscous polysaccharides, The first two authors contributed equally to this work. 1 Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota, USA; 2Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA; 3Imagenetix, Inc., San Diego, California, USA. Correspondence: Daniel D. Gallaher (dgallahe@umn.edu) Received 27 April 2011; accepted 4 October 2011; published online 17 November 2011. doi:10.1038/oby.2011.341 obesity | VOLUME 20 NUMBER 2 | february 2012 349 articles Intervention and Prevention by slowing the rate of gastric emptying and/or slowing glucose diffusion in the small intestine, and thereby reducing glucose absorption after a meal, may reduce adiposity. Changes in adiposity may be mediated by changing fuel utilization, specifically by changes in fatty acid oxidation. Isken et al. (6) have recently shown that mice fed a diet giving a high glycemic response had a blunting of fatty acid oxidation compared to mice fed a diet giving a low glycemic response and that this blunting preceded the increase in adiposity seen in this group. Thus, viscous fiber, by blunting the glycemic response to a meal, may increase fatty acid oxidation. At the molecular level, carnitine palmitoyltransferase 1B (CPT-1B) is the rate-limiting enzyme in fatty acid transport into the mitochondria and is considered the rate-limiting step in fatty acid oxidation (7). Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) interacts with many transcription factors, including PPAR’s, and serves as a nutrient sensing system that increases mitochondrial biogenesis and shifts substrate utilization toward fat and away from carbohydrate and enhances the commitment of skeletal muscle toward the more oxidative type I and IIa fibers, thus favoring fatty acid oxidation (8). PPARδ is also involved in oxidative metabolism, as its activation promotes β-oxidation (9). Induction of uncoupling protein 3 (UCP3) results in increased fatty acid oxidation (10) and thus also appears to play a role in facilitating fatty acid metabolism. Changes in fatty acid oxidation may be mediated by AMP-activated protein kinase (AMPK), a fuel sensor which plays a central role in regulation of fatty acid metabolism. Activation of AMPK leads to increases in fatty acid oxidation (11) and promotion of mitochondrial biogenesis (12). Since insulin decreases AMPK activation (13), we hypothesized that viscous dietary fibers, by blunting the postprandial plasma ­insulin response, would increase fatty acid oxidation, reduce adiposity, and improve mitochondrial function in skeletal muscle. We report here the effect of viscous dietary fiber on adiposity and plasma adipokine concentrations in rats. Additionally, we examined whether body composition changes were associated with changes in enzyme activity and gene expression related to muscle energetics and fuel utilization in liver. Methods and Procedures Animals Male Wistar rats (Harlan Sprague-Dawley, Indianapolis, IN) were housed individually in wire-bottom stainless steel cages in a temperature-controlled room (22 ± 1 °C) with a 12–12 h light–dark cycle (light 06:00–18:00). Animals were allowed free access to food and water. Animal housing and use complied with the University of Minnesota Policy on Animal Care. Initial body weights were 167 ± 2 g (~6 weeks of age). Diet composition Animals were adapted to a modification of the AIN-93G purified diet (14) for 1–3 days prior to feeding the experimental diets. Hydroxypropyl methylcellulose (HPMC; Dow Chemical, Midland, MI), a semisynthetic cellulosic ether available in several viscosity grades, and cellulose (Harlan-Teklad, Madison, WI) were used as the sources of dietary fiber. HPMC is soluble and highly viscous, but appears to completely resist fermentation. This is based on findings that feeding HPMC does not lower cecal pH, a marker of fermentation (N. Osterberg and D.D. 350 Gallaher, unpublished data) and that a closely related compound, methylcellulose, is not fermented (15). In contrast, purified cellulose is insoluble and has no viscosity, but like HPMC, cellulose is essentially nonfermentable. HPMC was provided by Imagenetix (San Diego, CA). The composition of the diets was as follows (g/kg): cornstarch, 347; casein, 200; dextrinized cornstarch, 115.2; sucrose, 87.3; corn oil, 150; fiber source, 50; AIN-93G mineral mix, 35; AIN-93G vitamin mix, 10; l-cystine, 3; choline bitartrate, 2.5. In addition, each diet contained 0.12% cholesterol and 0.0014% butylated hydroxyl-toluene as an antioxidant. The two diets contained either cellulose or a high-viscosity HPMC as the fiber source at 5% of the diet. As purified cellulose and HPMC are 100% dietary fiber, and the dietary fiber concentration was equal for both diets, the two diets were isocaloric, each containing 20.0 kJ/g, calculated based on a metabolizable energy content of 15.7 kJ/g for casein, 16 kJ/g for carbohydrates, and 38 kJ/g for fat. Experimental design Animals were divided into two dietary treatment groups, cellulose (n = 15) and HPMC (n = 30) and fed their respective diets for 6 weeks. Body weights and food intake were determined weekly. Body composition was determined 1 day prior to the initiation of the feeding trial and after week 5 of feeding by dual-energy X-ray absorptiometry (DXA; Lunar Prodigy Advance, GE Healthcare, Piscataway, NJ). After 5 weeks of feeding, animals were fasted overnight, and a blood sample was collected the following morning from the retro-orbital sinus. At the end of the feeding period, animals were fasted overnight and then presented a 5-g meal of their respective diets the following morning. Consumption of the meal was greater than 90% for all animals. Two and a half hours after giving the meal, animals were anesthetized by inhalation of isoflurane, opened by a midline incision, blood removed by cardiac puncture and collected into syringes containing EDTA (1 mg/ ml) and plasma collected after centrifugation. Liver and soleus (oxidative fiber) muscle was excised prior to removing fat pads (epididymal fat) and immediately frozen in liquid nitrogen and stored at −80 °C until analyzed. The small intestine was removed, and intestinal contents were collected by finger stripping and stored at 4 °C until viscosity was measured as described below, within 6 h of collection. Intestinal contents supernatant viscosity Contents were centrifuged at 19,500g for 45 min at 30 °C, the supernatant was collected, and viscosity of a 0.5-ml sample was measured at 37 °C with a Wells-Brookfield cone/plate viscometer, model LVT-CP (Wells-Brookfield Engineering Labs, Stoughton, MA), using a CP-51 cone. Intestinal contents supernatant viscosity was measured at 37 °C at all shear rates between 1.15 and 230 s−1 within 6–7 h of collection. Since all viscous dietary fibers display non-newtonian behavior (i.e., the viscosity changes with shear rate), viscosity values at each shear rate were plotted on a common log–log scale. The regression line was extrapolated to a common shear rate of 23 s−1 for all samples, and the resulting viscosity was expressed in millipascals seconds (mPa·s). Plasma adipokines, insulin, and glucose Plasma leptin, adiponectin (total), and insulin concentrations were measured using commercial rat-specific radioimmunoassay kits (Linco Research, St. Charles, MO). Plasma resistin was measured using a ­commercial rat-specific ELISA kit (B-Bridge International, Mountain View, CA). Plasma glucose was determined by a glucose oxidase method using a commercial kit (Autokit Glucose, Wako Chemicals USA, Richmond, WA). The homeostatic model assessment (HOMA) of insulin resistance was used to estimate insulin sensitivity (16), as HOMAIR = (FPI × FPG)/22.5, where FPI is fasting plasma insulin (mU/l) and FPG is fasting plasma glucose (mmol/l). Mitochondrial enzyme activities Citrate synthase (CS) and cytochrome C oxidase II activities were determined spectrophotometrically in muscle and liver homogenates using previously described methods (17). Briefly, ~50 mg of VOLUME 20 NUMBER 2 | february 2012 | www.obesityjournal.org articles Intervention and Prevention Immunoblot analysis AMPK activation was verified by immunoblot analysis of rat tissue extracts for the phosphorylated form of AMPK (Phospho-AMPKαThr172 (total α1 and 2; AMPK-p) as well as immunoblot of total AMPK (AMPK-t) probed as a control. To minimize the effects of hypoxia and other confounders, tissue samples were quickly immersed (before thawing in the immunoblot process) into a protease inhibitor cocktail containing adenine-9-β-d-arabinofuranoside hydrate, an AMP analog and competitive AMPK inhibitor (17). Briefly, protein (50 μg) from liver and muscle homogenates were resolved by SDS-PAGE and transferred to polyvinylidenedifluoride membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 2% BSA in TBS (25 mmol/l Tris, 135 mmol/l NaCl, 2.5 mmol/l KCl)/0.05% Tween 20 (TBST) for 1 h at room temperature. The membranes were assayed with pAMPK or total AMPK-specific antibodies (with 2% BSA in TBST overnight at 4 °C), followed by secondary antibodies conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA; all antibodies). Bands were visualized by enhanced chemiluminescence and quantified by laser densitometry in the linear range. Linearity of the detection system with this amount of protein loading was verified in experiments by assessment of β-actin protein expression. using the SAS System for Windows, release 9.1 (SAS Institute, Cary, NC). Intestinal contents supernatant variances were not normally ­distributed and therefore, the values were log-transformed prior to ­statistical analysis. Pearson correlations were determined between ­certain variables. P < 0.05 was taken for statistical significance. Results Body weights and food intake Animals consuming HPMC had a significantly lower final body weight, compared to the cellulose-fed group (220 ± 6 vs. 251 ± 8, respectively, P = 0.001). Additionally, the HPMC group displayed a lower growth rate beginning at week 3 and had a lower body weight plateau relative to the cellulose group (Figure 1). Overall, the average 24-h food intake did not differ significantly between the HPMC groups and cellulose group (22.8 ± 0.5 g/day vs. 23.3 ± 0.6 g/day, P > 0.05). Weekly 24-h food intake is shown in Figure 2. Food intake was slightly lower in the HPMC group in the first week but did not differ in subsequent weeks from the cellulose group, such that the estimated 500 450 Cellulose HV- HPMC * * 5 Final * 400 Body weight (g) skeletal muscle was weighed and diluted 20-fold and homogenized in extraction buffer (0.1 mol/l KH2PO4/Na2HPO4, 2 mmol/l EDTA, pH 7.2). CS activity was measured at 37 °C in 0.1 mol/l Tris-HCl (pH 8.3) assay buffer containing 0.12 mmol/l 5,5′-dithio-bis (2-nitrobenzoic acid) and 0.6 mmol/l oxaloacetate. After an initial 2-min absorbance reading at 412 nm, the reaction was initiated by the addition of 3.0 mmol/l acetyl-CoA, and the change in absorbance measured every 10 s for 7 min. Cytochrome C oxidase II activity was measured at 25 °C in 0.03 mol/l K-phosphate buffer containing reduced cytochrome c (2 mg/ml) and 4 mmol/l sodium hydrosulfite. The reaction was initiated by the addition of sample and the change of absorbance measured every 10 s for 5 min at 550 nm. Enzyme activity is expressed as μmol product/min/mg protein. * 350 300 250 200 * 150 Statistical analysis Group comparisons were carried out by analysis of variance, using least square means, due to differences in sample size between the groups, obesity | VOLUME 20 NUMBER 2 | february 2012 100 Initial 1 2 3 4 Week Figure 1 Body weight change in animals fed either cellulose or HPMCbased diets. Values represent mean ± s.e.m., n = 13 for cellulose and 26–27 for HPMC. HPMC, hydroxypropyl methylcellulose. 26 Cellulose Daily food intake (g/day) Gene expression Total RNA isolation (100 mg of muscle or liver) was undertaken using the method of Chomczynski and Sacchi (18). Briefly, tissue was homogenized in 2 ml of TRIZOL (acid-phenol method; Life Technologies, Grand Island, NY) and 500 μl of chloroform added to the homogenate, vortexed, and incubated at room temperature for 5 min. The mixture was centrifuged (13,000g, 15 min, 4 °C) and the aqueous phase transferred to a new tube. An equal volume of ice-cold isopropanol was added, incubated at room temperature for 10 min, RNA pelleted by centrifugation at 13,000g for 15 min at 4 °C and the supernatant decanted. The RNA was washed with 1 ml of 75% ethanol, centrifuged at 7,500g for 5 min at 4 °C. The pellet was then resuspended in 1 ml of 75% ethanol and further purified with RNeasy columns (Qiagen, Valencia, CA) to remove genomic DNA. Samples were redissolved in 35 μl of nuclease-free water. The quality and quantification of RNA was determined using a microfluidic Bioanalyzer spectrophotometer (Agilent, Foster City, CA) at 260/280 nm, and the average optical density ratio 260/280 was 1.91 ± 0.02 for both tissues. Real-time (q) RT-PCR was used to quantify the mRNA level of each gene (Applied Biosystems, Foster City, CA). Real-time PCR was carried out using 30 ng of total RNA(~0.3 ng mRNA) on a Bio-Rad I Cycler (Bio-Rad). PCR conditions were 48 °C for 30 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Each gene expression was normalized for cyclophilin A or RPLPO expression. Each sample was run induplicate, and the mean value was normalized for the control transcript level. 24 HV-HPMC 22 20 * 18 16 1 2 3 4 5 Week Figure 2 Food intake in animals fed either cellulose or HPMC-based diets. Values represent mean ± s.e.m., n = 13 for cellulose and 26–27 for HPMC. HPMC, hydroxypropyl methylcellulose. 351 articles Intervention and Prevention Table 1 Epididymal fat pad weight at the end of the experiment and body composition change over the first 5 weeks of feeding as measured by dual-energy X-ray absorptiometry Table 2 Plasma glucose, HOMA IR, and plasma hormone concentrations in the fasting or postprandial state Parameter Cellulose Parameter Glucose, fasting, mmol/l 7.95 ± 0.37 7.39 ± 0.18 Glucose, postprandial, mmol/l 15.18 ± 0.89 13.59 ± 0.46# Insulin, fasting, µU/ml 20.89 ± 1.11 19.35 ± 0.45 HOMAIR 7.32 ± 0.65 6.39 ± 0.25 Leptin, fasting, ng/ml 1.83 ± 0.13 1.00 ± 0.08*** Cellulose HPMC Epididymal fat pad weight, g (weight as percent of final body weight) 7.73 ± 0.43 (1.81 ± 0.09) 5.49 ± 0.28** (1.41 ± 0.06**) Adipose tissue gain, g (adipose tissue, % of final body weight) 83.9 ± 3.6 (29.3 ± 0.8) 65.5 ± 2.4* (26.3 ± 0.5)* Lean tissue gain, g (lean tissue, % of final body weight) Bone mineral gain, g (bone mineral, % of final body weight) 192.1 ± 5.7 (75.5 ± 1.1) 185.0 ± 4.3 (80.5 ± 0.6)* 7.4 ± 0.2 (2.6 ± 0.0) 6.4 ± 0.1* (2.6 ± 0.0) Values represent mean ± s.e.m., n = 13 for cellulose and 26–27 for HPMC. *P < 0.005. **P < 0.0001. total food intake did not differ between the HPMC and cellulose groups (724 g vs. 748 g, respectively, P = 0.29). Further, the area under the food intake curve also did not differ between the HPMC and cellulose groups (568.6 ± 11.4 vs. 582.2 ± 13.2 g/rat, respectively, P = 0.47). Intestinal contents supernatant viscosity The viscosity of the supernatants from the HPMC animals was significantly greater than those from the cellulose group, which had essentially no viscosity (1210 ± 221 vs. 3 ± 1 mPa·s, respectively, P = 0.001). Epididymal fat pad weight and body composition by DXA Fat pad weight, as either absolute weight or as a percentage of final body weight, was significantly less in the HPMC group relative to the cellulose group (Table 1). Table 1 shows the changes in tissue weight gain, and tissue weight as a percent of final body weight, over the first 5 weeks of the study period, as determined by DXA. Adipose tissue gain was less in the HPMC group relative to the cellulose group, as was adipose tissue expressed as a percent of final body weight (P < 0.002). Lean tissue gain did not differ between the groups. However, when expressed as a percent of final body weight, the HPMC group had significantly greater lean tissue than the cellulose group (P < 0.001). Bone mineral gain was greater in the cellulose group than the HPMC group (P < 0.005); however, when expressed as a percent of final body weight, there was no difference between the groups. Plasma hormones and glucose There were no statistically significant differences in plasma fasting and insulin concentrations between the cellulose and HPMC groups, although there was a trend for a lower postprandial glucose concentration in the HPMC group (P = 0.081) (Table 2). HOMAIR was lower in the HPMC group, but this difference did not achieve statistical significance (P = 0.11). Plasma adiponectin did not differ between the two groups (P = 0.16). However, fasting concentrations of both leptin and resistin were significantly lower (P = 0.001 and P = 0.005, respectively) in the HPMC group relative to the cellulose group. Postprandial plasma leptin concentration was 352 Leptin, postprandial, ng/ml Resistin, postprandial, ng/ml 4.27 ± 0.61 90.71 ± 4.45 Adiponectin, postprandial, µg/ml 1.54 ± 0.16 HPMC 2.61 ± 0.33* 70.65 ± 4.23** 1.82 ± 0.11 Values represent mean ± s.e.m., n = 13 for cellulose and 26–27 for HPMC. HOMA, homeostatic model assessment. *P = 0.012. **P = 0.005. ***P = 0.001. #P = 0.081 compared to cellulose group. Table 3 Enzyme activity in liver and muscle tissue in animals fed either cellulose or HPMC-based diets Tissue Cellulose HPMC Cytochrome C oxidase II (Complex IV) (µmol/min/mg protein) Liver Soleus muscle 5.40 ± 0.40 5.79 ± 0.11 74.42 ± 10.97 81.59 ± 7.89 Citrate synthase activity (µmol/min/mg protein) Liver 10.35 ± 0.76 Soleus muscle 7.35 ± 0.68 11.08 ± 0.21 9.01 ± 0.44* Values represent mean ± s.e.m., n = 13 for cellulose and 26–27 for HPMC. *P < 0.05 compared to cellulose group. also significantly reduced in the HPMC group compared to the cellulose group (P = 0.012). Liver and muscle enzyme activity Based on the body composition changes in the HPMC group, we next measured the activities of cytochrome C oxidase II (a marker of electron transport chain activity) and CS (a marker of mitochondrial mass and TCA cycle activity) in soleus (oxidative fiber) muscle (Table 3). Cytochrome C oxidase activity did not differ between the cellulose and HPMC groups in either the liver or soleus muscle. CS activity did not differ between the cellulose and HPMC groups in the liver. However, CS activity was significantly greater in the soleus muscle of the HPMC group compared to the cellulose group (P = 0.041). We also observed a trend for increased hepatic mtDNA content in the HPMC group relative to the cellulose group, but this did not reach statistical significance (280.0 ± 30.2 vs. 372.5 ± 24.7 (mitochondrial copy number/nuclear genome), respectively, P = 0.06). AMP kinase Activation of AMP kinase, estimated by the ratio of the immunologic determination of the relative amounts of the total and phosphorylated AMP kinase, was determined in soleus muscle and liver (Figure 3). The AMPK-p/AMPK-t was dramatically increased in the soleus muscle of the HPMC group compared to VOLUME 20 NUMBER 2 | february 2012 | www.obesityjournal.org articles Intervention and Prevention PCR (Table 4). Consistent with the increase in CS activity (Table 3), the expression levels of CPT-1B, PGC-1α, PPARδ, and UCP3 were all significantly greater in the soleus muscle in the HPMC group relative to the cellulose group. PPARα and MCD expression did not differ between the two groups. Liver. The expression of candidate genes involved in gluconeogenesis, free fatty acid oxidation, oxidative transcription, and mitochondrial energetics were determined by real-time PCR (Table 4). The expression level of PEPCK was significantly lower in the HPMC group relative to the cellulose group (P < 0.02). PGC-1α, PPARα, PPARδ, UCP3, and MCAD expression did not differ between the groups. AMPK-p/AMPK-total (A.U.) 10 Cellulose HPMC 8 ** 6 4 * 2 0 Liver Soleus muscle Figure 3 Ratio of phosphorylated AMP kinase to total AMP kinase in liver and soleus muscle in animals fed either cellulose or HPMC-based diets. Values represent mean ± s.e.m., n = 13 for cellulose and 26–27 for HPMC. *P = 0.004 compared to cellulose group; **P = 0.022 compared to cellulose group. HPMC, hydroxypropyl methylcellulose. Table 4 Expression of genes in soleus muscle and liver in animals fed cellulose or HPMC-based diets Gene mRNA/RPLPO mRNA (arbitrary units) Cellulose HPMC Soleus muscle CPT-1B 0.36 ± 0.03 0.45 ± 0.03* PGC-1α 0.33 ± 0.03 0.49 ± 0.05* PPARα 1.62 ± 0.61 0.94 ± 0.30 PPARδ 0.36 ± 0.03 0.45 ± 0.03* UCP3 33.0 ± 3.0 48.8 ± 4.8* MCAD 0.14 ± 0.06 0.14 ± 0.02 PEPCK 0.061 ± 0.011 PGC-1α 0.96 ± 0.21 1.50 ± 0.17 PPARα 0.0097 ± 0.0018 0.0068 ± 0. 0012 PPARδ 1.69 ± 0.33 2.08 ± 0.25 Liver 0.035 ± 0.004** UCP3 1.96 ± 0.50 1.67 ± 0.19 MCAD 0.015 ± 0.004 0.016 ± 0.002 Values are means ± s.e.m., n = 12 for cellulose group and 23–24 for the HPMC group. CPT-1B, carnitine palmitoyltransferase 1B; MCAD, medium-chain acyl-CoA dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; PPARα, peroxisome proliferatoractivated receptor-α; PPARδ, peroxisome proliferator-activated receptor-δ; UCP3, uncoupling protein 3. *P < 0.05. **P < 0.014. the cellulose group (P = 0.02), indicating greater AMPK activation in the HPMC group. AMPK activation was also increased in the livers of animals fed HPMC, relative to the cellulose group (P < 0.004), although the magnitude of the increase was less. Gene expression Soleus muscle. The expression of candidate genes involved in free fatty acid transport and oxidation, oxidative transcription, and mitochondrial energetics were determined by ­real-time obesity | VOLUME 20 NUMBER 2 | february 2012 Discussion Viscous dietary fibers are well established to have two important physiological effects, reducing the postprandial glucose response to a meal and lowering plasma cholesterol concentrations. In the series of experiments reported here, we measured adiposity using three different approaches: fat pad weight, plasma leptin concentration, and direct measurement of body composition by DXA. We present evidence that viscous dietary fibers reduce fat mass as well as plasma concentration of leptin and resistin. Previous work from our laboratory indicates that leptin concentration is strongly and inversely correlated to intestinal contents viscosity after feeding either nonfermentable (HPMC) or fermentable (oat β-glucans) dietary fibers (data not shown), suggesting that the reduction in adiposity observed in this study was proportional to intestinal contents viscosity produced by consumption of a viscous dietary fiber. This effect of fiber viscosity on adiposity is unlikely to be due to a decrease in fat absorption, as we have previously shown that glucomannan, a highly viscous dietary fiber, does not increase fecal fat excretion in rats (19). Our study stands in contrast to that of others, who found that feeding hamsters a high fat diet containing 4% HPMC reduced adipose tissue weight only in proportion to the body weight reduction, and found no significant decrease in plasma leptin concentrations compared to cellulose-fed hamsters (20), indicating no change in body composition. The reason for this inconsistency is unclear. Diet-induced reductions in adiposity, in the absence of changes in body weight, have been reported in rats and mice fed low glycemic index diets, relative to high glycemic diets (21), at least over the short-term. Interestingly, however, a recent study has shown that obesity-prone mice fed a diet of 10% guar gum, a viscous but highly fermentable dietary fiber, had an equivalent fat mass to mice fed insoluble (and presumably nonviscous) fiber up to 15 weeks of feeding, but had much greater fat mass with long-term feeding (from 27 weeks to 43 weeks) (22). This suggests that a large amount of fermentable fiber in the diet may nullify the reduction in adiposity produced by fiber viscosity and, over the long term, actually lead to an increase in adiposity. Similar to our finding of lower plasma leptin in animals with less adipose tissue, plasma leptin concentration was also lower in rats fed a low glycemic index diet (21). The nominal differences in body weight between the cellulose control and HPMC groups despite changes in adiposity 353 articles Intervention and Prevention may be attributed to increased lean body mass in the HPMC group (expressed as a percentage of final body weight) and an elevation in energy expenditure and an elevation in mitochondrial biogenesis. Consistent with this hypothesis, we found an increase in PGC-1α and PPARδ expression and greater CS activity in the muscle of the HPMC group. PGC-1α is a ­principal regulator of cellular energetics while PPARδ is linked to skeletal muscle mass gains and oxidative fiber type conversion (23). Thus, consumption of a viscous fiber appeared to lead to a significant change in body composition and contribute to muscle mass formation and/or preservation. As food intake was comparable between groups, differences in energy intake do not seem to explain the differences in body composition. AMPK acts as a sensor of cellular energy status and serves to regulate energy supply and demand. In response to energy deficit, the enzyme is activated by phosphorylation of Thr172 on the α-subunit in response to increases in intracellular AMP, whereupon it phosphorylates a number of downstream targets, thereby switching on energy-yielding pathways, such as ­glucose transport and fatty acid oxidation, and inhibiting pathways that consume ATP (24). Activation of AMPK increases muscle oxidative fiber type conversion and decreases adiposity (25), in part, by activation of PPARδ (25) and PGC-1α (12). Consistently, AMPK activation in the soleus muscle was significantly elevated in the HPMC group relative to the cellulose group, as well as key genes involved in lipid oxidation (CPT-1B and UCP3). The increase in PGC-1α is of particular note, as it directly coactivates a number of transcription factors, including PPARδ (9), which in turn activates UCP3 (23), as well as leads to increased mitochondrial biogenesis (26). Before β-oxidation can occur, long chain fatty acids must be bound to CoA, then translocated into the mitochondria by the enzyme complex CPT1. Downstream of CPT-1B, the upregulation of UCP3 in situations of increased lipid supply to the muscle is thought to play a role in fatty acid handling, possibly related to preventing mitochondrial oxidative stress (27). Collectively, these results suggest that the muscle of the animals fed HPMC was transcriptionally “remodeled” to increase mitochondrial content and favor lipid oxidation and a decrease in adiposity and gains in lean body mass. We hypothesize that the “fat-depleting” effects of the HPMC were mediated, in part, by an increase in muscle mitochondrial energetics, as evidenced by an increased expression of genes related to mitochondrial biogenesis, ­oxidative fiber type composition, lipid oxidation, and CS activity (marker of TCA cycle flux and mitochondrial biogenesis) (17). To our surprise, we observed an activation of AMPK in the liver of animals fed HPMC compared to cellulose-fed animals, but no modulation of mitochondrial markers. The mechanism(s) by which HPMC may activate hepatic AMPK are not clear, but may relate to the well-known decrease in postprandial plasma insulin concentrations induced by viscous fibers. Supportive of this hypothesis, insulin is known to decrease glucagon and inhibit AMP kinase activity in hepatocytes (28). Thus, a reduced postprandial insulin response in the HPMCfed animals may explain the greater AMPK activation in this 354 group. Consistent with the results from the current study, mice deficient in resistin show greater activation of AMPK compared to normal mice (29) suggesting reduced concentrations of plasma resistin in HPMC-fed animals may have contributed to the greater hepatic activation of AMPK. We also noted elevated hepatic AMPK phosphorylation was associated with reduced PEPCK expression (30), thereby favoring lower rates of gluconeogenesis (31). The effect of viscous dietary fiber on circulating concentrations of several adipokines was examined. Leptin has a number of physiological effects but, broadly speaking, acts to regulate food intake and energy expenditure by modulating neural ­circuits. Leptin and adiponectin both influence insulin ­sensitivity and whole body energy metabolism (32), and both adipokines have been linked to improvements in mitochondrial function in skeletal muscle (17). The change in body composition in animals fed viscous fiber was accompanied by decreases in the plasma concentrations of leptin in both the fasting and postprandial states and resistin in the fasting state. As leptin concentrations vary in proportion to adipose mass (33), the reduction in plasma leptin is consistent with the reduction seen in fat pad weight in the animals fed HPMC. Although the physiological function of resistin in humans remains uncertain, studies in rodents indicate a role in mediating hepatic (34) or muscle insulin resistance (35). As plasma resistin concentrations are increased in genetic models of obesity as well as diet-induced obesity (36), its reduction in the HPMC group relative to the cellulose group further supports the reduction in adiposity by viscous fiber. Adiponectin appears to be unique among the adipokines, in that its plasma concentration varies inversely with adiposity (37). Plasma adiponectin has an insulin sensitizing effect in both muscle and liver (38) and a thermogenic effect (enhanced lipid metabolism) in skeletal muscle (17). Recent studies in obese-diabetic Zucker Diabetic Fatty rats demonstrate ­feeding HPMC results in a significant elevation of plasma adiponectin relative to a group fed cellulose (D.A. Brockman, X. Chen, & D.D. Gallaher, unpublished data). However, we observed a nonsignificant increase in plasma adiponectin in the HPMC group, suggesting that the effect of viscous fiber on plasma adiponectin may depend on the underlying physiological state. We report for the first time that consumption of a viscous dietary fiber leads to a spectrum of metabolic changes that include a reduction in adiposity and an increase in lean body mass, accompanied by decreased plasma concentrations of the adipokines leptin and resistin, an increase in markers for fatty acid oxidation and mitochondrial biogenesis in muscle. Our results suggest that consumption of viscous dietary fiber may aid in reducing adiposity and thus yield health benefits beyond the well-established effects of lowering plasma cholesterol concentrations and improving plasma glucose control. Further investigation of the effect of viscous dietary fiber on adiposity and fuel utilization seems warranted. In particular, as our study was of relatively short duration, longer-term studies are needed to confirm the persistence of the effects noted in the present study. VOLUME 20 NUMBER 2 | february 2012 | www.obesityjournal.org articles Intervention and Prevention Acknowledgments This work was sponsored by Imagenetix, Inc., San Diego, CA, and the Minnesota Agricultural Experiment Station. Disclosure The authors declared no conflict of interest. © 2011 The Obesity Society REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Centers for Disease Control and Prevention. U.S. Obesity Trends. 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