Viscous Dietary Fiber Reduces Adiposity

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 ([email protected])
Received 27 April 2011; accepted 4 October 2011; published online 17 November 2011. doi:10.1038/oby.2011.341
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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
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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,
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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.
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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
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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
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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
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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
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