Effect of hydroxypropyl methylcellulose on obesity and glucose metabolism in a diet-induced obesity mouse model

Authors


Scott A. Young, 1897 Building, E-24, The Dow Chemical Company, Midland, MI 48667, USA.
Tel: +1 989 636 8728
Fax: +1 989 638 6443
Email: SAYoung@dow.com

Abstract

Background:  To investigate the effect of hydroxypropyl methylcellulose (HPMC) on weight loss and metabolic disorders associated with obesity using a high-fat diet-induced obese mouse model under a high-fat diet regimen.

Methods:  Obese male C57BL/6J (B6) mice were fed either a high-fat (60% kcal), low-fat (10% kcal), or high-fat diet plus HPMC (4% and 8%) for 5 weeks. Body, mesenteric adipose, and liver weights were determined at the end of the study. In addition, plasma cholesterol, insulin, glucose, adiponectin, and leptin were analyzed to determine the effects of HPMC. Hepatic and fecal lipids were measured to determine the effect of HPMC on lipid absorption and metabolism.

Results:  Supplementation of the high-fat diet with 4% and 8% HPMC resulted in significant weight loss in obese B6 mice. Furthermore, significant decreases were seen in adipose (30%–40%), liver weights (15%–26%), and concentrations of plasma cholesterol (13%–20%) and hepatic lipids (13%–36%). Supplementation with 8% HPMC led to significant improvements in glucose homeostasis and leptin concentrations. Reductions in plasma cholesterol, glucose, and insulin levels were strongly correlated with reduced leptin concentrations. Moreover, increases in fecal secretion of total bile acids, sterols, and fats indicated altered fat absorption when HPMC was incorporated in the diet.

Conclusion:  The data indicate that HPMC not only reduces body weight, but also normalizes the metabolic abnormalities associated with obesity and suggest that the effects of HPMC on glucose and lipid homeostasis in B6 mice are mediated by improvements in leptin sensitivity resulting from reduced fat absorption.

Introduction

The increase in obesity and related clinical pathologies to ‘epidemic’ levels in all parts of the world has been documented exhaustively in recent years and it is clear that multiple sociological and pharmaceutical approaches will have to be implemented to address the issue. Although adopting a lifestyle with improved diet quality, reduced caloric intake, and increased exercise would be an effective solution, it is clear that many individuals would benefit from dietary supplements or drugs that would support and enhance the necessary lifestyle changes.

Hydroxypropyl methylcellulose (HPMC) is a non-fermentable soluble dietary fiber that has been used in the manufacturing of many foods for several decades.1 It has a long safety record and generally recognized as safe (GRAS) status up to 20 g/day in the US.2 Several human trials have shown clear health benefits when the diet is supplemented with HPMC. For example, 2 g/day HPMC reduces plasma glucose and insulin levels in fasting studies3 and reduces the glycemic index when added directly to high index foods.4 Furthermore, numerous studies have shown that supplementation of the diet with medium to high viscosity HPMC over extended periods can reduce plasma total cholesterol, low-density lipoprotein–cholesterol (LDL-C) and very low-density lipoprotein–cholesterol (VLDL-C) while having only a minor effect on high-density lipoprotein–cholesterol (HDL-C).5–8 In addition, extensive studies in a Syrian Golden Hamster model have demonstrated increases in the fecal excretion of bile acids and neutral sterols,9 suggesting that the changes in plasma lipid may be due, at least in part, to interference with bile acid recycling. This has been confirmed by a recent study of hepatic gene expression levels in the hamster, in which upregulation of bile acid synthesis and export were observed.10

In preliminary studies, maturing hamsters on a high-fat diet supplemented with HPMC put on significantly less body weight than control animals, due primarily to reduced deposition of abdominal fat tissue and fat accumulation in the liver and skeletal muscle.11 This suggests that HPMC may also be of use in healthy weight management by reducing fat deposition.12 Herein, we describe the use of a broadly accepted obesity model to further characterize this observation.

The characteristic of C57BL/6J (B6) mice to develop human-like Type 2 diabetes symptoms when maintained on a high-fat diet for extended periods was first described in 1988.13 Similarities with the broader human metabolic syndrome have been documented14 and have been reviewed by Collins et al.15 After exposure to a diet consisting of 60% calories from fat for 16 weeks, this mouse strain reversibly develops obesity, hyperinsulinemia, hyperglycemia, and hypertension. Moreover, fat is specifically laid down in the mesentery, despite increased lipoprotein lipase activity levels and no hyperphagia.16 These effects are thought to be due to a failure of adrenergic control of adipocyte function15 and an increase in leptin resistance, which are modulated by environmental influences such as the nature and availability of the diet.17 This diet-induced obesity (DIO) model has been used extensively in elucidating underlying mechanisms of obesity,15,18 as well as in the testing of the efficacy of potential treatments.19

In the present study, we investigated the anti-obesity effects of 4% and 8% HPMC on obese B6 mice maintained on a high-fat diet over a period of 5 weeks. In addition, the effect of HPMC on glucose and lipid metabolism in the diet-induced obese mice was examined. The role of adipocytokines in the regulation of glucose and lipid metabolism associated with reduced fat accumulation by HPMC was further studied to delineate the effect of HPMC intake on metabolic risk factors associated with obesity.

Methods

Animal model and diets

Obese male B6 mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). These mice were fed a high-fat diet (60 kcal% fat, D12492; Research Diets, New Brunswick, NJ, USA) from 6 weeks of age. As the gold standard for the DIO model, B6 mice develop an obese phenotype after ad libitum access to a high-fat diet.13,15 Mice were housed individually in polycarbonate cages maintained at normal temperature (22 ± 4°C) with a relative humidity of 50 ± 15% and a light period from 0600 to 1800 h. Obese B6 mice at a starting age of approximately 18 weeks were acclimated on a 60 kcal% high-fat diet for 2 weeks. After acclimation, B6 mice were weighed and randomized into four diet groups of 10 mice each: two control groups, one fed a high-fat diet (60 kcal% fat) and the other fed a low-fat diet (10 kcal% fat, D12450B; Research Diets); and two treatment groups, both fed a high-fat diet but supplemented with either 4% or 8% (weight percentage of the diet) HPMC (K250M; lot no. VJ241907R1; Dow Chemical, Midland, MI, USA). The experimental diets supplemented with 4% or 8% HPMC were prepared by mixing 96% and 92% high-fat diet with 4% and 8% HPMC, respectively. Both the high-fat and low-fat synthetic diets contain 6.5% insoluble fiber. After the addition of 4% and 8% HPMC to the high-fat diets, the amount of insoluble fiber in these two diets is 6.2% and 5.9%, respectively. All mice were maintained on their assigned diets for 5 weeks. The animal study was conducted by Perry Scientific (PSI, San Diego, CA, USA) and adhered to the regulations outlined in the US Department of Agriculture DA Animal Welfare Act (nine CFR, Parts 1, 2, and 3) and the Institute for Laboratory Animal Research (ILAR) guidelines).20 The study protocol was approved by PSI’s Institutional Animal Care and Use Committee prior to initiation of the study.

Plasma and tissue collection

At the end of the study, mice were fasted for 12 h before being killed. Blood samples were collected by cardiac puncture and EDTA plasma samples were obtained after centrifugation of the blood sample at 2000 g for 30 min at 4°C. Plasma samples were stored at −80°C until analysis. Liver and visceral adipose tissues were weighed and frozen immediately in liquid nitrogen. The frozen liver samples were freeze-dried for lipid analysis.

Plasma biochemical determinations

Plasma triglycerides, total cholesterol, and free cholesterol levels were determined by enzymatic colorimetric assays using the Roche Diagnostics/Hitachi 914 Clinical Analyzer with assay kits from Roche Diagnostics (Indianapolis, IN, USA) and Diagnostic Chemicals (Oxford, CT, USA). In addition, concentrations of plasma lipoproteins, LDL-C, and HDL-C were determined using L-type LDL-C and L-type HDL-C assay kits from Roche Diagnostics and Wako Chemicals (Richmond, VA, USA), respectively. The VLDL-C levels were calculated as follows: total cholesterol – (HDL-C + LDL-C). Plasma concentrations of adiponectin (B-Bridge International, Sunnyvale, CA, USA), leptin (R&D Systems, Minneapolis, MN, USA), and insulin (Mercodia, Winston Salem, NC, USA) were measured using enzyme immunoassay methods according to the procedures provided with the kits used. In addition, fasting glucose levels were measured by collecting blood from each mouse by the tail-prick method. A drop of blood collected using a sterile needle was analyzed using a OneTouch Ultra meter with FastDraw test strips (Johnson and Johnson, Milpitas, CA, USA).

Analysis of hepatic lipids

Livers were collected, lyophilized, and milled to a fine powder before extraction of liver lipids using a Dionex accelerated solvent extractor (Dionex, Sunnyvale, CA, USA) with 75/25 hexane/2-propanol at 100°C and approximately 13.8 MPa. The solutions extracted were dried and weighed to determine the weight percentage of total lipids in the liver. Hepatic concentrations of triglycerides, total cholesterol, and free cholesterol were determined using colorimetric assays and a clinical analyzer, as described above.

Analysis of fecal lipids

Feces from each of the mice in the high-fat diet group and the high-fat diet groups supplemented with 4% and 8% HPMC were collected for two consecutive days at the end of the study. Fecal samples were lyophilized, milled, and extracted using a mixture of hexane and 2-propanol (3:2, v/v, 2% acetic acid) at approximately 13.8 MPa and 60°C for 30 min. The extracts were divided into two aliquots. One aliquot was analyzed for total bile acids, sterols, and fats (fatty acids, monoglycerols, diglycerols, and triglycerols) using a liquid chromatography method.9 Saturated and unsaturated fats were determined in the second aliquot by gas chromatography (GC) analysis. The fecal fats were derivatized to fatty acid methyl esters (FAME) following the American Oil Chemists’ Society (AOCS) official method with a few modifications.21 Briefly, a 9-mL aliquot of fecal lipid extract was evaporated under a nitrogen stream. The extract was then reconstituted in 300 μL of 0.5 mol/L NaOH in methanol. After hydrolysis for 5 min at 100°C, the sample was cooled, followed by methylation using 14% BF3 in methanol. The derivatized samples were analyzed by GC using an Agilent 6890 series GC (Agilent, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and a DB-23 analytical column (Agilent; 60 m × 0.25 mm i.d., 0.25 μm film thickness). The initial oven temperature was set at 200°C, held for 5 min, then raised to 250°C at 5°C/min and held for 5 min. Analytes were quantified with the EZChrom Elite (Version 3.2.1) software data system (Agilent). A calibration solution containing 2 mg/mL each of methyl palmitate (C16:0), methyl stearate (C18:0), methyl oleate (C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) and 11 μg/mL methyl erucate (Nu-chek Prep, Elysian, MN, USA) was prepared in heptane.

Statistical analysis

Results are presented as the mean ± SD. One-way analysis of variance followed by the Tukey–Kramer honestly significant difference (HSD) test was used for multiple comparisons. The body weight profiles of the diet groups were also analyzed by repeated-measure analysis. Differences were considered significant at < 0.05. Changes in the end-points are expressed relative to levels in the high-fat control group, except where noted otherwise. Pearson’s correlation coefficient was determined to investigate the relationship between various biomarkers and parameters. Regression using both caloric intake and dose level as variables was performed to determine the dose–response effects of end-points in the high-fat diet and 4% and 8% HPMC-supplemented groups after treatment. JMP 8.0.2 and SAS 9.2 (SAS Institute, Cary, NC, USA) were used for the statistical analyses.

Results

Food intake, body and tissue weights, and fasting plasma lipid profile

To determine whether HPMC supplementation can reduce high-fat diet-induced obesity and the accompanying metabolic disorders, B6 mice were fed high-fat diets supplemented with either 4% or 8% HPMC. The weight characteristics and caloric intakes of these two diet groups were compared with both the high-fat and low-fat diet control groups over 5 weeks of treatment. A significant reduction in body weight was observed for the 4% HPMC, 8% HPMC, and low-fat diet groups compared with the high-fat diet group (Fig. 1), despite a similar food intake in all four groups (Table 1). There was no significant difference in caloric intake between the high-fat diet and 8% HPMC-supplemented groups. Not surprisingly, the low-fat diet group had a significantly lower caloric intake compared with the high-fat diet control and 8% HPMC-supplemented groups owing to the lower caloric value of the low-fat diet (< 0.05). Feed efficiency was determined as [weight gain (g)/total calories consumed (kcal)] × 100. Even though the 4% HPMC-supplemented group did have a significantly lower caloric intake (< 0.05) than the high-fat diet control group, both the 4% and 8% HPMC-supplemented groups showed similar feed efficiency compared with the low-fat diet group, which was significantly lower than that of the high-fat diet control group (< 0.05). Based on multiple linear regression analysis, both caloric intake (= 0.0002) and HPMC dose (< 0.0001) had significant effects on body weight gain.

Figure 1.

 Body weight of male C57BL/6J mice in groups fed either the high-fat diet (◆), the low-fat diet (⋄) or the high-fat diet supplemented with 4% (bsl00066) or 8% (bsl00001) hydroxypropyl methylcellulose for 5 weeks. Data are the mean ± SE (= 10). Data points at each time point that do not share the same letter differ significantly (< 0.05).

Table 1.   Food intake, tissue weights, and plasma lipid content
 Dietary group
High-fat dietHigh-fat diet + 4% HPMCHigh-fat diet + 8% HPMCLow-fat diet
  1. Data are the mean ± SD (= 10). Values within rows with different superscript letters differ significantly (< 0.05).

  2. HPMC, hydroxypropyl methylcellulose; TG, triglycerides; TC, total cholesterol; FC, free cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein.

Feed (g)/day4.3 ± 1.2a3.3 ± 0.7a4.3 ± 1.1a3.8 ± 0.4a
Calories (kcal)/day22.8 ± 6.3a16.5 ± 3.3bc20.9 ± 5.3ab14.6 ± 1.6c
Weight gain (g)4.41 ± 1.70a−2.60 ± 2.36b−5.34 ± 1.09c−2.89 ± 2.46b
Feed efficiency0.59 ± 0.27a−0.46 ± 0.41b−0.80 ± 0.34b−0.58 ± 0.49b
Mesenteric adipose weight (g)1.44 ± 0.28a1.01 ± 0.33b0.83 ± 0.21b1.13 ± 0.23ab
Liver (g)1.70 ± 0.36a1.44 ± 0.24ab1.25 ± 0.22b1.43 ± 0.18ab
TG (mg/dL)74.5 ± 13.5a53.5 ± 9.5b60.7 ± 8.6ab71.6 ± 15.2a
TC (mg/dL)194 ± 21a168 ± 13b156 ± 8b157 ± 16b
FC (mg/dL)58 ± 6a56 ± 14ab62 ± 14a45 ± 5b
LDL/HDL0.11 ± 0.02a0.08 ± 0.01b0.09 ± 0.01b0.10 ± 0.01ab

Furthermore, supplementation of the high-fat diet with HPMC elicited a significant (< 0.05) reduction in mesenteric adipose tissue weight (Table 1). The mesenteric adipose weight for the low-fat diet group tended to be less than that of the high-fat diet group, although the difference did not reach statistical significance (= 0.07). Among the two HPMC-supplemented groups, only the 8% HPMC-supplemented group exhibited a significant reduction in liver weight compared with the high-fat diet group (< 0.05). Although 15.3% and 15.9% reductions in liver weight were observed in 4% HPMC-supplemented and low-fat diet groups, respectively, the differences compared with the high-fat diet group were not significant (= 0.18). Regression analyses revealed a significant dose effect of HPMC on reductions in adipose (<0.0001) and liver (= 0.0067) tissue weight, whereas the effect on caloric intake was not significant.

Plasma total cholesterol levels were 13.4% and 19.6% lower in the 4% and 8% HPMC-supplemented groups, respectively (< 0.05; Table 1). Similarly, plasma triglyceride concentrations were 28.2% (<0.01) and 18.5% (= 0.07) lower in the 4% and 8% HPMC-supplemented groups, respectively. Furthermore, a significant dose-dependent effect of HPMC was found for plasma total cholesterol (< 0.0001) and triglycerides (= 0.0139). Significant reductions in both total and free cholesterol levels were observed for the low-fat group; however, there was no difference in the triglyceride concentrations between low-fat and high-fat diet groups. The cholesterol-reducing effect of HPMC was further investigated by analyzing plasma lipoprotein profiles. Significant reductions in LDL-C were observed in the 4% and 8% HPMC-supplemented groups, as well as in the low-fat diet control group (31.4%, 32.5%, and 24.6%, respectively; < 0.05; Fig. 2). Similarly, reductions in VLDL-C of 38.9%, 38.2%, and 60.4% were found in the 4% and 8% HPMC-supplemented groups and low-fat diet group, respectively (< 0.05; Fig. 2). No differences in HDL-C were observed between the 4% HPMC-supplemented and high-fat diet groups, whereas significantly lower HDL-C levels were observed in the 8% HPMC-supplemented and low-fat diet groups (< 0.05). Moreover, HPMC supplementation at both 4% and 8% improved the LDL/HDL ratio to 0.083–0.089 (< 0.05) compared with the ratio in the high-fat diet control group, in which the LDL/HDL ratio was determined to be 0.11. The LDL/HDL ratio of the low-fat diet group did not differ significantly from that in the high-fat diet group. Similar to the dose–response effect of HPMC in reducing total cholesterol, HPMC also reduced VLDL- and HDL-C in a dose-dependent manner (VLDL: = 0.002; LDL: = 0.0359; HDL: < 0.0001).

Figure 2.

 Lipoprotein profile of C57BL/6J mice after 5 weeks feeding of either the high-fat diet (□), the low-fat diet (bsl00025) or the high-fat diet supplemented with 4% (bsl00023) or 8% (bsl00001) hydroxypropyl methylcellulose. Data are the mean ± SD (= 10). Bars not sharing the same letter differ significantly (< 0.05).

Hepatic lipid levels

Because liver weight was reduced in mice in the 8% HPMC-supplemented group, the hepatic lipid levels in this group were improved to levels approaching those seen in the low-fat diet group (Table 2). Total lipid levels in the liver were significantly reduced in the 8% HPMC-supplemented and low-fat diet groups by 36.4% and 61.9%, respectively (< 0.05). The reduction of total lipid content in the 8% HPMC-supplemented group accounts for approximately 90% of the reduction in liver weight. In addition, significant reductions in triglyceride levels were observed in both the 8% HPMC-supplemented and low-fat diet groups (41.1% and 73.9%, respectively; < 0.05). However, no significant differences in hepatic total cholesterol and free cholesterol levels were observed in any of the four diet groups. Dose-dependent effects of HPMC on hepatic total lipid levels (= 0.0029) and triglyceride levels (= 0.0031) were observed.

Table 2.   Effect of hydroxypropyl methylcellulose supplementation on liver lipids levels
 Dietary group
High-fat diet + 4% HPMCHigh-fat diet + 8% HPMCHigh-fat dietLow-fat diet
  1. Data are the mean ± SD (= 10). Values within rows with different superscript letters differ significantly (< 0.05).

  2. HPMC, hydroxypropyl methylcellulose; TG, triglycerides; TC, total cholesterol; FC, free cholesterol.

% Lipid38.7 ± 9.9ab27.5 ± 7.9bc44.6 ± 11.7a17.0 ± 4.3c
TG (mg/g)248 ± 83ab159 ± 86bc286 ± 88a75 ± 30c
FC (mg/g)5.0 ± 1.3a5.0 ± 1.3a5.4 ± 1.4a4.2 ± 0.6a
TC (mg/g)6.6 ± 1.0a7.3 ± 2.0a5.9 ± 0.4a5.9 ± 1.3a

Plasma glucose, insulin, adiponectin, and leptin concentrations

Fasting plasma glucose levels after 5 weeks of feeding are shown in Fig. 3a, indicating significant reductions in the 4% and 8% HPMC-supplemented and low-fat diet groups of 21.9%, 26.7%, and 26.5%, respectively (< 0.05). In addition to reduced glucose levels, the low-fat diet group also exhibited significantly lower insulin levels (66.4%; Fig. 3b), whereas the HPMC-supplemented groups exhibited a tendency for reduced insulin levels with a 57.5% reduction noted in the 8% HPMC-supplemented group (= 0.08). The dose-dependent effect of HPMC on plasma glucose (=0.0034) and insulin (= 0.039) was significant.

Figure 3.

 Plasma concentrations of (a) glucose, (b) insulin, (c) leptin, and (d) adiponectin in C57BL/6J mice after 5 weeks feeding of either the high-fat diet (□), the low-fat diet (bsl00021) or the high-fat diet supplemented with 4% (bsl00004) or 8% (bsl00001) hydroxypropyl methylcellulose. Data are the mean ± SD (= 10). Bars not sharing the same letter differ significantly (< 0.05).

To understand the role of adipocytokines in the regulation of glucose and lipid metabolism, fasting plasma adiponectin and leptin levels were determined (Fig. 3c,d). Supplementation of the high-fat diet with either 4% or 8% HPMC resulted in a significant decrease in circulating leptin levels compared with levels in the high-fat control group (< 0.05). The leptin levels in the HPMC-supplemented groups were equivalent to those in the low-fat diet group. The reduction in leptin levels ranged from 45% to 51% in the 4% HPMC-supplemented and low-fat groups compared with levels in the high-fat diet group. In the 8% HPMC-supplemented group, a further reduction in leptin of 77% was noted. Conversely, no significant differences in adiponectin concentrations were observed, except in the 8% HPMC-supplemented group, which showed a reduction of 33% (< 0.05) compared with the high-fat control group. Both leptin and adiponectin levels were modulated by HPMC in a dose-dependent manner (leptin: < 0.0001; adiponectin: = 0.0019).

Fecal lipid analysis

Regardless of similar caloric intakes among the high-fat diet groups (i.e. the high-fat diet and 8% HPMC-supplemented groups), significant reductions in body weight were observed in the HPMC-supplemented groups. This suggests that HPMC supplementation was able to alter energy absorption. To examine the effect of HPMC on fat absorption, fecal lipid content was analyzed (Fig. 4). Excretion of total bile acids was increased significantly by 6.8- and 13.3-fold in the 4% and 8% HPMC-supplemented groups, respectively (< 0.05). The excretion of fecal sterols was 1.8- and 1.5-fold higher in the 4% and 8% HPMC-supplemented groups, respectively (< 0.005 and = 0.078 compared with the high-fat diet group, respectively). The excretion of total fat, including fatty acids, monoacylglycerols, diacylglycerols, and triacylglycerols, in the 4% and 8% HPMC-supplemented groups was 78.08 ± 19.00 and 83.30 ± 21.14 mg/g feces, respectively, significantly higher (>3.0-fold) than that in the high-fat diet group (25.66 ± 8.09 mg/g feces). As shown in Fig. 4, both saturated and unsaturated fatty acid levels were significantly increased in the 4% and 8% HPMC-supplemented groups (< 0.05). Dose-dependent effects of HPMC on enhanced fecal fat and bile acid excretion (< 0.05) were also observed (Fig. 4).

Figure 4.

 Fecal excretion of saturated fatty acids (SFA), unsaturated fatty acids (UFA), bile acids (BA), and sterols in C57BL/6J mice after 5 weeks feeding of either the high-fat diet (□), or the high-fat diet supplemented with 4% (bsl00025) or 8% (bsl00001) hydroxypropyl methylcellulose. Data are the mean ± SD (= 10). Bars not sharing the same letter differ significantly (< 0.05).

Correlation between metabolic parameters and circulating adipocytokines

Correlations between body weight gain, plasma leptin, and metabolic parameter levels were evaluated to identify any relationships. There was a close correlation between body weight gain and plasma leptin (Table 3). Because adipose weights are proportional to body weights, there was also a significant positive correlation between leptin and adipose weight. As indicated in Table 3, reduced weight gain was strongly associated with an improvement in several obesity-related risk factors, such as insulin resistance (glucose and insulin levels), a reduction of circulating total cholesterol (as well as VLDL-C and LDL-C), and reduced liver accumulation of lipids. Similarly, significant correlations were found between leptin and fasting glucose, fasting insulin, plasma cholesterol, hepatic total lipids, and hepatic triglyceride levels. Although plasma concentrations of another adipocytokine, namely adiponectin, were positively correlated with adipose weight (=0.4436, = 0.0047), there was no significant correlation between adiponectin and body weight gain.

Table 3.   Relationships between body weight gain or plasma leptin levels and other metabolic parameters and hepatic lipid levels
VariableWeight gain (g)Leptin (ng/mL)
rPrP
  1. VLDL-C, very low-density lipoprotein–cholesterol; LDL-C, low-density lipoprotein–cholesterol; HDL-C, high-density lipoprotein–cholesterol.

Weight gain (g)0.7647<0.0001
Adipose weight (g)0.7098<0.00010.7490<0.0001
Fasting glucose (mmol/L)0.6815<0.00010.6734<0.0001
Fasting insulin (mU/L)0.47250.00420.59240.0002
Adiponectin (μg/mL)0.25460.11780.25760.1184
Plasma total cholesterol (mg/dL)0.56140.00050.7142<0.0001
VLDL-C (mg/dL)0.53960.00080.48600.0031
LDL-C (mg/dL)0.23710.17020.46130.0053
HDL-C (mg/dL)0.51770.00140.7429<0.0001
Liver total lipids (weight %)0.7404<0.00010.8465<0.0001
Liver triglycerides (mg/g)0.7377<0.00010.8302<0.0001

Discussion

The health benefits of dietary supplementation with the soluble fiber HPMC, including reductions in plasma cholesterol and postprandial glucose, have been reported previously in both animal and human studies.3–8,22 In addition to the hypocholesterolemic and hypoglycemic effects of HPMC, significant lower body weight gain, adipose weight, liver weight, and hepatic lipid levels have been observed in Syrian Golden hamsters.10,22 Because the animals used in those studies were maturing, HPMC was only effective on weight gain reduction, not weight loss. In the present study, we demonstrated for the first time that HPMC supplementation of a high-fat diet was able to reverse the diet-induced obesity of mature B6 mice after 5 weeks. It has been proposed that HPMC exerts its effects on weight gain and cholesterol by increasing the viscosity of the contents of the small intestine or by reducing bile acids in the intestine.23 The increased excretion of bile acids and neutral sterols leads to reduced intestinal absorption of cholesterol. In addition, bile acids are involved in facilitating dietary fat processing in the intestine through micelle formation. Owing to the reduction in the availability of bile acids as a result of their enhanced excretion following HPMC supplementation, limited fat absorption leads to a lower amount of fat and lipid accumulated in adipose tissue and liver. The present study extended observations reported from a hamster study,9 showing that, in B6 mice, HPMC supplementation of high-fat diets increases the fecal excretion of bile acids and neutral sterols by 6–13-fold and approximately 1.5-fold, respectively. In addition, we observed a greater than threefold increase in total fecal fat resulting from the increased excretion of both saturated and unsaturated fatty acids. Higher lipid excretion in the 8% HPMC-supplemented group compared with the 4% HPMC-supplemented group was associated with a greater weight loss. These findings demonstrate that HPMC lowers lipid absorption in the gastrointestinal tract, resulting in a 30%–42% reduction in mesenteric adipose weight following supplementation with 4% and 8% HPMC, respectively. Furthermore, significant reductions in liver weight and liver lipid levels were observed, with values similar to those in the low-fat diet control group. As a non-fermentable dietary fiber, the liver triglyceride-lowering effect of HPMC is not mediated by direct inhibition of lipogenesis. However, the increase in fecal bile acid excretion has been linked previously to regulation of bile acid, cholesterol, and triglyceride metabolism in hamsters fed a high-fat diet supplemented with HPMC.10 The reduction in circulating and hepatic triglyceride levels was modulated by increased expression of genes involved in fatty acid oxidation (PPARα and ACOX) and decreased expression of fatty acid synthesis genes (SCD1 and FAS).10 In addition to its effects in altering lipid metabolism in liver, the effects of HPMC on overall triglyceride storage and mobilization in adipose tissue and major lipid depots of the body will provide additional insight into the mechanisms underlying HPMC-induced changes in fat absorption and metabolism.

Diet-induced obesity in B6 mice has been studied extensively. The risk factors associated with obesity are linked, in part, to the fat-induced hyperplasia of adipocytes, particularly in the mesenteric fat pad.13,14 Consequently, altered expression of adipocytokines secreted by adipose tissue regulates both glucose and lipid metabolism. For example, most adipocytokines, such as leptin and tumor necrosis factor-α, are increased, whereas adiponectin levels are decreased in obesity and Type 2 diabetes. Leptin resistance is associated with increased adiposity in diet-induced obesity in that the leptin level is elevated but functions with reduced efficacy owing to impaired signaling and transport.24,25 This leads to over accumulation of triacylglycerol in the muscle and liver, which is associated with insulin resistance.26 In the present study, we showed that dietary HPMC supplementation was able to reduce adipose tissue, liver weight, liver lipids, and triglycerides in a dose-dependent manner. Moreover, the reductions in adipose weight and hepatic lipids and triglycerides were associated with decreased leptin levels. Even though both leptin and adiponectin are involved in glucose and lipid metabolism, only leptin levels were found to be strongly correlated with improved insulin and glucose levels following HPMC supplementation. The correlation between plasma concentrations of insulin and leptin is consistent with previous reports in mice and humans.24,27 Therefore, these results indicate that HPMC supplementation of a high-fat diet is able to improve glucose metabolism by reducing lipid storage in insulin-sensitive tissues via improved leptin regulation.

In contrast with leptin, adiponectin levels are decreased in obesity, diabetes, and coronary heart diseases in human and animal models.28,29 However, such correlations between adiponectin and body/tissue weight have not been observed consistently in B6 mice.30,31 In the present DIO mouse study, significant decreases were observed in adiponectin levels in the 8% HPMC-supplemented diet group compared with the high-fat diet group. The low-fat diet group did not exhibit significant differences in adiponectin compared with the high-fat diet group. The lack of any significant increase in total plasma adiponectin levels following supplementation of the high-fat diet with HPMC is consistent with other studies using the same mouse model and similar diet regimens.32 Evidence of reduced adiponectin receptor levels in insulin-sensitive tissues, such as the muscle and liver, has been reported as a potential mechanism underlying the insulin-sensitizing effect of adiponectin in this mouse model.30 Alternatively, circulating concentrations of high molecular weight (HMW) adiponectin multimer instead of total adiponectin concentrations have been reported as an indicator of obesity and diabetes.33 Because the mechanism underlying the effect of HPMC on adiponectin was not elucidated, further studies evaluating the regulation of adiponectin and other adipocytokines following improvements in glucose and lipid metabolism are warranted. Moreover, changes in proinflammatory cytokine levels following HPMC supplementation could be a potential mechanism underlying the effects of HPMC in inflammatory and metabolic disorders.

Supplementation of the high-fat diet with HPMC decreased the atherogenic lipid profile in B6 mice, similar to hamsters and humans. Specifically, HPMC not only reduced plasma total cholesterol and triglyceride levels in DIO mice, but also significantly reduced LDL-C and VLDL-C in a dose-dependent manner. Furthermore, significant reductions in liver triglycerides and lipid concentrations in B6 mice were observed following HPMC supplementation; both of these are important indicators of whole-body energy homeostasis. The reduction in hepatic lipids and triglycerides was strongly correlated with increased bile acid and fat excretion. These results suggest that HPMC exerts its hypocholesterolemic effects by altering dietary lipid absorption and metabolism. In addition to reduced hepatic triglyceride levels, hamsters on an HPMC-supplemented high-fat diet exhibited significant reductions in hepatic cholesterol compared with the DIO mouse model.10 Hamsters have been widely used as a model in which to study the effects of various drugs and/or interventions on cholesterol, lipoprotein metabolism, and atherosclerosis. The activity of enzymes regulating hepatic cholesterol and lipid metabolism in the liver may differ between hamsters and B6 mice. However, positive effects of HPMC on energy homeostasis have been demonstrated in both models.

In conclusion, the results of the present study indicate that supplementation of a high-fat diet with HPMC is effective in altering dietary lipid absorption and inducing weight loss similar to that seen in mice on a low-fat diet without reducing caloric intake. Moreover, the improvements observed in insulin, glucose, and leptin concentrations following HPMC supplementation strongly associated with lower adipose fat mass. Normalized adipocytokine levels are also involved in the regulation of glucose and lipid homeostasis leading to improvements in dyslipidemia and insulin resistance. The present study further extends the dietary use of HPMC from the prevention of dyslipidemia-related diseases to the treatment of insulin resistance and obesity.

Acknowledgments

The authors are grateful to Dr Wenyu SU (Engineering Sciences, The Dow Chemical Company, Midland, MI, USA) for performing repeated-measures analysis of variance for the weight gain data. The authors thank Demetrius DIELMAN, Margaret COVINGTON, and Suzanne LEHR (Analytical Sciences, The Dow Chemical Company, Midland, MI, USA) for technical assistance.

Disclosure

No part of the work has been published before, except in abstract form. The authors declare that they have no conflict of interest.

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