C2
`
`J Pharm Pharmaceut Sci (wvvw. cspsCanada.org) 8 (3):400-408, 2005
`
`Disodium Ascorbyl Phytostanyl
`Phosphates
`(FM-VP4)
`reduces
`plasma cholesterol concentration,
`body weight and abdominal fat
`gain within a dietary-induced
`obese mouse model
`
`, Verica Risovic 1
`Norbert A. Looije 1
`, David J.
`Stewart'', Daniel Debeyer2
`, James Kutney2 and
`Kishor M. Wasan 1
`•
`
`1Division of Pharmaceutics and Biopharmaceutics,
`Faculty of Pharmaceutical Sciences, University of
`British Columbia, Vancouver BC, Canada & 2Forbes
`Medi-Tech Inc, Vancouver B.C., Canada
`
`Received March 19, 2005,Revised June 15, 2005,
`Accepted August 15, 2005, Published August 24. 2005.
`
`ABSTRACT
`
`Purpose: The purpose of this study was to determine
`if Disodium Ascorbyl Phytostanol Phosphates (FM(cid:173)
`VP4) alters animal body weight and plasma lipid
`levels in a dietary-induced obese mouse model.
`Methods: Twenty-four C57BL6 mice (28 days old)
`were housed individually and fed a standard mouse
`diet for 2 weeks upon arrival. After 2 weeks the
`animals were weighed and divided in 4 groups of
`similar average weight, and the groups received a
`low fat (10% kcal from fat) and high fat (45% kcal
`from fat) diet with or without FM-VP4 (2% w/w) for
`12 continuous weeks. Food, water and caloric intake
`and body weight were recorded on a daily basis
`throughout the duration of the study. Following the
`12th week of the study all animals were humanely
`sacrificed and blood and abdominal fat pads were
`harvested for further analysis. Plasma cholesterol,
`triglyceride, AST I ALT and creatinine levels were
`measured using enzymatic kits. Results: There is a
`significant difference in weight gain between the
`low-fat diet and the low-fat diet+ 2% w/w FM-VP4
`treatment groups (P<0.05), as well as between the
`high-fat diet and the high-fat diet+ 2% w/w FM-VP4
`treatment groups (P<0.05). However, the reduction
`of weight gain of the high-fat diet + 2% FM-VP4
`treatment group compared to the high-fat group was
`51 %, while the reduction in weight gain between the
`low-fat diet+ 2% w/w FM-VP4 treatment group and
`the low-fat diet group was 17% over the duration of
`the study. No significant differences in food and
`
`400
`
`water inta.kes, serum creatinine and AST/ALT levels
`were observed between
`the
`four groups. No
`significant differences in caloric intake between the
`low-fat diet and the low-fat diet+ 2% w/w FM-VP4.
`However, a significant difference in caloric intake
`between high-fat diet and the high-fat diet+ 2% w/w
`FM-VP4 treatment groups was observed. In addition,
`significant reductions in plasma cholesterol levels
`and abdominal fat pad weight between diet alone and
`diet + FM-VP4 treatment groups were observed.
`Conclusions: These findings suggest that FM-VP4
`may have potential weight-loss and cholesterol
`lowering activity in both High Fat and Low Fat Diets
`treated groups.
`
`INTRODUCTION
`
`The obesity epidemic has been recognized by the
`World Health Organization as one of the top 10
`global health problems (I). Worldwide, more than
`one billion adults are ovenveight and over 300
`million are obese
`(1). Most countries are
`experiencing dramatic increases in obesity.
`the
`is
`a condition associated with
`Obesity
`accumulation of excessive body fat resulting from
`chronic imbalance of energy whereby the intake of
`energy exceeds expenditure. The excess body fat
`predisposes an obese individual to chronic diseases
`such as coronary heart disease, type 2 diabetes'.
`diseases of the gall bladder and cancer (2, 3). The
`high incidence of obesity, its multifactorial nature
`and the scarcity of adequate therapeutics have fuelled
`an increase in anti-obesity drug-related research.
`Although a number of pharmacological approaches
`have been
`investigated
`in
`recent years,
`few
`therapeutically
`effective products have
`been
`developed (2).
`Our laboratory has been investigating the lipid
`lowering and anti-atherosclerotic effects of a novel
`cholesterol absorption inhibitor, FM-VP4 (disodium
`ascorbyl phytostanyl phosphates, FM-VP4, Figure
`1), in several animal models (4-9). In several of these
`studies, we noted
`that FM-VP4 administration
`caused a decrease in body weight without any
`observable liver or kidney toxicity or changes in
`
`Corresponding Author: Dr. Kishor M. Wasan
`Faculty of Pharmaceutical Sciences, University of
`British Columbia, 2146 East Mall Avenue
`'
`Vancouver, BC, Canada, V6T 1 Z3
`kwasan@interchange.ubc.ca
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`
`food or water intake (7, 8). Although several studies
`have described the effects of plant sterols/stanols on
`significantly decreasing total plasma and lipoprotein
`cholesterol levels between 10-20% at doses bet\¥een
`1-5 g/day ( l 0-24), there is no evidence for any
`weight loss properties. Therefore, our observation of
`non-toxic weight loss appears to be specific for FM(cid:173)
`VP4. However, to date, weight loss studies with FM(cid:173)
`VP4 have not been conducted in a dietary-induced
`obese animal model. Therefore, the purpose of this
`study was
`to determine
`if disodium ascorbyl
`phyiostanol phosphates (FM-VP4) alters animal
`body weight and plasma lipid levels in a dietary(cid:173)
`induced obese mouse model receiving a low-( l 0%
`kcal from fat) and high-fat diet (45% kcal from fat).
`
`MATERIALS AND METHODS
`
`Chemicals
`
`Disodium ascorbyl phytostanyl phosphates (FM(cid:173)
`VP4; Lot number: 81699 BRI FM-VP4-06; Figure l)
`was prepared by the chemistry group of Forbes
`Medi-Tech Inc. Research Laboratories. FM-VP4 is a
`semi-synthetic esterified phytostanol derivative,
`produced as
`the disodium salt. The two major
`components of FM-VP4 are disodium ascorbyl
`campesta:nyl phosphate and disodium ascorbyl
`sitostanyl phosphate. The powdered active ingredient
`was stored at 4°C and to date has been demonstrated
`to be stable for up to 12 months under these
`conditions. The
`low and high
`fat diets were
`purchased from Research Diets Inc. (New Jersey,
`USA; See Table l for the complete composition of
`each diet) (25).
`
`Dietmy-Induced Obese i\.-fouse Afodel
`
`Four-week-old male C57BL6 mice were purchased
`from Charles River Laboratories, Quebec, Canada.
`These animals normally exhibit significant weight
`gain following the consumption of a high fat-diet
`(45% kcal from fat) over a 12-week period (25).
`
`Experimental design
`
`Twenty-four C57BL6 mice (4 weeks old) were
`obtained from Charles River Laboratories (Montreal,
`Quebec, Canada). Upon arrival the animals were
`acclimatized by being housed individually and fed a
`standard mouse diet for 2 weeks. Housing consisted
`of a 12 h light/ dark cycle at a constant temperature
`
`(21°C ± 2) and humidity. After 2 weeks the animals
`were weighed and divided in 4 groups (11=6 for first
`9 weeks, n=5 for weeks 9-12; one mouse was
`randomly sacrificed at week 9 for genotyping) of
`similar average weight, and the groups received a
`low fat-diet (l 0 % kcal from fat); high fat-diet ( 45%
`kcal from fat); low fat-diet+ 2% (w/w) FM-VP4 or a
`high fat-diet+ 2% (w/w) FM- VF4 respectively for
`12 consecutive weeks (Table l). Allocation of
`treatment to each group was randomly detem1ined
`before the start of the study. Homogeneity of groups
`was validated on the criteria of body weight, plasma
`cholesterol and plasma triglyceride on the day of
`randomization.
`Food, water and caloric intakes (calculated
`based on the amount of food consumed daily and the
`nutritional information provided in table 1) intakes
`and body weight were recorded for all animals on a
`
`daily basis. Follo\ving the 121h week of the study
`animals were humanely sacrificed using a C02
`chamber and blood and abdominal fat pads were
`harvested for further analysis. Plasma cholesterol,
`triglyceride, serum creatinine and AST I ALT levels
`were determined using enzymatic kits (Boehringer
`Mannheim, Germany) as previously described (7,8)
`
`Diet Preparation and Animal Care
`
`Diet preparation was carried out at Research Diets
`(Table 1) and FM-VP4 was incorporated into the diet
`as previously published (5,7,8). The Animal Care
`Committee of the University of British Columbia
`approved the study. The concentration of FM-VP4 in
`food was confim1ed at the beginning of study. 15-20
`grams of the control food was collected in glass
`containers, labelled accordingly and stored at 4°C.
`Considering that the average daily food intake was
`estimated to be 3 g, mice were administered about 60
`mg FM-VP4 each day, equivalent to 2% (w/w) of the
`diet.
`
`Collection of blood and harvesting of abdominal
`fat pads
`
`At the end of the study mice were sacrificed using
`taken from the right
`C02 gas and blood was
`ventricle.
`Blood
`cells were
`pelleted by
`centrifugation
`and
`plasma was
`harvested.
`Abdominal fat pads were removed from the abdomen
`of each mouse and weighed. The abdominal fat pads
`(this is the fat from the dorsal abdomen region of the
`mouse) were removed by a member of the animal
`
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`care unit at UBC who was blinded to the dose group
`of each individual mouse and used the dissection
`technique from the work of Henry et al. (26).
`
`Statistical Analysis
`
`Results were expressed as mean ± SD (standard
`deviation). Statistical analysis were conducted using
`(PCANOVA; Human
`an analysis of variance
`
`Dynamic Systems) and assuming unequal variance
`(Newman Keuls post-hoc test) was used to assess the
`differences between the FM-VP4 treatment groups
`and the untreated control group for body weight,
`abdominal fat weight, plasma lipids, food, water and
`
`R
`
`R = CH3 {campestanol)
`R = C;,H5 (sitostanol)
`
`T
`
`Figure l. Chemical Strncture ofDisodium Ascorbyl Phytostanyl Phosphates (FM-VP4)
`
`Table 1: The Composition of the Low Fat; High Fat; Low Fat+ 2% (w/w) FM-VP4 and High Fat+ 2% (w/w)
`FM-VP4 diets and their caloric content
`Diet Ingredient
`Low Fat
`
`High Fat
`
`LowFat+2%
`FM-VP4
`gm
`kcal
`200
`800
`800
`3
`12
`12
`315
`1260
`1260
`35
`140
`140
`350
`1400
`1400
`50
`0
`0
`25
`225
`225
`20
`180
`180
`10
`0
`0
`0
`13
`0
`0
`0
`5.5
`0
`16.5
`0
`10
`40
`40
`0
`0
`2
`0
`20
`0
`0
`0.025
`0
`0
`0.025
`0
`0
`0
`0
`4057
`1075
`4057
`Low Fat+ 2% FM~V p 4
`gm%
`kcal%
`18.9
`20
`66
`70
`10
`4.2
`3.77
`100
`
`Casein, 80 Mesh
`L-Cystein
`Com Starch
`Maltodextrin I 0
`Sucrose
`Cellulose, BW200
`Soybean Oil
`Lard
`Mineral mix, S 10026
`DiCalciurn Phosphate
`Calcium Carbonate
`Potassium Citrate, lH='O
`Vitamin mix, VlOOO l
`Choline Bitartrate
`FM-VP4
`FD&C Yellow Dye #5
`FD&C Red Dye #40
`FD&C Blue Dye # l
`TOTAL
`Diet
`
`gm
`200
`3
`315
`35
`350
`50
`25
`20
`IO
`13
`5.5
`16.5
`10
`2
`0
`0.05
`0
`0
`1055
`F at
`I.ow
`gm%
`kcal%
`19.2
`20
`70
`67.3
`IO
`4.3
`3.85
`100
`
`Protein
`Carbohydrate
`Fat
`TOTAL
`Kcal/gm
`
`kcal
`
`402
`
`High Fat+
`2%FM-VP4
`gm
`kcal
`
`gm
`200
`3
`72.8
`100
`172.8
`50
`25
`177.5
`10
`13
`5.5
`16.5
`IO
`2
`0
`0
`0.05
`0
`858
`.hF
`Hig
`at
`gm%
`kcal%
`23.7
`20
`41.4
`35.l
`23.6
`44.9
`100
`4.73
`
`kcal
`
`800
`200
`800
`12
`12
`3
`291
`72.8
`291
`400
`100
`400
`691.2
`691.2
`172.8
`0
`0
`50
`225
`25
`225
`1597
`177.5
`1598
`0
`0
`10
`0
`0
`13
`0
`0
`5.5
`0
`0
`16.5
`IO
`40
`40
`0
`0
`2
`0
`0
`20
`0
`0
`0
`0
`0.05
`0
`0
`0
`0
`4057
`878
`4057
`F f ~-VP4
`F at+2%
`High
`gm%
`kcal%
`23.1
`20
`40.5
`35. l
`23.1
`44.9
`100
`4.62
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`
`--·«·---- Hgh F~t
`
`50
`
`40
`
`130
`ol
`!;'ii
`.._ ..
`~--·n
`.jlll.; _
`OS>
`~
`
`10
`
`Figure 2: Average weekly
`weight of male C57Bl6 mice on
`either Low Fat; High Fat; Low Fat
`+ 2% (w/w) FM-VP4 or High Fat
`+ 2% (w/w) FM-VP4 diet. Week
`1 - 9, 11=6; week 10- 12. n=S.
`
`0
`
`0
`
`2
`
`4
`
`8
`6
`'lime ('weeks)
`
`10
`
`12
`
`intake, AST I ALT and sernm creatmme
`caloric
`levels. A p-value of less than 0.05 indicated a
`significant difference between treated and untreated
`groups.
`
`RESULTS
`
`Total body and abdominal fat pad weight
`
`The weight gain of the four groups is depicted in
`Figure 2. This figure clearly shows that weight gain
`of the High Fat group was higher throughout the
`entire duration of the experiment compared to all the
`other groups while all groups started at the same
`average weight, this finding is significant from week
`2 until the end of the experiment (p<0.05 ). Even
`though there was a significant difference in weight
`gain between the Low Fat and the Low Fat + 2%
`(vdw) FM-VP4 groups (P<0.05), as well as between
`the High Fat and the High Fat+ 2% (w/w) FM-VP4
`groups (P<0.05), the difference in the weight gain
`between the High Fat and High Fat+ 2% (w/w) FM(cid:173)
`VP4 group was much more profound than between
`the Low Fat and the Low Fat+ 2% (w/w) FM-VP4
`group. The reduction of weight gain of the High Fat
`+ 2% (w/w) FM-VP4 group compared to the High
`Fat group was 51 %, while the reduction in weight
`gain ben;v-een the Low Fat+ 2% (w/w) FM-VP4 and
`the Low Fat group was 17%.
`Both groups, which have been fed the FM-VP4
`enriched diets, followed a very similar weight pattern
`throughout
`the entire experiment despite
`the
`difference in caloric content of the diet, and there
`
`was no significant difference in average weight
`between the
`two groups
`throughout
`the entire
`experiment.
`The weight of the abdominal fat pads of the
`treatment groups is depicted in Figure 3. The weight
`of the abdominal fat pads of the High Fat group was
`increased by 115% compared to the Low Fat group
`as a result of the high fat content of their diet. The
`weight of the abdominal fat pads of the High Fat +
`2% (w/w) FM-VP4 group has decreased by 70%
`compared to
`the High Fat group and by 38%
`compared to the Low Fat group. The Low Fat+ 2%
`(w/w) FM-VP4 group compared to the High Fat+
`2% (w/w) FM-VP4 group did not significantly differ
`in weight of the fat pads, otherwise all differences
`between groups were
`statistically
`significant
`(P<0.05).
`
`4.0
`
`l 3.0
`"'" j 2.0
`:I:
`
`1.0
`
`00
`
`Low Fat
`
`High fat
`
`LowF.~t +
`2~-", VP4
`
`High Fat+
`2'%VP4
`
`Figure 3: Weight of the abdominal fat pads in grams of
`male C57Bl6 mice on either Low Fat; High Fat; Low Fat
`+ 2% (w/w) FM-VP4 or High Fat + 2% (w/w) FM-VP4
`diet. a. P<0.05 vs. Low Fat; b. P<0.05 vs. High Fat; c.
`P<0.05 vs. Low Fat + 2% FM-VP4; d. P<0.05 vs. High
`Fat+ 2'% FM-VP4.
`
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`
`Food, caloric and water intake
`
`The patterns of food intake and of caloric intake of
`the four treatment groups are depicted in Tables 2
`intake were steady
`and 3. Food and caloric
`throughout the experiment for all treatment groups.
`But even though it is not as evident from the data of
`the food intake, the caloric intake of the High Fat
`group was slightly higher compared to all other
`
`treatment groups throughout the dmation of the study
`(Table 3). In weeks 6 through 10 this difference in
`caloric intake is even significant (p<0.05) compared
`to the High Fat+ 2% (w/w) FM-VP4 group that has
`a similar caloric content per gram diet (Table 1). This
`was an unexpected result since food with the same
`to be eaten
`caloric content
`is expected
`111
`approximately the same amount by the animals.
`
`Table 2: Average weekly food intake in grams of male C57Bl6 mice on either Low Fat High
`Fat; Low Fat+ 2% (w/w) FM-VP4 or High Fat+ 2'% (w/w) FM-VP4 diet.
`Group I Week
`High Fat+ 2%
`Low Fat
`High Fat
`Low Fat+ 2%
`FM-VP4
`FM-VP4
`2.6± 1.0
`2.5 ± 0.8
`3.5 ± 0.7
`3.3 ± 0.5
`3.1 ± 0.5
`3.2 ± 0.4
`3.0 ± 0.4
`3.1±0.6
`2
`3
`2.9± 0.3
`3.2± 0.2
`2.8± 0.5
`3.1±0.3
`3.2 ± 0.3d
`3.0 ± 0.4
`3.7 ± 0.3a.c
`3.2 ± 0.2d
`4
`3.3 ± 0.4 d
`2.7 ± 0.2a.c
`3.2 ± 0.3 d
`3.0 ± 0.3
`5
`2.8 ± 0.2
`3.2 ± 0.3
`3.2 ± 0.4
`3.3 ± 0.4
`6
`2. 7 ± 0.2"b.c
`3.3 ± 0.2 d
`3.3 ± 0.3 d
`3.2±0.4d
`7
`2.7±0.2b.c
`3.3±0.2d
`3.3±0.4d
`3.0 ± 0.5
`8
`2.7 ± 0.2aJu
`3.2 ± 0.2 d
`3.2 ± 0.3 d
`3.1±0.4 cl
`9
`2.9 ± 0.2
`3.2 ± 0.3
`3.5 ± 0.3
`3.3 ± 0.5
`10
`3.0 ± 0.5
`3.2 ± 0.5
`3.3 ± 0.5
`3.4± 0.5
`11
`2.8 ± 0.3
`3.2 ± 0.3
`3.1 ± 0.3
`3.2 ± 0.5
`12
`a. P<0.05 vs. Low Fat; b. P<0.05 vs. High Fat; c. P<0.05 vs. Low Fat+ 2'% FM-VP4; d. P<0.05 vs.
`High Fat+ 2%, FM-VP4.Average weekly food intake is given in grams.
`
`2
`3
`4
`5
`6
`
`Table 3: Average weekly caloric intake in kilocalories of male C57Bl6 mice on either Low Fat;
`High Fat; Low Fat+ 2% (w/w) FM-VP4 or High Fat+ 2~o (w/w) FM-VP4 diet.
`Group I Week
`Low Fat
`High Fat
`Low Fat+ 2%
`High Fat+ 2%
`FM-VP4
`FM-VP4
`16.5 ± 3.2c
`9.8± 3.0
`12.6 ± 4.6
`11.8 ± 2.1
`11.9 ± 1.5
`14.2 ± 2.5
`14.4 ± 2.0
`12.l±l.3
`12.0 ± 0.7
`13.5 ± 1.5
`13.2 ± 2.4
`12.2 ± 1.3
`12.0 ± 0.9
`12.7 ± L5
`14.1±2.1
`12.2 ± 1.3
`12.0 ± l.3b
`14.2 ± l.3c
`12.5 ± l.O
`12.6 ± 1.5
`15.1±1.9a_c.d
`12.6 ± l.6b
`12.0 ± l.Ob
`12.8 ± l.Ob
`15.4± l.5a.c,d
`12.3±1.4b
`12.5± 0.8b
`12.6± 0.9b
`'"I
`I
`15.3±2.0",c.cl
`12.4 ± 0. 9b
`12.6±1.lb
`ll.7± I.Su
`8
`15.2±1. 6a.c,d
`12.7 ± l.Ob
`11.9± l.5b
`12.0 ± 0.9b
`9
`16.4±1.3"-c<l
`13.4± I.Oh
`12.8 ± l.9h
`12.3 ±Li'
`10
`12.2 ± 1.7
`13.8 ± 2.3
`15.8 ± 2.5
`13.2 ± 2.0
`11
`l 2.0 ± 1.1
`l3.0 ± 1.4
`14.6 ± 1.6
`12.3 ± 2.0
`12
`a. P<0.05 vs. Low Fat: b. P<0.05 vs. High Fat; c. P<0.05 vs. Low Fat+ 2% FM-VP4; ct. P<0.05
`vs. High Fat+ 2% FM-VP4, Average weekly caloric intake is given in kilocalories.
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`High Fat+ 2% FM-VP4
`
`Table 4: Average weekly water intake in milliliters of male C57Bl6 mice on either Low Fat; High Fat; Low
`Fat+ 2% (w/w) FM-VP4 or High Fat+ 2% (w/w) FM-VP4 diet.
`Group I Week
`Low Fat
`High Fat
`Low Fat+ 2%
`FM-VP4
`4.8 ± 1.9
`4.8 ± 1.5
`5.5 ± 1.2
`5.4 ± 1.3
`6.2 ± 1.5
`6.3 ± l.4
`5.6 ± 1.5
`5.8 ± 1.8
`2
`7.4 ± 1.7
`7.4 ± 1.5
`6.2 ± 1.3
`6.5 ± 1.4
`3
`6.3 ± 1.5
`5.9 ± 1.3
`6.5 ± 1.1
`7.1 ± 1.2
`4
`6.6 ± 1.7
`6.9 ± l.2
`7.0 ± l.3
`6.1±1.2
`5
`5.9±0.6b
`4.7±0.6"
`5.3 ± 0.4
`5.2 ± 0.5
`6
`4.6 ± 0.2
`4.5 ± 0.5
`4.5 ± 0.4
`4.5 ±0.2
`7
`5.0 ± 0.3
`5.0 ± 0.5
`4.6 ±0.3
`5.0 ± 0.6
`8
`4.9 ± 0.9
`4.7 ± 0.6
`4.9±0.8
`5.1±0.4
`9
`4.6 ± 0.4
`4.5 ± 0.2
`4.8 ± 0.5
`4.5 ± 0.1
`12
`a. P<0.05 vs. Low Fat; b. P<0.05 vs. High Fat; c. P<0.05 vs. Low Fat+ 2% FM-VP4; d. P<0.05 vs. High Fat
`+ 2% FM-VP4. Average \veekly water intake is given in milliliters. Weeks l 0 and l l were not detennined.
`
`Water intake was measured on a daily basis for
`the first 34 days while it was measured twice a week
`for the remainder of the experiment. The weekly
`averaged data and statistical significance of
`comparison of each group is given in Table 4. Water
`intake of the groups did vary on a week to week
`basis but the differences in water intake between the
`treatment groups were not significant during the
`entire experiment. Water intake was not measured in
`weeks 10 and 11, but since there was no difference in
`the water intake between groups for all the other
`weeks(weeks 1-9 and week 12) it might be assumed
`that the water intake in these 2 weeks were not
`significantly different.
`
`Plasma total cholesterol and triglyceride levels
`
`The effects of FM-VP4 on plasma total cholesterol
`and triglyceride levels are shown in Figures 4 and 5
`respectively. The animals of the High Fat group
`displayed a significant increase
`in
`total plasma
`cholesterol of the High Fat + 2% (w/w) FM(cid:173)
`cholesterol as compared to the animals of the Low
`Fat group as would be expected (p<0.05). The total
`VP4 group was significantly lower compared to the
`High Fat group by 32.2% (p<0.05), while the total
`plasma cholesterol concentration of the Low Fat +
`2% (w/w) FM-VP4 group was significantly lower
`compared to the Low Fat group by 45.l % (p<0.05).
`These results are in accordance with previously
`obtained results suggesting that FM-VP4 decreases
`
`plasma cholesterol levels following chronic oral
`administration (5,7,8).
`
`:...:.::.d.
`
`300
`
`~ 200
`~
`1100
`
`0
`
`Low Fat .;.
`2% FM-VP4
`
`lhgh Fat +
`2% FM-VP'1
`
`Figure 4: Total plasma cholesterol levels in mg/dl of
`male C57Bl6 mice on either Low Fat; High Fat; Low Fat
`+ 2% (w/w) FM-VP4 or High Fat+ 2% (w/w) FM-VP4
`diet. a. P<0.05 vs. Low Fat; b. P<0.05 vs. High Fat; c.
`P<0.05 vs. Low Fat+ 2% FM-VP4; d. P<0.05 vs. High
`Fat+ 2%, FM-VP4.
`
`levels were not
`triglyceride
`The plasma
`significantly different between High Fat versus High
`Fat + 2% (w/w) FM-VP4 treatment groups (Figure
`5). However, significant lowering of the triglyceride
`levels between the Low Fat + 2% (w/w) FM-VP4
`group compared to the High Fat and the Low Fat
`groups respectively (p<0.05) was observed.
`
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`
`High Fat+ 2% FM-VP4
`
`Table 4: Average weekly water intake in milliliters of male C57Bl6 mice on either Low Fat; High Fat; Low
`Fat+ 2% (w/w) FM-VP4 or High Fat+ 2% (w/w) FM-VP4 diet.
`Group I Week
`Low Fat
`High Fat
`Low Fat+ 2%
`FM-VP4
`4.8 ± 1.9
`4.8 ± 1.5
`5.5 ± 1.2
`5.4 ± 1.3
`6.2 ± 1.5
`6.3 ± l.4
`5.6 ± 1.5
`5.8 ± 1.8
`2
`7.4 ± 1.7
`7.4 ± 1.5
`6.2 ± 1.3
`6.5 ± 1.4
`3
`6.3 ± 1.5
`6.5 ± 1.1
`7.1 ± 1.2
`5.9 ± 1.3
`4
`6.9 ± l.2
`7.0 ± l.3
`6.6 ± 1.7
`6.1±1.2
`5
`5.9±0.6b
`4.7±0.6"
`5.3 ± 0.4
`5.2 ± 0.5
`6
`4.6 ± 0.2
`4.5 ± 0.5
`4.5 ± 0.4
`4.5 ±0.2
`7
`5.0 ± 0.3
`5.0 ± 0.5
`4.6 ±0.3
`5.0 ± 0.6
`8
`4.9 ± 0.9
`4.7 ± 0.6
`4.9±0.8
`5.1±0.4
`9
`4.6 ± 0.4
`4.5 ± 0.2
`4.8 ± 0.5
`4.5 ± 0.1
`12
`a. P<0.05 vs. Low Fat; b. P<0.05 vs. High Fat; c. P<0.05 vs. Low Fat+ 2% FM-VP4; d. P<0.05 vs. High Fat
`+ 2% FM-VP4. Average \veekly water intake is given in milliliters. Weeks l 0 and l l were not detennined.
`
`150.00 ...
`
`ij'
`]'. 100.0(1
`
`1
`~ .50.00
`...
`
`0.00
`
`Low Fat
`
`Hi&hFat
`
`2: ... ¢ FM:.-\rf'4
`
`2:-~ F.M-V'F'4
`
`Figure 5: Total plasma triglyceride levels in mg/dl
`of male C57Bl6 mice on either Low Fat; High Fat;
`Low Fat + 2% (w/w) FM-VP4 or High Fat + 2%
`(w/w) FM-VP4 diet. a. P<0.05 vs. Low Fat; b.
`P<0.05 vs. High Fat.
`
`Plasma creatinine and AST/ALT levels
`
`No significant differences in plasma creatinine, AST
`and ALT levels were observed for all four groups in
`this study (data not shmvn).
`
`DISCUSSION
`
`The pmpose of this experiment was to elucidate if
`FM-VP4 reduced body weight gain in a dietary(cid:173)
`induce obese animal model. The 2% (w/w) FM-VP4
`dose chosen in this study was based on our previous
`
`406
`
`findings reported in gerbils (7,8) and ApoE deficient
`mice (5). Jn those studies we have reported that FM(cid:173)
`VP4 at 2% (w/w) significantly reduced body weight
`gain (gerbils only) (8) and plasma lipid levels
`(gerbils and mice) (5,8) without any side effects. In
`addition, F1'1-VP4 is well tolerated even at a high
`daily dose ( l 00 mg/day) \Nithout producing diarrhoea
`or other gastrointestinal
`intolerance signs
`(8).
`Furthermore, since FM-VP4 comprises vitamin C
`(ascorbic acid) and phytostanyl moieties, covalently
`linked by a phosphodiester bridge, it is possible that
`the effects reported in these studies might be due to
`stanols
`to
`inhibit
`the ability of unesterified
`cholesterol abs011)tion, or the combined effect of free
`ascorbate and unesterified stanols following cleavage
`of FM-VP4 into its component parts by digestive
`lipases. Thus, we have compared the effects of FM(cid:173)
`VP4 with equivalent amounts of ascorbic acid or
`phytostanols given individually or together in the
`diet in the ApoE deficient mouse study and found
`minimal effects of these components of FM-VP4 (5).
`Therefore, only FM-VP4 was used in this study.
`We observed that the largest effect could be seen
`bet\veen animals of the High Fat group compared
`with animals of the High Fat + 2% (w/w) FM-VP4
`group, which suggests that FM-VP4 indeed has a
`weight gain reducing effect when it is administered
`to animals that have a high percentage of fat in their
`diet. In the 12 weeks this experiment lasted there is a
`reduction of weight gain of 51 % between the High
`Fat and the High Fat+ 2% (w/w) FM-VP4 groups,
`while the weight gain between the Low Fat and the
`Low Fat+ 2% (w/w) FM-VP4 group was reduced by
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`17%. Interestingly the food and caloric intake of the
`animals in the High Fat group were slightly higher
`than the animals of all other groups including the
`High Fat + 2% (w/w) FM-VP4 group, suggesting
`that FM-VP4 might have an appetite suppressing
`effect since the caloric content of the two High Fat
`diets are comparable (Table 1). This is fmther
`supported by the observation that in all of the
`comparisons beyond week five of the study there
`were reductions in food intake in the High Fat+ 2%
`(w/w) FM-VP4 group compared to the High Fat
`group. However, this finding was not observed in the
`Low Fat control and FM-VP4 treatment groups
`suggesting
`that FM-VP4 may only
`inhibit
`the
`gastrointestinal absorption of excessive fat. Further
`studies to explain these findings are required.
`There was also a significant decrease in total
`plasma cholesterol
`levels and
`the weight of
`abdominal fat pads between the High Fat and High
`Fat+ 2% (w/w) FM-VP4 groups (Figures 2 and 3).
`The observation that the caloric intake was lower in
`the High Fat+ 2% (w/w) FM-VP4 group compared
`to the High Fat group could have impacted the
`difference in body weight gain (Figure 2), weight of
`the abdominal fat pads (Figure 3) and plasma
`cholesterol
`levels (Figure 4)
`in
`these animals.
`However, the finding that the High Fat + 2%i (w/w)
`FM-VP4 group also showed a significant decrease in
`the values of the obesity parameters compared to the
`Low Fat group with an observed lower caloric
`intake, shows that the difference in weight gain can
`be attributed to the effect ofFM-VP4.
`the FM-VP4
`The
`reduced weight gain
`in
`treatment groups did not seem detrimental to the
`animals. All the animals were healthy and active and
`showed no signs of any discomfort due to the
`treatment. The only group that showed signs of less
`activity
`throughout
`the
`last 3 weeks of the
`experiment was the High Fat group, possibly due to
`their high body weight. Interestingly the animals of
`the Low Fat+ 2% (w/w) FM-VP4 and the High Fat+
`2%
`(w/w)
`FM-VP4
`groups
`had
`similar
`characteristics concerning weight gain, plasma
`cholesterol and weight of the abdominal fat pads.
`This observation suggests that FM-VP4 may inhibit
`the uptake of excessive amounts of cholesterol, but it
`does not hinder the uptake of essential amounts of
`cholesterol needed by the body to function properly.
`Future studies to investigate this are warranted.
`In conclusion, FM-VP4 displayed potential
`weight-loss and cholesterol lowering properties in
`both High Fat and Low Fat Diets treated groups.
`
`407
`
`These results warrant fmther investigation in a dose
`response study to see if FM-VP4 will show· as potent
`anti-obesity effects in more clinically relevant doses.
`
`ACKNOWLEDGEMENTS
`
`this project was provided by a
`Funding for
`Collaborative Research Development Grant from the
`National Sciences and Engineering Research Council
`of Canada and Forbes Medi-Tech Inc. (#CRDPJ
`305231-03 to KMW).
`
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