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Unité de Biochimie Toxicologique et Cancérologique, Université Catholique de Louvain, UCL-PMNT 7369-B-1200 Brussels, Belgium
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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KEY WORDS: • fructan • triglycerides • lipogenesis • rat • liver • oligofructose
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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) and animals
(de Deckere et al. 1995
, Overton et al. 1994
, Roberfroid and Delzenne 1998
, Tokunaga et al. 1986
). The mechanism of their
lowering effect on serum lipids remains incompletely elucidated.
Oligofructose (OFS)3
is a natural food ingredient that can also be prepared by enzymic
hydrolysis of chicory inulin. This commercially available OFS is called
Raftilose. Because of the presence of ß-linkages, OFS is undigestible
in the upper gastrointestinal tract. Consequently, it enters the
cecum/large bowel intact, where it is largely fermented and causes
proliferation of selected anaerobic bacteria, mostly bifidobacteria
(Wang and Gibson 1993
).
Feeding rats a diet supplemented with 10 g/100 g OFS significantly
lowers triacylglycerol (TAG) and phospholipid (PL) serum concentrations
(Delzenne et al. 1993
). This article will review
probable biochemical mechanism(s) accounting for the hypolipidemic
effect of OFS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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Raftilose P95, a mixture of glucosyl-(fructosyl)n-fructose (64%) and (fructosyl)m-fructose (36%) with an average degree of polymerization of 4.8, was used as the source of OFS. It was a gift from Orafti (Tirlemont, Belgium).
Animals.
Male Wistar rats ICOPS-WY IOPS from Iffa Credo (Les Oncins,
France), weighing initially ~120 g, were housed individually on a
12-h dark/12-h light alternating schedule. Rats were assigned to one of
two experimental groups. Treated rats received diets containing 10
g/100 g Raftilose P95. The basal diet was obtained from a
commercial source (UAR A04, UAR, Villemoisson-sur-Orge, France). The
composition of all diets is described in the legend of Table 1. The measurement of hepatic enzyme activities and metabolites, and
of fatty acid synthase mRNA was performed on tissue samples taken at
0900 h, corresponding to the postprandial period. The entire cecum
was excised, weighed and stored at -20°C.
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Serum and liver triacylglycerols, nonesterified fatty acids,
phospholipids, cholesterol and glucose were measured using enzymatic
kits (Sopar-Biochem, Brussels, Belgium). The same kits were used for
liver lipids analysis, after chloroform-methanol extraction
according to Folch et al. (1957)
. Insulin
concentrations were determined using a RIA kit from Novo Nordisk
(Bagsvaerd, Denmark). Immunoreactive glucose-dependent
insulinotropic polypeptide (GIP) was measured in serum by double
antibody RIA with human synthetic GIP as standard (Morgan et al. 1978
). Glucagon-like peptide 1 (GLP-1) was analyzed in
cecal extracts by double antibody RIA as previously described
(Elliott et al. 1993
, Knapper et al. 1995
). The esterification pathway was assessed by measuring
glycerol-3-phosphate hepatic content (Hohorst 1965
) and
glycerol-3-phosphate acyltransferase (GPAT) as well as
Mg2+-dependent phosphatidate phosphohydrolase (PAP)
activities (Bates et al. 1977
, Cheng and
Saggerson 1978
). The oxidation of fatty acyl-SCoA was
assessed by measuring carnitine palmitoyl transferase (CPT I)
(Schäfer et al. 1993
). Fatty acid synthesis was
assessed by measuring acetyl-coenzyme A carboxylase (ACC)
(Maeda et al. 1976
), ATP citrate lyase (ATPCL)
(Takeda et al. 1969
), fatty acid synthase (FAS)
(Linn 1981
), malic enzyme (ME) (Hsu and Lardy 1969
) and glucose-6-phosphate deshydrogenase (G6PDH)
(Löhr and Waller 1974
) activities. Protein was
assayed by the method of Lowry et al. (1951)
, using
bovine serum albumin (BSA) as standard. All enzymes assays were linear
with respect to protein concentration. Total liver RNA was isolated
with an acid guanidinium-thiocyanate-phenol-chloroform mixture
(Chomczynski and Sacchi 1987
) after liver glycogen
removal (Sambrook et al. 1989
). Northern blot analysis
was performed as previously described (Brichard et al. 1994
). To normalize the amount of total RNA loaded on each
lane, specific mRNA levels were expressed relative to those of 18S rRNA
assessed by methylene blue staining of the blot.
Statistical analysis.
Statistical analysis of the results was performed by two-way ANOVA testing for the interaction time vs. treatment and using Statview 512+ (Abacus Concepts, Berkeley, CA) as software. Student's t test was used to compare results at one particular time point.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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summarizes the results obtained by comparing the influence of OFS
given at the final concentration of 10 g/100 g in several diets, i.e.,
a standard diet (AO4), a fiber-free diet (semisynthetic diet
without any digestible compound, except OFS), or a high fat diet known
to induce hypertriglyceridemia and hypercholesterolemia. In all of
these protocols, the constant response was a decrease in TAG
concentration. This triglyceride (TG)-lowering effect was significant
(P < 0.05) after 1 wk of feeding for the standard diet
and lasted for up to 16 wk (P < 0.05); it was
significant as well in the postprandial as in the postabsorbtive period
(Fiordaliso et al. 1995
). Analysis of serum
lipoproteins, after ultracentrifugation of the serum collected from
food-deprived rats, showed that OFS feeding affected only the VLDL
fraction, whereas the composition of both LDL and HDL remained
unchanged (Fiordaliso et al. 1995
).
Because a positive relationship exists between VLDL-TG hepatic
output and liver lipogenesis, OFS was hypothesized to reduce de novo
liver fatty acid synthesis (lipogenesis). This hypothesis was first
investigated by measuring incorporation of
14C-acetate (a lipogenic precursor) into TG in
hepatocytes isolated from control and OFS-fed rats. OFS feeding
significantly reduced TG synthesis and secretion from acetate in
isolated hepatocytes (Fig. 1
) (Kok et al. 1996b
).
|
). Fatty acid esterification is also known to depend on the
availability of glycerol-3-phosphate and fatty acyl-CoA. Table 2
provides evidence that hepatic glycerol-3-phosphate concentrations were
significantly higher in OFS-fed rats than in controls. This
relative increase in glycerol-3-phosphate liver content might be due to
a decrease in its utilization for fatty acid esterification. Indeed,
the daily administration of OFS slighly but significantly reduced the
hepatocyte capacity to esterify 14C-palmitate
into TAG (Fiordaliso et al. 1995
).
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). The hypothesis of an increased fatty acid catabolism
to explain a lower availability is unlikely. The activity of CPT I, the
rate-limiting enzyme in the hepatic mitochondrial ß-oxidation
(MacGarry et al. 1978
), was not increased in the liver
of OFS-fed rats (Table 2)
. Moreover, Kok et al. (1993)
have
previously shown that mitochondria-enriched fractions prepared from
the liver of OFS-fed rats had the same capacity to oxidize various
fatty acids as those prepared from untreated rats.
Fatty acid synthesis is controlled by several key enzymes that respond
coordinately to physiologic stimuli. The activities of all lipogenic
enzymes were found to be lower in the livers of OFS-fed rats
compared with controls (Table 2)
, suggesting that OFS feeding could
decrease lipogenic flux and thus liver TAG-VLDL secretion capacity
(Arbeeny et al. 1992
, Gibbons 1990
).
The coordinate inhibition of all of the lipogenic enzymes as well as
the fact that the activity of FAS is regulated only by the protein
content, essentially at the transcriptional level (Hillgartner et al. 1995
), suggests that OFS might decrease the lipogenic
flux in the liver by reducing the expression of the genes coding for
lipogenic enzymes. FAS mRNA was reduced by 40% in OFS-fed rats
compared with controls, as was found for FAS activity (Table 2)
.
Collectively, the TAG-lowering effect of OFS in rats appears to be due to its antilipogenic action in the liver, i.e., by reducing the activity and possibly the expression of all lipogenic enzymes.
Other possible mechanisms.
Whatever metabolic change is responsible for the hypolipidemic effect,
one must still explain how a nondigestible carbohydrate can regulate
systemic lipid metabolism if, as shown in previous studies
(Fiordaliso et al. 1995
, Kok et al. 1998a and 1998b
), it has no significant increasing effect on the fecal
excretion of lipids.
By analogy with fermentable dietary fibers such as pectin
(Roberfroid 1993
), and knowing their effect on the
physiology of the gastrointestinal tract, it could be possible that
dietary OFS modifies the kinetics of absorbtion of dietary
carbohydrates, leading to modifications of both serum glucose and
hormones (insulin, glucagon). Moreover, dietary modulation of lipogenic
activity is often linked to modifications of glucose and/or insulin
serum levels. Indeed, acarbose, an intestinal glucosidase inhibitor
that delays starch digestion, reduces glucose absorption and
postprandial glycemia and insulinemia, and also decreases FAS activity
in the liver (Maury et al. 1993
). Similarly, resistant
starch decreases serum TG concentration in rats (de Deckere et al.
1995
), reduces FAS activity by 50 and 20% in adipose tissue and liver,
respectively, and concommitantly lowers postprandial insulin response
by 30% without affecting glucose response (Takase et al. 1994
).
Recently, OFS ingestion was reported to reduce postprandial glycemia
and insulinemia by 17 and 26%, respectively (Table 3) (Kok et al. 1996b
). This could explain the lower
FAS activity and mRNA. The transcription level of FAS is recognized to
be primarily activated by glucose and insulin (Hillgartner et al. 1995
). Studies are now in progress to analyze the putative
involvement of glucose and insulin homeostasis in the hypolipidemic
effect of oligofructose.
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). Serum GIP concentrations are
enhanced in OFS-fed rats (Table 3)
. OFS increases GLP-1 twofold in
the cecum (218 ± 28 vs. 120 ± 7 pmol/cecum in
OFS-fed and control rats, respectively) (P < 0.01). This latter increase is a consequence of cecal hypertrophy and
might be related to the trophic effects of short-chain fatty acids
produced by OFS fermentation in the cecocolon (Roberfroid 1993
). |
CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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).
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FOOTNOTES |
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3 Abbreviations used: ACC, acetyl-coenzyme A
carboxylase; ATPLC, ATP citrate lyase; BSA, bovine serum albumin; CPT
I, carnitine palmitoyl transferase; FAS, fatty acid synthase; GIP,
glucose-dependent insulinotropic polypeptide; GLP-1,
glucagon-like peptide 1; GPAT, glycerol-3-phosphate
acyltransferase; G6PDH, glucose-6-phosphate dehydrogenase; ME, malic
enzyme; OFS, oligofructose; PAP, phosphatidate phosphohydrolase; PL,
phospholipid; TAG, triacylglycerol; TG, triglyceride.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONREFERENCES |
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