(Hypertension. 1999;34:1007-1011.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Potential Role of Glycerol Leading to Rat Fructose Hypertension
From the Department of Physiology, School of Medicine, Buenos Aires University (P.F.D., M.I.R., L.E.A., I.J.d.l.R.); Center for Endocrinological Research, R. Gutierrez Pediatric Hospital (I.A., S.N., E.D.); Austral University (S.N.); and Favaloro University (L.C.), Buenos Aires, Argentina.
Correspondence to Dr Ignacio J. de la Riva, Depto de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155. 7mo Piso, (1121) Buenos Aires, Argentina. E-mail idelariv{at}fmed.uba.ar
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Abstract |
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Abstract—A fructose-enriched diet promotes hypertension in rats. We thought that an enhancement of the glycolytic and/or lipid disorder (s) that raise blood pressure could be the cause. Therefore, we studied 4 groups of Sprague-Dawley rats (±200 g): (1) control rats received a standard diet and tap water; (2) the glycerol group of rats received a standard diet and 0.54 mol/L glycerol in tap water; (3) the fructose group was given a fructose-enhanced diet (chow had 55% fructose instead of dextrin) and tap water; and (4) the fructose-glycerol group was given the fructose-enhanced diet and 0.54 mol/L glycerol in drinking water. At the end of the second week, the findings were as follows. Blood pressure was 149±2 mm Hg in the fructose-glycerol group versus 129±2 (P<0.001), 131±2 (P<0.001), and 140±3 (P<0.005) mm Hg in the control, glycerol, and fructose groups, respectively. Insulinemia was higher in the fructose-glycerol group than the control (P<0.001), glycerol (P<0.001), and fructose groups (P<0.001); triglyceridemia was higher in the fructose-glycerol (P<0.02), fructose (P<0.05), and glycerol groups (P<0.02) than the control group. Thoracic aorta rings showed a lower ED50 to 12,13-phorbol dibutyrate in the fructose-glycerol group than in the control (P<0.001), glycerol (P<0.002), and fructose groups (P<0.001). In conclusion, glycerol-fructose administration resulted in hypertriglyceridemia, hyperinsulinemia, and increased vascular sensitivity to 12,13-phorbol dibutyrate (with respect to the control group), and significantly greater expression of protein kinase C
and ßII (with respect to the glycerol group).
Key Words: fructose • hypertension • insulin • glycerol • triglycerides
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Introduction |
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In experimental animals, fructose overload can raise blood pressure (BP) and cause hypertriglyceridemia, hyperinsulinemia, impaired glucose tolerance, and insulin resistance in several target tissues.1 2 3 4 5 In this regard, many investigators have suggested that increased insulin levels and/or resistance may be causally related to elevated BP.6 7 This possibility is supported by reports correlating hyperinsulinemia with hypertension.1 8 However, hyperinsulinemia and hypertension have been described as independent factors by other authors.7 9 Consequently, the underlying mechanisms of these 2 conditions are, as yet, undefined.
The lipid dysmetabolism that accompanies fructose overload is another possible cause of hyperinsulinemia and hypertension. In fact, a vicious cycle between hypertriglyceridemia and insulin resistance exists.10 Prentki and Corkey11 studied type II diabetes mellitus and proposed that hyperinsulinemia could be explained by alterations in the glycolytic pathway and lipid metabolism (hypertriglyceridemia is one of the features of a lipid disorder). This metabolic abnormality is followed by an increase of long-chain acyl coenzyme A, which results in an elevation of insulin secretion. The hypertriglyceridemia associated with fructose overload seems to result from lower plasma extrahepatic triglyceride lipase activity and greater VLDL-triglyceride secretion rates.12 Similarly, increased levels of glycerol in the diet induce hypertriglyceridemia in the rat as a result of lower triglyceride clearance after decreased lipoprotein lipase activity.13 Lee et al14 found that raised levels of glucose increase diacylglycerol, which in turn activates protein kinase C (PKC) in the cultured vascular cells and in the aorta, heart, and other tissues from streptozotocin-diabetic rats15 16 17 ; PKC can also modulate contractions of vascular smooth-muscle cells.18 19 The liver and kidneys, which are formally recognized as the main organs responsible for glycerol metabolism (which leads to glyconeogenesis), account for less than 50% of endogenous glycerol clearance.20 This finding indicates that other tissues (putatively including the vascular tissue) that contain glycerol kinase in low concentrations may participate in its metabolism and, thus, potentiate diacylglycerol synthesis.
Accordingly, the objective of this study was to observe the effect of glycerol supplementation on BP to determine to what extent lipid and/or glycolytic pathways are primarily involved in fructose-caused hypertension.
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Methods |
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Animals
Male Sprague-Dawley rats (Animal Facilities, Biochemistry College, Buenos Aires University) that weighed 180 to 230 g at the start of the study were used. Animals were maintained in a room at 22±2°C, and the air was adequately recycled. Initially, all rats were fed a commercial standard laboratory chow (Asociación Cooperativas Argentinas) with the following composition (wt/wt): 20% proteins, 3% fat, 2% fiber, 6% minerals, and 69% starch and vitamin supplements. After 2 weeks, animals were identified by ear marks and randomly divided into 4 groups: (1) the control group continued on the standard diet and was given tap water to drink; (2) the glycerol group received the same solid diet and a glycerol solution (0.54 mol/L) was added to the tap water; (3) the fructose group received a special diet containing 14% starch and 55% fructose, instead of the 69% starch of the standard diet, and tap water to drink; and (4) the fructose-glycerol group received the same diet as group 3 and the 0.54 mol/L glycerol solution to drink. All groups were examined after 14 days.
BP and Body Weight Measurement
Rats were acclimated to the procedure of BP measurement at 1.00
PM twice a week starting 1 week before dietary manipulation
and continuing through the experimental period. Indirect
systolic BP was determined as previously
reported.21 The mean of 3 consecutive stable readings was
used as the measurement of the systolic BP of each rat for that
day, and the average BP of the last 2 readings (11th and 13th days of
the study) were used for statistical comparisons. The procedure for
analyzing the BP data was similar to that reported by Hwang et
al.1 In addition, rats were weighed before dietary
manipulation and at the end of the study.
Plasma Assays
Animals fasted for 5 hours and then were anesthetized
with an intraperitoneal injection of pentobarbital
(60 mg/kg body weight); 90 minutes later, blood samples were drawn from
the retro-ocular plexus. The samples were immediately
centrifuged and frozen at -20°C until assayed for glucose,
triglycerides, and insulin. Glucose (Kit Winner Glycemia
HK, UV) and triglyceride (Boehringer Mannheim
GPO-PAP, enzymatic method kit) levels were measured by
spectrophotometric methods (Automatic Analyzer, Abbott Spectrum
CCX). Insulin was determined in plasma samples by radioimmunoassay
using the method of Herbert et al.22
Catecholamines in the Artery Wall
The catechols in tissue homogenates from the
abdominal aorta were determined by high-pressure liquid
chromatography with electrochemical detection, as
reported previously.23
PKC Western Blots in the Artery Wall
Descendent thoracic rat aorta segments were removed and
homogenized in 0.3 mL of ice-cold
homogenization buffer. PKC, ßII, and 
were
determined by immunoblotting, as previously
described.24 One sample from each of the 4 rat groups was
processed in parallel in each gel. The autoradiograms
were quantified by densitometric scanning. Values from the treated
animals were expressed as percentages of a single control value.
Contractility of Aorta Rings
Batches of 4 rats (1 from each group) were killed daily by
decapitation. The thoracic aorta was harvested, placed into cold Krebs
solution, and prepared for contractility
recording, as previously reported.21 Thereafter,
the effect of 10-5 mol/L nitroprusside on
baseline ring tension was observed. In other groups of rings, the dose
response to 12,13-phorbol dibutyrate (PDBu; 5 ·
10-8 to 5 · 10-6
mol/L) was determined.
Statistical Analysis
Results are expressed as mean±SEM, and the significance level
was P<0.05. Comparisons of data between different groups
were made by 1-way ANOVA followed by a Newman-Keuls post hoc test when
significance was indicated.
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Results |
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Effect of Fructose and Glycerol on Arterial Pressure
After 14 days of experimental intervention, BP was as follows (mm Hg): control, 129±2; glycerol, 131±2; fructose, 140±3; and fructose-glycerol, 149±2. Significant differences were observed for the fructose group versus control (P<0.005) and glycerol groups (P<0.005) and for the fructose-glycerol group versus control (P<0.001), glycerol (P<0.001), and fructose groups (P<0.005) (Figure 1).
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Fasting Glycemia, Triglyceridemia, and
Insulinemia
On day 14, glycemia values for the glycerol, fructose, and
fructose-glycerol groups were not significantly different from the
control group. However, the glycemia concentration was significantly
lower in the fructose-glycerol rats when compared with the fructose
rats (P<0.05). Plasma triglyceride
concentrations increased significantly with respect to the control
group in all treated groups. Plasma insulin concentrations were
significantly different from all other groups only in fructose-glycerol
rats (Figure 2).
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Vascular Contractility
No change in basal tone was discovered by the administration of
10-5 mol/L nitroprusside. However,
dose-response curves with PDBu showed a significantly lower
ED50 (higher sensitivity) in aorta rings from the
fructose-glycerol group with respect to those from the control
(P<0.001), glycerol (P<0.002), and fructose
(P<0.001) groups. Maximum tension was significantly higher
only in the fructose group (Figure 3).
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Significant differences
versus control (glycerol, P<0.05; fructose,
P<0.05; and fructose-glycerol, P<0.02).
Responses of fructose-glycerol (P<0.05) and glycerol
groups (P<0.05) were significantly different than those
of fructose group. §Responses from fructose-glycerol
(P<0.05) and glycerol groups (P<0.05)
were significantly different than those of fructose group. Right,
ED50 of PDBu expressed as -log mol/L (Mean±SEM).
*Fructose-glycerol group was significantly different from the other
groups.
Catecholamines in the Artery Wall
No significant differences were observed among groups;
nevertheless, with the exception of dopamine in the fructose group, all
mean values were lower than in the control group
(Table).
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PKC Western Blot in Artery Wall
PKC expression was significantly lower in the glycerol group
than the control and fructose-glycerol groups, and PKCßII expression
was significantly lower in the glycerol group than the
fructose-glycerol group. No significant changes in PKC were detected
between groups (Figure 4).
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Discussion |
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Although diabetes has been thoroughly investigated at the clinical and experimental levels, little attention has been paid to prior metabolic disorders and hypertension, which may ensue from excessive carbohydrate intake. The experimental model studied here is currently under intensive research; it mimics the risk factors of sweet-toothed subjects in the general population, ranging from "resistant" to "prediabetic" individuals.
Cardiac output is reportedly within normal limits in the fructose-overload experimental model1 ; thus, peripheral vascular resistance must be increased to account for a high BP. Therefore, in addition to general metabolic disorders, this article examines some factors concerning the target organ, ie, the vascular wall. Glycerol supplementation was done to enhance the metabolic disorders leading to hypertension.
Peral de Bruno et al25 reported that aorta rings from rats after 10 weeks of a fructose diet have an increased basal tone, as shown by the presence of a relaxing effect to 10-5 mol/L nitroprusside. In contrast, the present experiments failed to disclose changes in basal tone by nitroprusside in vitro after 2 weeks of fructose overload. Sensitivity to PDBu stimulation was significantly greater in vessels from our fructose-glycerol rats (Figure 3), although at a concentration of 7.27 · 10-7 mol/L, both the glycerol and fructose groups showed significantly greater vascular responses than controls. Such findings agree with the raised BP (putatively in peripheral resistance) that developed after 2 weeks both in the fructose and fructose-glycerol groups. Moreover, results from the fructose-glycerol group in particular support the potential effect of glycerol; BP, insulinemia, and vascular sensitivity to PDBu were all significantly greater when fructose was supplemented with 0.54 mol/L glycerol in drinking water.
The maximal tension to PDBu that developed was lower in the fructose-glycerol and glycerol groups and higher in the fructose group when compared with controls. This prompted us to investigate whether the different maximal responses to the direct PKC activator PDBu correlated with a different expression of PKC in the thoracic aorta within the studied groups. However, only in the glycerol group did a direct relationship between changes in the maximal PDBu-induced response and PKC expression exist.
Concerning catecholamines, an increased systemic sympathetic tone has been reported in fructose-overloaded rats.26 27 In this regard, although not significantly different among groups, all mean values in the aorta wall were lower in glycerol-supplemented groups (Table); these results render the increase in local catecholamine content in these groups unlikely. This suggestion is of particular interest for the fructose-glycerol rats in view of their greater incidence of hypertension.
In conclusion, our results showed the following: (1) oral glycerol
administration per se (glycerol group) was accompanied by
hypertriglyceridemia (as high as in other
groups), normal insulinemia, and decreased thoracic aorta PKC
expression; (2) in the fructose group,
hypertriglyceridemia was again present,
but rats failed to show hyperinsulinemia; (3) when
glycerol was administered with fructose (fructose-glycerol group),
hypertriglyceridemia,
hyperinsulinemia, increased vascular sensitivity to
PDBu, and significantly greater values of PKC and ßII expression
with respect to the glycerol group were simultaneously
present and accompanied by greater BP values with respect to the
other 3 experimental groups. However, the mechanism linking glycerol
potentiation to fructose overload remains unclear.
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Acknowledgments |
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This work was supported by grants from the National Research Council (Consejo Nacional de Investigaciones Cientificas y Tecnicas [CONICET]) and the University of Buenos Aires, Argentina. Drs Armando, PhD, Norwicki, PhD, and de la Riva, PhD, are senior investigators from CONICET, Argentina.
Received May 17, 1999; first decision June 15, 1999; accepted July 14, 1999.
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