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Laboratory of Nutrition Physiopathology, Centre National de la Recherche Scientifique, ESA 7059, University Paris 7 (M.-H.G., B.P.), F-75251 Paris 05, France; and Laboratory of Experimental Medicine, Brussels Free University (O.S., L.L., A.S., W.J.M.), B-1070 Brussels, Belgium
Address all correspondence and requests for reprints to: Prof. W. J. Malaisse, Laboratory of Experimental Medicine, Brussels Free University, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail: malaisse{at}med.ulb.ac.be
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Abstract |
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Introduction |
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The major aim of the present study was to compare the metabolism of D-glucose and/or D-fructose in both isolated pancreatic islets and parotid cells from either GK or control rats. The insulin secretory response to the two hexoses was also investigated. The selection of this theme is based on the following four series of considerations.
First, the comparison between pancreatic islets and parotid gland may be relevant to the respective contributions of insulin-producing and extrapancreatic cells to the perturbation of glucose homeostasis in the GK rats, the parotid cells being considered as an example of functionally glucose-unresponsive extrapancreatic cells.
Second, the study of the catabolism of D-glucose and/or D-fructose was inspired by recent acquisitions on the metabolic interaction between these two hexoses in pancreatic islets. Thus, in addition to current knowledge on the reciprocal effects of the two sugars on their respective rate of phosphorylation by hexokinase, it was recently shown that D-glucose increases D-fructose phosphorylation by glucokinase (9) and preferentially stimulates the oxidation of the ketohexose in intact islets (10).
Third, although fructokinase activity is also present in pancreatic islet homogenates (11), the respective contribution of this enzyme and the hexokinase isoenzymes (9, 10) to the overall rate of D-fructose phosphorylation in intact islets exposed to the ketohexose in the absence or presence of D-glucose remains to be assessed.
Last, the proposal was recently made that the insulinotropic action of D-fructose may not be entirely accounted for by its oxidative catabolism in islet cells (12). In this respect, it was considered of interest to compare the metabolic and secretory responses to the ketohexose in islets from either control or GK rats.
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Materials and Methods |
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Animals
Sixty-four control rats and 67 GK rats with mean body
weights of 226 ± 10 g (n = 64) and 268 ± 7 g
(n = 67), respectively, were given free access to food (KM-04-k12,
Pavan Service, Oud Turnhout, Belgium) and tap water up to the time of
death. The higher body weight (P < 0.001) of the GK
rats coincided with both a higher prevalence of male than female rats
(73.1 vs. 14.0%) and a somewhat greater mean age (18.1
± 0.6 vs. 
14.2 ± 0.7 weeks) of the diabetic
compared with the normal animals. The GK rats, males and both males and
females, were obtained from two colonies maintained in Paris and
Brussels, respectively. In 1 series of experiments related to insulin
release, the male Paris GK rats were compared with sex- and age-matched
Wistar rats raised in parallel. In the other experiments, however,
female Wistar rats (197 ± 3 g; n = 55) obtained from B
& K Ltd. (Hull, UK) were used for comparison with GK rats of both
sexes. Note that from data collected within the same series of
experiments and in agreement with a prior report (3), no significant
difference linked to sex could be detected in plasma glucose
[14.2 ± 0.6 mM (n = 22) and 14.2 ± 0.7
mM (n = 9) in male and female GK rats, respectively]
or insulin [2.82 ± 0.31 ng/ml (n = 32) and 1.84 ±
0.41 ng/ml (n = 9) in male and female GK rats, respectively]
levels.
Blood collection and preparation of pancreatic islets and parotid
cells
The animals were killed by decapitation, and blood was collected
in heparinized tubes. After centrifugation, the plasma was removed and
stored at -20 C for further glucose (13) and insulin (14)
determinations. Islets were isolated from the pancreases of 3–4
control or GK rats using a collagenase (Roche Molecular Biochemicals, Mannheim, Germany) digestion technique (15) and
subsequently separated from the remaining exocrine tissue by
hand-picking under a stereomicroscope. Hanks’ solution saturated with
O2 (95%) and CO2 (5%) was used during the
isolation procedure. The freshly isolated islets were immediately used
for experiments. In parallel, 1–2 groups of 15 islets each from
control or diabetic rats were sonicated (3 times, 5 sec each time) in
0.25 ml redistilled water. An aliquot (25 µl) of this aqueous
homogenate was mixed with 1.0 ml phosphate buffer (0.1 M;
pH 7.0) containing 10 mg/ml BSA and then stored at -20 C for further
determination of insulin content, as reported previously (15). The
remaining homogenate was used to measure the protein content of islet
cells according to the method of Lowry et al. (16). Cells
from parotid gland were prepared by the procedure of Mangos et
al. (17).
D-glucose and
D-fructose uptake
The apparent distribution space of
D-[U-14C]glucose or
D-[U-14C]fructose was measured using an oil
filtration technique described previously (18). Briefly, groups of 15
islets each were incubated for 5–20 min at 37 C in 80 µl
bicarbonate-buffered medium (15) supplemented with 5 mg/ml BSA and also
containing 3HOH and either 2 mM
L-[1-14C]glucose or 20 mM
D-[U-14C]glucose or
D-[U-14C]fructose. Then, the islets were
separated from the medium by centrifugation through a layer (150 µl)
of silicone oil (Versilube F-50, General Electrics, Waterford, NY) into
60 µl of a solution of CsCl (640 mM) in diluted HCl (50
mM). The tip of the tube containing the islet pellet was
removed with a scalpel and examined for its radioactive content.
D-Glucose and
D-fructose metabolism
The conversion of
D-[5-3H]glucose or
D-[5-3H]fructose to 3HOH and the
production of 14CO2 from
D-[1-14C]glucose,
D-[6-14C]glucose,
D-[U-14C]glucose,
D-[1-14C]fructose,
D-[6-14C]fructose, or
D-[U-14C]fructose were measured over a
120-min incubation period at 37 C in 40 µl bicarbonate-buffered
medium (15) containing 5 mg/ml BSA and the appropriate
D-glucose or D-fructose concentration mixed
with a tracer amount of both 3H- and
14C-labeled corresponding hexoses, using 15–20 islets or
2 x 105 parotid cells/sample. The incubation was
halted by adding 20 µl citrate-NaOH buffer (0.4 M; pH
4.9) containing KCN (5 mM), rotenone (10 µM),
and antimycin A (10 µM). The
14CO2 and 3H2O formed
during the incubation were determined by liquid scintillation counting;
after that, the 14CO2 was trapped in 0.25 ml
hyamine hydroxide (Packard, Downers Grove, IL) during a 60-min
incubation period at 37 C, and the recovery of
3H2O in 5 ml H2O was completed over
a further incubation of 22 h at 20 C, as described previously
(19). The acidified medium containing the islets was stored at -20 C
and later examined for its content of 14C-labeled acidic
metabolites and amino acids separated by ion exchange chromatography
using AG1-X8 (chloride form; Bio-Rad Laboratories, Inc.,
Hercules, CA) and Dowex 50 (H+ form; Fluka Chemical Co., Buchs, Switzerland) resins, as previously described (20, 21).
Insulin release
For measuring insulin release, groups of seven or eight islets
each were incubated for 120 min at 37 C in 1.0 ml of our usual
bicarbonate-buffered medium (15) containing 5 mg/ml BSA and
equilibrated against a mixture of O2 (95%) and
CO2 (5%). Test agents were added as indicated. The amount
of insulin released in the medium was measured by RIA, as described
previously (15).
Data analysis
All results, including those already mentioned, are presented as
the mean (±SEM) together with either the degree of freedom
(df) or the number of observations (n), each of which refers to an
individual animal or a group of either islets or parotid cells. The
statistical significance of differences between mean values was
assessed using Student’s t test. Such differences are
usually based on the comparison of data collected in nearly equal
numbers within the same experiment(s). The SEM on the sum,
difference, product, or ratio between distinct mean values was
calculated as previously described (22, 23).
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Results |
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). The insulinogenic index (i.e. the
paired ratio between plasma insulin/D-glucose
concentration) and the islet protein and insulin content as well as the
paired ratio between the latter two variables were not significantly
different, however, in the two groups of rats (Table 1).
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, the apparent distribution space of
D-[U-14C]glucose or
D-[U-14C]fructose (20.0 mM each),
when expressed relative to the paired 3HOH space, exceeded
(P < 0.001 in all cases) that of
L-[1-14C]glucose. After only 5 min of
incubation, the paired ratio between the distribution space of the
uniformly 14C-labeled hexoses and that of 3HOH
was not significantly different (P > 0.35) in islets
exposed to either D-[U-14C]glucose or
D-[U-14C]fructose. After 20 min of
incubation, however, such a paired ratio was higher (P
< 0.001) for aldohexose than for ketohexose. It indeed increased
significantly between the 5th and 20th minute of incubation in islets
exposed to D-[U-14C]glucose
(P < 0.001) while failing to do so in the islets
exposed to D-[U-14C]fructose
(P > 0.3). This difference is probably attributable at
least in part to the higher rate of D-glucose than
D-fructose catabolism (see below), resulting in a larger
intracellular accumulation of radioactive metabolites of the hexose in
the former than in the latter case.
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). In both control and GK rats,
D-fructose (10.0 mM) provoked only minor
changes in the utilization of
D-[5-3H]glucose. In this respect, there was
no significant difference between control and diabetic rats; the
overall mean value for the ketohexose-induced decrease in
D-[5-3H]glucose utilization did not exceed
5.2 ± 3.4% (df = 226).
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D-[U-14C]Glucose
and D-[U-14C]fructose
oxidation
The mean absolute value for the oxidation of
D-[U-14C]glucose (10.0 mM) was
not significantly different in control and GK rats (Table 4). Relative to the paired value for
D-[5-3H]glucose utilization, however, the
oxidation of D-[U-14C]glucose was somewhat
lower in GK rats than in control animals. Indeed, in the range of
hexose concentrations between 10.0–16.7 mM, such a paired
ratio averaged 35.9 ± 1.4% in GK rats (n = 28) compared
with 40.1 ± 1.3% in normal animals (P < 0.05;
n = 23). In both control and GK rats, the oxidation of
D-[U-14C]glucose failed to be affected
significantly by D-fructose (10.0 mM), as
judged from either the absolute values for the oxidation of the
aldohexose or the paired ratio between
D-[U-14C]glucose oxidation and
D-[5-3H]glucose utilization.
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). Such a difference remained highly
significant (P < 0.005 or less) in both control
and GK rats when the oxidation of the hexoses was expressed
relative to the paired value for the utilization of
D-[5-3H]hexoses.
The absolute value for the oxidation of
D-[U-14C]fructose was higher in GK rats than
in control animals. For instance, in the experiments summarized in
Table 4, the measurements made in GK rats averaged 127.2 ± 7.2%
(n = 123; P < 0.005) of the corresponding values
found under the same experimental conditions (i.e. in the
absence or presence of D-glucose) in control animals
(100.0 ± 4.3%; n = 86). Such a difference was not observed,
however, when the oxidation of
D-[U-14C]fructose was expressed relative to
the paired value for D-[5-3H]fructose
utilization. As a matter of fact, the paired ratio between
D-[U-14C]fructose oxidation and
D-[5-3H]fructose utilization was, on
occasion, lower in GK rats than in control animals (e.g. in
the experiments illustrated in Fig. 1).
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It should be underlined that in islets from either control or GK rats incubated in the presence of both D-glucose and D-fructose (10.0 mM each), the paired ratio between D-[U-14C]fructose oxidation and D-[5-3H]fructose utilization was higher (P < 0.05 or less) than that between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization. This difference is likely to reflect at least in part the phosphorylation of D-fructose by fructokinase (7), in which case the catabolism of the C1-C2-C3 moiety of the ketohexose may well exceed that of its C4-C5-C6 moiety, which contains the 3H atom present on the C5 in D-[5-3H]glucose.
As illustrated in Fig. 1, the concentration-response relationship for
the enhancing action of D-glucose on
D-[U-14C]fructose oxidation, relative to
D-[5-3H]fructose utilization, displayed a
comparable pattern in islets from control and GK rats, with a maximal
response to the aldohexose at a concentration of D-glucose
close to 10.0 mM.
D-[1-14C]glucose
and D-[6-14C]glucose
oxidation
In absolute terms, the oxidation of
D-[1-14C]glucose or
D-[6-14C]glucose (10.0 mM) by
islets from GK rats was not significantly different from that found,
within the same series of experiments, in islets from normal rats
(Table 5). In both normal and GK rats, it
failed to be significantly affected by D-fructose (10.0
mM).
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In both control and GK rats, the oxidation of D-[1-14C]glucose, when expressed relative to the paired value for D-[5-3H]glucose utilization, exceeded (P < 0.001) that of D-[6-14C]glucose.
D-fructose (10.0 mM) failed to significantly affect the paired ratio between D-[1-14C]glucose or D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization in both control and GK rats.
As judged from these data, the participation of the pentose phosphate pathway in the total metabolism of D-glucose (24) was not significantly different in control and GK rats in either the absence or presence of D-fructose. The ketohexose, however, slightly decreased (P < 0.06) the fraction of D-glucose metabolism that occurs by the pentose cycle, from 3.5 ± 0.3% to 2.3 ± 0.5% (pooled values in control and GK rats).
D-[1-14C]Fructose
and D-[6-14C]fructose
oxidation
In islets from normal rats, the oxidation of
D-[1-14C]fructose exceeded that of
D-[6-14C]fructose, and in the absence of
D-glucose, the apparent fractional contribution of the
pentose shunt to the generation of CO2 and
D-glyceraldehyde-3-phosphate was higher than that found in
islets exposed to an equimolar concentration of D-glucose
(10.0 mM). Moreover, the aldohexose decreases the apparent
fraction of D-fructose metabolism occurring by the pentose
cycle (10).
In several respects, a comparable situation was found in GK rats (Table 6). Thus, in either the absence or
presence of D-glucose (10.0 mM), the oxidation
of D-[1-14C]fructose (also 10.0
mM) exceeded that of
D-[6-14C]fructose, as judged by either the
absolute values for 14CO2 output or the paired
ratio between such an output and the generation of 3HOH
from D-[5-3H]fructose (P <
0.001 in all cases).
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At variance with the situation found in control rats, however, D-glucose failed to decrease the apparent fractional contribution of the pentose phosphate pathway to the catabolism of D-fructose, with mean values of 5.7 ± 1.3% and 5.9 ± 1.5% in the absence and presence of the aldohexose, respectively. The former percentage was higher (P < 0.05) than that found for D-glucose metabolism in islets from GK rats (see above), such a difference being again comparable to that found in normal rats (10).
D-[3,4-14C]Fructose
oxidation
It was recently reported that in islets from normal rats exposed
to 10 mM D-fructose, the paired ratio between
D-[3,4-14C]fructose oxidation and
D-[5-3H]fructose utilization was higher than
that found in the case of D-[1-14C]fructose,
D-[2-14C]fructose or
D-[6-14C]fructose (10). Likewise, the
enhancing action of D-glucose (10.0 mM) on the
oxidation of D-[3,4-14C]fructose was higher
than that found with the other three 14C-labeled
tracers.
In islets from GK rats, D-glucose (10.0 mM) also increased (P < 0.001) the ratio between D-[3,4-14C]fructose oxidation and D-[5-3H]fructose utilization from 34.8 ± 1.7% (n = 15) to 58.2 ± 1.8% (n = 16). As in normal rats, the former percentage was higher (P < 0.005 or less) than that found with D-[1-14C]fructose or D-[6-14C]fructose (see above). In the GK rats, the ratio of D-[3,4-14C]fructose oxidation in the presence/absence of D-glucose, as judged from the paired comparison between 14CO2 and 3HOH production, averaged 1.68 ± 0.09 (df = 29), compared with 1.32 ± 0.06 (df = 44) and 1.63 ± 0.10 (df = 44) in the case of D-[1-14C]fructose and D-[6-14C]fructose, respectively. The hierarchy for the relative magnitude of the enhancing action of D-glucose on the oxidation of the different 14C-labeled tracers of D-fructose was thus also comparable in GK and normal rats (10).
Generation of 14C-labeled acidic
metabolites and amino acids
Whether in control or GK rats, the net production of
14C-labeled acidic metabolites and amino acids
from D-[U-14C]glucose was not affected
significantly by the incorporation of D-fructose in the
incubation medium (Table 7, upper panel). Like the
utilization of D-[5-3H]glucose, the
generation of radioactive acidic metabolites from
D-[U-14C]glucose was higher
(P < 0.001) in GK rats than in control animals.
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The production of 14C-labeled acidic metabolites from D-[U-14C]fructose appeared quite variable. No significant production could be detected in three of six series of experiments. When measurable, it was not significantly different in the absence or presence of D-glucose and was slightly, but not significantly (P < 0.07), higher in GK than control rats.
The production of 14C-labeled acidic metabolites and amino
acids from D-[1-14C]glucose or
D-[6-14C]glucose also tended to be higher in
GK rats than control rats (Table 7,
lower panel). Pooling all available data, in the GK rats it
averaged 154.1 ± 8.5% (n = 80; P < 0.001)
of the mean corresponding value found in control animals (100.0 ±
7.0%; n = 86) in the case of the 14C-labeled acidic
metabolites and 120.4 ± 3.5% (n = 75; P <
0.001) compared with a control value of 100.0 ± 3.3% (n =
90) in the case of the radioactive amino acids. The presence of
D-fructose tended, as a rule, to slightly decrease the net
production of acidic metabolites and amino acids from either
D-[1-14C]glucose or
D-[6-14C]glucose. Pooling all available data
obtained for both metabolites with both tracers in both control and GK
rats, the measurements made in the presence of the ketohexose averaged
89.4 ± 2.9% (n = 116; P < 0.05) of the
mean corresponding value found in its absence (100.0 ± 3.8%;
n = 161).
In the islets from GK rats, D-glucose failed to significantly affect the net generation of 14C-labeled amino acids from either D-[1-14C]fructose (7.0 ± 0.8 and 7.8 ± 0.3 pmol/islet·120 min in the absence and presence of D-glucose, respectively; n = 23–24) or D-[6-14C]fructose (6.8 ± 0.8 and 7.8 ± 0.5 pmol/islet·120 min in the absence and presence of D-glucose; n = 23–24). The aldohexose, however, decreased (P < 0.001) the net production of radioactive amino acids from D-[3,4-14C]fructose from 3.3 ± 0.3 to 1.8 ± 0.2 pmol/islet·120 min (n = 16 in both cases). The latter finding and the fact that the net formation of 14C-labeled amino acids was lower (P < 0.005 or less) in the islets exposed to D-[3,4-14C]fructose rather than D-[1-14C]fructose or D-[6-14C]fructose are consistent with the knowledge that 14CO2 is generated from D-[3,4-14C]fructose in the reaction catalyzed by pyruvate dehydrogenase and that D-glucose preferentially stimulates mitochondrial oxidative events such as the decarboxylation of pyruvate in the same reaction (25).
Insulin release
In fair agreement with several prior observations
(26, 27, 28, 29), the basal value for insulin output was higher
(P < 0.001) in GK than normal rats (Table 8). In normal rats, insulin release was
augmented (P < 0.01 or less) above basal value by
2-ketoisocaproate (10 mM), D-glucose (7.0 and
10.0 mM), and, in the presence of the aldohexose,
D-fructose (10.0–30.0 mM). In the absence of
D-glucose, however, D-fructose (10.0
mM) failed to significantly affect insulin output
(P > 0.9).
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The insulinotropic action of D-fructose was even more severely affected than that of D-glucose in the islets of GK rats, as documented by the following findings. First, in islets from GK rats, D-fructose at concentrations of 10.0 and 20.0 mM failed to augment significantly insulin output evoked by 10.0 mM D-glucose, at variance with the situation found in islets from control rats. Second, in the presence of 30.0 mM D-fructose, the output of insulin relative to that recorded in the sole presence of 10.0 mM D-glucose averaged no more than 147.9 ± 14.4% (n = 24) in GK rats (n = 28) compared with 194.0 ± 14.9% in control animals (P < 0.025; n = 24).
The comparison between the results obtained at 7.0 mM D-glucose in control rats and 10.0 mM D-glucose in GK rats clearly document that the poor secretory response to D-fructose found in the latter animals cannot be blamed on the fact that the absolute value for glucose-stimulated insulin release was lower in GK rats than in normal animals.
Experiments in parotid cells
To compare the situation found in pancreatic islets to that
prevailing in functionally glucose-unresponsive cells, the metabolism
of D-[U-14C]glucose and/or
D-[U-14C]fructose (10.0 mM
each) was also investigated in parotid cells (Table
9).
In control rats, the generation of 14CO2 and 14C-labeled amino acids and acidic metabolites from D-[U-14C]glucose was not significantly affected by D-fructose. The oxidation of D-[U-14C]fructose and its conversion to 14C-labeled amino acids were not vastly different from those found with D-[U-14C]glucose. The net production of radioactive acidic metabolites was much lower, however, in cells exposed to D-[U-14C]fructose than in those exposed to D-[U-14C]glucose (P < 0.001). Unlabeled D-glucose severely decreased the metabolism of D-[U-14C]fructose (P < 0.02 or less); the relative magnitude of this effect was less pronounced (P < 0.001) in the case of 14C-labeled acidic metabolites than for either 14CO2 or radioactive amino acids.
In most respects, the situation found in parotid cells of GK rats was similar to that just described in the case of control animals. The sole obvious difference between control and GK rats consisted of a higher yield (P < 0.001) of 14C-labeled metabolites from D-[U-14C]glucose in the latter than in the former animals.
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Discussion |
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Although the production of 3HOH from D-[5-3H]glucose; the net generation of 14C-labeled acidic metabolites from D-[U-14C]glucose, D-[1-14C]glucose, and D-[6-14C]glucose; and, to a lesser extent, the net production of radioactive amino acids from the latter two tracers were all higher in islets from GK rats than in those from control animals, the oxidative response to D-glucose was obviously impaired in the diabetic animals. Indeed, the paired ratio between D-[U-14C]glucose, D-[1-14C]glucose, or D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization was always lower in GK rats than in control animals.
As a rule, D-fructose failed to significantly affect the catabolism of D-glucose. At the most, the ketohexose slightly decreased the fraction of D-glucose metabolism that occurs by the pentose cycle and the production of radioactive acidic metabolites and amino acids from 14C-labeled D-glucose.
The metabolism of D-fructose differed from that of D-glucose in several respects. The experiments conducted in islets from control rats indicate that this could not be attributed to any difference in the net uptake of the two hexoses during the first 5 min of incubation, confirming a prior observation (30).
The first difference between the metabolism of D-glucose and D-fructose consisted in the lower rate of catabolism of the ketohexose than aldohexose, both tested at the same concentration of 10 mM.
Second, D-glucose obviously affected the metabolism of D-fructose. It decreased the production of 3HOH from D-[5-3H]fructose, but increased the paired ratio between D-[U-14C]fructose, D-[1-14C]fructose, D-[6-14C]fructose, or D-[3,4-14C]fructose oxidation and D-[5-3H]fructose utilization. These contrasting effects of the aldohexose on the metabolism of the ketohexose could be due to the following interactions between these two monosaccharides. First, D-glucose severely inhibits the phosphorylation of D-fructose by the low Km hexokinase (10), fails to affect the phosphorylation of D-fructose by fructokinase (11), and augments the phosphorylation of D-fructose by glucokinase (9). The latter effect and the participation of fructokinase in the phosphorylation of D-fructose may account for the relatively modest effect of D-glucose on D-[5-3H]fructose utilization by intact islets, as distinct from the marked D-glucose-induced inhibition of D-fructose phosphorylation by the low Km hexokinase. Second, the preferential stimulation by D-glucose of D-fructose oxidation, relative to its utilization, is likely to reflect the effect of the aldohexose to favor mitochondrial oxidative processes, relative to total glycolytic flux (25). In this respect, it should be underlined that the catabolism of the exogenous hexoses, and hence the release of insulin, are much higher in islets exposed to D-glucose (with or without D-fructose) than in islets exposed solely to D-fructose, in which case the output of insulin is not higher than the basal value, at least under the present experimental conditions.
Third, when the islets were exposed to both D-glucose and D-fructose, the paired ratio between D-[U-14C]fructose oxidation and D-[5-3H]fructose conversion to 3HOH was higher than the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization. This situation is probably attributable to the participation of fructokinase in the phosphorylation of D-fructose. Indeed, such a process will result in the generation of D-[2-3H]glyceraldehyde from D-[5-3H]fructose, and a sizable fraction of this tritiated triose could well escape further catabolism in the islets. Assuming radioisotopic equilibration between the intracellular pools of D-[U-14C]glucose 6-phosphate and D-[U-14C]fructose 6-phosphate, the comparison between the paired ratios for the conversion of the D-[U-14C]hexoses to 14CO2 and that of the D-[5-3H]hexoses to 3HOH in the islets from control or GK rats exposed to both D-glucose and D-fructose indicates that the contribution of fructokinase accounts for at least 21.8 ± 5.5% of the total rate of phosphorylation of the ketohexose. These considerations also imply that the formation of 3HOH from D-[5-3H]fructose sensibly underestimates the true phosphorylation rate of the ketohexose. Therefore, the fractional contribution of the pentose phosphate pathway to the metabolism of D-fructose, as calculated by the equation proposed by Katz and Wood (24), is overestimated. As a matter of fact, when allowance was made for the estimated contribution of fructokinase to the phosphorylation of D-fructose (see above), the fractions of D-fructose and D-glucose metabolized via the pentose cycle were no more different from one another in islets of GK rats exposed to aldohexose than those in islets of rats exposed to ketohexose.
In all of these respects, there was no significant difference between
control and GK rats. The concentration-response relationship for the
effect of D-glucose on
D-[U-14C]fructose oxidation was also
comparable in the control and diabetic animals (Fig. 1). The sole
difference between control and GK rats consisted of the fact that the
absolute values for D-[5-3H]fructose
conversion to 3HOH or
D-[U-14C]fructose oxidation, like those for
D-[5-3H]glucose utilization, were higher in
the latter than in the former animals. However, such was not the case
when the oxidation of D-[U-14C]fructose was
expressed relative to the paired value for
D-[5-3H]fructose conversion to
3HOH.
In the islets of GK rats, the preferential impairment of the oxidative response to D-glucose coincided with a lower secretory response to the aldohexose. The secretory response to 2-ketoisocaproate, which is also dependent on the mitochondrial catabolism of this secretagogue, was similarly impaired in the islets of GK rats. However, despite the fact that the metabolism of D-fructose, in the absence or presence of D-glucose, was essentially comparable in islets from GK and control rats, the insulinotropic action of the ketohexose, relative to that of the aldohexose, was again impaired in the diabetic animals. This finding provides further support for the view that the secretory response to D-fructose may not be entirely accounted for by its nutritional value in the islet cells (12). It further suggests that regardless of its precise molecular determinant(s), the modality of D-fructose insulinotropic action that is not linked to its role as a fuel in insulin-producing cells is also altered in GK rats.
The results obtained here in parotid cells are compatible with
the knowledge that at a 10.0-mM concentration of the
hexoses, the phosphorylation of D-fructose by hexokinase is
virtually abolished by D-glucose, whereas
D-fructose fails to affect significantly the
phosphorylation of D-glucose (10). Moreover, the present
data suggest that D-fructose may also be phosphorylated by
fructokinase in parotid cells. The absolute rate of
D-fructose phosphorylation by fructokinase can be estimated
by the residual generation of 14C-labeled metabolites from
D-[U-14C]fructose in the presence of
D-glucose, taking into account the fact that a large
fraction of D-[U-14C]glyceraldehyde formed
from D-[U-14C]fructose 1-phosphate may well
escape further catabolism or be converted to
[U-14C]glycerol in the reaction catalyzed by
glycerol-1-dehydrogenase (Fig. 2). The
latter reaction, by consuming NADH, may account for the fact that in
the sole presence of D-[U-14C]fructose, the
net generation of radioactive acidic metabolites (e.g.
L-[U-14C]lactate), when expressed
relative to the total generation of 14C-labeled
metabolites, was much lower than that in parotid cells exposed to
D-[U-14C]glucose. A comparable situation was
found in parotid cells from GK rats, except that the total production
of 14C-labeled metabolites from
D-[U-14C]glucose was higher
(P < 0.001) in GK rats than in control animals. This
illustrates that the metabolism of D-glucose is not
affected adversely in the parotid cells of GK rats. Moreover, the
fractional conversion of D-[U-14C]glucose to
14CO2, 14C-labeled amino acids, and
acidic 14C-labeled metabolites was virtually identical in
control and GK rats.
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Acknowledgments |
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Footnotes |
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Received April 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-D-glucose
pentaacetate: metabolic aspects. Mol Gen Metab 64:135–147[CrossRef][Medline]
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