1 Diabetes and Metabolism Unit, Evans Department of Medicine and Department of Physiology, Boston University Medical Center, Boston, Massachusetts 02118; 2 Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia; 3 Diabetes and Nutrition Research Group, Institute of Experimental Endocrinology, Slovak Academy of Sciences, 83306 Bratislava, Slovak Republic; and 4 Department of Biochemistry, Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel
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ABSTRACT |
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In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E641-E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism.
acetyl-coenzyme A carboxylase; malate; insulin resistance; hyperglycemia; hyperinsulinemia; glucose sensing
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INTRODUCTION |
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MALONYL-CoA is a negative regulator of fatty acid oxidation by virtue of its ability to inhibit carnitine palmitoyltransferase I (CPT I). Thus, in liver, when malonyl-CoA levels are high, CPT I is inhibited, leading to a decreased rate of fatty acid oxidation and an increased rate of glycerolipid synthesis (22, 23). Conversely, when malonyl-CoA levels are low, CPT I is relatively uninhibited and fatty acid oxidation is high (25). Recent studies have shown that substantial increases in malonyl-CoA occur in rat soleus muscle when it is incubated for 20 min with a medium enriched in glucose and insulin or glucose and acetoacetate (37). These increases in malonyl-CoA were observed in the absence of stable changes in the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme for malonyl-CoA formation; however, they correlated with increases in the whole cell concentration of citrate (37), an allosteric activator of ACC and a precursor of its substrate cytosolic acetyl-CoA (14), and of malate, and to a remarkable degree (r = 0.95) with the sum of the concentrations of citrate and malate (37). Because malate is an antiporter for citrate efflux from the mitochondria (21), it was suggested that an increase in its concentration, if it occurred in the cytosol, could cause a redistribution of intracellular citrate, resulting in an even greater increase in its concentration in the cytosol.
Whether this relationship between cytosolic citrate and malonyl-CoA, proposed on the basis of studies in incubated muscle, occurs in vivo is not known. To address this question, the concentrations of malonyl-CoA, citrate, and malate and the activity of ACC were measured in rat muscle in vivo under conditions in which the concentrations of insulin and/or glucose in plasma were increased. The conditions studied include glucose and insulin infusions, a euglycemic-hyperinsulinemic clamp, and the fa/fa rat. In addition, similar measurements were performed in denervated (inactive) rat muscle and in rat muscle of starved rats after refeeding, both of which have increased levels of malonyl-CoA.
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MATERIALS AND METHODS |
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Animals
Rats used for studies carried out in Australia were obtained from the in-house Garvan Institute animal colony and for studies carried out in the US from Charles River Breeding Laboratories (Wilmington, MA), unless otherwise noted. For individual experiments, the nutritional state of the rats and details of their handling before muscle sampling are described in Table 1.
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Experimental Protocols
Australian studies.
glucose infusions.
Adult male Wistar rats weighing ~350 g were maintained in separate
cages in a room with a controlled 12:12-h light-dark cycle (light on at
0600). They were fed standard laboratory chow. To carry out chronic
infusions, cannulas were placed in the right jugular vein and left
carotid artery, exteriorized at the vertex of the head, and attached to
a swiveling infusion device, as previously described (38). Three days
after the surgery, rats were infused via the carotid cannula
(peristaltic roller pump, Watson-Marlow model 101U/R, Falmouth, UK)
according to one of the following protocols: 50% glucose in water
(50% dextrose, Viaflex, Baxter, Sydney, Australia) at a glucose
infusion rate of 40 mg · kg1 · min
1
(~1.6 ml/h) for 4 days; an equal volume of normal saline for 3 days
and then glucose for 1 day; or with saline for 4 days (11, 18). At the
end of the infusions, the rats were given a lethal injection of
pentobarbital sodium (Lethabarb, 150 mg/kg iv), and the red quadriceps
muscles were excised, freeze-clamped, and stored at
70°C for
subsequent analysis.
American studies.
euglycemic-hyperinsulinemic clamp.
Male Sprague-Dawley rats weighing ~300-350 g each were
anesthetized by xylazine hydrochloride (10 mg/kg body wt) and ketamine hydrochloride (75 mg/kg body wt) and were fitted with chronic artery
and jugular cannulas. Clamp studies were conducted 72 h after catheter
implantation in the unrestrained sedentary conscious state after an
overnight fast, as described by Oakes et al. (26). Human insulin
(Novolin, Novo Nordisk) was infused continuously at a constant rate of
6.4 mU · kg1 · min
1
for 90 min. The arterial blood glucose concentration was clamped at the
basal fasting level with a variable rate 30% (wt /vol) glucose
infusion. At the end of the clamp, rats were given a lethal injection
of pentobarbital (110 mg/kg body wt iv), and tissues were rapidly
removed, freeze-clamped, and stored at
70°C for subsequent analysis.
Slovak Republic studies. euglycemic-hyperinsulinemic clamp. Male Wistar Charles River Deutschland rats weighing 300-350 g were housed in wire mesh cages in a room controlled for temperature (22 ± 2°C) and light (12:12-h light-dark cycle; lights off at 1800) and were fed the commercially available/VELAZ Prague, Czech Republic/standard laboratory rat chow ST 1 ad libitum. The clamp method was exactly the same as described in the US studies.
Assays
For the Australian studies, plasma glucose was determined by the immobilized glucose oxidase method (YSI 23 AM glucose analyzer, Yellow Springs Instruments, Yellow Springs. OH) and plasma insulin by a double-antibody radioimmunoassay that utilizes a polyclonal antiserum and purified rat insulin as standard (16). For the studies carried out in Slovakia, plasma glucose was measured with a Beckman Glucose Analyzer (Fullerton, CA), and for studies carried out in the US, it was measured by the hexokinase/glucose-6-phosphate dehydrogenase method (5). In both countries, plasma insulin was measured by radioimmunoassay with a kit from Linco Research (St. Louis, MO) and a rat insulin standard.Malonyl-CoA was determined radioenzymatically (25, 35) and citrate and malate fluorometrically in neutralized perchloric acid extracts of whole muscle, as described previously (20). Glycogen was determined as described elsewhere (28). Acetyl carnitine was analyzed by the radioisotopic (14C label) method of Cooper et al. (8), and long-chain fatty acyl (LCFA) carnitine was analyzed according to Pace et al. (27). LCFA-CoAs were separated and quantified by HPLC (17, 26) by use of a Waters Nova-Pak C18 4-µM reverse-phase column (3.9 × 100 mm) with a linear gradient of 40-62% acetonitrile with 25 mM KH2PO4 (pH 5.3). Peaks were detected at 254 nm and individually quantitated by comparing peak area with that of an internal standard. The LCFA-CoA content was calculated as the sum of the six major CoA species, namely palmitoyl (16:0), palmitoleoyl (16:1), linolenoyl (18:3), linoleoyl (18:2), oleoyl (18:1), and stearoyl (18:0).
ACC activity was assayed as described by Vavvas et al. (41). Briefly,
frozen muscle was powdered under liquid
N2, homogenized in a buffer
(containing 30 mM NaHEPES, pH 7.4; 2.5 mM EGTA; 3 mM EDTA; 32%
glycerol; 20 mM KCl; 40 mM -glycerophosphate; 40 mM NaF; 4 mM
NaPPi; 1 mM
Na3VO4;
0.1% Nonidet P-40; 2 mM diisopropyl fluorophosphate; 2 mM PMSF; 5 µM
aprotinin, leupeptin, and pepstatin A; and 1 mM dithiothreitol), and
centrifuged at 100,000 g for 40 min.
ACC was immunoprecipitated from the supernatant with monoclonal antibody (7AD3) directed against the
NH2 terminus of the muscle isoform
ACC
(kindly provided by Dr. Lee
Witters, Dartmouth Medical School), and ACC activity was determined by
the
14CO2
fixation method (41).
Statistics
Results are expressed as means ± SE for the indicated number of muscles. Statistical differences between multiple groups were determined by ANOVA followed by the Student-Neuman-Keuls multiple comparison test or by an unpaired t-test with Bonferroni modification. ![]() |
RESULTS |
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Effect of 1 and 4 Days of Glucose Infusion on Malonyl- CoA, Citrate, and Malate Concentrations and ACC Activity
Infusion of rats with glucose for 1 day resulted in large increases in plasma glucose and insulin and an increase in the concentration of malonyl-CoA in muscle from 2 nmol/g (saline control) to 3.5 nmol/g (Table 2). Infusion of glucose for an additional 3 days resulted in a further increase in the concentration of malonyl-CoA to 4.7 nmol/g, despite a return of plasma glucose to control values and a nearly 30% decrease in plasma insulin. ACC activity and the whole cell concentration of citrate were not significantly increased in either group of glucose-infused rats; however, large increases in malate were observed in both sets of animals (Table 2). An extremely close correlation (r = 0.85, P < 0.01) was observed between the concentration of malonyl-CoA and the sum of the concentrations of citrate and malate in the two groups (Fig. 1A), a finding very similar to that previously described in rat muscles incubated with insulin, glucose, and acetoacetate in varying combinations (37).
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Concentrations of Malonyl-CoA, Citrate, and Malate in Muscle After Insulin Infusion, After a Euglycemic-Hyperinsulinemic Clamp, After Denervation, and in the fa/fa Rat
Data for rats infused with insulin and sufficient glucose to maintain euglycemia (hyperinsulinemia-euglycemic clamp) for 90 min are presented in Table 3. During the clamp, plasma insulin was 218 µU/ml (Slovakia) and 232 µU/ml (US), and plasma glucose was maintained at preinfusion levels. In both studies, twofold increases in malonyl-CoA and citrate were observed (Table 3). Levels of malate tended to be higher; however, the increases in its concentration were not statistically significant at the end of the 90-min clamp. As shown in Fig. 1B, the concentration of malonyl-CoA correlated closely with the whole cell concentration of citrate-malate.
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Infusion of insulin via implants for 5 h resulted in a marked decrease
in plasma glucose from 6.2 to 1.4 mM and a 60% increase in the
concentration of malonyl-CoA in the soleus muscle (3.1 vs. 5.1 nmol/g).
The latter was associated with a nearly twofold increase in the
concentration of citrate (180 vs. 300 nmol/g) but no change in the
concentration of malate (Table
4A).
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A somewhat different pattern was observed in denervated muscle. As reported previously (36), the concentration of malonyl-CoA in the soleus muscle 24 h after sciatic nerve section was increased approximately twofold to 3.4 nmol/g in the absence of a change in either plasma glucose or insulin. In contrast to the insulin-infused rats, the concentration of citrate in these muscles was not increased, but the concentration of malate was increased nearly twofold (105 vs. 185 nmol/g). No increase in the activity of immunoprecipitated ACC was observed in the denervated rats (Table 4B), although the possibility that one was missed because of anesthesia, diminishing contractile activity, and increasing ACC activity in control rats was not ruled out.
The concentration of malonyl-CoA was also increased in soleus muscles of the fa/fa rat (1.9 vs. 2.9 nmol/g) in keeping with the high plasma insulin levels in these rodents (Table 4C). As in denervated muscles, the increase in malonyl-CoA was associated with a twofold increase in the concentration of malate but no change in citrate. ACC activity was also unchanged. As in the glucose infusion experiments, a significant correlation (r = 0.8, P < 0.05) was observed between the concentration of malonyl-CoA and the sum of the concentrations of citrate and malate in the four groups (Fig. 1B).
The Starved-Refed Rat
In agreement with previous reports, the concentration of malonyl-CoA in muscle was low in a 48-h-starved rat (24, 44) and increased with refeeding (44). Thus the concentration of malonyl-CoA in the soleus of a 48-h-starved rat was 0.9 nmol/g (vs. 2.0 nmol/g in a typical rat fed ad libitum), and it increased to 1.7 and 2.4 nmol/g, respectively, after 3 and 24 h of refeeding (Table 5). As expected, both plasma glucose (1.4-fold) and insulin (10-fold) were higher after refeeding (data not shown), as was muscle glycogen. No increase in muscle citrate was observed at 3 h, and only a modest, albeit statistically significant, increase was seen at 24 h. The concentration of malate was not increased at either time point, nor was the sum of the concentrations of citrate and malate. The concentration of acetylcarnitine, a potential alternative to citrate as a source of cytosolic acetyl-CoA (19), was also unchanged (Table 5). In contrast, the concentration of LCFA-CoA, which was 7.4 nmol/g in the 48-h-starved rat, decreased to 5.8 and 5.1 nmol/g, respectively, after 3 and 24 h of refeeding (Table 5). The concentrations of individual LCFA-CoAs, including linoleoyl, oleyl, palmitoyl, and palmitoleoyl, showed a similar pattern of change (data not shown).
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DISCUSSION |
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The notion that the activity of the muscle isoform of ACC
(ACC), and secondarily the
concentration of malonyl-CoA, can be regulated by changes in the
cytosolic concentration of citrate was based on studies in which rat
soleus muscles were incubated with insulin, glucose, and acetoacetate
in various combinations (37). As noted earlier, the increase in
malonyl-CoA in these muscles was not accompanied by an increase in
assayable ACC
activity. Rather, it correlated with
increases in the whole cell concentrations of citrate
(r = 0.8) and/or malate, and in all
instances with the sum of citrate-malate
(r = 0.95) (Table
6). Our interpretation of these findings
was that ACC is responding to an increase in the cytosolic
concentration of citrate that is reflected more closely by an increase
in the sum of the whole cell concentrations of citrate and malate than
of citrate alone (37). Although supported by a substantial body of
correlative evidence in skeletal muscle, the pancreatic
-cell (31),
and other tissues, it remains to be proven whether this correlation
reflects a cause-and-effect relationship (33).
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The results of the present study suggest that a similar correlation between changes in citrate, malate, and malonyl-CoA occurs in skeletal muscle in vivo. Thus increases in the sum of the whole cell concentrations of citrate and malate, because of an increase sometimes in one of these metabolites and sometimes in both of them, correlated closely with increases in the concentration of malonyl-CoA in a variety of conditions. These included infusions of glucose for 1 and 4 days, insulin infusion for 5 h, a euglycemic-hyperinsulinemic clamp, the fa/fa rat, and denervation (Figs. 1, A and B). Significant increases in malonyl-CoA, citrate, and malate have also been observed in humans studied during a euglycemic-hyperinsulinemic clamp (4). The common denominator in all of these situations, except for denervation, was a sustained increase in plasma levels of insulin and/or glucose. We would propose that, in the absence of an increase in energy expenditure, such elevations in plasma insulin and glucose eventually lead to increases in glucose uptake and metabolism by muscle that exceeds its need for glucose as a fuel. An increase in the cytosolic concentration of citrate in such a circumstance could be considered part of an autoregulatory mechanism that restrains the additional use of glucose (by partially inhibiting phosphofructokinase) as a fuel (21, 30), as well as the oxidation of fatty acids (via inhibition of CPT I by malonyl-CoA) (36). The finding of an increase in the sum of citrate and malate in denervated muscle suggests that a decrease in energy expenditure could also alter fuel metabolism by this mechanism.
The hypothetical mechanism by which the apparent cytosolic
concentration of citrate [i.e., the sum of whole cell citrate
plus malate] increases in skeletal muscle, heart (30, 34), brain (32),
and the pancreatic -cell (31) in response to changes in their fuel
supply and hormonal milieu has recently been reviewed (33). It has been
suggested that malate functions in this mechanism as
1) an antiporter for citrate efflux
from the mitichondria and 2) the
form in which oxaloacetate, generated in the cytosol from transamination of aspartate (by pyruvate) and the ATP citrate-lyase reaction (which also generates cytosolic acetyl-CoA), enters the mitochondria to replete and expand its pool of tricarboxylic acid cycle intermediates.
Why increases in malonyl-CoA concentration are sometimes paralleled by an increase in the concentration of whole cell citrate and at other times by an increase in whole cell malate in muscle remains to be determined. Increases in citrate predominate in muscles exposed to high concentrations of glucose or insulin for only a few hours, whereas after longer periods of glucose infusion and in the two chronic models (i.e., denervation and the fa/fa genotype), the major change was an increase in malate (Table 6). This suggests that the early response of the muscle cell to a perceived oversupply of fuel (glucose) is an increase in citrate, but that later this is supplanted by an increase in malate. The nature of the adaptation responsible for this transition is not known; however, it is noteworthy that in all of the chronic models studied here, the ability of insulin to stimulate glucose transport into muscle and/or its incorporation into glycogen was depressed (6, 9, 11). In other words, the muscle was insulin resistant. Further studies are needed to determine whether this insulin resistance in some way causes the changes in citrate and malate, or vice versa (21, 30).
Alterations in the apparent cytosolic concentration of citrate are not
the sole determinant of changes in the concentration of malonyl-CoA in
skeletal muscle. An increasing body of evidence suggests that the
decrease in malonyl-CoA in muscle during exercise is due to an increase
in the ratios of AMP to ATP and/or creatine (Cr) to Cr-phosphate (Cr-P)
(29), which in turn increases the activity of an AMP-activated protein
kinase (AMPK) that phosphorylates and inhibits ACC (Ref. 12, reviewed
by Ref. 29). As recently shown by Vavvas et al. (41), the
AMP-CrP-mediated mechanism can diminish the assayable activity of
ACC during contraction within
seconds by activating the
2-isoform of AMPK. These
investigators also demonstrated that modulation of ACC by this kinase
supersedes regulation by citrate. Similar findings, as well as an
increase in free AMP, have been reported by Hutber et al. (13), who
demonstrated that this type of regulation also occurs in skeletal
muscle in vivo during exercise (43).
The results of the present study suggest the existence of a third
mechanism for the regulation of malonyl-CoA in rat muscle in vivo. Thus
soleus muscles showed a marked increase in malonyl-CoA levels in rats
fed for 3 or 24 h after 48 h of starvation, despite the absence of an
increase in the sum of the concentrations of citrate and malate or, in
agreement with previous reports (10, 44), an increase in ACC activity
(data not shown). The cause of the increase in malonyl-CoA in such
muscles remains to be determined. One possibility is that it is due to
an increase in cytosolic concentration of citrate not reflected by our
measurements. Another is that it is related to a decrease in the
cytosolic concentration of LCFA-CoA. LCFA-CoA is an allosteric
inhibitor of ACC in skeletal
(39) and cardiac (40) muscle and liver (2) and, where examined (liver
and cardiac muscle), this effect is countered by citrate (2, 40). In
the present study, whole cell LCFA-CoA levels were diminished after
refeeding by 31%, in keeping with the observation that the
concentration of this metabolite is lower in muscle of fed than that of
starved rats (7). If this decrease in LCFA-CoA concentration is
reflected in the cytosol, an increase in ACC activity in situ would be
expected. That such a mechanism operates in vivo is suggested by the
studies of Awan and Saggerson (3), in which they demonstrated a
40-60% decrease in the concentration of malonyl-CoA in the
perfused rat heart when its LCFA-CoA concentration was increased by
45% after a 45-min perfusion with 0.5 mM palmitate. Interestingly, the
decrease in malonyl-CoA in this study occurred under conditions in
which cytosolic citrate should have been substantially increased (i.e.,
perfusion with insulin and glucose in addition to FFA) (30), suggesting
that the effect of LCFA-CoA or a related metabolite superseded that of citrate.
One can only speculate as to why the sum of the concentrations of citrate and malate (i.e., apparent cytosolic citrate) does not increase in muscle during refeeding after a fast. Our belief is that two factors contribute to this. 1) A major part of the glucose taken up by muscle during the early phase of refeeding is used for glycogen synthesis (Table 5). 2) Physiological adaptations after the first few hours of refeeding (i.e., decreased food intake and, secondarily, plasma glucose and insulin levels) decrease muscle glucose uptake when a normal rat eats spontaneously. Such adaptations are presumably either not present or they are abnormal during glucose and insulin infusions and in hyperinsulinemic-hyperglycemic rodents.
Finally, this discussion has focused on factors regulating malonyl-CoA synthesis. In contrast, little is known about the disposition of malonyl-CoA in skeletal muscle, although a malonyl-CoA decarboxylase (3, 15) and a fatty acid elongation system have been demonstrated in heart (3) and a malonyl-CoA decarboxylase with a cytosolic location in skeletal muscle (1). Whether these enzymes are regulated by nutritional or hormonal factors has not been explored. That these or similar enzymes operate in skeletal muscle is suggested by the observation that malonyl-CoA levels can diminish by as much as 30-50% within minutes when a muscle is deprived of fuel (36) or is made to contract vigorously (41).
In conclusion, the results suggest that increases in the concentration of malonyl-CoA in skeletal muscle, induced in vivo by sustained increases in plasma glucose and/or insulin or by inactivity (denervation), are attributable to increases in the cytosolic concentration of citrate. In contrast, the mechanism responsible for the increase in malonyl-CoA in muscle during refeeding after a fast remains to be determined.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge Ted Kurowski, Virendar Kaushik, Donna Wilks, Kuet Li, Alica Mitkova, and Silvia Kuklova for technical assistance and Dr. Yasuo Ido for help in setting up the acetylcarnitine assay.
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FOOTNOTES |
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This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-19514 and DK-49417 and grants from the Juvenile Diabetes Foundation (to N. B. Ruderman), the National Health and Medical Research Council of Australia (to E. W. Kraegen), and the Slovak Diabetes Association (to E. Sebokova). E. Sebokova was also the recipient of a Fulbright Visiting Professor Scholarship.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and correspondence: A. K. Saha, Diabetes and Metabolism Unit, Boston Univ. Medical Center, 88 E. Newton St., E-307, Boston, MA 02118 (E-mail: aksaha{at}bu.edu).
Received 1 October 1998; accepted in final form 22 February 1999.
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