1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; and 2University of Wisconsin Children's Diabetes Center, Madison, Wisconsin 53706
Submitted 7 January 2003 ; accepted in final form 18 April 2003
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ABSTRACT |
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ATP-sensitive K+ channel; 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid
Four present hypotheses regarding the KATP channel-independent pathway are as follows. 1) As a result of anaplerosis, glucose induces an increase in mitochondrial citrate, cytosolic citrate, and cytosolic malonyl-CoA. The resulting malonyl-CoA-induced inhibition of carnitine palmitoyltransferase (CPT)-1 and diversion of fatty acids away from oxidation result in the generation of increased amounts of long-chain acyl-CoA and other potential signaling molecules in the cytosol (10, 14, 44). 2) Glucose induces an increase in the production in the mitochondria of glutamate, which is exported to the cytosolic compartment. In the cytosol, glutamate sensitizes the secretory machinery to Ca2+ perhaps by an action on the granules (38). 3) Changes in adenine nucleotide concentrations are regulators of the KATP channel-independent pathway (49). 4) Protein acylation is involved (53, 56).
In the present study, we reexamined the idea that only nutrients can
activate the KATP channel-independent pathway and that anaplerosis
is essential to it by studying the effects of glucose, leucine,
2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), which is a
nonmetabolizable analog of leucine, and glutamine. These agents were studied
alone and in combination. Leucine is the most potent amino acid in stimulating
insulin release and is thought to be so because of its ability to activate
glutamate dehydrogenase (GDH) in addition to its metabolism as a fuel
(20,
27,
50). BCH is a selective
activator of GDH, and this appears to be the sole mechanism by which it
stimulates insulin secretion
(16,
20,
27,
39,
43,
50). The provision of leucine
and glutamine mimicks the pyruvate dehydrogenase and pyruvate carboxylase
pathways, respectively, of glucose metabolism by providing mitochondrial
acetyl-CoA and ATP, and mitochondrial intermediates (anaplerosis),
respectively (Fig. 1). The
combination of these two compounds reproduced the effect of glucose to
activate both the KATP channel-dependent and KATP
channel-independent pathways. Most importantly, it was found that the
nonnutrient BCH alone (without glutamine) was sufficient to replicate the
effect of glucose on the KATP channel-independent pathway of
-cell signaling, and it did so without any suppression of fatty acid
oxidation or overall increase in esterification. Our data identify signals
from the tricarboxylic acid cycle as key activators of the KATP
channel-independent pathway.
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MATERIALS AND METHODS |
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Isolation of pancreatic islets. Male Sprague-Dawley rats (250350 g) were used in this study and had access to unlimited food and water. After CO2 asphyxiation, the pancreas was surgically removed, and the islets were isolated by collagenase digestion (31).
Insulin secretion under perifusion conditions. Krebs-Ringer bicarbonate-HEPES buffer [KRBH; containing (in mM) 129 NaCl, 5 NaHCO3, 4.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 10 HEPES at pH 7.4, and 0.1% bovine serum albumin (BSA)] was used for the insulin secretion studies. The perifusion protocol was slightly modified from that originally described (26). Briefly, 20 islets were placed into 70-µl perifusion chambers. An equilibration period of 4045 min with KRBH and 2.8 mM glucose at 37°C was followed by the test period. Samples were collected at 1- or 2-min intervals at a flow rate of 1 ml/min. Insulin was measured by RIA using a charcoal separation method (21). The nutrient and BCH concentrations in the various experiments are specified in the figure legends and the term basal glucose refers to a concentration of 2.8 mM glucose. When the KATP channel-independent pathway was studied, 250 µM diazoxide and 40 mM KCl were added simultaneously with the test agents.
Insulin secretion under static incubation conditions. Batches of five size-matched islets per tube were used. The islets were preincubated in KRBH buffer for 60 min and then incubated in the presence and absence of the test agents for 60 min. At the end of the incubations, samples were taken and kept at -20°C for RIA.
Measurement of palmitate oxidation. The method was as previously reported (12, 56). In brief, paired groups of 15 islets were incubated at 37°C for 1 h in 200 µl of KRBH buffer, to which were added 0.2 µCi [1-14C]palmitate, 0.8 mM L-carnitine, BSA (to a final concentration of 1%), and test agents as indicated. At the end of the incubations, islet metabolism was stopped by the addition of 200 µl of 0.5 M HCl to the incubation mixture. 14CO2 was collected in 300 µl of 1 M NaOH and measured by liquid scintillation spectrometry.
Measurement of palmitate incorporation into lipids. The method was similar to that used previously (12). Rat pancreatic islets were preincubated in KRBH buffer containing 2.8 mM glucose for 60 min. Groups of 60100 islets were then incubated with 200 µl of KRBH containing 0.8 mM L-carnitine, 0.2 µCi [1-14C]palmitate, and the test compounds (glucose, BCH, leucine, diazoxide, and KCl) in Eppendorf tubes in a 37°C incubator for 60 min. At the conclusion of the incubation, the media were removed and the islets washed twice with 0.3 ml of ice-cold PBS. Immediately after addition of 200 µl of 0.2 M NaCl, the islet pellets were frozen in liquid N2. After thawing, 750 µl of chloroform-methanol (2:1) and 50 µl of 0.1 N KOH were added and the samples vortexed vigorously for 2 min. After centrifugation at 2,000 g for 20 min, the bottom lipid-soluble layer was washed once with 200 µl of methanol-water-chloroform (48:47:3), and incorporation of radiolabeled palmitate into lipids was quantified by liquid scintillation counting.
In some experiments, islet samples were incubated for 60 min in the presence of test substances supplemented with L-carnitine and 0.2 µCi [1-14C]palmitate, as described above. After two washes in PBS, 200 µl of chloroform and 100 µl of methanol-HCl (100:1) were added and the samples stored overnight at 4°C. The following day, 75 µl of double-distilled H2O were added to each of the samples, and after vigorous vortexing and a 10-min centrifugation at 4°C the upper aqueous phase was removed. This step was repeated once, and the organic (lower) phase was dried down under liquid N2. Dried-down samples were reconstituted in a mixture of chloroform-methanol (1:1), and a lipid carrier mixture was added to each of the samples consisting of tri-, di-, and monoacylglycerols, phospholipids, and palmitic acid. After separation by thin-layer chromatography [solvent system: n-hexane-diethyl ether-methanol-acetic acid (90:20:2:3)], the plates were sprayed with 2',7'-dichlorofluorescein, and single spots were visualized under UV light. Individual spots were cut out, transferred to glass vials, and after addition of 0.5 ml of methanol and 5 ml of BioSafe2 scintillation fluid, radioactivity was determined by liquid scintillation counting.
Metabolite assays. Batches of 100 rat islets were incubated for 30 min at 37°C in KRBH in the presence and absence of the various insulin secretagogues used for the insulin secretion studies. Metabolites were measured by alkali-enhanced fluorescence of NAD(P)(H), as previously described (37).
Statistical analysis. All data were subjected to one-way ANOVA.
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RESULTS |
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To distinguish between the effects of leucine metabolism and the activation of GDH in the generation of the responses, similar paired experiments were performed using BCH instead of leucine. The results are shown in Fig. 2B. Glucose and glutamine gave similar data to those in Fig. 2A. Interestingly, BCH reproduced the effect of leucine both alone and in combination with glutamine. Glutamine was without effect on secretion, whereas the combination of glutamine and BCH mimicked the second phase of glucose-stimulated insulin.
These data are in accord with the idea that both arms of glucose metabolism, the production of acetyl-CoA and ATP, and the provision of TCA cycle intermediates are required for the full biphasic response to glucose. Additionally, the fact that BCH alone mimicked the effect of leucine suggests that activation of GDH is the dominant effect of leucine and that its metabolism under these conditions is of little importance in stimulus-secretion coupling.
In the next experiments, we examined the effects of glucose, leucine and
glutamine, and BCH and glutamine on the KATP channel-independent
pathway of -cell signaling (see Fig.
3). In this paradigm, the first phase of the glucose response is
mimicked by the depolarizing action of KCl, whereas the KATP
channel-independent pathway provides the second phase. The response to glucose
and KCl was slightly biphasic, with a peak response after 5 min, a slight
nadir, and a subsequent rising second phase. The responses to leucine plus
glutamine and BCH plus glutamine in the presence of KCl and diazoxide were
similar to each other, and both stimulated biphasic insulin secretion to a
significantly greater extent than glucose. Again, similar results were
obtained when dimethyl glutamate was used instead of glutamine (data not
shown). Thus activation of the TCA cycle by provision of substrate and
activation of GDH mimics the effect of glucose on the KATP
channel-independent pathway. The greater response seen with the combination of
glutamine or dimethyl glutamate and either leucine or BCH compared with that
of glucose is likely due to the activation of GDH by leucine and BCH and the
resulting greater influx of
-ketoglutarate into the TCA cycle.
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In a third series of experiments, leucine, BCH, and glutamine alone were tested for their effects on the KATP channel-independent pathway. The results are shown in Fig. 4. Remarkably, over the first 20 min (minutes 1030), leucine and BCH alone stimulated insulin secretion via the KATP channel-independent pathway to a significantly greater extent than glucose. Subsequently, the responses to glucose and BCH were similar, whereas the response to leucine was slightly, but not significantly, less than that of glucose.
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Although leucine provides acetyl-CoA and activates GDH, the result obtained with BCH demonstrates that activation of GDH alone is sufficient to reproduce the effects of glucose on the KATP channel-independent pathway. Neither glutamine nor dimethyl glutamate (not shown) had any effect on secretion, because the effects of glutamine plus KCl or of dimethyl glutamate plus KCl were the same as those of KCl alone.
The importance of the activation of the TCA cycle can be seen in the response to succinate used as succinic acid methyl ester to study the effects of mitochondrial metabolism. Succinate stimulated biphasic insulin release comparably to glucose (Fig. 5A) and strongly stimulated the KATP channel-independent pathway in the presence of KCl and diazoxide (Fig. 5B).
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We next determined the effects of the various secretagogues on palmitate oxidation. The results of five paired experiments performed in the absence and presence of 40 mM KCl and 250 µM diazoxide are summarized in Fig. 6. In the absence of KCl and diazoxide, palmitate oxidation was reduced 42% by 16.7 mM glucose (P < 0.05) and 27% by 20 mM leucine (P < 0.05). In sharp contrast, 20 mM BCH slightly increased palmitate oxidation (13%, P < 0.05). When the measurements were repeated in the presence of 40 mM KCl and 250 µM diazoxide, the results were essentially the same. Palmitate oxidation was reduced 51% by 16.7 mM glucose (P < 0.01) and 51% by 20 mM leucine (P < 0.01). BCH under these conditions had no effect. The importance of these data is that, of all the compounds tested, BCH failed to decrease palmitate oxidation despite its similar effect on insulin secretion and ability to stimulate the KATP channel-independent pathway. This effect of BCH had been reported previously (39), but its significance to the mechanisms of augmentation could not have been realized at that time.
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When glutamine and the combinations of either leucine or BCH plus glutamine were examined, palmitate oxidation was reduced 58% by 10 mM glutamine (P < 0.01), 64% by 20 mM leucine plus 10 mM glutamine (P < 0.001), and 41% by 20 mM BCH plus 10 mM glutamine (P < 0.05). In the presence of 40 mM KCl and 250 µM diazoxide, palmitate oxidation was reduced 61% by glutamine (P < 0.001), 61% by leucine plus glutamine (P < 0.001), and 49% by BCH plus glutamine (P < 0.01). The results are shown in Fig. 6.
The effects of 16.7 mM glucose, 20 mM BCH, and 20 mM leucine on palmitate incorporation into lipids were determined, and the results are presented in Fig. 7. Glucose at 16.7 mM and 20 mM leucine both increased the esterification of palmitate (P < 0.005 for both), whereas 20 mM BCH had no effect. Similar results were obtained when the studies were performed in the presence of 40 mM KCl and 250 µM diazoxide (again P < 0.005 for both glucose and leucine).
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Additional studies were performed in an attempt to detect small changes that might have been obscured by the method that measures the incorporation of [14C]palmitate into total lipids. Thus, after the islets were labeled in the presence of [14C]palmitate and the same stimuli as in Fig. 7 over a 30-min period, lipids were separated by thin-layer chromatography into various lipid subclasses. Glucose significantly increased the incorporation of palmitate into phospholipids (P < 0.05) and triglycerides (P < 0.05) but not into monoacylglycerols or diacylglycerols (DAG). Leucine increased the incorporation into phospholipids (P < 0.001), monoacylglycerols (P < 0.05), and triglycerides (P < 0.001), but not into DAG. BCH had no significant effect on the incorporation of palmitic acid into any of the four subclasses. These data appear to rule out a potential mechanism for the KATP channel-independent pathway involving increased DAG synthesis (55) and activation of DAG-binding proteins such as Munc-13 that are involved in and can augment exocytosis (9, 11, 46). The esterification results are shown in Fig. 8.
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In seeking additional insight into the results obtained, we determined
intracellular levels of malate, citrate, glutamate, and -ketoglutarate.
The results are presented in Table
1. In HIT-T15 cells, the increase in citrate and malonyl-CoA
levels during glucose-stimulated insulin release results in inhibition of
CPT-1, diversion of long-chain acyl-CoAs from oxidation in the mitochondria,
and presumed increased levels of acyl-CoA in the cytosol
(10). We found no change of
citrate levels in rat pancreatic islets in response to any of the
secretagogues studied (Table
1). BCH decreased glutamate levels in the absence or presence of
KCl and diazoxide, presumably due to the allosteric activation of GDH and
enhanced conversion of glutamate to
-ketoglutarate
(37). In line with this,
-ketoglutarate levels were increased by BCH. Because BCH does not
stimulate insulin release by its own metabolism, the decrease in glutamate and
increase in
-ketoglutarate levels reflect the increased metabolism of
endogenous fuel required to supply the TCA cycle, the KATP
channel-independent signaling pathway and subsequent exocytosis. Because
diazoxide, which maintains the KATP channel in the open state, did
not prevent the alterations in metabolite levels, the changes induced by BCH
were independent of KATP channel effects. Incubation with 10 mM
glutamine, which alone has no effect on insulin secretion, increased glutamate
levels eightfold, consistent with previous results
(37).
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Finally, to address the question whether increased TCA flux alone or long-chain acyl-CoA accumulation alone is sufficient to recapitulate the KATP channel-independent pathway, the effect of the CPT-1 inhibitor etomoxir was tested alone under the KCl-diazoxide-2.8 mM glucose paradigm, and also in the presence of KCl-diazoxide-2.8 mM glucose plus 3, 10, or 20 mM BCH. In these experiments, 10 µM etomoxir had no effect alone (control 1.03 ± 0.08% of content released vs. etomoxir 1.24 ± 0.12%, n = 11) or in the presence of 3 mM BCH (control 1.92 ± 0.18% of content released/60 min vs. 2.24 ± 0.33%, n = 9), 10 mM BCH (3.72 ± 0.61% of content released/60 min vs. 3.84 ± 0.56%, n = 5), or 20 mM BCH (4.26 ± 0.38% of content released/60 min vs. 5.06 ± 0.70%, n = 5). In contrast, 10 and 20 mM BCH alone significantly augmented insulin release (control 1.03 ± 0.08% of content released/60 min vs. 10 mM BCH 3.72 ± 0.61%, and vs. 20 mM BCH 4.26 ± 0.38%, n = 5; P < 0.001 for both). Thus inhibition of CPT-1 and long-chain acyl-CoA accumulation is insufficient to reproduce the effects of the KATP channel-independent pathway. In contrast, activation of the TCA cycle is sufficient.
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DISCUSSION |
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These results emphasize the critical role of the mitochondria in generating
the signals necessary for stimulation of insulin secretion via the
KATP channel-independent pathway. Both anaplerosis and activation
of GDH provide -ketoglutarate and other downstream intermediates. It
seems likely that
-ketoglutarate, or possibly some metabolite derived
from
-ketoglutarate, gives rise to the signals necessary to activate
the KATP channel-independent pathway. In support of this, succinate
(used as the methyl ester) also induces biphasic insulin release and strongly
stimulates the KATP channel-independent pathway in the presence of
KCl and diazoxide. Knowledge that simple activation of GDH by BCH is all that
is necessary for the KATP channel-independent pathway should be
extremely helpful in the identification of the complete signaling system.
The results presented here appear to be incompatible with two current
hypotheses concerning the signaling mechanisms underlying the KATP
channel-independent pathway. The anaplerosis/malonyl-CoA/long-chain acyl-CoA
hypothesis requires that glucose induces an increase in the production of
mitochondrial citrate, cytosolic citrate, and cytosolic malonyl-CoA. The
resulting malonyl-CoA-induced inhibition of CPT-1 and diversion of fatty acids
away from oxidation in the mitochondria results in the generation of increased
amounts of cytosolic long-chain acyl-CoA and other potential signaling
molecules (10,
12,
14,
15,
42,
44,
58). Despite the large amount
of evidence in favor of this hypothesis since it was first proposed
(14), the hypothesis is
currently controversial for the following reasons. Expression of malonyl-CoA
decarboxylase in INS-1 cells, which decreased malonyl-CoA levels, impaired the
ability of glucose to suppress fatty acid oxidation and decreased the
incorporation of fatty acid into lipid but failed to affect glucose-stimulated
insulin secretion (3,
42). Second, triacsin C, an
inhibitor of long-chain acyl-CoA synthetase, reduced fatty acid oxidation and
incorporation of glucose into lipids but had no effect on glucose-stimulated
insulin release (3). A third
reason is that much of the evidence in favor of the hypothesis has been
obtained from work on clonal -cell lines that do not exhibit the
KATP channel-independent pathway
(22,
42,
52) and that may display
different metabolic pathways to those of the native
-cell. With respect
to the data in the present study, we could not show an increase in citrate
levels in response to glucose or other combinations of agents that stimulate
the KATP channel-independent pathway. Furthermore, BCH, which
strongly activates this pathway, fails to reduce palmitate oxidation or to
increase palmitate incorporation into lipids as does glucose. Although our
findings are difficult to reconcile with a hypothesis that involves a rise in
citrate and malonyl-CoA and a decrease in fatty acid oxidation, the data
provided by the authors of this hypothesis with respect to the need for
anaplerosis (10,
14,
44) remain strong and are
supported by our data.
A second hypothesis states that glucose induces an increase in the
production in the mitochondria of glutamate, which is exported to the
cytosolic compartment and sensitizes the secretory machinery to
Ca2+ (38). This
hypothesis is also controversial
(5,
37,
57). Evidence in favor is the
report that glucose generated glutamate from -cell mitochondria, that a
membrane-permeant glutamate analog sensitized the glucose-induced secretory
response, and that glutamate added directly to permeabilized INS-1 cells
increased Ca2+-stimulated insulin secretion. Note again, however,
that the INS-1 cell used does not manifest the KATP
channel-independent pathway. In addition, the hypothesis is supported by
experiments in which overexpression of glutamate decarboxylase (GAD) in INS-1
cells was used to reduce cytosolic glutamate levels. In these cells, insulin
secretion in response to 15 mM glucose was reduced by 37% compared with
controls, whereas the secretory response to KCl was unaffected. Similar
results were obtained in perifused rat pancreatic islets after adenovirus
transfection of GAD (47). The
evidence against this hypothesis is that we here and others
(37) report that glucose does
not increase the levels of intracellular glutamate. Also, as has been shown
here and by others (5,
37), glutamine raises
glutamate levels markedly without stimulating insulin release and without
augmenting stimulated insulin release in the presence of KCl and diazoxide.
Dimethyl glutamate similarly had no effect on secretion, as already reported
(57). However, the fuel effect
of both glutamine and dimethyl glutamate can be detected when GDH is activated
by leucine or BCH.
Our data do not address the third hypothesis, that changes in adenine and
or guanine nucleotide concentrations are regulating the KATP
channel-independent pathway
(49). However, the activity of
the KATP channel-independent pathway was unaltered under conditions
in which the ATP-to-ADP ratio was decreased
(2). In this study, chronic
exposure of islets to leucine resulted in impaired glucose-stimulated insulin
secretion and was associated with a decreased ATP-to-ADP ratio relative to
control islets. This defect was presumed responsible for the impaired
secretion because of the failure of glucose to decrease KATP
channel activity and depolarize the -cell. When the effect of glucose
via the KATP channel-independent pathway was studied in the
presence of KCl and diazoxide, the response was unaffected and was the same as
in control islets. This is the most direct evidence yet available against the
hypothesis that adenine nucleotides have a major role in the control of the
KATP channel-independent pathway. With respect to guanine
nucleotides, depletion of cellular GTP by 40% by treatment of
-cells
with mycophenolic acid also had no effect on the KATP
channel-independent pathway when studied under the KCl-diazoxide paradigm
(28). These data appear to
preclude a major controlling role for adenine and guanine nucleotides in the
KATP channel-independent pathway.
In conclusion, in the presence of elevated [Ca2+]i, activation of GDH and increased activity of the TCA cycle provide all the signals necessary for activation of the KATP channel-independent pathway. Neither decreased fatty acid oxidation nor esterification into lipids is necessary for BCH to mimic the effect of glucose on the pathway. The nature of the signals emanating from the TCA cycle that augment insulin secretion via the KATP channel-independent pathway are unknown and remain to be determined. With respect to the effect of the signals, our hypothesis is that they accelerate the rate at which docked granules achieve the immediately releasable state (52).
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DISCLOSURE |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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