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Originally published In Press as doi:10.1074/jbc.M307921200 on December 1, 2003

J. Biol. Chem., Vol. 279, Issue 9, 7470-7475, February 27, 2004
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Chronic High Glucose Lowers Pyruvate Dehydrogenase Activity in Islets through Enhanced Production of Long Chain Acyl-CoA

PREVENTION OF IMPAIRED GLUCOSE OXIDATION BY ENHANCED PYRUVATE RECYCLING THROUGH THE MALATE-PYRUVATE SHUTTLE*

Ye Qi Liu{ddagger}, Jacob A. Moibi, and Jack L. Leahy§

From the Division of Endocrinology, Diabetes, and Metabolism, University of Vermont, Burlington, Vermont 05405

Received for publication, July 21, 2003 , and in revised form, October 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In islet {beta}-cells, the high expression of pyruvate carboxylase and the functional importance of the downstream anaplerosis pathways result in a unique characteristic whereby high glucose and fatty acids both increase production of a key fatty acid metabolite, long chain acyl-CoA, for signaling and enzyme regulation in {beta}-cells. We showed previously in islets that pyruvate dehydrogenase (PDH) activity is lowered by excess fatty acids (the so-called Randle effect). We have now investigated PDH activity and pyruvate metabolism in islets after 48-h culture at 16.7 mmol/liter glucose. Active PDH Vmax was lowered 65% by 48 h of high glucose, and this effect was markedly attenuated by co-culture with triacsin C, which inhibits acyl-CoA synthase. Despite the large reduction in PDH activity, glucose oxidation was twice normal. The reason was continued metabolism of pyruvate through pyruvate carboxylase (Vmax, 83% of control) and diversion of flux through the pyruvate-malate shuttle. The result was a 3-fold increase of the pyruvate concentration that overcame the lowered PDH activity by mass action as shown by glucose oxidation measured with [6-14C]glucose being twice normal. In addition, glucose-induced insulin secretion was 3-fold increased after 48 h of high glucose, and this effect was totally blocked by co-culture with triacsin C. These results show that a unique feature of islet {beta}-cells is not only fatty acids but also excess glucose that impairs PDH activity. Also, a specialized trait of {beta}-cells is a long chain acyl-CoA-mediated defense mechanism that prevents a reduction in glucose oxidation and consequently in insulin secretion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular and biochemical basis for how glucose stimulates insulin secretion is not well understood. A hypothesis with considerable experimental support pertains to pyruvate metabolism through pyruvate carboxylase (PC),1 the so-called anaplerosis pathway (1, 2). Several downstream metabolites are proposed to be effectors of glucose-induced insulin secretion. Best known is malonyl-CoA, which inhibits fatty acid oxidation so that long chain acyl-CoA (LC-CoA) accumulates (3, 4) and stimulates exocytosis directly (5) or acts indirectly via complex lipid formation (6, 7), protein kinase C activation (8), or protein acylation (9). Another proposed effector is production of cytoplasmic reducing equivalents through the malate-pyruvate (10) and citrate-pyruvate shuttles (11). Attesting to the importance of the anaplerosis pathway are studies of islets or clonal {beta}-cells with impaired glucose-induced insulin secretion because of being cultured with excess fatty acids (12–14) or from rodent models of type 2 diabetes (15, 16) that show lowered expression of PC or downstream enzymes. Thus, interest in the anaplerosis pathways has mostly focused on potential direct regulatory effects on insulin secretion.

We studied {beta}-cells exposed to states of excess fatty acids in which glucose-induced insulin secretion is preserved, and we have identified another potential regulatory role for PC and its downstream pathways. We cultured islets for 4 days with 0.25 mmol/liter oleate and 5.5 mmol/liter glucose, and we observed that glucose oxidation was increased despite pyruvate dehydrogenase (PDH) activity being lowered by 30% (17). We made a similar finding in isolated islets from insulin-resistant, hyperlipidemic, nondiabetic Zucker fatty rats (18). The effect of excess fatty acids to lower PDH activity was first described by Randle et al. (19) in heart, skeletal muscle, and liver and subsequently was shown by others to occur in islets (20, 21). The mechanism is an increase in PDH kinase activity that deactivates PDH (19). In muscle, glucose oxidation, thus ATP production, is lowered because PDH is the sole pathway for mitochondrial pyruvate metabolism. Islets are more complex because of the presence and high activity of PC (22). In our studies of fatty acid-associated lowering of PDH activity (17, 18), PC activity was unaffected or slightly increased, and flux through the malate-pyruvate shuttle was more than doubled, as was the concentration of pyruvate. We proposed from these findings that islets are protected against the mitochondrial dysmetabolism that occurs with excess fatty acids in other tissues because of having an alternate pyruvate metabolism pathway through PC and that diversion through this pathway results in augmented flux through the pyruvate shuttles, which raises the level of cytoplasmic pyruvate to an extent that overcomes the lowered PDH activity through mass action.

Long term culture of islets and {beta}-cells under high glucose conditions is another state of increased anaplerosis (23). The effect on pyruvate metabolism is unclear. One study noted no change in PDH mRNA or protein expression (23), whereas another showed that PDH activity was lowered (24). These results are reminiscent of the PDH deactivation induced by excess fatty acids. Fatty acids and high glucose both increase production of LC-CoA (3). We showed previously that phosphofructokinase activity was enhanced in 4-day fatty acid-cultured islets and 2-day high glucose-cultured islets, with both of those effects blocked by inhibition of acyl-CoA synthetase using triacsin C (25, 26). Whether the PDH deactivation related to excess fatty acids is similar and consequently occurs in islets with long term high glucose is unknown.

The current study investigated pyruvate metabolism in rat islets cultured 48 h at 16.7 mmol/liter glucose. We confirmed inhibition of PDH activity and showed substantial prevention by triacsin C. Also, glucose oxidation and glucose-induced insulin secretion were enhanced despite the lowered measured PDH activity in association with a marked increase in the malate-pyruvate shuttle and a tripling of the pyruvate concentration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Islet Culture—Islets were isolated from Sprague-Dawley rats (Taconic, Germantown, NY) by an adaptation of the method of Gotoh et al. (27): pancreas duct infiltration with collagenase, Histopaque gradient separation, and hand picking. They were cultured 48 h at 37 °C in humidified air and 5% CO2 in RPMI 1640 medium supplemented with 5.5 or 16.7 mmol/liter glucose, 10% newborn calf serum, 2 mmol/liter glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin (all from Invitrogen). Triacsin C (0.5 µg/ml) was included in the 48-h culture where stated in the text. Following the culture period, islet extracts were made for measurement of enzyme activities, or the islets were preincubated for 30 min in KRB that contained 5.5 mmol/liter glucose and 0.07% BSA and then incubated either for an additional 60 min at various glucose concentrations for analysis of metabolites or secreted insulin or for 90 min for the glucose metabolism studies. DNA was measured using the method of Labarca and Paigen (28), and protein was measured by a commercial kit that used BSA as standard (Bio-Rad). To avoid confusion over terminology in the text, islet groups in terms of the 48-h culture conditions are referred to as "normal glucose" and "high glucose," and references to the post-culture incubation studies include the actual utilized glucose concentrations.

Enzyme Activities—Glucose phosphorylation was measured in islet extracts as described previously (29) using a method based on quantifying conversion of NAD+ to NADH by exogenous glucose-6-phosphate dehydrogenase (30). Islet homogenates were centrifuged at 12,000 x g for 10 min, and the supernatants were incubated at 10 glucose concentrations (0.03–100 mmol/liter) to measure glucose phosphorylation. Maximal velocity (Vmax) and Michaelis-Menten constant (Km) values for glucokinase and hexokinase were calculated by linear regression from an Eadie-Scatchard plot and 10 cycles of the method of Spears et al. (31) to identify the activity of each enzyme.

Active PDH was measured as described previously (17). Islets (300) were homogenized on ice in 0.3 ml of 50 mmol/liter HEPES, pH 7.5, 0.2 mmol/liter KCl, 3 mmol/liter EDTA, 5 mmol/liter dithiothreitol, 0.1 mmol/liter N{alpha}-p-tosyl-L-lysine chloromethyl ketone, 0.1 mmol/liter trypsin inhibitor, 0.02 trypsin inhibitory units/ml aprotinin, 2% rat serum, 0.25% (v/v) Triton X-100; then freeze-thawed three times; and passed through a 0.5-ml insulin syringe 10 times. Extract (50 µl) and 50 µl of reaction buffer (50 mmol/liter HEPES, pH 7.5, 1 mmol/liter MgCl2, 3 mmol/liter NAD, 0.4 mmol/liter thiamine pyrophosphate, 0.4 mmol/liter CoA, 2 mmol/liter dithiothreitol, 0.1% Triton X-100, 7.5 units/ml lipoamide dehydrogenase, 1 mmol/liter pyruvate, 0.1 µCi of [1-14C]pyruvate) were added to a cup inside a rubber-stoppered 20-ml scintillation vial that contained a center well with filter paper. Following incubation at 37 °C for 20 min, the reaction was stopped by injecting 200 µl of 1 N HCl into the cup. CO2 was trapped in the filter paper by injecting 100 µlof1 N KOH into the center well. For PDH activity, we set 1 unit equal to 1 µmol of CO2/min.

PC was measured according to the method of MacDonald et al. (15). Islet homogenate (5 µg) was incubated in 100 µl of reaction buffer (2 mmol/liter Na3-ATP, 2.5 mmol/liter NaHCO3, 10 mmol/liter MgCl2, 100 mmol/liter KCl, 1 mmol/liter dithiothreitol, 8 mmol/liter pyruvate, 0.2 mmol/liter acetyl-CoA, 2 µCi of [14C]NaHCO3) at 37 °C for 30 min. The reaction was stopped by 50 µl of 10% trichloroacetic acid followed by liquid scintillation counting.

Malate dehydrogenase was measured as described previously (17). Islets (100) were homogenized in 100 µl of 10 mmol/liter HEPES, pH 7.4, 250 mmol/liter sucrose, 2.5 mmol/liter EDTA, 2 mmol/liter cysteine, 0.02% BSA. Extract (10 µl) was added to 0.82 ml of 0.12 mol/liter glycine, pH 10, 100 µl of 0.85 mol/liter L-malate, pH 7, 67 µl of 37.5 mmol/liter NAD. Absorbance at 340 nm was measured for 20 min.

Malic enzyme was measured as described previously (17). Islets (100) were sonicated in 100 µl of 50 mmol/liter triethanolamine, pH 7.4, 3 mmol/liter MnCl2, 0.02% BSA. Extract (50 µl) or NADPH standard (1–12 nmol) was added to 0.95 ml of prewarmed reaction buffer (50 mmol/liter triethanolamine, pH 7.4, 3 mmol/liter MnCl2, 0.02% BSA, 0.1 mmol/liter NADP, 1 mmol/liter L-malate at 37 °C). Fluorescence was measured for 20 min at excitation 340 nm and emission 420 nm.

Metabolites—Following the 48-h culture and 30-min preincubation, islets were incubated 60 min at 37 °C in prewarmed and oxygenated KRB that contained the stated glucose concentrations in the text or figure, followed by biochemical analysis.

Glucose 6-phosphate (Glc-6-P) concentration was measured as described previously (25). Islets underwent rapid lysis after the 60-min incubation using 10 µl of 40 mmol/liter NaOH and were placed on ice for 10 min; then 3 µl of 0.15 mol/liter HCl was added with incubation at 75 °C for 20 min to destroy cellular enzymes to ensure stability of the Glc-6-P. Extract (20 islets) was added to 15 µl of reaction buffer (0.15 mol/liter Tris/HCl, pH 8.1, 20 µmol/liter NADP, 0.02 units/ml grade 1 glucose-6-phosphate dehydrogenase from yeast (Roche Applied Science)) and incubated for 30 min at 28 °C in a shaking water bath followed by addition of 4 µl of 1 mol/liter NaOH and incubation at 75 °C for 20 min to destroy any remaining NADP. The formed NADPH was amplified by the cycling method by adding 100 µl of reagent (9 units/ml type II glutamate dehydrogenase, 5 mmol/liter {alpha}-ketoglutaric acid, 1 mmol/liter Glc-6-P, 6 units/ml glucose-6-phosphate dehydrogenase) for 60 min at 38 °C, followed by heating to 100 °C for 10 min to stop the reaction. A sample (100 µl) was transferred to a UV cuvette containing 1 ml of 0.006 units/ml 6-phosphogluconate dehydrogenase in 0.15 mol/liter Tris/HCl, pH 8.1, 30 µmol/liter NADP, 0.1 mmol/liter EDTA, 30 mmol/liter ammonium acetate, 5 mmol/liter MgCl2, and let stand at room temperature for 30 min. The formed 6-phosphogluconate was measured by fluorometer at 340 nm excitation and 420 nm emission, and islet Glc-6-P concentration was determined from Glc-6-P standards (1–20 pmol).

Malate and pyruvate were measured as described previously (17). Islets (300) were lysed in 150 µl of 2 mol/liter perchloric acid on ice for 20 min and then centrifuged for 10 min at 12,000 x g. Supernatant was neutralized with 3 mol/liter KHCO3 and recentrifuged at 12,000 x g. Malate and pyruvate standards were prepared in perchloric acid. 50 µl of extract or malate standard (0.1–1 nmol) was added to 250 µl of reaction buffer (20 mmol/liter 2-amino-2-methyl propanol, pH 9.9, 2 mmol/liter glutamate, 50 µmol/liter NAD, 1 µg of aspartate aminotransferase, 2.5 µg of malate dehydrogenase) for 20 min at 30 °C. Nonmetabolized NAD was removed by addition of 0.25 ml of potassium phosphate, pH 11.9, and incubation at 60 °C for 15 min, followed by addition of 12 µl of 1 mol/liter imidazole and another 15 min at 60 °C. Fluorescence was measured at excitation 340 nm and emission 465 nm. 30 µl of extract or pyruvate standard (20–200 pmol of sodium pyruvate) was added to 100 µl of reaction buffer (50 mmol/liter imidazole, pH 7, 0.6 mmol/liter ascorbate, 0.2 mg/ml BSA, 6 µmol/liter NADH, 0.125 units/ml lactate dehydrogenase) at room temperature for 20 min. Nonmetabolized NADH was removed by addition of 20 µl of 2 N HCl at room temperature for 30 min followed by 1 ml of 6 N NaOH for 10 min at 60 °C. Fluorescence was measured at excitation 360 nm and emission 460 nm.

Oxaloacetate was measured as described previously (17). Islets (50) were lysed 20 min in 40 µl of 0.25 mol/liter perchloric acid at –20 °C followed by sonication and addition of 20 µl of 0.94 mol/liter KOH. Extract (30 µl) or oxaloacetate standard in perchloric acid (0.2–2.0 pmol) was added to 200 µl of reaction buffer (75 mmol/liter K2PO4, pH 7.4, 80 nmol/liter [acetyl-3H]acetyl-CoA, 50 µg/ml citrate synthase) at room temperature for 60 min. The reaction was stopped by 600 µl of charcoal mixture (8 g of charcoal, 38 g of citric acid monohydrate, 120 ml of 95% ethanol) followed by centrifugation at 12,000 x g and liquid scintillation counting of the supernatant.

Citrate concentration was measured as described previously (26). Islets (200) were lysed with trichloroacetic acid and placed on ice followed by centrifugation to remove precipitated proteins. Supernatant was neutralized by ether extraction five times, lyophilized, and resolubilized in 100 µl of H2O. Extract (20 µl) or citrate standard (0.1–2 nmol) was added to 0.1 mol/liter Tris/HCl buffer, pH 7.6, 40 µmol/liter ZnCl2, 3 µmol/liter NADH, 0.4 µg/ml malate dehydrogenase (final volume of 0.5 ml). Fluorescence was determined at 340 nm excitation and 465 nm emission, and then the measurements were repeated 5 min after adding 10 µl of citrate lyase solution, with the {Delta} value representing the citrate content.

Islet Glucose Metabolism and Insulin Secretion—Glucose utilization and oxidation were determined with D-[5-3H]glucose and [U-14C]glucose as described previously (32). Experiments were also performed with [6-14C]glucose and [3,4-14C]glucose based on the principle that the 6-isomer enters the citric acid cycle as [2-14C]acetyl-CoA by way of PDH, whereas the 3,4-isomer reflects pyruvate metabolism through both PDH and PC (33). Insulin secretion was measured in duplicate vials of 10 islets following the 60-min incubation in KRB, 10 mM HEPES, 0.5% BSA, glucose (2.8, 8.3, and 16.7 mM) at 37 °C in a shaking water bath. Islets were sedimented by gentle centrifugation, and insulin in the medium was measured by insulin radioimmunoassay. Islet insulin content was measured post-sonication and -extraction in acid ethanol.

Malate Release from Isolated Mitochondria—The method is previously described (17). For mitochondrial isolation, islets (400) were homogenized in 0.4 ml of 5 mmol/liter potassium HEPES, pH 7.5, 230 mmol/liter mannitol, 70 mmol/liter sucrose, and centrifuged at 600 x g for 5 min to sediment the nuclear and cell debris, followed by recentrifugation of the supernatant at 5,500 x g for 10 min to sediment the mitochondria. The sedimented mitochondria were resuspended in 120 µl of ice-cold buffer (5 mmol/liter potassium HEPES, pH 7.3, 5 mmol/liter K2PO4, 5 mmol/liter KHCO3, 2 mmol/liter Na2ADP, 230 mmol/liter mannitol, 70 mmol/liter sucrose, with or without 10 mmol/liter pyruvate) and kept on ice. Malate release was measured using the method of MacDonald (10). The mixture was placed at 37 °C, and 50-µl samples were taken at 0 and 10 min. Samples were centrifuged at 14,000 x g for 2 min followed by addition of 15 µl of 0.92 M perchloric acid to the supernatant, return of the pH to 7.0 with 1 mol/liter KOH, and recentrifugation at 14,000 x g for 2 min. Malate was measured using the method of Sener et al. (34). Supernatant (40 µl) or malate standard (0–30 pmol) was added to 200 µl of reaction buffer (100 mmol/liter Tris/KCl, pH 8.0, 1 mmol/liter NAD, 0.2 mmol/liter [3H]acetyl-CoA, 20 µg/ml malate dehydrogenase from pig heart, 60 µg/ml citrate synthase from pig heart) at room temperature for 60 min. The final product of [3H]citrate was separated with 600 µl of charcoal mixture (120 ml of 95% ethanol, 8 g of charcoal, 38 g of citrate acid monohydrate) and centrifugation at 14,000 x g for 5 min followed by liquid scintillation counting of the supernatant.

Data Presentation—All data are expressed as mean ± S.E. Statistical significance was determined by the unpaired Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of 48-h High Glucose Culture on Islet Glucose Metabolism—Islet protein (0.73 ± 0.01 ng/islet in normal glucose-cultured islets versus 0.72 ± 0.01 ng/islet in high glucose-cultured islets, n = 4, p = NS), DNA (21 ± 1 ng/islet in normal glucose-cultured islets versus 20 ± 1 ng/islet in high glucose-cultured islets, n = 5, p = NS), and insulin (33 ± 5 ng/islet in normal glucose-cultured islets versus 31 ± 5 µg/islet in high glucose-cultured islets, n = 6, p = NS) contents were unchanged by the high glucose culture.

We (35) and others (30) have reported that islet glucokinase expression and activity are increased by long term exposure to high glucose. As expected, this effect was noted in this study (glucokinase Vmax of 11.7 ± 0.9 mol of glucose/kg of DNA/90 min in high glucose-cultured islets versus 5.6 ± 0.5 mol of glucose/kg of DNA/90 min in normal glucose-cultured islets, p < 0.001; glucokinase Km of 18.8 ± 3.9 mmol/liter glucose in high glucose-cultured islets versus 13.0 ± 0.5 mmol/liter glucose in normal glucose-cultured islets, p = NS; hexokinase Vmax of 3.8 ± 0.5 mol of glucose/kg of DNA/90 min in high glucose-cultured islets versus 3.6 ± 0.3 mol of glucose/kg of DNA/90 min in normal glucose-cultured islets, p = NS; hexokinase Km of 0.08 ± 0.02 mmol/liter glucose in high glucose-cultured islets versus 0.10 ± 0.01 mmol/liter glucose in normal glucose-cultured islets, p = NS; n = 4). It is also well known that chronic high glucose increases gene expression and consequently activity of phosphofructokinase (26, 36). These dual effects resulted in no change in the regulation of the Glc-6-P concentration in the high glucose-cultured islets as shown in Fig. 1; the Glc-6-P concentrations of both groups of cultured islets were identical after the 60-min incubations at 5.5 and 16.7 mmol/liter glucose.



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  		FIG. 1.
Glucose 6-phosphate concentration in islets following 48-h culture at 5.5 or 16.7 mmol/liter glucose. Freshly isolated rat islets were cultured 48 h, preincubated 30 min at 5.5 mmol/liter glucose, and then incubated 60 min at 5.5 mmol/liter (open bars) or 16.7 mmol/liter glucose (filled bars). Data are mean ± S.E., and the number of experiments is 4. G6P, glucose 6-phosphate.

 
Islet glucose utilization (Fig. 2A) and glucose oxidation (Fig. 2B) were also measured. The high glucose-cultured islets had increases at 8.3 and 27.7 mmol/liter glucose compared with the normal glucose-cultured islets, with the -fold increase for utilization being 40–50% versus more than a doubling for oxidation.



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  		FIG. 2.
Glucose utilization (A) and oxidation (B) in islets following 48-h culture at 5.5 mmol/liter (open circles) or 16.7 mmol/liter glucose (filled circles) determined with D-[5-3H]glucose (n = 4) and [U-14C]glucose (n = 4), respectively. Freshly isolated rat islets were cultured 48 h, preincubated 30 min at 5.5 mmol/liter glucose, and then incubated 90 min at 2.8, 8.3, or 27.7 mmol/liter glucose. Data are mean ± S.E.

 
PDH Activity—The high glucose culture lowered active PDH Vmax by 65% (Fig. 3A). This effect was substantially prevented by co-culture with triacsin C. A time course study in Fig. 3B showed an increase in PDH activity after 2 h of 16.7 mmol/liter glucose that was back to base line at 6 h and then subnormal at 24 and 48 h.



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  		FIG. 3.
Pyruvate dehydrogenase activity in islets following 48-h culture at 5.5 or 16.7 mmol/liter glucose with or without 0.5 µg/ml triacsin C. A, freshly isolated rat islets were cultured for 48 h, followed by measurement of PDH activity as described in the text (n = 5). µU, microunit. B, time course of high glucose effects on islet PDH activity. All islet groups were cultured a total of 48 h. During that period, the glucose concentration was switched from 5.5 to 16.7 mmol/liter to result in the shown number of h of high glucose. Data are mean ± S.E., and the number of experiments is 3. *, p < 0.05 compared with value at 0 h.

 
Pyruvate Metabolism—In contrast to PDH, the Vmax of PC was only minimally lowered by the high glucose culture (83% of control islets) (Table I). Our prediction based on our studies of lowered islet PDH activity associated with excess fatty acids (17, 18) was that there would be diversion of pyruvate metabolism through PC that would result in increased flux through the malate-pyruvate shuttle and consequently augment the pyruvate concentration as an explanation for the increased glucose oxidation. This shuttle consists of mitochondrial metabolism of pyruvate to oxaloacetate by PC, conversion to malate by malate dehydrogenase, and malate export to the cytoplasm followed by malic enzyme conversion back to pyruvate. We tested our prediction by measuring activities of these enzymes, concentrations of the metabolites, and malate efflux from isolated mitochondria.


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  		TABLE I
Enzyme activities in 48-h high glucose-cultured (16.7 mmol/liter) and normal glucose-cultured (5.5 mmol/liter) rat islets

The values in parentheses refer to the number of experiments that were performed.

 
In the high glucose-cultured islets, malate dehydrogenase activity was unchanged, and malic enzyme Vmax was 40% increased compared with the normal glucose-cultured islets (Table I). Metabolite concentrations were measured after the 48-h culture, preincubation, and 60 min at 5.5 or 16.7 mmol/liter glucose (Fig. 4). In the high glucose-cultured islets, pyruvate and malate concentrations were markedly increased after the 60 min of 16.7 mmol/liter glucose (p = 0.002 and p = 0.025, respectively) but not 5.5 mmol/liter glucose (Fig. 4, A and B). Oxaloacetate was more complicated with increases at both 5.5 and 16.7 mmol/liter glucose (p = 0.001 for both), although the -fold increase at 16.7 mmol/liter was smaller than for pyruvate and malate (Fig. 4C). Malate efflux from isolated mitochondria was 6-fold increased in the high glucose-cultured islets (Fig. 5).



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  		FIG. 4.
Metabolite concentrations in islets following 48-h culture at 5.5 or 16.7 mmol/liter glucose. Freshly isolated rat islets were cultured for 48 h, preincubated 30 min at 5.5 mmol/liter glucose, and then incubated 60 min at 5.5 mmol/liter (open bars) or 16.7 mmol/liter glucose (filled bars). A, pyruvate. B, malate. C, oxaloacetate (OAA). D, citrate. Data are mean ± S.E. The number of experiments is 4 for all groups.

 



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  		FIG. 5.
Malate release from isolated mitochondria in islets following 48-h culture at 5.5 mmol/liter (open bars) or 16.7 mmol/liter glucose (filled bars). Mitochondrial malate efflux was measured over 10 min with or without 10 mmol/liter pyruvate in the incubation buffer. Data are mean ± S.E., and the number of experiments is 6.

 
An alternate metabolic pathway for oxaloacetate besides conversion to malate is citrate synthase-induced production of citrate and distal metabolism through malonyl-CoA or recycling to pyruvate through the pyruvate-citrate shuttle. However, we have reported previously that citrate synthase activity is lowered in high glucose-cultured isles (26). In agreement with this finding, in contrast to the other metabolites, citrate levels were not increased in the high glucose-cultured islets (Fig. 4D).

Glucose Oxidation Using [6-14C]- and [3,4-14C]Glucose—We next tested the prediction that flux through PDH was augmented despite the lowered measured PDH activity. This was done by comparing glucose oxidation of intact cells using 6- and 3,4-isomers of [14C]glucose; the 6-isomer is mostly metabolized through PDH, whereas the 3,4-isomer reflects overall pyruvate oxidation (33). Islets were assessed for 14CO2 production during 90-min incubations at 5.5 and 16.7 mmol/liter glucose that followed the 48-h culture and preincubation (Fig. 6A is the 6-isomer, and Fig. 6B is the 3,4-isomer). Glucose oxidation as measured with both tracers was increased in the high glucose-cultured islets. Moreover, the patterns of the increase were identical for both tracers. Thus, the proportional flux through PDH assessed as the 6-tracer result expressed as a percentage of the 3,4-tracer (overall oxidation) was 40 ± 5% at 5.5 mmol/liter glucose and 46 ± 2% at 16.7 mmol/liter glucose in the normal glucose-cultured islets and was 42 ± 7% and 41 ± 6%, respectively, in the high glucose-cultured islets (p = NS between any group). These results verify the prediction that flux through PDH is increased in the high glucose-cultured islets in an absolute sense despite the markedly lowered measured PDH activity.



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  		FIG. 6.
Glucose oxidation using [6-14C]glucose (A) and [3,4-14C]glucose (B) in islets following 48-h culture at 5.5 or 16.7 mmol/liter glucose. Freshly isolated rat islets were cultured 48 h, preincubated 30 min at 5.5 mmol/liter glucose, and then incubated 90 min at 5.5 mmol/liter (open bars) or 16.7 mmol/liter glucose (filled bars). Data are mean ± S.E. The number of experiments is 3.

 
Effect of Triacsin C on Insulin Secretion and Pyruvate Concentration—We next tested the effect of adding 0.5 µg/ml triacsin C during the 48-h culture on post-culture glucose-induced insulin secretion. Glucose-induced insulin secretion was 3-fold increased after 48 h of high glucose (Fig. 7A, islets cultured at normal glucose, and Fig. 7B, islets cultured at high glucose). Addition of triacsin C to the 48-h culture had no effect on glucose-induced insulin secretion in the normal glucose-cultured islets but totally eliminated it in the high glucose-cultured islets. As expected, the post-culture pyruvate concentration following a 60-min incubation at 16.7 mmol/liter glucose behaved in a similar fashion, as triacsin lowered it only in the high glucose-cultured islets (normal glucose-cultured islets, 1.41 ± 0.08 pmol/islet without triacsin C and 1.31 ± 0.08 pmol/islet with 0.5 µg/ml triacsin C, p = NS; high glucose-cultured islets, 3.88 ± 0.50 pmol/islet without triacsin C and 2.67 ± 0.47 pmol/islet with 0.5 µg/ml triacsin C, p = 0.033, n = 4 for all groups).



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  		FIG. 7.
Effect of co-culture with triacsin C on glucose-induced insulin secretion from islets following 48-h culture at 5.5 or 16.7 mmol/liter glucose. Freshly isolated rat islets were cultured 48 h at 5.5 mmol/liter (A) or 16.7 mmol/liter glucose (B) with (filled bars) or without (open bars) 0.5 µg/ml triacsin C and then incubated 60 min at 2.8, 8.3, or 16.7 mmol/liter glucose, all without triacsin C. Data are mean ± S.E. The experimental number for each group is 6.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show that in islets, chronic high glucose impairs PDH activity in the manner that is uniquely attributed to excess fatty acids in other tissues (so-called glucose fatty acid cycle of Randle). This statement is based on the following observations. First, we established previously that this effect occurs with excess fatty acid in islets (17, 20), plus the mechanism was shown by others to be that of the glucose fatty acid cycle, namely, augmented PDH kinase activity that inactivates PDH (20). Second, in muscle, the fatty acid effect takes between 12 and 18 h to occur based on starvation studies (19), which agrees with the results in this study. Actually, the time course in this study was more complex, as PDH activity increased and then fell, likely as a result of the known rapid activation of PDH in islets by glucose (37). Third, if PDH deactivation causes the subnormal PDH activity, then PDH gene expression and cellular level should be unaltered as was shown in high glucose-cultured INS cells (23). Fourth, and most telling, triacsin C prevented to a large extent the high glucose-induced reduction of PDH activity. Triacsin C inhibits fatty acyl-CoA synthetase (38–40). Cellular metabolism of fatty acids entails oxidation or esterification to fatty acyl-CoA and complex lipids. Considerable interest has focused on LC-CoA as a mediator of cellular signaling through modulating gene expression (41) or protein allosteric regulation (42). In {beta}-cells, LC-CoA regulatory effects are proposed for insulin secretion secondary to protein acylation (9), protein kinase C activation (8), complex lipid formation (6, 7), or direct mediation (5). A unique feature of {beta}-cells is that high glucose enhances the production of cytoplasmic LC-CoA because of the atypical expression of PC such that 50% of mitochondrial pyruvate metabolism normally occurs through PC (10, 22); additionally, its downstream product malonyl-CoA allosterically inhibits fatty acid oxidation through carnitine palmitoyltransferase I (3, 4). Long term exposure to high glucose augments LC-CoA production through up-regulation of gene expression of fatty acid synthase and acetyl-CoA carboxylase (23). Thus, a specialized feature of {beta}-cells is the ability of chronic high glucose and chronic excess fatty acids to both enhance LC-CoA production and thus to duplicate LC-CoA-mediated signaling and functional changes. We demonstrated this for up-regulation of phosphofructokinase activity and lowered citrate synthase activity by showing that triacsin C prevented these effects in fatty acid-cultured and high glucose-cultured islets (25, 26). The current results support a third shared effect, namely inhibition of PDH activity. It is notable that another study reported inhibition of islet PDH activity following chronic exposure to excess fatty acids and also high glucose, although the mechanism was unknown (24).

Why was glucose oxidation not impaired in the high glucose-cultured islets, as is typical for lowered PDH activity related to the glucose fatty acid cycle in other tissues (19)? We propose that the reason was mass action of an increased pyruvate concentration on flux through PDH and that a key contributor to this increase was enhanced trafficking through the malate-pyruvate shuttle. Support for this conclusion was our finding in the high glucose-cultured islets that activities of the shuttle enzymes were minimally lowered to increased (depending on the enzyme) in contrast to the two-thirds reduction for PDH, plus the shuttle metabolites were increased following 60 min at 16.7 mmol/liter glucose including a 3-fold higher pyruvate concentration. Increased shuttle trafficking was confirmed by the 6-fold increase in malate release from isolated mitochondria. Flux through PDH was tested with [6-14C]glucose and shown to be increased in the high glucose-cultured islets in the same proportion as that measured with [3,4-14C]glucose and [U-14C]glucose. Another relevant observation is the effect of chronic high glucose (also chronic excess fatty acids) to inhibit citrate synthase activity (26), as this will divert oxaloacetate metabolism away from malonyl-CoA production. Thus, our results are in agreement with our prior studies using excess fatty acids (17, 18) and show that a constellation of specialized features in {beta}-cells results in the observed protection against a lowering of glucose oxidation when PDH activity is decreased, namely the atypical expression of PC, the effect of chronic high glucose to impair citrate synthase activity, and the presence of the malate-pyruvate shuttle for pyruvate recycling. Additional support for our conclusions is the observation that triacsin C eliminated the increased glucose-induced insulin secretion in the high glucose-cultured islets, which confirms there is a LC-CoA-mediated protection against high glucose-induced {beta}-cell dysfunction.

Additional aspects of the experimental findings need consideration regarding this proposed schema. The first is that the malate-pyruvate shuttle is not the only source of the increased pyruvate concentration in the high glucose-cultured islets, but our findings suggest it is dominant. Chronic high glucose augments islet glucokinase (30, 35) and phosphofructokinase activities (26, 36), so glycolysis is enhanced as we observed in this study. However, the -fold increase in glycolysis was less than 2-fold as opposed to the 6-fold increase in mitochondrial malate release. Also, in the islets cultured with normal glucose conditions, short term exposure to high glucose (60 min at 16.7 mmol/liter glucose) caused a large increase in glycolysis but a very small increase of the pyruvate concentration. Thus, the observed 3-fold increase in the pyruvate concentration in the high glucose-cultured islets highlights the important contribution of the recycling source for pyruvate. The second issue regards PDH, which is a highly regulated enzyme complex by way of allosteric activators and repressors as well as intrinsic phosphorylation (43). The PDH reaction product, acetyl-CoA, exerts feedback inhibition that would be expected to be operative in the high glucose-cultured islets because of the enhanced flux through PDH (in an absolute sense). In contrast, oxidation of fatty acids is another important source for acetyl-CoA in {beta}-cells, and an opposite (stimulatory) effect on PDH would be expected from the reduced fatty acid oxidation, which is part of the increased anaplerosis that occurs in {beta}-cells with chronic high glucose (23). Thus, regulation of islet PDH activity after long term high glucose is multifactorial and highly complex. Regardless, the substantial albeit incomplete recovery of PDH activity with triacsin C clearly indicates a key role for LC-CoA in the lowered PDH activity.

In summary, we propose based on the findings in this study plus our results in states of excess fatty acids (17, 18) that a specialized feature of {beta}-cells is that chronic high glucose and excess fatty acids both enhance anaplerosis-induced LC-CoA production and thus have identical functional effects related to LC-CoA signaling. The current study focused on inhibition of PDH activity. In other tissues, this effect is uniquely linked to excess fatty acids as opposed to our results in islets. Also unique for {beta}-cells is failure of this effect to reduce glucose oxidation and consequently insulin secretion. The mechanism of this protection appears to result from the atypical high expression of PC and diversion of pyruvate metabolism to pyruvate recycling, which causes a buildup of cellular pyruvate to well above the normal level, which overcomes the reduction in PDH activity. The potential relevance of this protection relates to the growing importance being assigned to the anaplerosis pathways in the regulation of insulin secretion, including hypersecretion states of {beta}-cell adaptation (3, 4, 10, 44–46). Furthermore, this concept is in perfect agreement with studies of failed {beta}-cell adaptation in vitro and in vivo that have observed lowered expression of PC or downstream enzymes (12–16). On the other hand, it should not be inferred from our results that chronic high glucose and excess fatty acids always induce similar effects on islet signaling and function. We have focused on similarities that we postulate stem from regulation by LC-CoA. Differences also are known, presumably reflecting alternate regulatory mechanisms. For instance, high glucose up-regulates gene expression of the key enzyme in malonyl-CoA production, acetyl-CoA carboxylase (23, 47), but fatty acids reduce it (12). Also, carnitine palmitoyltransferase I gene expression is increased by fatty acids (48) but unaffected by glucose (23). These differences highlight the complexity of nutrient effects on the {beta}-cell.


   FOOTNOTES
 
* This work was supported by Grants DK56818 and P20 RR/DE17702 from the National Institutes of Health and the Centers of Biomedical Research Excellence Program, NCRR, National Institutes of Health, respectively (to J. L. L. and Y. Q. L., respectively) and by grants from the American Diabetes Association (to J. L. L. and Y. Q. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a Junior Faculty Award from the American Diabetes Association. Present address: Kosair Children's Hospital Research Institute, Dept. of Pediatrics, University of Louisville School of Medicine, 570 S. Preston St., Suite 304, Louisville, KY 40202. Back

§ To whom correspondence should be addressed: University of Vermont College of Medicine, Given C331, Burlington, VT 05405. Tel.: 802-656-2530; Fax: 802-656-8031; E-mail: jleahy{at}zoo.uvm.edu.

1 The abbreviations used are: PC, pyruvate carboxylase; LC-CoA, long chain acyl-CoA; PDH, pyruvate dehydrogenase; BSA, bovine serum albumin; NS, not significant. Back


   ACKNOWLEDGMENTS
 
We thank Yun Long for expert technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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