Enhanced rat
-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway
Y. Q. Liu,1,2
J. Han,1
P. N. Epstein,1,2 and
Y. S. Long1
1Kosair Children's Hospital Research Institute, Department of Pediatrics, and 2Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky
Submitted 10 September 2004
; accepted in final form 24 October 2004
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ABSTRACT
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Islet
-cell proliferation is a very important component of
-cell adaptation to insulin resistance and prevention of type 2 diabetes mellitus. However, we know little about the mechanisms of
-cell proliferation. We now investigate the relationship between pyruvate carboxylase (PC) pathway activity and islet cell proliferation 5 days after 60% pancreatectomy (Px). Islet cell number, protein, and DNA content, indicators of
-cell proliferation, were increased two- to threefold 5 days after Px. PC and pyruvate dehydrogenase (PDH) activities increased only
1.3-fold; however, islet pyruvate content and malate release from isolated islet mitochondria were approximately threefold increased in Px islets. The latter is an indicator of pyruvate-malate cycle activity, indicating that most of the increased pyruvate was converted to oxaloacetate (OAA) through the PC pathway. The contents of OAA and malate, intermediates of the pyruvate-malate cycle, were also increased threefold. PDH and citrate content were only slightly increased. Importantly, the changes in cell proliferation parameters, glucose utilization, and oxidation and malate release were partially blocked by in vivo treatment with the PC inhibitor phenylacetic acid. Our results suggest that enhanced PC pathway in Px islets may have an important role in islet cell proliferation.
islets of Langerhans; islet cell proliferation; pyruvate-malate cycle; phenylacetic acid
PYRUVATE IS CONVERTED TO OXALOACETATE (OAA) by pyruvate carboxylase (PC) and to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex (59). PDH is dominant in most cell types; however, PC and PDH levels are almost the same in normal pancreatic
-cells, and they provide equivalent glycolytic input to the tricarboxylic acid (TCA) cycle (32, 37). However, in islets of genetically obese animals, PDH activity usually declines, but PC activity is maintained, at least during the early stage of obesity (26). This is consistent with our previous in vitro finding (30) that treatment of islets with fatty acids resulted in inhibition of PDH activity but little change, or a modest increase, in PC activity. Both PDH and PC activities and mRNA expression, however, are severely suppressed after overt type 2 diabetes develops (21, 39, 61). Therefore, PC is expected to be important in
-cell adaptation to insulin resistance during obesity and prediabetes.
Adaptation of the
-cell includes enhanced function and proliferation. Our previous observations (26) indicated that PC may be involved in
-cell adaptation. In
-cells, pyruvate is converted to OAA, then to malate, and back to pyruvate by PC, malate dehydrogenase, and malic enzyme, respectively; this forms a cycle referred to as the pyruvate-malate shuttle (33) or pyruvate cycling (31). This cycle generates three important intermediates: OAA, malate, and NADPH. OAA can be used for aspartate synthesis (44) and NADPH for lipid and fatty acid synthesis (9, 16). NADPH is also antiapoptotic (6, 52) and important for insulin secretion (33, 35). Malate release from mitochondria is necessary for the pyruvate-malate shuttle, and is also an indicator of activity in this shuttle (26, 30, 33). Another important function of PC is to restore lost TCA cycle intermediates by producing OAA from pyruvate. This replenishment is called anaplerosis (35). Farfari et al. (11) reported that PC regulates insulin secretion by the pathway of anaplerosis. Other evidence also supports the role of anaplerosis in insulin secretion. For example, anaplerosis provides more of the TCA cycle intermediate
-ketoglutarate via the glutamate dehydrogenase pathway, which significantly increases insulin secretion (2, 24). We (26) reported that inhibition of PC activity by phenylacetic acid (PAA), an inhibitor of PC (3, 11), suppresses anaplerosis and almost completely inhibits insulin secretion from normal and obese nondiabetic Zucker rat islets. Protein and lipid synthesis provides resources for cell proliferation. A major component of the adaptation of the
-cell to insulin resistance is an increase in
-cell mass by enhancing cell proliferation. OAA and NADPH are important products for protein and lipid synthesis, respectively. We (26) found that increased PC activity is associated with increasing
-cell mass during insulin resistance in the Zucker fatty rat. Additional information supports an essential role of PC in proliferation of other cell types. PC activity and protein synthesis are increased in dividing mammalian cancer cells (25), suggesting that the PC pathway provides resources for cell proliferation and growth. Mutations in the PC molecule provide additional evidence to link PC to cell proliferation. The Gancedo group [Blazquez et al. (4) and Stucka et al. (53)] reported that a PC-deficient strain of yeast cannot grow in glucose-ammonium medium; however, this strain can grow in medium with glucose and aspartate. The most likely reason for these results is that aspartate can replenish the TCA cycle by providing OAA via transamination with
-ketoglutarute (4). Other reports show that inactivation of the chromosomal PC gene in wild-type Corynebacterium glutamicum led to severely reduced growth rate on lactate, indicating that PC is essential for growth (47). In contrast, overexpression of PC in C. glutamicum results in increasing glutamate and lysine production (48). These studies indicate that the PC pathway and anaplersosis can be important regulators of cell proliferation.
However, little is known about PC regulation of pancreatic
-cell proliferation. We (28) have reported that islet cell proliferation is enhanced after 60% pancreatectomy (Px) with increases in protein and lipid synthesis. We also investigated
-cell adaptation in obese nondiabetic Zucker rat islets. This rat is insulin resistant with increased
-cell mass and higher islet PC activity (26). The current study investigated the relationship between PC pathway activity and cell proliferation in Px rat islets.
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MATERIALS AND METHODS
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Rats and Px.
The principles of animal laboratory care under the guidelines of both National Institutes of Health and the University of Louisville's Animal Care Committee were followed strictly. Male Sprague-Dawley rats (Taconic, Germantown, NY) weighing 120150 g were used for this research. Px was performed using our previously described method (23). Rats were killed for islet isolation 1, 2, and 5 days or 4 wk after surgery. Px rats started to gain body weight from day 2, and the rate of weight gain was one-half that of the sham-operated (Sham) group at day 5 after Px, as we previously reported (18). Blood glucose and serum insulin levels were normal up to 4 wk after surgery in Px rats (29). For in vivo PAA treatment, some rats were injected intraperitoneally with 0.5 g·kg1·day1 PAA 2 days before surgery (both Sham and Px) and continuously treated until the day before islet isolation. Blood glucose and body weight in PAA-treated rats were almost same as in the nontreated group.
Islet isolation.
Islets were isolated from rats by an adaptation of the method of Gotoh et al. (13), using pancreas duct infiltration with collagenase, Histopaque gradient separation, and hand picking. Before the measurements were made, islets were cultured for 1 h at 37°C in humidified air and 5% CO2 in RPMI 1640 supplemented with 5.5 mmol/l glucose and 10% newborn calf serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from GIBCO, Grand Island, NY).
Islet cell number and protein and DNA contents.
For counting islet cell number, two batches of 20 typical-size islets from one rat pancreas were separately digested with 200 µl of 0.5% trypsin-EDTA medium for 30 min at 37°C. After separation into single cells by gentle shaking, 10 µl of cell solution were loaded onto a cell-counting chamber and counted under inverted microscopy. The counting was repeated five times for each batch of islets. Six pancreata were used for cell number counting for each point data. DNA was measured by the method of Labarca and Paigen (20) and protein by a commercial kit using BSA as standard (Bio-Rad, Hercules, CA).
PC activity assay.
PC was measured according to the method of MacDonald et al. (39). Ten microliters of islet homogenate 10 (5 µg of protein) were incubated in 40 µl of reaction buffer (in mmol/l: 2 Na3-ATP, 2.5 NaHCO3, 10 MgCl2, 100 KCl, 1 dithiothreitol, 8 pyruvate, and 0.2 acetyl-CoA and 2 µCi [14C]NaHCO3/ml) at 37°C for 30 min. The reaction was stopped by addition of 50 µl of 10% trichloroacetic acid followed by overnight air drying and liquid scintillation counting.
PC protein determination.
The level of PC protein was measured using the method of MacDonald et al. (36) with modification. One hundred islets were homogenized in 50 µl of homogenization buffer containing 20 mmol/l Tris·HCl (pH 7.5), 137 mmol/l NaCl, 100 mmol/l NaF, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 50 mg/ml aprotinin, 50 mg/ml leupeptin, and 10% glycerol and centrifuged at 13,000 rpm for 15 min. The supernatants were assayed for protein concentration, and equal amounts of protein samples were denatured in Laemmli buffer containing 0.1 mol/l DTT. Proteins in cell lysates were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 1% milk and 1% bovine serum albumin in Tris-buffered saline (20 mmol/l Tris·HCl, pH 8.0, 0.15 mol/l NaCl) and incubated with 2 µCi of 125I-labeled streptavidin in the same solution containing 0.01% Tween 20. After 1 h, the membrane was washed twice for 15 min in blocking solution and once in Tris-buffered saline and then exposed to X-ray film. PC protein was recognized as a 116,000-Da band on autoradiographs of nitrocellulose blots. PC protein content was quantified by a computerized scanner with Scion Image.
Islet glucose utilization and oxidation.
Glucose utilization and oxidation were performed by previously described methods (30) with D-[5-3H]glucose (for utilization) or [U-14C]glucose (for oxidation) (Amersham, Arlington Heights, IL).
Active PDH activity.
Active PDH activity was measured as described (30), using [1-14C]pyruvate (Amersham). One unit of active PDH activity was consider to equal formation of 1 µmol CO2/min.
Malate release from isolated mitochondria.
The method has been previously described (30). In brief, islet mitochondria were obtained from 400 islets. Malate release was measured using the method of MacDonald (33). Supernatant (40 µl) or malate standard (030 pmol) was added to 200 µl of reaction buffer (100 mmol/l Tris·KCl, pH 8.0, 1 mmol/l NAD, 0.2 mmol/l [3H]acetyl-CoA, 20 µg/ml malate dehydrogenase from pig heart, and 60 µg/ml citrate synthase from pig heart) at room temperature for 60 min. The final product of [3H]citrate was separated by mixing 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 rpm for 5 min followed by liquid scintillation counting of the supernatant.
Malate and pyruvate contents.
Islets (300) were lysed in 150 µl of 2 mol/l perchloric acid (PCA) on ice for 20 min and then centrifuged for 10 min at 12,000 g. Supernatant was neutralized with 3 mol/l KHCO3 and centrifuged again at 12,000 g. Malate and pyruvate standards were prepared in PCA. Malate was measured by the method of MacDonald (33). Pyruvate was measured by a previously described method (27).
OAA content.
Islets (50) were lysed for 20 min in 40 µl of 0.25 mol/l PCA at 20°C followed by sonication and addition of 20 µl of 0.94 mol/l KOH. OAA was measured by a previously described method (30).
Citrate content.
Islets were lysed with trichloroacetic acid, placed on ice, and centrifuged to remove precipitated proteins. The supernatant was neutralized by ether extraction five times, lyophilized, and resolubilized in 100 µl of H2O. Citrate was measured by a previously described method (30).
Data presentation and statistical methods.
All data are expressed as means ± SD. Unless otherwise stated, the listed n values represent the number of experiments performed using islets from separate isolation and Px days. Statistical significance in results obtained from Sham and Px islets was compared by Student's t-test. ANOVA was used for comparing the changes among three groups and over. A value of P < 0.05 was considered significant.
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RESULTS
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Islet cell number, protein content, and DNA content after Px.
Three days after Px, all animals preserved normal blood glucose, serum insulin, and body weight, as we previously reported (28, 29). In this study, we monitored cell number, protein content, and DNA content to assess cell proliferation at 1 day, 5 days, and 4 wk after Px (Fig. 1). All of these parameters were dramatically increased (3-fold) 5 days after Px and remained elevated
1.3-fold at 4 wk. These results were consistent with our previous report of increased islet bromodeoxyuridine incorporation (a marker for DNA synthesis and cell proliferation) at 5 days (2.6-fold increase) (28). The protein and DNA contents after 4 wk were consistent with our previous report (29). These data demonstrate that islet cells initiate proliferation 1 day after Px and undergo rapid proliferation for 5 days to adapt to the loss of
-cells. Proliferation decelerates over the next 24 wk. Because the peak changes were seen 5 days after Px (which we confirmed at several time points between 2 and 10 days after Px; data not shown), we performed most of the following studies using 5-day Px islets.

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Fig. 1. Changes in pancreatic islet cell number (A), protein content (B), and DNA content (C) after 60% pancreatectomy (Px). d, Days; w, weeks. *P < 0.05, #P < 0.001 vs. sham operated (Sham); P < 0.001 between the 2 indicated groups. Data are means ± SD; n = 6.
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Two- and five-day Px islet PC activity and protein level.
To understand the mechanism by which cell proliferation was increased, we measured PC activity in 2- and 5-day Px islets (Fig. 2). Surprisingly, PC activity was increased only 1.3-fold at 2 and 5 days after Px, which is much less than the increase in islet DNA and protein content (2.3-fold increase). The increased rates were small but significant (P < 0.05 vs. Sham at both 2 and 5 days). In vivo treatment with the PC inhibitor PAA suppressed islet PC activity to normal levels in Px mice. However, PAA did not affect PC activity significantly in Sham mice, suggesting that this dose of PAA was not toxic to normal tissue and that newly synthesized PC in Px islets is most sensitive to this dose of PAA. We tested PC protein level in 5-day Px islets to see whether Px affects PC expression. The result (Fig. 3) demonstrated that PC expression levels were not changed by Px, indicating that Px affects PC activity only, not protein expression.

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Fig. 2. Pyruvate carboxylase (PC) activity is elevated by Px, and this is blocked by in vivo treatment with phenylacetic acid (PAA; 0.5 g·kg1·day1). *P < 0.05 between the indicated groups. Data are means ± SD; n = 6.
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Fig. 3. PC protein levels are unchanged in 5-day Sham and Px islets. PC bands were detected with 125I-labeled streptavidin, as described in MATERIALS AND METHODS. Each lane in A was loaded with 20 µg of protein extract obtained from islets isolated from 1 animal. Similar results were obtained in 3 independent experiments. B: PC protein content quantification. Data are means ± SD. NS, no significant difference between Sham and Px.
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Glucose utilization and oxidation in 5-day Px islets.
We measured glucose utilization and oxidation in 5-day Px islets, as shown in Fig. 4. Glucose utilization and oxidation were significantly increased in these islets, demonstrating that glycolytic flux was accelerated; this result is consistent with our previous findings (28, 29). The increases in glucose utilization and oxidation were significantly reduced by PAA, indicating that stimulation of metabolism was dependent on PC activity. As shown in Fig. 2, increased PC activity was inhibited to normal levels by PAA; however, the increases in glucose utilization and oxidation were not completely suppressed to normal levels, suggesting that the PDH pathway (Table 1) can maintain part of the increased metabolism when PC is inhibited by PAA.

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Fig. 4. Effect of in vivo PAA treatment (0.5 g·kg1·day1) on glucose utilization (A) and oxidation (B) in Sham and 5-day Px islets. Freshly isolated islets were incubated at 2.8 or 16.7 mmol/l glucose containing D-[5-3H]glucose or [U-14C]glucose. *P < 0.01 for Px vs. Sham. Data are means ± SD; n = 4.
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Pyruvate, OAA, and malate content.
Increased glycolysis is expected to increase pyruvate production. Our results (Table 1) demonstrated that, 5 days after Px, pyruvate content was increased threefold. Because increased pyruvate can be metabolized by PC or PDH, we measured the products from these two pathways to test which pathway metabolized pyruvate. First, we tested the PC pathway by measuring OAA and malate content. As shown in Table 1, these two products were significantly (3-fold) increased in Px islets. These results clearly indicate that increased pyruvate is metabolized by the PC pathway, because only the PC pathway can increase OAA and malate content (33). This result also indicates that anaplerosis is enhanced.
PDH and citrate content in 5-day Px islets.
Next, we tested whether the increased pyruvate was metabolized by the PDH pathway by measuring active PDH (aPDH) activity and its product citrate. There was a slight increase in aPDH activity (1.4-fold; Table 1). Citrate content was also modestly increased (1.5-fold; Table 1). These data demonstrated that only a small part of the increased pyruvate was metabolized by the PDH pathway.
Malate release from mitochondria in 5-day Px islets.
To confirm that the increased pyruvate was metabolized by the PC pathway, we measured 10-min malate release from mitochondria. This is an indicator of pyruvate-malate shuttle activity (33). Malate release was significantly increased (3-fold) in Px islets, and
50% of this release was inhibited (P < 0.025) by in vivo PAA treatment (Fig. 5). The increased malate release was consistent with the results in Table 1 showing elevated OAA and malate content. It is also consistent with a role for the PC pathway in enhanced cell proliferation in Px islets.

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Fig. 5. Malate release (10 min) from mitochondria is increased in 5-day Px islets, and this is partially blocked by PAA treatment (0.5 g·kg1·day1). Procedures to measure malate release from mitochondria are described in MATERIALS AND METHODS. Data are means ± SD; n = 6.
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Inhibition by in vivo PAA treatment of Px-induced increase in islet cell number, DNA content, and protein content.
The results shown in Fig. 1 demonstrated that Px islet cells underwent rapid proliferation 15 days after Px. The results shown in Fig. 5 and Table 1 indicate that PC pathway activity was increased at the same time points. This close association suggests that the PC pathway may be important for Px islet cell proliferation. To obtain evidence to support this proposal, we treated animals with PAA (0.5g·kg1·day1 ip) in vivo to see whether inhibition of the PC pathway would downregulate cell proliferation. PAA produced a partial but significant inhibition of both the PC pathway (Fig. 5) and all of the parameters of cell proliferation: islet cell number, protein, and DNA content (Fig. 6). This strongly indicates that PC is involved in the Px-induced increase in cell proliferation. The Sham mice treated with PAA demonstrated a small reduction in PC activity (Fig. 6) and a small but insignificant drop in the parameters of cell proliferation.

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Fig. 6. Effects of in vivo PAA treatment on cell number, protein content, and DNA content in islets isolated 5 days after Px. Experimental methods are the same as used for Figs. 1 and 2. Data are means ± SD; n = 4.
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DISCUSSION
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PC pathway and pyruvate metabolism in Px islets.
PC is abundant in the two tissues that carry out gluconeogenesis, liver and kidney (8), but it is almost absent in nongluconeogenic tissues (49). In normal pancreatic islets, almost 0.4% of total protein is PC (34). Because pancreatic islets contain no phosphoenolpyruvate carboxylase (38), which is needed for gluconeogenesis,
-cell PC must be involved in an alternate function, pyruvate entry into the TCA cycle. In normal
-cells, PC and PDH are expressed in almost equal levels; thus pyruvate enters the TCA cycle by these two pathways in equal amounts (37). Our data demonstrated that Px induced an increase of pyruvate entry into the TCA cycle primarily through the PC pathway. The evidence is as follows. First, glycolytic flux was accelerated, as shown in Fig. 4. This result is consistent with our previous reports (28, 29) showing increased glucokinase (GK) and phosphofructokinase activities. Second, pyruvate content (Table 1) was increased threefold, which may come from a combination of increased glycolysis and malate recycling. We propose that the increase in pyruvate content drives increased pyruvate metabolism through PC. Third, OAA and malate contents were also increased threefold (Table 1), consistent with the elevation in pyruvate content. MacDonald (33) has shown that increased OAA and malate can come only from the PC pathway, not the PDH pathway. Fourth, pyruvate-malate shuttle activity was enhanced threefold (Fig. 5), which is consistent with the threefold increase in pyruvate and malate content (Table 1). Fifth, PDH activity and its product citrate were increased by only 4050% (Table 1), not enough to account for the elevated pyruvate metabolism.
The small increase in PC activity measured in Px islets (Fig. 2) was surprising compared with the large increase in OAA, malate, and cell proliferation. However, we have recently observed (27) that malate release can rise threefold despite a 17% reduction in PC activity in islets exposed to high glucose. Those earlier observations and our current results imply that islets possess sufficient PC activity to sustain increased pyruvate conversion to OAA in the presence of elevated pyruvate content. PC activity of Px islets is much lower than that in proliferative islets from obese Zucker rats. This may be because in the Px rat the time for compensation is short (several days) and there is no insulin resistance; in contrast, in Zucker rats the time course is much longer (several months), and the animals have severe insulin resistance. Our 60% Px model is different from the 90% Px model. The latter is hyperglycemic, and PC mRNA was significantly decreased 4 wk after 90% Px (21, 22), suggesting that PC was downregulated by hyperglycemia. An important role for hyperglycemia in downregulation of PC is supported by similar findings in islets cultured with high glucose (27).
In the present study, PC activity was measured by the radiometric method described by MacDonald (39). This method, which relies on PC-catalyzed 14CO2 fixation in a reaction mixture containing [14C]NaHCO3, is completely specific for PC. The commonly used procedure (43), which relies on detection of NADH production, does not discriminate between PC and lactate dehydrogenase (LDH). We confirmed this lack of specificity by using pure PC (Sigma), pure LDH (Sigma), or mixtures of the two (unpublished observation). Because our previous results (26) used the NADH method to find an increase in PC activity in islets from obese Zucker rats, we reexamined those results with the more specific radiometric method (39). The new assays revealed a 2.3-fold increase in PC activity from obese Zucker rats (25.1 ± 3.3 nmol·min1·mg islet protein1; n = 4) compared with PC activity in control lean Zucker rat islets (11.1 ± 2.2 nmol·min1·mg islet protein1; n = 4, P < 0.001). The fold increase in PC activity is the same as what we found by the previous method, but the absolute values are lower than our previous results (26). The method of determining PC activity by measuring NADH production has been widely used by many authors for determination of PC (10, 15, 50, 58) and LDH activity (1, 14, 17, 40, 60) (only a partial list) in many different tissues. Because this method cannot discriminate between PC and LDH activities, the results reflect the total activity of PC plus LDH, which can be misleading when interpreted as only PC or LDH.
PC pathway links pyruvate-malate shuttle and anaplerosis to cell proliferation in Px islets.
The importance of PC activity for
-cell proliferation was indicated in the current study. The conversion of pyruvate to OAA, then to malate, and back to pyruvate forms the pyruvate-malate shuttle (33). This cycle generates three important intermediates for cell proliferation: OAA, malate, and NADPH. These are required for
-cell proliferation, as OAA is used for protein and lipid synthesis (9, 16, 44), malate for production of NADPH, and NADPH for lipid and fatty acid synthesis (9, 16). The marked increase in
-cell proliferation after Px coincided temporally with a threefold increase in activity of the pyruvate-malate shuttle. The increase in pyruvate-malate shuttle activity was shown to be important for the stimulation of proliferation, since its partial inhibition with PAA produced a similar decline in proliferation. As we have previously reported (26), PC has an essential role in
-cell adaptation and prevention of diabetes. In insulin-resistant, obese Zucker rat islets, we found that PC activity increased 2.3-fold, malate release from mitochondria increased 3-fold, and islet
-cell mass increased 3.9-fold. A recent report showed that an approximately fourfold increase in anaplerotic flux could be attributed to an increase in PC flux in INS-1
-cell line (7). Our previous findings (28) demonstrated that protein and DNA biosynthesis in Px islets was significantly increased. Increased protein synthesis might require higher OAA, and our data showed that OAA content in Px islets was increased threefold, which is consistent with the fold increase in cell number, protein, and DNA content in Px islets. Enhanced DNA synthesis also utilizes increased pentose phosphate pathway (PPP) activity, as we described previously (5, 28). This may explain why PAA could not completely inhibit cell DNA synthesis in Px islets (Fig. 6C).
PDH and pyruvate metabolism in the
-cell.
In most tissues, PDH is the predominant enzyme for entry of pyruvate into the TCA cycle, whereby PDH regulates the rate of glucose oxidation (46). Because
-cell glucose flux is critical to cell function and insulin secretion, it is crucial that the
-cell possess a stable means to maintain this flux. However, PDH activity of the
-cell can be readily inhibited: high glucose exposure for 2 days dramatically reduces islet PDH activity to 30% of control (27). Treatment of islets with the free fatty acid oleate results in inhibition of PDH activity and decreased levels of citrate (30). Also, PDH activity declines significantly in prediabetic or obese rat islets (26). PC activity in the
-cell is more robust than PDH is; it is resistant to inhibition by high glucose or oleate, and it increases in prediabetic or obese rat islets (26). Thus one reason that the
-cell possesses high PC activity may be to stabilize the ability to carry glucose carbons into the TCA cycle.
Other factors involved in islet cell proliferation.
In addition to PC and other pathways discussed above, GK and the PPP may be important regulators for islet cell proliferation after Px. GK can accelerate glycolysis. GK is the flux-controlling enzyme for glycolysis in
-cells and, as such, serves as the "gatekeeper" for metabolic signaling (41) and is thought to be responsible for glucose-stimulated insulin secretion (42). GK is expressed in both
- and
-cells in developing fetal rat islets (56). In diabetic rat islets, GK activity (45) and mRNA (21) are greatly reduced. In pancreatic
-cell GK knockout (GK/) mice,
-cell insulin synthesis and function are affected, which leads to low birth weight and diabetes (54, 55). In our previous studies, GK activity was significantly increased in post-Px proliferative islets (28, 29). We propose that increased GK in Px islets elevates glucose 6-phosphate, which promotes PPP and entry of pyruvate into the TCA cycle; thus more protein, DNA, and pyruvate are synthesized. Increasing PPP activity produces more ribose phosphate that is used for DNA and RNA synthesis (19, 51). We have shown that PPP activity and DNA synthesis were significantly increased in Px islets (28). One other factor that regulates islet cell proliferation is Akt. Akt is a serine/threonine kinase and is critical in insulin-induced metabolism of glucose and lipid (12, 57). We have reported that Akt expression in 60% Px islets was significantly increased (18). The fact that many regulatory factors contribute to islet cell proliferation may explain why PAA treatment produced only a partial blockade of proliferative changes in Px islets.
In summary, we have shown that Px increases islet glycolysis, PC activity, pyruvate-malate shuttle flux, and cell proliferation. In vivo treatment with the PC inhibitor PAA partially blocks all these changes. This suggests that PC regulates or sustains islet cell proliferation by enhancing pyruvate-malate shuttle activity and anaplerosis. We propose and summarize the metabolic pathways in a schema (Fig. 7) to explain how PC may maintain elevated
-cell function and proliferation. Increased PC cycling activity generates more OAA and malate for anaplerosis and causes the release of more malate into the cytosol for production of NADPH. The latter can be used for lipid and fatty acid synthesis and insulin secretion. OAA is needed for protein synthesis. Our data suggest that the PC pathway regulates cell proliferation in Px islets by increasing the production of intermediates from the pyruvate-malate shuttle and from anaplerosis.

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Fig. 7. Proposed metabolic scheme for explaining Px islet cell proliferation. Px increases glucokinase activity, glucose utilization, and pyruvate production and increases pentose phosphate pathway activity and ribose phosphate production for DNA synthesis. Increased PC cycling activity by Px generates more malate; thus more malate releases from mitochondrion into cytosol to produce more NADPH that can be used for lipid and fatty acid synthesis, and insulin secretion. This cycling also provides more oxaloacetate (OAA) for protein synthesis through aspartate. PDH, pyruvate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; Ac-CoA, acetyl-CoA; CS, citrate synthase. Dark lines mean increase.
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GRANTS
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This work was supported by grants to Y. Q. Liu from the National Institutes of Health (P20 RR-DE17702 from the COBRE Program of the National Center for Research Resources) and the American Diabetes Association (Junior Faculty Award).
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ACKNOWLEDGMENTS
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We acknowledge Dr. Michael MacDonald for the discussion on PC activity assay.
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FOOTNOTES
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Address for reprint requests and other correspondence: Y. Q. Liu, Kosair Children's Hospital Research Institute, Dept. of Pediatrics, Univ. of Louisville School of Medicine, 570 South Preston St., Suite 304, Louisville, KY 40292 (E-mail: yqliu001{at}gwise.louisville.edu)
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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