Glycerol-Stimulated Proinsulin Biosynthesis in Isolated Pancreatic Rat Islets via Adenoviral-Induced Expression of Glycerol Kinase Is Mediated via Mitochondrial Metabolism

l-[³⁵S]methionine (1,200 Ci/mmol), d-[U-¹⁴C]glucose (250–360 mCi/mmol), and [U-¹⁴C]glycerol (125–180 mCi/mmol) were purchased from DuPont-New England Nuclear (Boston, MA). [¹²⁵I]-insulin (374 mCi/mg) was a gift from Eli Lilly (Indianapolis, IN). Pansorbin was purchased from Calbiochem (La Jolla, CA). Rat insulin was measured by radioimmunoassay using a kit from Linco (St. Louis, MO). Unless otherwise stated, all other chemicals were purchased from Sigma Chemical or Fisher Scientific (Fair Lawn, NJ) and were of the highest grade and purity available.

Pancreatic islets of Langerhans were isolated from male 150-g Sprague-Dawley rats by collagenase digestion and Histopaque-Ficoll density gradient centrifugation as described previously (21). Batches of 800 isolated islets were placed in 60-mm diameter Petri dishes in 5 ml of RPMI-1640 medium containing 11 mmol/l glucose supplemented with 10% (vol/vol) fetal calf serum, 200 units/ml penicillin, and 200 mg/ml streptomycin for 1–2 h at 37°C in 95% air/5% CO₂. Purified replication-deficient recombinant adenovirus capable of expressing Escherichia coli glycerol kinase (AdV-CMV-GlyK) or β-galactosidase (AdV-CMV-βGal) in primary cells was prepared as described previously (20). First, 10 μl of AdV-CMV-GlyK or AdV-CMV-βGal stock virus preparations (1 × 10¹³ pfu/ml) was added to a batch of 800 isolated islets and incubated for 1 h at 37°C. The medium was then replaced with fresh RMPI-1640 medium, and the islets were incubated for an additional 16 h at 37°C. This procedure generated a 25-fold multiplicity of infection, equivalent to that used in previous studies for AdV-CMV-GlyK–mediated glycerol-kinase expression (20). After islets had been infected with AdV-CMV-GlyK or AdV-CMV-βGal and subsequently cultured for 16 h, they were collected and assessed for proinsulin biosynthesis and insulin secretion as described below.

Glycerol-kinase activity was measured essentially as previously described (20,22). Briefly, 100 AdV-CMV-GlyK– or AdV-CMV-βGal–infected pancreatic islets were suspended in an extraction buffer consisting of 50 mmol/l HEPES, 40 mmol/l KCl, 11 mmol/l MgCl₂, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, and 1% Triton X-100 (pH 7.8) and frozen in liquid nitrogen. Islet-cell suspensions were thawed on ice and sonicated (25 W, 10 s), and cell debris was pelleted by centrifugation at 20,000g for 15 min at 4°C. Samples of islet extract supernatant (10 μg of protein) was added to a 50-μl total volume assay buffer containing 100 mmol/l Tris/HCl (pH 7.2), 5 mmol/l ATP, 10 mmol/l MgCl₂, 100 mmol/l KCl, 2.5 mmol/l dithiothreitol, 4 mmol/l glycerol, and [U-¹⁴C]glycerol (10 μCi/ml) and incubated for 2 h at 25°C. The reaction was terminated by the addition of 100 μl of 97% ethanol, 3% methanol. Fifty microliters of the terminated reaction mix was spotted onto circular DEAE filters (Schleieicher & Schuell, Keene, NH) and air-dried using a filtration manifold (Millipore, Bedford, MA). The filters then were washed five times with 100 ml of distilled water and then air-dried. Dried filters were placed in scintillation vials and counted in 10 ml of BioSafe II scintillation mixture (Research Products International, Mount Prospect, IL).

Essentially, a method previously described for MIN6 cells was followed (12). For each observation, ∼50 AdV-CMV-GlyK– or AdV-CMV-βGal–infected islets were preincubated in 1.5-ml microcentrifuge tubes in 300 μl of Krebs-Ringer bicarbonate buffer, 16 mmol/l HEPES, and 0.1% (wt/vol) bovine serum albumin (BSA) containing 2.8 mmol/l glucose for 1 h at 37°C. Cells were centrifuged at 800g for 2 min, resuspended, then incubated for 1 h in 300 μl of the same buffer containing insulin secretagogues as indicated. The last 20 min of this incubation period was in the presence of 100 μCi/ml [³⁵S]methionine to monitor protein biosynthesis (12,18,21). For assessment of insulin secretion in the same cells, a 200-μl aliquot of this second incubation medium was collected and centrifuged at 1,500g for 2 min to remove debris, and the supernatant was saved and stored at −80°C pending analysis of insulin secreted by radioimmunoassay. The islets then were washed in 500 μl of phosphate-buffered saline containing 0.1% (wt/vol) BSA, then resuspended in 300 μl of lysis buffer and lysed as described previously (12,18,21). A 5-μl aliquot of the resulting lysate was stored at −80°C pending analysis of intracellular insulin content by radioimmunoassay. Another 5-μl aliquot of the lysate was taken for assessment of total protein synthesis by trichloroacetic acid (TCA) precipitation as described previously (18,21). Subsequently, all cell lysates were equilibrated for equivalent levels of total protein synthesis before analysis of proinsulin biosynthesis so that specific regulation of proinsulin synthesis above that of general protein synthesis in the β-cell could be assessed (18,21). The remaining equilibrated cell lysate was subjected to specific (pro)insulin immunoprecipitation for assessment of proinsulin biosynthesis, with analysis by alkaline-gel electrophoresis, fluorography, and densitometric scanning as described (18,21).

The ratio of α-glycerophosphate (α-GP) to dihydroxyacetone phosphate (DHAP) was assessed as described previously (12). Briefly, groups of 500 AdV-CMV-GlyK– or AdV-CMV-βGal–infected rat islets were incubated for 1 h at 37°C in 300 μl of Krebs-Ringer bicarbonate buffer, 16 mmol/l HEPES, and 0.1% (wt/vol) BSA containing 2.8 mmol/l glucose, 16.7 mmol/l glucose, or 5 mmol/l glycerol as indicated. At the end of this incubation period, the islets were centrifuged at 500g for 3 min, and the supernatant was removed and stored at −80°C pending radioimmunoassay for insulin secretion. The islets were washed in phosphate-buffered saline, then resuspended in 400 μl of ice-cold 5 mmol/l HEPES-NaOH buffer (pH 7.2) containing 60 mmol/l sucrose, 190 mmol/l mannitol, 15 mmol/l KCl, 3 mmol/l KH₂PO₄, 1 mmol/l MgCl₂, and 0.5 mmol/l EDTA and homogenized on ice using 25 strokes of a Potter homogenizer. The homogenate then was centrifuged (1,500g, 10 min, 4°C) to remove nuclear and whole cells. The supernatant was removed, and the resultant pellet was resuspended in 400 μl of the same buffer and rehomogenized as above. The two resultant supernatants were combined. A 10-μl aliquot of the homogenate was stored at −80°C pending analysis of protein content, and the remaining homogenate was deproteinized with 6N HClO₄ and stored at −80°C. Before analysis, the samples were neutralized to pH 7.2 with 5 mol/l K₂CO₃, and the protein precipitate was removed by centrifugation at 10,000g for 10 min at 4°C. The α-GP and DHAP levels were measured by fluorometric enzyme assays as previously outlined (12,23).

Groups of either 200 AdV-CMV-GlyK– or AdV-CMV-βGal–infected rat islets were incubated for 1 h at 37°C in 300 μl of Krebs-Ringer bicarbonate buffer, 16 mmol/l HEPES, and 0.1% (wt/vol) BSA containing 2.8 mmol/l glucose, 16.7 mmol/l glucose, or 0.1–5.0 mmol/l glycerol as indicated. The islets then were centrifuged at 500g for 3 min, and a 150-μl aliquot of the incubation medium was removed for analysis of lactate output as previously described (12,20,24). The islets were resuspended in lysis buffer and analyzed for protein content as outlined (12,18,21).

Glycerol and glucose oxidation assays were performed as previously described (25,26). Briefly, groups of 25–30 islets were suspended in 200 μl of Krebs-Ringer bicarbonate buffer, 16 mmol/l HEPES, and 0.1% (wt/vol) BSA containing 2.8 mmol/l glucose, 16.7 mmol/l glucose, or 0.1–5.0 mmol/l glycerol in rounded bottom cups sealed with a rubber-sleeved stopper. Then, ∼2 × 10⁵ cpm of [U-¹⁴C]glucose in samples containing glucose as a substrate or [U-¹⁴C]glycerol in samples containing glycerol as a substrate were added to the islet suspensions. The cups were then sealed within a 20-ml borosilicate glass scintillation vial using a rubber-sleeved stopper and incubated for 2 h at 37°C. Then islet-cell oxidation was halted by the addition of 20 μl of sodium phosphate buffer (pH 6.0) containing 50 mmol/l rotenone to the islets in the cups via the sleeved stoppers. Afterward, 100 μl of HClO₄ was added to the islets in the cup and 300 μl of 1 mol/l benzethonium was added to the bottom of the scintillation vials via the appropriate sleeved stoppers. The samples then were incubated for an additional 2 h at 37°C in a shaking water bath. Then the seals were removed and the cups were discarded, and 10 ml of BioSafe II scintillation cocktail was added to the vials. The vials then were kept at 25°C overnight before scintillation counting.

Protein was assayed by the bicinchoninic acid method (Pierce, Rockford, IL). Data are presented as means ± SE. Statistical significance between groups was analyzed using Student’s t test; P ≤ 0.05 was considered significant.

Recombinant adenovirus-mediated gene transfer to isolated rat pancreatic islets in vitro gave a >80% efficiency of gene transfer to the islet cells, similar to that previously described (27,28). Infection of isolated rat islets with AdV-CMV-GlyK gave a glycerol-kinase activity of 360 ± 42 nmol glycerol 3-phosphate/mg protein per h (n = 5) at 16 h after infection, comparable with that previously observed in INS-1 cells (20). Little glycerol-kinase activity (<1.5 nmol glycerol 3-phosphate/mg protein per h) was found in either uninfected or AdV-CMV-βGal–infected controls at 16 h after infection. This was supportive of a recent study in which it was demonstrated that there is negligible glycerol-kinase activity in either isolated rat pancreatic islets or pancreatic β-cell line INS-1, unless glycerol-kinase protein expression is introduced via AdV-CMV-GlyK (20).

In control AdV-CMV-βGal–infected pancreatic islets, glycerol did not induce an increase in proinsulin biosynthesis (Fig. 1A) or insulin secretion (Fig. 1B), and AdV-CMV-βGal–infected islets oxidized glycerol minimally (Fig. 1C). Similar results were found in uninfected control islets (data not shown). However, in AdV-CMV-GlyK–infected islets, glycerol specifically stimulated proinsulin biosynthesis above that of total protein synthesis (Fig. 1A), and there was significant glycerol-induced insulin secretion (Fig. 1B). Moreover, AdV-CMV-GlyK–infected islets demonstrated a dose-dependent increase in glycerol oxidation that was not apparent in AdV-CMV-βGal–infected control islets (Fig. 1C). Glycerol-stimulated proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK–infected islets was independent of the presence of glucose (Figs. 1A and B). The threshold concentration of glycerol required to specifically increase the rate of proinsulin biosynthesis above that of total protein synthesis in AdV-CMV-GlyK–infected islets was between 0.25 and 0.5 mmol/l glycerol (Fig. 1A). Half-maximal stimulation of proinsulin biosynthesis was achieved at 0.6 ± 0.1 mmol/l glycerol (n = 5) that reached a maximum stimulation at 1 mmol/l glycerol, sixfold (P ≤ 0.002) above that at basal 0.1 mmol/l glycerol or 2.8 mmol/l glucose (Fig. 1A). Maximum stimulation of proinsulin biosynthesis at >1 mmol/l glycerol was not significantly different from that at 16.7 mmol/l glucose in AdV-CMV-GlyK– or AdV-CMV-βGal–infected islets (Fig. 1A). Glycerol-induced proinsulin biosynthesis was most likely occurring at the translational level as established previously for a 1-h stimulatory period (12,15,16,18,21). Preproinsulin mRNA levels remained the same in AdV-CMV-GlyK–infected islets incubated for 1 h at either 0.25 mmol/l or 2 mmol/l glycerol and likewise at 2.8 mmol/l or 16.7 mmol/l glucose (data not shown). Moreover, an inhibitor of transcription, actinomycin-d (10 μg/ml) (17,21), had no effect on glycerol-stimulated proinsulin biosynthesis (data not shown), as observed previously for glucose-induced proinsulin biosynthesis (12,17,18,21).

The threshold concentration of glycerol required to stimulate insulin secretion in AdV-CMV-GlyK–infected islets was higher than that required for glycerol-stimulated proinsulin biosynthesis: between 1.0 and 1.5 mmol/l glycerol (Fig. 1B). Half-maximal stimulation of insulin secretion in AdV-CMV-GlyK–infected islets was achieved at 1.7 ± 0.2 mmol/l glycerol (n = 5), which reached a maximum stimulation at 5 mmol/l glycerol, fourfold (P ≤ 0.01) above that at a basal 0.1 mmol/l glycerol or 2.8 mmol/l glucose (Fig. 1B). A similar pattern of glycerol-induced insulin secretion has been observed in AdV-CMV-GlyK–infected INS-1 cells (20).

The threshold concentration of glycerol required to gain significant glycerol oxidation in AdV-CMV-GlyK–infected islets was between 0.25 and 0.5 mmol/l glycerol (Fig. 1C), similar to that required to initiate glycerol-stimulated proinsulin biosynthesis (Fig. 1A). A maximum rate of glycerol oxidation in AdV-CMV-GlyK–infected islets was achieved above 5 mmol/l glycerol, which was 12-fold higher (P ≤ 0.001) than that at basal 0.25 mmol/l glycerol and 4-fold (P = 0.005) above basal glucose oxidation at 2.8 mmol/l glucose (Fig. 1C). Half-maximal glycerol oxidation was observed at 1.4 ± 0.2 mmol/l glycerol (n = 3; Fig. 1C).

In both AdV-CMV-GlyK– and AdV-CMV-βGal–infected islets was a 2.5-fold increase (P = 0.02) in lactate output at a stimulatory 16.7 mmol/l glucose compared with that at a basal 2.8 mmol/l glucose (Fig. 2). In contrast, between 0.2 and 2 mmol/l glycerol, there was no significant change in lactate output from either AdV-CMV-GlyK– or AdV-CMV-βGal–infected islets compared with that at a basal 2.8 mmol/l glucose or 0.2 mmol/l glycerol (Fig. 2). At 5 mmol/l glycerol, a slight increase was observed in lactate output in AdV-CMV-GlyK–infected islets, compared with the basal rate in AdV-CMV-βGal–infected control islets; however, this was not statistically significant (Fig. 2). In the added presence of 1 mmol/l α-cyanohydroxycinnaminic acid (α-CHC), an inhibitor of the plasma membrane and mitochondrial lactate/pyruvate transporter (12,29), lactate output generally was reduced by ∼70% (P = 0.05) in both AdV-CMV-GlyK– and AdV-CMV-βGal–infected islets whether incubated at 0.2–5 mmol/l glycerol or at 2.8 mmol/l or 16.7 mmol/l glucose, confirming α-CHC as an inhibitor of lactate export (data not shown).

An indication of α-GP shuttle activity can be gained by measuring the intracellular levels of α-GP and DHAP, which has been proposed as being reflective of a cytosolic NAD⁺/NADH ratio (30,31,32). In AdV-CMV-GlyK– and AdV-CMV-βGal–infected islets incubated at 16.7 mmol/l glucose, there was a significant increase in DHAP levels compared with that at a basal 2.8 mmol/l glucose (P = 0.05; Table 1). In contrast, α-GP levels did not change significantly in the same islets, so the DHAP:α-GP ratio increased significantly upon increasing the glucose concentration from 2.8 to 16.7 mmol/l glucose (P = 0.05; Table 1) as observed previously (12,32). In AdV-CMV-βGal–infected islets incubated at 2 mmol/l glycerol, the levels of DHAP and α-GP as well as the DHAP:α-GP ratio did not change significantly from AdV-CMV-GlyK– or AdV-CMV-βGal–infected islets incubated at 2.8 mmol/l glucose (Table 1). However, AdV-CMV-GlyK–infected islets that were incubated at 2 mmol/l glycerol levels of DHAP increased twofold over that in AdV-CMV-βGal–infected control islets incubated at 2 mmol/l glycerol levels (P = 0.01; Table 1). In addition, α-GP levels were increased by >50% in AdV-CMV-GlyK–infected islets versus AdV-CMV-βGal–infected control islets incubated at 2 mmol/l glycerol (P = 0.05; Table 1). It should be noted that DHAP and α-GP levels in AdV-CMV-GlyK–infected islets incubated at 2 mmol/l glycerol were higher than those in AdV-CMV-GlyK– or AdV-CMV-βGal–infected islets incubated at 2.8 or 16.7 mmol/l glucose (Table 1). Notwithstanding, the DHAP:α-GP ratio in AdV-CMV-GlyK–infected islets incubated at 2 mmol/l glycerol was significantly elevated compared with AdV-CMV-βGal–infected control islets incubated at 2 mmol/l glycerol (P = 0.02) (Table 1), to a similar extent as that observed in islets incubated at a stimulatory 16.7 mmol/l glucose (Table 1) (12). DHAP levels, α-GP levels, and the DHAP:α-GP ratio were not affected in AdV-CMV-GlyK– or AdV-CMV-βGal–infected islets incubated at 2 mmol/l glycerol in the added presence of 1 mmol/l α-CHC (Table 1).

Pyruvate entry into mammalian cells and mitochondria can be inhibited by α-CHC (12,29). In the presence of 1 mmol/l α-CHC, 16.7 mmol/l glucose-stimulated proinsulin biosynthesis was inhibited significantly in both AdV-CMV-GlyK– and AdV-CMV-βGal–infected islets by ∼60% (P = 0.01) (Fig. 3A). Glucose-induced insulin secretion also was inhibited significantly in AdV-CMV-GlyK– and AdV-CMV-βGal–infected islets by ∼60% (P = 0.005) (Fig. 3B). In AdV-CMV-GlyK–infected islets, 2 mmol/l glycerol-induced proinsulin biosynthesis was inhibited significantly by ∼75% in the presence of 1 mmol/l α-CHC (P = 0.002) (Fig. 3A), as was 2 mmol/l glycerol-induced insulin secretion (P = 0.002) (Fig. 3B). Glycerol did not induce proinsulin biosynthesis or insulin release in AdV-CMV-βGal–infected islets; 1 mmol/l α-CHC had no effect on the basal rate of proinsulin biosynthesis or insulin release observed at 2 mmol/l glycerol in these control islets (Fig. 3B). Finally, it was observed that the rate of glycerol oxidation at 2 mmol/l glycerol in AdV-CMV-GlyK–infected islets was inhibited significantly from 335 ± 54 pmol (n = 3) CO₂ produced from glycerol/islet/h to 87 ± 14 (n = 3) pmol CO₂/islet/h in the added presence of 1 mmol/l α-CHC (P = 0.01).

It has been shown that negligible glycerol-kinase activity is present in pancreatic β-cells, but recombinant adenovirus-mediated expression of glycerol kinase in pancreatic β-cells (INS-1) can “engineer” glycerol-stimulated insulin release (33). This study demonstrated that expression of glycerol kinase in primary isolated pancreatic rat islets can introduce not only glycerol-induced insulin release but also a parallel glycerol-induced increase of proinsulin biosynthesis at the translational level. This reaffirms the notion that the β-cell is poised exquisitely so that nutrients that stimulate insulin secretion also are able to increase specifically and rapidly the rate of proinsulin biosynthesis to maintain intracellular insulin stores at optimal levels (2,12).

Glycerol can be taken up by mammalian cells in proportion to the extracellular glycerol concentration (33,34). The expression of glycerol kinase at suitably high levels in pancreatic islet β-cells would generate α-GP from glycerol, which then could be converted to DHAP and enter the lower half of glycolysis, as DHAP is in equilibrium with glyceraldehyde-3-phosphate (33). This study found that the intracellular levels of DHAP and α-GP were markedly increased in AdV-CMV-GlyK–infected islets compared with AdV-CMV-βGal–infected control islets when incubated with glycerol, which resulted in a significant increase in the DHAP:α-GP ratio (similar to stimulatory 16.7 mmol/l glucose concentration [Table 1]). The oxidation of α-GP to DHAP could be achieved by a NAD⁺-linked cytosolic glycerol phosphate dehydrogenase and/or via a mitochondrial FAD-linked glycerol phosphate dehydrogenase in the glycerophosphate shuttle. Formation of DHAP from α-GP by the cytosolic glycerol phosphate dehydrogenase is an equilibrium reaction that should not alter the DHAP:α-GP ratio (35). In contrast, oxidation of newly formed α-GP to DHAP by the FAD-linked glycerol phosphate dehydrogenase in the mitochondrial loop of the glycerophosphate shuttle does increase in the DHAP:α-GP ratio (5,14,20,35). Thus, because glycerol induced a significant increase in the DHAP:α-GP ratio in AdV-CMV-GlyK–infected islets, it was likely that a larger proportion of the DHAP was generated from glycerol (via α-GP) by the FAD-linked glycerol phosphate dehydrogenase in the mitochondrial loop of the glycerophosphate shuttle. A glycerophosphate shuttle–mediated increase in the DHAP:α-GP ratio would reflect an increase in the islet β-cell cytosolic NAD⁺/NADH ratio, as well as provide a means for delivering reducing equivalents to the mitochondrial electron transport chain, at the FADH-linked complex-II, for subsequent generation of ATP (14,35). As such, increased NADH generated by increased glyceraldehyde-3-phosphate dehydrogenase activity as a consequence of increased DHAP glycolytic metabolism (5) could be compensated for efficiently by a coincidental increase in NAD⁺ production from increased glycerophosphate shuttle activity so that glycolysis does not become hindered (4,5,36). Such NADH reoxidation in glycolysis is mediated by lactate dehydrogenase in mammalian cells, but primary pancreatic β-cells have very low lactate dehydrogenase activity, so NAD⁺ production for glycolysis is mediated mostly through glycerophosphate shuttle activity (4,11,36). This is consistent with the fact that glycerol does not increase lactate output in AdV-CMV-GlyK–infected islets, as observed in this study. It was shown previously in the pancreatic β-cell line (INS-1) that AdV-CMV-GlyK–mediated expression of glycerol kinase could introduce glycerol-induced insulin secretion with a concomitant increase in lactate output (20). However, insulinoma-derived β-cell lines contain high levels of lactate dehydrogenase activity (37,38), so cytosolic NAD⁺ levels in INS-1 cells would likely be replenished via lactate production, lessening the need for increased glycerophosphate shuttle activity.

Notwithstanding, sufficient expression of glycerol kinase in pancreatic islets can generate meaningful levels of a glycolytic intermediate, DHAP, from glycerol for a nutrient fuel–induced stimulation of proinsulin biosynthesis and insulin secretion. It was interesting to note that the threshold concentration for glycerol-stimulated proinsulin biosynthesis was lower (0.25–0.5 mmol/l glycerol; Fig. 1A) as compared with that for stimulation of insulin secretion (1.0–1.5 mmol/l glycerol; Fig. 1B) in AdV-CMV-GlyK–infected islets. This was reflective of a lower threshold glucose concentration required to stimulate proinsulin biosynthesis translation (2–4 mmol/l glucose) versus insulin secretion (4–6 mmol/l glucose) (1), as well as that for pyruvate-stimulated proinsulin biosynthesis (0.13–0.2 mmol/l pyruvate) versus insulin secretion (0.2–0.3 mmol/l pyruvate) in MIN6 cells (12). It follows that the different threshold concentrations observed for glucose regulation of proinsulin biosynthesis versus insulin release do not necessarily lie at the level of glucose sensing, as proposed previously (1), but downstream in the generation of secondary metabolic stimulus-response coupling signals. Indeed, it was apparent that glycerol metabolism was required for a stimulus-response coupling of glycerol-induced proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK–infected pancreatic islets. The glycerol dose-response curve for glycerol-induced proinsulin biosynthesis paralleled that for glycerol oxidation in AdV-CMV-GlyK–infected islets, similar to that for glucose-induced proinsulin biosynthesis in normal pancreatic islets (1). Moreover, inhibition of glycerol oxidation by α-CHC similarly inhibited glycerol-induced proinsulin biosynthesis and insulin secretion in AdV-CMV-GlyK–infected islets to that for glucose-induced proinsulin biosynthesis and insulin secretion in control islets (Fig. 3). This suggested that in glycerol-stimulated AdV-CMV-GlyK–infected islets, the α-GP generated from glycerol kinase was being converted to DHAP (mostly via mitochondrial glycerol phosphate dehydrogenase, as discussed above) and entering the lower half of glycolysis to generate pyruvate. Pyruvate then can be either transported into mitochondria for entrance into the Krebs Cycle and subsequent oxidation or converted to lactate via lactate dehydrogenase, resulting in increased lactate output. However, as glycerol did not significantly increase lactate output from AdV-CMV-GlyK–infected islets, the pyruvate generated from glycerol was targeted preferentially for mitochondrial metabolism. This also is consistent with the relatively low lactate dehydrogenase activity and high pyruvate carboxylase activity in primary pancreatic islet β-cells (11,36). It also should be noted that addition of α-CHC did not affect a glycerol-induced increase in the DHAP:α-GP ratio in AdV-CMV-GlyK–infected islets, despite the α-CHC–mediated inhibition of glycerol oxidation and glycerol-induced proinsulin biosynthesis and insulin release. As such, it seemed unlikely that the glycerophosphate shuttle per se was generating downstream secondary signals to stimulated proinsulin biosynthesis and insulin release (4,12), although it cannot be ruled out that other mitochondrial shuttles could be playing a stimulus-coupling role in β-cells (5,8,13). However, other studies that used alternative experimental approaches have indicated that the glycerophosphate shuttle is not involved directly in generating stimulus-coupling signals for nutrient-stimulated insulin secretion in the β-cell (4,9,12).

In summary, this study indicated that in AdV-CMV-GlyK–infected primary β-cells, both glycerol-induced proinsulin biosynthesis and insulin release could be introduced in parallel, which were probably controlled from a secondary metabolic coupling signal(s) generated from mitochondria, via anaplerosis and/or glycerol oxidation, in agreement with previous studies that examined nutrient regulation of insulin release (4,11,12,13,14,38). It is not clear whether the same mitochondrial-derived metabolic stimulus-coupling signal controls both proinsulin biosynthesis translation and insulin secretion, and further experimentation is required. However, this study demonstrates further that the pancreatic β-cell has evolved a mechanism whereby nutrients coregulated proinsulin biosynthesis and insulin secretion, even when that nutrient is not normally metabolized unless engineered to do so by introduction of an enzyme that usually is not expressed. This provides a means to replenish insulin stores rapidly to optimal levels by the same stimulus that triggers insulin secretion.

This work was supported by grants from the National Institutes of Health (DK47919 and DK50610) and the Juvenile Diabetes Foundation International.

We thank Drs. Chris Newgard and Richard Noel at the University of Texas Southwestern Medical Center at Dallas for provision of the recombinant adenovirus-expressing glycerol kinase.