INTRODUCTION

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Maturity-onset diabetes of the young (MODY)¹ (1), an autosomal dominant form of non-insulin-dependent diabetes mellitus in which affected subjects develop hyperglycemia generally before the age of 25 years (1, 2), may be due to mutations in the transcription factor hepatocyte nuclear factor-1 (HNF-1) (3). These mutations appear to cause diabetes by inducing alterations in -cell function. Insulin secretion is abnormally low in individuals with HNF-1 diabetes, non-diabetic subjects with mutations in HNF-1 demonstrate reduced insulin secretory responses to high glucose, and insulin levels may be reduced even under basal conditions (4, 5).

The mechanisms whereby mutations in the gene for HNF-1 lead to abnormalities in insulin secretion have not been determined. We have demonstrated previously that mice lacking both alleles of the HNF-1 gene (HNF-1 (/)) have substantially reduced insulin secretory responses to glucose compared with wild type mice (HNF-1 (+/+)) whereas heterozygous HNF-1 (HNF-1 (+/)) are normal (6). In this study, experiments have been performed in wild type mice, and in heterozygous and homozygous HNF-1-deficient mice (7), to define the nature of the defects present in -cell signaling pathways that are responsible for reduced insulin secretory responses to glucose in HNF-1 diabetes.

	EXPERIMENTAL PROCEDURES

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Pancreatic Islet and -Cell Isolation-- Pancreatic islets were isolated from 6-12-week-old HNF-1 (/) mice and their age-matched heterozygote (+/) and wild type (+/+) controls by collagenase digestion and differential centrifugation through Ficoll gradients using a modification of procedures previously described (8). Islets were either cultured overnight prior to experiments performed to measure insulin secretion or plated on glass coverslips and cultured for 48-96 h for measurements of intracellular calcium ([Ca²⁺]_i). Single -cells used for membrane current recordings were obtained from islets dispersed by trituration (200 strokes through a 200-µl pipette tip) in Ca²⁺ - and Mg²⁺-free Hank's solution at room temperature. Cells were plated on glass coverslips and maintained in culture for 2-6 days. All cultures were performed using RPMI 1640 medium supplemented with fetal bovine serum, 11.6 mM glucose, 100 microunits/ml penicillin, and 100 µg/ml streptomycin as described previously (9). Medium was replaced every third day.

Insulin Secretion from Isolated Perifused Pancreatic Islets-- Following a base-line sampling period during which insulin concentrations were measured at 1-min intervals, the following secretagogues were added sequentially to the perifusate (a modified Krebs-Ringer buffer (KRB) containing (in mM) 119 NaCl, 4.7 KCl, 2 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 2 glucose) for a period of 10-15 min: D-glyceraldehyde (24 mM), methyl pyruvate (24 mM), tolbutamide (100 µM), and KCl (20 mM). Insulin concentrations were measured in the effluent perifusate every third minute and every minute for 3 min immediately after a change in perifusion conditions. DNA was measured in aliquots of islets from the same mice and the insulin secretory responses expressed as picomoles/liter/µg of DNA. The average insulin concentration in the effluent perifusate during administration of each secretagogue was calculated for each individual experiment, and the means for each secretagogue were compared between HNF-1 (+/) and (/) islets. Since glyceraldehyde administration was associated with a very pronounced and brief first phase of insulin secretion and a much lower second phase, the mean peak insulin secretory response (the single point of highest response) was compared between groups.

Assay Methods-- Insulin concentrations were measured by a double-antibody radioimmunoassay using a rat insulin standard. The intra-assay coefficient of variation for this technique is 7%. All samples were assayed in duplicate. DNA content of pancreatic islets was quantified in aliquots of 20 islets from three to four (+/) and (/) mice using a fluorimetric assay as described previously (10).

Intracellular Ca²⁺ Concentration in Pancreatic Islets-- [Ca]_i was estimated using fluorescence imaging techniques previously used by our laboratory (11).

Membrane Current Recordings-- The bathing solution contained (in mM): 137 NaCl, 4.7 KCl, 2 CaCl₂, 1.2 Mg SO₄, 1.2 KH₂PO₄, 10 HEPES (pH 7.45), 2-10 glucose, and the -cell was dialyzed with an internal solution containing (in mM): 120 KCl, 5 NaCl, 10 EGTA, 0-5 MgATP, 10 HEPES (pH 7.2). The resistance of the patch pipette ranged between 1.0 and 2.5 megohms. Care was taken during the break-in procedure to minimize the negative pressure utilized to gain access, as this increased the lifetime of the recordings. No series resistance compensation was used. Experiments were performed at room temperature. Current was normalized by reference to the cell capacitance. -Cells were identified by a combination of morphometric criteria and the presence of nutrient-suppressible ATP-sensitive K⁺ currents. Average values are expressed as mean ± S.E.

NAD(P)H Fluorescence Measurements-- Mutant and control islets were maintained in culture as described above for 48-96 h to allow adherence to coverslips. Using a fluorescence imaging system, cellular autofluorescence was excited by ultraviolet light at 360 nm and the emitted fluorescence intensity was measured at 460 nm. Experiments were performed at 37 °C in islets that were perifused with KRB. The relative increase in fluorescence intensity was obtained by dividing the fluorescence intensity following stimulation with secretagogues by the mean fluorescence intensity measured during the base-line resting condition (KRB + 2 mM glucose).

Measurements of Islet Glycolytic Flux-- Islet glycolytic flux was determined by measuring the formation of ³H₂O from D-[5-³H]glucose (12). Duplicate batches of 10 islets from HNF-1 (+/+), (+/), and (/) mice were incubated in 100 µl of KRB containing either 2 or 14 mM glucose, 5 mM HEPES, 0.2% w/v bovine serum albumin, and 1 µCi of D-[5-³H]glucose. The incubations were carried out in 1-ml Eppendorf tubes inside 20-ml glass scintillation vials containing 1 ml of distilled water and fitted with airtight rubber seals. Following a 30-min pre-incubation, in KRB containing 2 mM glucose, incubations were performed for 120 min at 37 °C with continuous shaking. Islet metabolism was stopped by the addition of 0.1 ml of 0.1 M HCl, injected into the inner tube through the rubber seal. The sealed scintillation vials were subsequently left at room temperature overnight to allow equilibration of the ³H₂O produced by the islets with the water in the outer vials. Liquid scintillation fluid was then added to the outer tube and mixed, and the radioactivity was quantified using a scintillation analyzer (Tricarb 2200CA, Packard Instrument Co., Downer's Grove, IL). The recovery of ³H from known amounts of ³H₂O was 40 ± 5% under these experimental conditions. The rate of glycolytic flux was corrected for recovery and expressed as picomoles of glucose/islet/2 h.

Magnesium Green Fluorescence Measurements-- Magnesium Green (MgG) fluorescence was measured in islets as an indirect measure of intracellular ATP. ATP has a greater affinity than ADP for Mg²⁺ (13, 14). Therefore, as intracellular ATP increases, the cytosolic ionized Mg²⁺ concentration falls, reflected by a reduction in fluorescence intensity in cells loaded with MgG. Control and mutant islets were isolated and maintained in culture on coverslips, as described above, for 48-96 h. Islets were loaded with MgG acetoxymethylester (5 µM, Molecular Probes, Eugene, OR) for 30 min at 37 °C in a humidified incubator. Islets were perifused as described previously (11) using oxygenated KRB containing variable concentrations of glucose and ketoisocaproic acid (KIC), and fluorescence intensity was measured using a fluorescence imaging system as described above. Fluorescence was excited by ultraviolet light at 495 nM, and emission was measured at 535 nM. The relative decrease in fluorescence intensity was obtained by dividing the maximum decrease in fluorescence intensity following stimulation with secretagogues by the mean fluorescence intensity measured during the base-line equilibration period (KRB + 2 mM glucose).

Measurement of Islet ATP Content-- ATP content of pancreatic islets was measured by a quantitative bioluminescence assay using a modification of a previously described protocol (15). Experiments were performed on islets from HNF-1  (+/+), and (/) mice that had been incubated overnight in RPMI containing 11.6 mM glucose. On day 2, groups of 15 islets were hand-picked and pre-incubated in KRB containing 2 mM glucose for 45 min. Since the HNF-1 (/) islets were smaller than the (+/+) islets, the smaller control islets were picked for these experiments. Following the pre-incubation, islets were transferred to separate tubes containing 0.5 ml of KRB with either 2 or 14 mM glucose and incubated for 5 min. After incubation, 0.5 ml of ice-cold 10% trichloroacetic acid was added and the tubes were vortex-mixed and kept on ice for 15 min. Following centrifugation, a fraction of the supernatant was removed and mixed with 1.5 ml of diethyl ether to remove the trichloroacetic acid. After vortex mixing, the ether phase was discarded and the procedure was repeated three times. The final extracts were diluted 1:1 with an ATP assay buffer and the ATP concentration of the supernatant was assayed using an ATP bioluminescence kit (FL-AA, Sigma) as per the manufacturer's instructions. ATP was measured in duplicate 0.1-ml aliquots from each sample, mixed with 0.1 ml of the ATP assay mix containing luciferase and luciferin. ATP is consumed and light emitted when luciferase catalyzes the oxidation of D-luciferin. The emitted light was measured using a luminometer (Monolight 1500, Analytical Luminescence Laboratories, San Diego, CA).

Statistical Analysis-- Results are expressed as mean ± S.E. The statistical significance of differences between animal groups was evaluated using the Wilcoxon rank-sum test or one-way analysis of variance where appropriate. Differences were considered to be significant at p < 0.05.

	RESULTS

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We have previously demonstrated that insulin secretion, in response to an increase in glucose concentration from 2 to 26 mM, in islets from HNF-1 (/) animals is substantially (approximately 85%) lower than in islets from both HNF-1 (+/+) or (+/) animals (6). In order to further define the insulin secretory defect in the HNF-l (/) mouse, isolated pancreatic islets were stimulated with secretagogues that provoke insulin release by distinct mechanisms. Increases in extracellular glucose concentration normally initiate insulin secretion from pancreatic -cells by inducing closure of ATP-sensitive K⁺ channels (KATP) via an increase in the cytosolic ATP/ADP ratio (16, 17). This leads to membrane depolarization and influx of Ca²⁺ through voltage-dependent Ca²⁺ channels. The elevation in [Ca²⁺]_i is a necessary prerequisite for insulin granule exocytosis. Sulfonylurea drugs like tolbutamide stimulate insulin secretion by directly blocking KATP (18). As was the case with glucose, tolbutamide produced a significantly smaller secretory response in HNF-1 (/) compared with (+/) islets (0.17 ± 0.05 versus 0.4 ± 0.05 pmol/liter/ng of DNA, p < 0.05, Fig. 1). On the other hand, direct depolarization of islet -cells using 20 mM KCl induced equivalent responses in (/) and (+/) islets (0.19 ± 0.04 versus 0.25 ± 0.03 pmol/liter/ng of DNA, p > 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Insulin secretory responses to glyceraldehyde, methylpyruvate, tolbutamide, and KCl. Following a period of equilibration of the pancreatic islets, 24 mM D-glyceraldehyde (6-20 min, A), 24 mM methyl pyruvate (36-50 min, B), 100 µM tolbutamide (66-80 min, C), and 20 mM KCl (96-105 min, D) were administered in the presence of 2 mM glucose (upper panel) in islets from HNF-1 (/) () and HNF-1 (+/) () mice. The peak insulin secretory response to glyceraldehyde and the mean insulin secretory response to the other secretagogues were compared (lower panel) in the HNF-1 (/) (hatched bars) and HNF-1 (+/) (open bars) islets. The peak response to glyceraldehyde and the response to tolbutamide were significantly reduced in the HNF-1 (/) islets (p < 0.01, Wilcoxon rank-sum test). Data represent the mean ± S.E. of seven observations in the HNF-1 (+/) mice and five in the (/) mice.

Stimulation of islets from HNF-1 (/) mice with glyceraldehyde, a metabolic intermediate that enters glycolysis distal to glucose 6-phosphate, produced only 30% of the peak secretory response seen in (+/) islets when applied to the (/) islets (0.24 ± 0.06 versus 0.73 ± 0.18 pmol/liter/ng of DNA; p < 0.05) (Fig. 1A). In addition, the area under the curve for [Ca²⁺]_i in (/) islets in response to glyceraldehyde was 28% of the (+/) value (Table I). These data suggest that specific steps in the early signal transduction process stimulated by glucose were impaired in the HNF-1 (/) islets. The observation that KCl induced equivalent increases in [Ca²⁺]_i in HNF-1 (/) and (+/) islets (Table I) suggests that later steps responsible for the elevation in [Ca²⁺]_i were unaffected. We have previously shown that 14 mM glucose failed to elevate [Ca²⁺]_i in the (/) islets (6).

View this table: [in this window] [in a new window]   Table I [Ca]i responses to glyceraldehyde, methyl pyruvate, tolbutamide, and KCl [Ca2+]i was measured in islets using fura-2 dual wave excitation photometry. Fluorescence emitted at 510 nM was measured using a fluorescence imaging system. The incremental area under the [Ca2+]i curve (AUC) in response to each secretagogue was calculated and the mean ± S.E. AUCs are presented. The n values represent the number of animals studied in each group. *, p < 0.05 compared to HNF-1 (+/) mice.

In order to further evaluate alterations in proximal steps in the glucose-stimulated stimulus-secretion coupling pathway, we measured changes in KATP currents (I_KATP) in single pancreatic -cells utilizing standard whole-cell voltage clamp techniques. Exposure of single -cells to stimulatory concentrations of glucose ordinarily results in a rapid suppression of I_KATP. HNF-1 (/) -cells displayed large amplitude I_KATP that were fully activated on break-in or progressively increased during the 5-10 min dialysis period (Fig. 2A). By contrast, in HNF-1 (+/) and (+/+) -cells the amplitude of I_KATP initially was small or completely absent, then increased and subsequently decreased rapidly during dialysis (Fig. 2, B and C). On average, the HNF-1 (/) cells had about 90-fold larger I_KATP than the controls: 175.4 ± 5.8 pA/pF (n = 12) compared with 2.1 ± 0.8 pA/pF (n = 8) (Fig. 3D).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Characterization of outward K+ currents in pancreatic -cells from HNF-1 (/) and (+/) mice. A-C, depolarizing pulses of 160 ms in duration were delivered at 10-mV increments from a holding potential of 60 to +40 mV. Shown are currents recorded immediately following a break in (left panel), and the 600 s of (right panel), intracellular dialysis. Recordings were made in the presence of 10 mM glucose (A and B) and 2 mM glucose (C). D, time course of KATP current activation (measured by pulsing from 60 to +40mV) expressed as current density subsequent to break-in in the HNF-1 (/) and HNF-1 (+/) -cells. The inset shows the biphasic alteration in KATP current in the (+/) -cells on a magnified current density scale.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Characterization of K-ATP currents in pancreatic islets from HNF-1 (/) mice. A, MgATP causes a dose-dependent suppression of KATP current. A family of I KATP was produced by pulsing from 60 mV in 10-mV increments to 30 mV in the presence of 500, 100, and 10 µM MgATP. B, concentration-response curve of the (/) (upper panel) and (+/) -cells. Note the similar IC50. The (+/) curves were obtained in cells bathed in 2 mM glucose. C, IKATP in HNF-1 (/) islets shows normal sulfonylurea sensitivity. Application of 1 mM tolbutamide leads to complete suppression of IKATP activated by a ramp protocol from 120 to +20 mV. Note the predicted change in holding current induced by tolbutamide (open circle, control; solid circle, tolbutamide). D, exposure of HNF-1 (+/) islets to the ATP synthesis inhibitor, sodium azide (Na azide) reproduces the changes in IKATP seen in (/) -cells. Azide induced large increases in IKATP using the same ramp in voltage from 120 to +20 mV as in C (solid square, azide).

To determine whether alterations of KATP were responsible for the reduced glucose responsiveness in the HNF-1(/) -cells, different concentrations of ATP or NADH were applied via the patch pipette to block I_KATP. In the (/) -cells, I_KATP had normal sensitivity to ATP and NADH: the IC₅₀ for ATP was approximately 75 µM (Fig. 3, A and B), while both ATP and NADH caused complete current suppression at 500 µM (n = 6), in accordance with published data (17), and confirmed in the case of ATP in (+/) -cells bathed in 2 mM glucose. Furthermore, both HNF-1 (/) and (+/) -cells were equivalently suppressed by 1 mM tolbutamide (Fig. 3C) (18). These findings suggested that the glucose insensitivity of HNF-1 (/) -cells might be related to an inability to produce ATP following the metabolism of glucose. This hypothesis was supported by the finding that -cells from (+/) animals bathed in 10 mM glucose, together with the ATP synthesis uncouplers sodium azide (Fig. 3D) or FCCP (data not shown) (19, 20) produced I_KATP of a similar magnitude to those seen in HNF-1 (/) -cells bathed in 10 mM glucose alone.

To further define the defect in ATP production, we studied the effect of a number of specific substrates and inhibitors of ATP synthesis. The methyl ester of pyruvate, a potent insulin secretagogue that generates large amounts of mitochondrial ATP (21, 22), was used to specifically stimulate mitochondrial ATP production. Exposure of the HNF-1 (/) -cells to 20-50 mM methyl pyruvate induced an immediate (<20 s) suppression of I_KATP (Fig. 4), which was reversible following addition of sodium azide, demonstrating that the effect is mediated through a stimulation of mitochondrial ATP production. Leucine, another stimulator of mitochondrial ATP synthesis (23), elicited similar effects on I_KATP in the homozygous mutant -cells that were reversed by sodium azide (data not shown). These results suggest that the ability of mitochondria to generate ATP from leucine or methyl pyruvate is unaffected by the loss of HNF-1 expression. Consistent with the effectiveness of mitochondrial substrates in suppressing I_KATP, methyl pyruvate produced equivalently robust elevation in [Ca²⁺]_i in the mutant and control islets (Table I). Furthermore, the mean insulin secretory responses to methyl pyruvate were not significantly different in HNF-1 (+/) and (/) islets (0.64 ± 0.04 versus 0.44 ± 0.12 pmol/liter/ng of DNA; p > 0.05) (Fig. 1B). Iodoacetic acid (IAA) inhibits glycolysis at the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preventing NADH and glycolytic ATP generation (24). When perifused prior to glucose, 1 mM IAA markedly attenuated the insulin secretory response to glucose in control islets ((+/) or (+/+)), rendering them as unresponsive to glucose as the HNF-1 (/) islets (Fig. 5).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Stimulators of mitochondrial ATP production suppress IKATP in HNF-1 (/) -cells. A family of KATP currents before (left) and after (right) exposure to methyl pyruvate are shown. Middle panel shows ramps during wash-in of reagent. Note that the methyl pyruvate largely suppressed IKATP, unlike the effects of glycolytic substrates. Similar results were obtained with leucine (data not shown). Family and ramp protocols are as described in Fig. 3.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   The effect of IAA on insulin secretory responses to glucose. Responses of islets from four control (two (+/) and two (+/+)) mice to a single-step increase in glucose concentration (upper panel) from 2 to 14 mM (open bar) were measured in the absence of IAA and following perifusion of 1 mM IAA (hatched bar) for 15 min prior to and during a second challenge with 14 mM glucose. The mean insulin secretory response to glucose in the presence (hatched bar) and absence (open bar) of IAA were compared (lower panel). The mean response was significantly reduced by exposure to IAA (p < 0.01, Wilcoxon rank-sum test).

Glycolysis generates ATP directly and indirectly via production of NADH. To determine if the impairment of ATP production is secondary to failure to reduce NAD⁺ to NADH, NAD(P)H generation by islets in response to glucose was estimated using fluorescence imaging. Fluorescence intensity at 460 nm increased by 72% and 71% over basal levels in response to 14 mM glucose in the HNF-1 (+/+) (Fig. 6, A and D) and (+/) islets (data not shown) but only by 23% in the (/) islets (p < 0.05; Fig. 6, B and D). IAA also abolished NAD(P)H responses of (+/+) islets to glucose (Fig. 6C) to levels similar to those seen in (/) islets with glucose alone (Fig. 6B). Methyl pyruvate (24 mM) induced a 2.1-fold increase in NAD(P)H in HNF-1 (/) islets, which tended to be greater than the responses in (+/+) islets (1.7-fold increase) (Fig. 7, A, B, and D). That the large increases in NAD(P)H in both (/) and (+/+) islets induced by methyl pyruvate occurred by stimulation of Krebs cycle metabolism was supported by the lack of effect of iodoacetate on methyl pyruvate-induced increases in NAD(P)H (Fig. 7, C and D).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   NAD(P)H responses to glucose in HNF-1 (/) and in (+/+) islets. Representative examples of NAD(P)H fluorescence measurements in response to a step increase in glucose concentration from 2 to 14 mM (filled bars) in HNF-1 (+/+) islets (A), HNF-1 (/) islets (B), and HNF-1 (+/+) islets before and after perifusion of 1 mM iodoacetic acid (hatched bar, C). Perifusate contained 2 mM glucose unless indicated. The relative increase in fluorescence induced by glucose (D) was 68% lower in (/) islets (hatched bar) than in controls (open bar, p < 0.05) and the response to glucose in (+/+) islets was attenuated by IAA (solid bar on the right, p < 0.01).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   NAD(P)H responses to methylpyruvate in HNF-1 (/) and (+/+) islets. Representative examples of NAD(P)H fluorescence measurements in response to stimulation with 14 mM glucose (filled bar) followed by 24 mM methylpyruvate (open bars) in the presence of 2 mM glucose in HNF-1 (+/+) islets (A) and (/) islets (B). Responses to 24 mM methylpyruvate were also measured in (+/+) islets before and after perifusion of 1 mM IAA (hatched bar, C). Methylpyruvate induced a 2.1-fold relative increase in NAD(P)H fluorescence in (/) islets (hatched bar) compared with 1.7-fold rise in (+/+) islets (open bar, p > 0.05, D). Following exposure to IAA, methylpyruvate produced a 1.6-fold increase in NAD(P)H (solid bar, p > 0.05 compared with +/+ islets in the absence of IAA).

To document a defect in cytosolic glucose metabolism, flux of glucose through glycolysis was estimated by measuring the formation of ³H₂O from tritiated glucose in islets from (+/+), (+/), and (/) mice. The average glycolytic flux in islets from HNF-1 (/) islets (12.7 ± 3.6 pmol/islet/2 h) was not different from (+/+) and (+/) islets (13.7 ± 2.6 and 15.1 ± 3.1, respectively) under basal conditions (2 mM glucose). Glucose utilization in islets incubated at 14 mM glucose was increased by 49.9 ± 10.3 pmol/islet/2 h and 48.6 ± 9.8 pmol/islet/2 h in HNF-1 (+/+) and (+/) islets, respectively. In contrast, the increase in glycolytic flux in response to stimulation with 14 mM glucose in HNF-1 (/) islets was significantly attenuated (26.1 ± 1.3 pmol/islet/2 h, p < 0.05 compared with both control groups) (Fig. 8A). Since a greater rate of glucose utilization could in theory be due to the larger size of the control islets, the respective mean relative increases in glucose utilization in the different mouse groups at 14 mM glucose over the values measured at 2 mM glucose were compared. Glucose utilization in HNF-1 (+/+) islets was 5.4 ± 0.9-fold greater at 14 than at 2 mM glucose compared with 4.7 ± 0.8-fold in (+/) islets (p > 0.05 compared with (+/+) islets) and 2.6 ± 0.5-fold in the (/) islets (p < 0.05 compared with (+/+) islets; Fig. 8B).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Measurements of glucose utilization in HNF-1 (+/+), (+/), and (/) islets. Glucose utilization was determined by measuring the formation of 3H2O from tritiated glucose in batches of 10 HNF-1 (+/+), (+/), and (/) islets incubated in duplicate for 120 min in KRB containing either 2 or 14 mM glucose and a tracer dose of D-[5-3H]glucose. Rates of glycolytic flux were similar at 2 mM glucose (not shown). The absolute increase in glucose utilization (A) as glucose concentration was increased from 2 to 14 mM in HNF-1 (+/+) islets (49.9 ± 10.3 pmol/islet/2 h, open bar) was similar to that in HNF-1 (+/) islets (48.6 ± 9.8 pmol/islet/2 h, hatched bar), and both were greater than in HNF-1 (/) islets (26.1 ± 1.3 pmol/islet/2 h, solid bar; *, p < 0.05 compared with (+/+) and (+/)). Compared with glycolytic flux at 2 mM glucose, glucose utilization at 14 mM glucose was 5.4 ± 0.9-fold higher (B) in HNF-1 (+/+) islets, 4.7 ± 0.8-fold higher in (+/) islets (p > 0.05, compared with (+/+) and 2.6 ± 0.5-fold higher in the (/) islets; #, p < 0.05 compared with (+/+)). Data are the mean ± S.E. of six experiments in each group.

These results predict a reduction in ATP production in response to glucose in the (/) islets. In order to determine if this occurred, changes in Magnesium Green fluorescence were measured in islets from HNF-1  (+/+), (+/), and (/) mice in response to stimulation by 14 mM glucose and 10 mM KIC (a stimulator of mitochondrial metabolism) in the presence of 2 mM glucose (Fig. 9). The expected small reduction in MgG fluorescence was consistently seen in islets from (+/+) and (+/) mice. The average maximal decrease in fluorescence intensity in the (+/+) islets in response to a step increase from 2 to 14 mM glucose was 13.5 ± 1.7% and in the (+/) mice was 10.7 ± 2.6%. Although these differences within each of the groups represented statistically significant differences from base-line, the magnitude of the change in fluorescence was not different between the (+/+) and (+/) islets. In contrast, glucose did not induce a significant change (2.4 ± 1.0%) in fluorescence intensity in the (/) islets. The average change in fluorescence was significantly less than in control islets (p < 0.03 compared with (+/) and p < 0.005 compared with (+/+) islets; Fig. 9C). In contrast, there was no difference in the response to 10 mM KIC in the three groups. The average maximal decrease in fluorescence intensity in response to KIC was 13.6 ± 1.8, 15.5 ± 1.6, and 14.5 ± 2.1 in the (+/+), (+/), and (/) islets, respectively (p > 0.05; Fig. 9D).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Measurement of Magnesium Green fluorescence in HNF-1 (+/+), (+/), and (/) islets. Representative examples of MgG fluorescence measurements from HNF-1 (+/+) islets (A) and HNF-1 (/) islets (B) stimulated by an increase in glucose concentration from 2 to 14 mM (open bar) followed by exposure to 10 mM KIC administered in the presence of 2 mM glucose (hatched bar). Perifusate contained 2 mM glucose unless indicated. CCCP (10 µM), an uncoupler of oxidative phosphorylation, which results in reduced ATP levels, was added for the last 5 min of each experiment (solid bar). The average reduction in MgG fluorescence intensity in response to glucose stimulation was 13.5 ± 1.7% in HNF-1  (+/+) islets (open bar; n = 8 mice), 10.7 ± 2.6% in (+/) islets (hatched bar; n = 6, p > 0.05 compared with (+/+)) and 2.4 ± 1.0% in (/) islets (solid bar; n = 5; p < 0.05 compared with (+/+) and (+/)) (C). Changes in MgG fluorescence intensity in response to KIC were similar in all groups (D).

Islet ATP content was also measured in islets from HNF-1  (+/+) and (/) mice, incubated at 2 and 14 mM glucose, using a quantitative bioluminescence assay (Fig. 10). The results demonstrated a failure of islet ATP content to increase in HNF-1 (/) islets, following incubation at the higher glucose concentration. Thus, ATP content of HNF-1 (+/+) islets incubated at 14 mM glucose was higher than in islets incubated at 2 mM glucose (1.3 ± 0.2 compared with 1.0 ± 0.2 pmol/islet (n = 11), p < 0.05 using paired t test). In contrast, the ATP levels in the (/) islets actually demonstrated a small decrease, which was not statistically significant (0.5 ± 0.1 pmol/islet compared with 0.6 ± 0.1 pmol/islet (n = 5), in islets incubated at 14 mM and 2 mM glucose, respectively; p > 0.05).

View larger version (11K): [in this window] [in a new window]   Fig. 10.   ATP content in HNF-1 (+/+) and (/) islets. ATP content was measured in islets from HNF-1 (+/+) and (/) following incubation at 2 (open bars) and 14 (hatched bars) mM glucose, using a quantitative bioluminescence assay. The ATP content of HNF-1 (+/+) islets was 30% higher in islets cultured at 14 mM glucose compared with islets cultured at 2 mM glucose (n = 11 mice, p < 0.05). In contrast, the ATP content of HNF-1  (/) islets was 10% lower in islets incubated at 14 mM glucose than in islets incubated at 2 mM glucose (n = 5 mice, p > 0.05).

	DISCUSSION

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This study was undertaken to further characterize the defect in -cell function in HNF-1 (/) mice. We show that there is a defect in ATP production in response to glucose in (/) -cells related to failure to generate NADH from glycolytic substrates.

We have demonstrated that glucose (6) and glyceraldehyde-induced insulin secretion is attenuated in HNF-1 (/) islets while the secretagogue effects of methyl pyruvate and KCl-induced depolarization are normal. Although insulin secretion following application of tolbutamide was reduced in islets from homozygous mutant mice, it is likely that this is secondary to a defect in ATP production. This hypothesis accords with the observation that the sensitivity of KATP to sulfonylureas is low in cells with impaired ATP production (25, 26).

To determine whether there was a specific abnormality in the KATP channel or its regulation, direct measurements using electrophysiological techniques were undertaken using the secretagogues whose mode of action is summarized in Fig. 11. -Cell membrane depolarization induced by high glucose is initiated by closure of the KATP channel via an increase in the ATP/ADP ratio (16, 17). Using standard whole-cell voltage clamp configuration recordings, we demonstrated that the ability of glucose to induce closure of KATP channels in the HNF-1 (/) mouse -cells was significantly reduced. Since the KATP current was blocked by the addition of ATP and NADH to the cell, the failure to generate ATP appears to account for the reduced glucose responsiveness of the KATP channel in HNF-1 (/) mouse -cells. This conclusion is supported by the finding that treatment of control islets with uncouplers of oxidative phosphorylation, FCCP and sodium azide, generated large KATP currents, similar to those seen in the (/) cells.

View larger version (24K): [in this window] [in a new window]   Fig. 11.   Metabolic signaling pathways in the -cell. The figure shows the fate of representative secretagogues: glucose and glyceraldehyde enter glycolysis, methyl pyruvate (me-pyruvate) generates ATP after entering the tricarboxylic acid (TCA) cycle in the mitochondrion and tolbutamide depolarizes the -cell membrane after binding to the sulfonylurea receptor. An increase in the ATP/ADP ratio leads to depolarization of the -cell membrane by closing the KATP channel. Also shown are the sites of action of two inhibitors of energy production; IAA inhibits glycolysis at the level of GAPDH, and FCCP inhibits the terminal steps of oxidative phosphorylation (Ox-Phos). Other abbreviations used include glyceraldehyde 3- phosphate (GAH-P) and 1,3-bisphosphoglycerate (P-GP).

To determine whether ATP formation in HNF-1 (/) islets in response to glucose stimulation is reduced, two approaches were utilized. The first technique involved the measurement of Magnesium Green fluorescence in islets. This method of measuring ATP takes advantage of the fact that ATP has a greater affinity for Mg²⁺ than ADP so that the intracytoplasmic [Mg²⁺] decreases when ATP is formed (13, 14). Using this technique, we have demonstrated a significantly smaller decrease in MgG fluorescence intensity in (/) islets compared with control islets in response to glucose stimulation, thus providing indirect evidence to support the hypothesis that there is a defect in ATP generation in response to glucose in HNF-1 (/) islets. Second, we measured the ATP content of islets from HNF-1 (+/+) and (/) islets incubated at 2 and 14 mM glucose using a quantitative bioluminescence assay. Whereas the ATP content of HNF-1 (+/+) islets increased in response to glucose stimulation, no increase was found in the (/) islets. Taken together, these data support the hypothesis that there is a defect in ATP production by HNF-1 (/) islets in response to glucose stimulation.

Glucose-induced inhibition of I_KATP has previously been shown to depend on glycolysis, distal to the generation of glyceraldehyde 3-phosphate (27), a conclusion consistent with the observation that glyceraldehyde is an effective secretagogue in normal islets. In particular, the production of NADH by GAPDH and its consequent conversion into ATP by mitochondria is critical to normal glucose signaling in -cells (27, 28). A defect in glucose-induced insulin secretion in HNF-1 (/) islets caused by the inability of glucose metabolism to produce ATP could be the consequence of reduced NAD(P)H responses to glucose in these islets. In agreement with these predictions, whereas glucose induced a robust elevation in NAD(P)H in (+/) and (+/+) islets, the rise in NAD(P)H in the HNF-1 (/) islets was markedly attenuated.

Failure of glucose-induced ATP generation could result from a defect occurring at several points in the pathway of glucose metabolism, which occurs in two phases: glycolysis in the cytosol and oxidation of the resulting NADH in the mitochondria. Methyl pyruvate was used to determine at which site the defect occurs in HNF-1 (/) islets. Although pyruvate alone does not stimulate insulin secretion (29), probably due to its restricted transport into the mitochondrion, methyl pyruvate freely permeates the mitochondrial membrane and thereby generates a large amount of ATP that closes the KATP channel in normal -cells (28). In fact, methyl pyruvate at various concentrations suppressed I_KATP in HNF-1 (/) islets. In addition, leucine, an amino acid that is metabolized in mitochondria and produces ATP, was also capable of suppressing KATP currents in the homozygous mutant -cell. Furthermore, the reduction in MgG fluorescence intensity in (/) islets stimulated by 10 mM KIC was equivalent to that in control islets suggesting that mitochondrial ATP generation is intact in the mutant islets. Taken together, these data indicate that under appropriate conditions the mitochondria in the (/) -cell are capable of synthesizing ATP and suggest that the defect in HNF-1 (/) -cells is either in generating or transporting NADH into mitochondria.

Since decreased production of ATP could be secondary to reduced formation of NADH, NAD(P)H fluorescence was measured in islets following stimulation with glucose and methyl pyruvate. The increase in fluorescence after glucose stimulation was significantly lower in the (/) islets than in control islets, whereas the responses to methyl pyruvate are similar. These findings confirm that mitochondrial NADH generation is not reduced in islets from HNF-1 (/) mice, again suggesting that cytoplasmic NADH generation or its transfer into the mitochondrion is abnormal. The demonstration of reduced glucose utilization in HNF-1 (/) islets in response to glucose stimulation also supports the hypothesis that the impairment in ATP generation in mutant islets results from a defect in cytosolic glucose metabolism. The ³H of the fifth carbon of the labeled glucose yields a tritiated water molecule during the reaction catalyzed by enolase (30, 31), implying that the defect in glycolytic signaling is proximal to the enolase reaction. Although the precise nature of the signaling defect remains elusive, it is possible that a reduction in the expression of one or more of the enzymes of the glycolytic pathway proximal to the enolase step accounts for the defects observed in glucose-stimulated signal transduction.

The HNF family of transcription factors has been implicated in the regulation of key glycolytic enzymes in the liver (32, 33), and expression of multiple HNF subtypes including HNF-1 has been reported in pancreatic islets and insulinoma cells (34, 35). The ability of mitochondrial substrates such as methyl pyruvate to 1) increase NAD(P)H, 2) block KATP, 3) elevate intracellular [Ca²⁺]_i, and 4) stimulate insulin secretion in the HNF-1 (/) islets holds out the hope that similar agents could have therapeutic benefit in the treatment of HNF-1 diabetes or perhaps even other forms of type 2 diabetes.