Insulin secretion and differential gene expression in glucose-responsive and -unresponsive MIN6 sublines

Kohtaro Minami1, Hideki Yano1, Takashi Miki1, Kazuaki Nagashima1, Chang-Zheng Wang1, Hiroko Tanaka1, Jun-Ichi Miyazaki2, and Susumu Seino1

1 Department of Molecular Medicine, Chiba University Graduate School of Medicine, Chiba 260 - 8670; and 2 Department of Nutrition and Physiological Chemistry, Graduate School of Medicine, Osaka University, Suita 565 - 0871, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have established two sublines derived from the insulin-secreting mouse pancreatic beta -cell line MIN6, designated m9 and m14. m9 Cells exhibit glucose-induced insulin secretion in a concentration-dependent manner, whereas m14 cells respond poorly to glucose. In m14 cells, glucose consumption and lactate production are enhanced, and ATP production is largely through nonoxidative pathways. Moreover, lactate dehydrogenase activity is increased, and hexokinase replaces glucokinase as a glucose-phosphorylating enzyme. The ATP-sensitive K+ channel activity and voltage-dependent calcium channel activity in m14 cells are reduced, and the resting membrane potential is significantly higher than in m9 cells. Thus, in contrast to m9, a model for beta -cells with normal insulin response, m14 is a model for beta -cells with impaired glucose-induced insulin secretion. By mRNA differential display of these sublines, we found 10 genes to be expressed at markedly different levels. These newly established MIN6 cell sublines should be useful tools in the analysis of the genetic and molecular basis of impaired glucose-induced insulin secretion.

pancreatic beta -cells; mRNA differential display; MIN6-m9; MIN6-m14


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TYPE 2 DIABETES IS A MULTIFACTORIAL disorder characterized by both impaired insulin secretion from pancreatic beta -cells and impaired insulin action at the target tissues, but it has not been determined which defect is primary (1). However, evidence has accumulated, suggesting that defective beta -cell function itself is important in the pathogenesis of type 2 diabetes (23). For example, several types of type 2 diabetes have been found to be caused by mutations of genes expressed in beta -cells that are distinct from the genes involved in insulin action. These include several subtypes of maturity-onset diabetes of the young (MODY 1-5). MODY 1-5 are caused by mutations in hepatocyte nuclear factor (HNF)-4alpha (39), glucokinase (36), HNF-1alpha (40), insulin promoter factor 1 (IPF-1) (29), and HNF-1beta (4), respectively, all of which are related to pancreatic beta -cell function, including insulin synthesis and secretion and beta -cell development. In addition, maternally transmitted mitochondrial gene mutations are thought to cause diabetes due to impaired insulin secretion, possibly because of a defect in ATP production (35). Because insulin secretion is critical in the regulation of blood glucose concentrations, impaired glucose-induced insulin secretion due to alteration of the genes expressed in pancreatic beta -cells that are involved in insulin synthesis and/or insulin secretion could well contribute to the development of type 2 diabetes.

One way to identify the genes associated with impaired glucose-induced insulin secretion would be to compare the genes expressed in the beta -cells of normal subjects with those of type 2 diabetic patients who have impaired secretion, but this is not practical, because pancreatic islets from human subjects are difficult to obtain. Alternatively, islets from model animals such as the Goto-Kakizaki (GK) rat can be used for this purpose (5). Another approach is to compare gene expression between two different beta -cell lines that exhibit different insulin secretory profiles, for example, between a cell line showing normal insulin response and one showing impaired response.

The MIN6 cell line is one of the few beta -cell lines that retain insulin secretory response to glucose and other secretagogues (7, 19), and it has been used extensively in studies of the mechanisms of insulin secretion. During experiments, however, a sudden loss of glucose-induced insulin secretion from MIN6 cells during the course of the passages is sometimes noticed, possibly due to an outgrowth of cells with a poor response to glucose or a reduced expression of the genes responsible for glucose-induced insulin secretion. Accordingly, subcloning of MIN6 cells exhibiting different insulin-secretory responses to glucose might facilitate comparison of the physiological properties and the expression of the genes associated with impaired insulin response to glucose.

In the present study, we have established two sublines of MIN6 cells that exhibit different insulin-secretory properties and metabolic features. In addition, we have compared gene expression between the two sublines by use of mRNA differential display and found 10 genes expressed at markedly different levels. These two sublines of MIN6 cells should be useful for clarifying the molecular and genetic basis of impaired glucose-induced insulin secretion.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subcloning of MIN6 cells. MIN6 cells were cultured in DMEM containing 25 mM glucose, 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 100 mg/l streptomycin sulfate, and 60.5 mg/l penicillin G under a humidified condition of 5% CO2-95% air at 37°C. Subcloning of MIN6 cells was performed by the limiting dilution method. Briefly, the cells were diluted and cultured in a petri dish to form separate colonies originating from a single cell. Each colony was then picked up and further cultured in a new dish to obtain a cloned cell. The cells were then screened by an index of glucose-stimulated insulin secretion. The criterion was a significant increase in insulin secretion by 25 mM glucose compared with that by 3 mM glucose.

Measurement of insulin secretion. Cells (1 × 105 cells/well, 48-well plate) were seeded and precultured in DMEM for 2 days. The cells were preincubated for 30 min in HEPES-balanced Krebs-Ringer bicarbonate buffer (KRH: 119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM MgCl2, 1.19 mM KH2PO4, 25 mM NaHCO3, and 10 mM HEPES, pH 7.4) containing 0.5% BSA (7) with 5 mM glucose and then were incubated for 2 h with various concentrations of glucose or alpha -ketoisocaproate (KIC). Released insulin was measured by ELISA (Mitsui Pharmaceuticals, Tokyo, Japan). The amount of insulin secretion was normalized by the cellular protein content, rather than by cell number or DNA content, because the protein contents of m9 and m14 cells were practically identical (m9: 0.21 ± 0.029, m14: 0.20 ± 0.010 mg cellular protein/106 cells, means ± SE; n = 5-7).

Measurement of glucose and lactate concentrations in culture media. Cells were cultured at a density of 1 × 105 cells/well in a 48-well plate. After 1, 3, and 5 days of culture, aliquots of the medium were removed and deproteinized by the addition of 0.3 M perchloric acid (PCA). The centrifuged supernatant was used for measurement of lactate and glucose with appropriate kits (Roche Diagnostics, Mannheim, Germany).

Measurement of cellular ATP content and respiratory chain-driven ATP synthesis. Cells were incubated for 2 h in the presence or absence of an uncoupler of oxidative phosphorylation in mitochondria, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma, St. Louis, MO), with or without glucose. The cells then were washed twice with ice-cold KRH, solubilized with 100 µl of cell culture lysis reagent (Promega, Madison, WI), and the lysates were collected. The amount of ATP was measured with an ATP bioluminescence assay kit (Roche Diagnostics) according to the manufacturer's instructions.

Respiratory chain-driven ATP synthesis in digitonin-permealized cells was measured as previously described (32). Briefly, detached cells were suspended in Ham's F-10 medium and kept at room temperature for 30 min. Cells were washed and resuspended in 150 mM KCl, 25 mM Tris · HCl, 2 mM EDTA, 10 mM KH2PO4, 1 mM ADP, 0.1% BSA, 20 µg/ml digitonin, 10 mM malate, and 10 mM glutamate (pH 7.4). Reactions were continued for 10 min at 37°C, were terminated by adding 0.5 M (final concentration) PCA, and were chilled on ice for 15 min. The cells were then harvested, and the amount of ATP was measured as described above. To eliminate non-mitochondria-derived ATP production, ATP production in the presence of 10 µM CCCP was subtracted from that in the absence of CCCP.

Assay of enzyme activities. The activity of lactate dehydrogenase (LDH) was determined as follows. Briefly, cell extracts were incubated in a glycylglycine buffer (pH 10.0) with 1 mM lactate, 5 mM NAD, 50 mM glutamate, and 10 unit/ml glutamate-pyruvate transaminase at 25°C. Velocity of NADH formation was monitored by reading absorbances at 340 nm. Enzyme activity was expressed as nanomoles of NADH per minute per milligram of protein.

For determination of glucose-phosphorylating activity, disrupted cells were centrifuged, and supernatants were incubated in a triethanolamine buffer (pH 7.4) containing 0.5 mM NAD, 5 mM ATP, 1 unit/ml glucose-6-phosphate dehydrogenase, and 0.5 or 50 mM glucose at 30°C. Velocity of NADH formation was monitored by reading absorbances at 340 nm. Enzyme activity was expressed as nanomoles of NADH per minute per milligram of protein.

Electrophysiological analyses. Whole cell recordings of ATP-sensitive K+ current were performed as described previously (11). The extracellular solution contained 135 mM NaCl, 5 mM KCl, 5 mM CaCl2, 2 mM MgSO4, 5 mM HEPES, and 3 mM glucose (pH 7.4). The pipette solution contained 107 mM KCl, 11 mM EGTA, 2 mM MgSO4, 1 mM CaCl2, and 11 mM HEPES (pH 7.2).

The membrane potentials of the cells were measured by the perforated patch-clamp method in the current clamp mode (2). The extracellular solution contained 125 mM NaCl, 5 mM KCl, 1.3 mM KH2PO4, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 2.8 mM glucose (pH 7.4). The pipette solution contained 130 mM potassium aspartate, 10 mM KCl, 10 mM EGTA, 10 mM MOPS (pH 7.2), and 100 µg/ml nystatin.

Whole cell Ba2+ currents through the voltage-dependent calcium channels (VDCCs) were recorded as described (11). Briefly, Ba2+ was used as a charged carrier for measurement of VDCC currents. The extracellular solution contained 40 mM Ba(OH)2, 20 mM 4-aminopyridine, 90 mM tetraethylammonium hydroxide, 10 mM tetraethylammonium chloride, 140 mM methanesulfonate, and 10 mM MOPS (pH 7.4). The pipette solution contained 10 mM CsCl, 130 mM cesium aspartate, 10 mM EGTA, 5 mM Mg-ATP, and 10 mM MOPS (pH 7.2). Cells were maintained at a holding potential of -60 mV, and square pulses of 400-ms duration at potentials between -40 and +70 mV in steps of 10 mV were applied every 4 s. Recordings were performed with the use of the EPC-7 amplifier (List Electronics, Darmstadt, Germany).

Measurement of intracellular calcium concentration. Cells were loaded with 2 µM fura 2 acetoxymethyl ester (Dojindo, Kumamoto, Japan) for 50 min in the incubation buffer containing 154 mM NaCl, 6.2 mM KCl, 3.3 mM CaCl2, 1.5 mM KH2PO4, 1.6 mM MgSO4, 12.4 mM NaHCO3, 20 mM HEPES, and 2.8 mM glucose (pH 7.4) and then were mounted on the stage of the microscope. The perifusion rate was ~1 ml/min at 37°C. Intracellular calcium concentration ([Ca2+]i) was measured by a dual-excitation wavelength method (340/380 nm) as described (11). Fluorescence emission at 510 nm was monitored, and the ratio calculation was digitized every 10 s by a computerized image processor (Argus-50/CA; Hamamatsu Photonics, Hamamatsu, Japan).

mRNA differential display. mRNA differential display was performed with a commercial kit (Takara, Otsu, Japan) according to the manufacturer's instructions. Differentially expressed bands of interest were extracted from the gel and reamplified by PCR and were then cloned into pGEM-T Easy Vectors (Promega). The obtained clones were then subjected to RNA blot analysis. Sequencing was performed with the ABI PRISM dye terminator cycle sequencing FS ready reaction kit and DNA sequencer model 377 (Applied Biosystems, Foster City, CA). The sequences were compared with those in the database at the National Center for Biotechnology Information by means of the BLAST network service.

RNA blot analysis. Ten micrograms of total RNA from m9 and m14 cells were subjected to formaldehyde-agarose gel electrophoresis. The RNA was transferred to a nylon membrane and ultraviolet cross-linked. The membrane was hybridized with alpha -[32P]dCTP-labeled probes corresponding to cDNA of mouse glucokinase (GenBank Acc. No. L38990, nt 98-652), mouse hexokinase (J05277, nt 1464-1903), hamster sulfonylurea receptor 1 (SUR1; L40623, nt 3126-4049), mouse Kir6.2 (U73626, nt 753-1427), mouse GLUT-1 (M23384, nt 473-885), mouse GLUT-2 (X15684, nt 297-955), mouse NADH dehydrogenase subunit 1 (J01420, nt 2761-3061), mouse cytochrome c oxidase subunit 2 (J01420, nt 7016-7500), or mouse beta -actin (X03672, nt 571-1170). Megaprime random primer labeling kit (Amersham-Pharmacia Biotech, Uppsala, Sweden) was used for labeling the probes. The cDNAs obtained by mRNA differential display were similarly labeled and were used as probes for hybridizations. Blots were exposed to Kodak X-OMAT AR film (Eastman-Kodak, Rochester, NY) at -70°C.

Statistical analysis. Statistical significance of difference between the two groups was determined by unpaired Student's t-test. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subcloning of cells from MIN6 cells. We subcloned a total of 42 cell lines from MIN6 cells and screened them by an index of glucose-induced insulin secretion. Among these, two cell lines, designated m9 and m14, were selected as a good responder and a poor responder, respectively, to glucose. The difference in morphology between these two cell lines is that m14 cells are generally round, whereas m9 cells have an irregular shape (Fig. 1). Both cell lines were established at 18 passages.


View larger version (71K):
[in this window]
[in a new window]
 
Fig. 1.   Phase-contrast photomicrograph of m9 and m14 cells. Cells were grown on a tissue culture dish and photographed through a microscope (×100). A: MIN6-m9 cells (good responders to glucose); B: MIN6-m14 cells (poor responders to glucose).

Insulin secretory properties. Insulin secretion from m9 cells was stimulated by glucose in a concentration-dependent manner (Fig. 2A) comparable to that from the original MIN6 cells (7). The cells also secreted insulin by KIC, a nonglucose insulin secretagogue, in a concentration-dependent manner (Fig. 2B). Moreover, as in normal islets, the stimulatory effects of glibenclamide and acetylcholine on insulin secretion were observed in m9 cells (data not shown). In contrast, insulin secretion from m14 cells responded poorly to increments of both glucose (Fig. 2A) and KIC (Fig. 2B). However, in the presence of 30 mM KCl, m14 cells secreted almost the same amount of insulin as m9 cells (Fig. 2C). Figure 2D shows the effect of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, on glucose-induced insulin secretion in m9 and m14 cells. High glucose-induced insulin secretion was potentiated by 100 µM IBMX similarly (two- to threefold increases vs. that in the absence of IBMX) in both sublines. Furthermore, forskolin, an activator of adenylate cyclase, and 8-bromoadenosine 3',5'-cyclic monophosphate, a membrane-permeable analog of cAMP, had effects similar to those of IBMX (data not shown). These properties of insulin secretion in both cell lines were retained through at least 40 passages.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2.   Insulin secretory properties of m9 and m14 cells. Cells were precultured for 2 days in a 48-well plate and preincubated for 30 min in BSA-HEPES-balanced Krebs-Ringer bicarbonate buffer with 5 mM glucose. Incubation was performed in the presence of the indicated concentrations of glucose (A) or alpha -ketoisocaproate (KIC; B) for 2 h. Plasma membrane depolarization was induced by addition of 30 mM KCl with 250 µM diazoxide in the presence of 2.8 mM glucose (C). cAMP-mediated potentiation of glucose-induced insulin secretion was measured in the presence of 100 µM IBMX (D) with 25 mM glucose. Released insulin was determined by ELISA. Values are means ± SE (n = 4). ** P < 0.01.

Metabolic features. Although glucose consumption by m14 cells was much greater than that by m9 cells (Fig. 3A), the increment of cellular ATP content after stimulation of 25 mM glucose was not significantly different between the sublines (Fig. 3B). The ATP content of m9 cells was greatly reduced by 10 µM CCCP, an uncoupler of oxidative phosphorylation in mitochondria, but the compound did not affect ATP production in m14 cells (Fig. 3B). In accordance with this observation, a nonoxidative glucose metabolite lactate in m14 media was significantly higher than in m9 cell media (Fig. 3A). Cellular LDH activity was also significantly higher in m14 cells (Table 1).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Metabolic features of m9 and m14 cells. A: changes in lactate and glucose concentrations in the culture media. Cells were seeded onto a 48-well plate at day 0. At days 1, 3, and 5, aliquots of each medium were removed, and the concentrations of lactate and glucose were determined. Data are means ± SE (n = 6). B: cellular ATP contents. Cells were precultured and preincubated as described above. Incubation was performed in the presence or absence of 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), with or without 25 mM glucose for 2 h. Cells were lysed, and ATP contents were determined by a bioluminescence method. C: respiratory chain-driven ATP synthesis. Digitonin-permeabilized cells were incubated with 20 mM malate and 20 mM glutamate for 10 min. ATP production in the presence of 10 µM CCCP was subtracted. Data are means ± SE (n = 7). * P < 0.05, **P < 0.01. NS, difference is not significant.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Enzyme activities in MIN6 sublines

To examine the functional capacity of the mitochondrial oxidative phosphorylation system, ATP synthesis driven by the respiratory chain was measured in digitonin-permeabilized m9 and m14 cells. When stimulated with malate and glutamate, CCCP-sensitive ATP production (ATP production without CCCP minus the production with 10 µM CCCP) in m14 cells was not reduced but rather was enhanced compared with that in m9 cells (Fig. 3C). The expression of mitochondrial genes of m9 and m14 cells, as assessed by NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit 2 mRNA, was not different (Fig. 4A).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Expression of mRNAs that are critically involved in glucose-induced insulin secretion. Total RNA (10 µg) isolated from the cells was electrophoresed (m9 on left lane, m14 on right lane) and transferred onto a nylon membrane. A: mitochondrial genes. ND1 and COX2 indicate NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit 2, respectively. B: glucose-phosphorylating enzymes. GK and HK indicate glucokinase and hexokinase, respectively. C: ATP-sensitive K+ (KATP) channel subunits, SUR1 and Kir6.2. D: glucose transporters GLUT-1 and GLUT-2. beta -Actin was hybridized as internal control (bottom, each box).

RNA blot analysis revealed the expression of high Michaelis-Menten constant (Km) glucose-phosphorylating enzyme glucokinase mRNA to be markedly decreased in m14 cells compared with m9 cells (Fig. 4B, top), whereas low Km enzyme hexokinase mRNA was markedly increased in m14 cells (Fig. 4B, bottom). In the presence of 50 mM glucose (representing the activity of glucokinase plus hexokinase), the difference in glucose-phosphorylating activity was not significant between m9 and m14 cells, whereas in the presence of 0.5 mM glucose (representing hexokinase activity), the activity was significantly higher in m14 cells (Table 1). Glucokinase activity (glucose-phosphorylating activity at 50 mM glucose minus the activity at 0.5 mM glucose) was extremely low in m14 cells (Table 1).

In addition, we also examined the mRNA levels of glucose transport proteins GLUT-1 and GLUT-2. GLUT-1 mRNA expression was detected in both m9 and m14 cells, whereas GLUT-2 expression was not detected in either of the cell lines (Fig. 4D). To confirm the level of GLUT-2 mRNA expression in these cell lines, we performed RT-PCR. GLUT-2 PCR products from m9 and m14 cDNAs were only faintly detected after 30 cycles of amplification, whereas GLUT-1 PCR products were clearly detected after 25 cycles of amplification (data not shown). There was no significant difference in the expression of GLUT-1 and GLUT-2 mRNA between m9 and m14 cells.

Electrophysiology and [Ca2+]i. Because insulin secretion is triggered by an elevation of [Ca2+]i through activation of VDCCs resulting from membrane depolarization by closure of ATP-sensitive K+ (KATP) channels, we next examined the electrophysiological features and [Ca2+]i of the two cell lines. As shown in Fig. 5A, the normalized peak KATP channel conductance of m14 cells was significantly lower than that of m9 cells, indicating that the number of functional KATP channels on the plasma membrane is less in m14 cells. However, mRNA levels of Kir6.2 and SUR1, the components of the beta -cell KATP channels (6), were not significantly different in m9 and m14 cells (Fig. 4C). The resting membrane potential, which is determined primarily by KATP channels in beta -cells (18), was significantly higher in m14 than in m9 cells (Fig. 5B).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Electrophysiological properties and intracellular calcium concentrations ([Ca2+]i) in m9 and m14 cells. A: normalized peak KATP channel conductance. Data are means ± SE (n = 12). B: resting membrane potential. The membrane potentials of the cells were measured by the perforated patch-clamp method in the current clamp mode. Data are means ± SE (n = 10). C: [Ca2+]i change in the cells. [Ca2+]i was measured using fura 2 acetoxymethyl ester. Representative examples are shown. D: the current-voltage relationships of voltage-dependent calcium channels. Data are means ± SE (n = 12). * P < 0.05, m9 vs. m14.

[Ca2+]i was elevated dramatically in response to 25 mM glucose in m9 cells, whereas no significant change in [Ca2+]i was observed in m14 cells (Fig. 5C). Furthermore, the current-voltage relationships of VDCCs in m9 and m14 cells are shown in Fig. 5D. The VDCC inward currents in m14 cells are significantly less than those in m9 cells.

mRNA differential display. To investigate differences in gene expression between m9 and m14 cells, we performed mRNA differential display of these cell lines. By use of 216 combinations of anchored and arbitrary primers, 98 unique bands were detected by differential RT-PCR. Among these differentially displayed bands, RNA blot analysis showed 10 genes to be differentially expressed between the two cell lines (Fig. 6). As shown in Table 2, the sequences of five clones represent known genes. Clones g-1, g-2, and g-3 were underexpressed in m14 cells and represent stanniocalcin (STC), delta-like protein precursor (DLK)/preadipocyte factor 1 (Pref-1), and KIAA0480, respectively. Clones p-1 and p-2 were overexpressed in m14 cells and represent Na+/H+ exchanger 3 kinase A regulatory protein (E3KARP) and CCK-B receptor, respectively. Four matched only expressed sequence tag (EST) sequences, and one did not match any sequences in the GenBank database.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 6.   Expression of the genes identified by mRNA differential display. Total RNA (10 µg) isolated from the cells was electrophoresed (m9 on left lane, m14 on right lane) and transferred onto a nylon membrane. A: the genes strongly expressed in m9 cells; B: the genes expressed exclusively in m14 cells. Numbers on the right indicate sizes of transcripts (kb). beta -actin was hybridized to confirm equal loading (bottom, each box).


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Summary of differentially expressed genes

RNA blot analysis of the unknown genes. To determine tissue expression of the unknown genes that are differentially expressed between m9 and m14 cells, RNA blot analysis of mouse tissues was performed (Fig. 7). Clone g-4 was expressed in ovary, brain, kidney, and testis. Clone g-5 was expressed in ovary and testis. Expression of clone p-3 was observed in ovary, lung, kidney, intestine, and testis. Clone p-4 was expressed exclusively in brain. No hybridizing signals were detected in clone p-5 (data not shown).


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 7.   Tissue expression of the unknown genes that are differentially expressed between m9 and m14 cells. Total RNA (10 µg) isolated from mouse tissues was blotted and hybridized. Lane 1, ovary; lane 2, brain; lane 3, heart; lane 4, lung; lane 5, liver; lane 6, kidney; lane 7, skeletal muscle; lane 8, intestine; lane 9, testis; lane 10, pancreas. Numbers on the right indicate sizes of transcripts (kb).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we have established and characterized two sublines derived from MIN6 cells. One, designated m9, has features similar to those observed in the original MIN6 cells (7, 19) and retains glucose-induced insulin secretion after repetitive passages. The other, designated m14, has impaired glucose-induced insulin secretion. Thus both a glucose-responsive and a glucose-unresponsive cell line have been cloned from a single beta -cell line, facilitating comparison of the physiological properties and the expression of the genes associated with impaired glucose-induced insulin secretion.

In m14 cells, KIC-induced insulin secretion was also impaired, and cellular ATP was generated largely through nonoxidative pathways, metabolic features similar to those of mitochondrial DNA-depleted beta -cell lines (12, 28, 32). However, because the mitochondrial gene expression of m14 cells was not different from that of m9 cells (Fig. 4A) and direct activation of mitochondrial ATP production in digitonin-permeabilized cells was not reduced in m14 cells (Fig. 3C), it is not likely that the loss of KIC-induced insulin secretion or the increase in nonoxidative ATP production found in m14 cells is the result of mitochondrial dysfunction.

In accordance with the enhancement of nonoxidative ATP production, glucose consumption and lactate production were markedly enhanced in m14 cells (Fig. 3A). Glucose enters pancreatic beta -cells through specific transport proteins and is metabolized mainly in the glycolytic pathway, generating pyruvate as a substrate of the tricarboxylic acid cycle in mitochondria. In a nonoxidative state, pyruvate is converted to lactate by LDH, and lactate is then released from the cells. In general, however, because beta -cells express little LDH activity, most glucose-derived pyruvate enters the oxidative pathway (24, 26). In contrast, m14 cells exhibited significantly higher LDH activity than m9 cells (Table 1), which could lead to a diversion of pyruvate from mitochondrial oxidation. This might cause the acceleration of glucose metabolism in glycolysis due to compensation for reduced mitochondrial ATP generation. Interestingly, it has been reported that low LDH activity is important in beta -cell glucose sensing through unidentified mechanisms and that stable overexpression of LDH attenuates glucose-induced insulin secretion in MIN6 cells (42). Thus the impaired glucose-induced insulin secretion in m14 cells could be due in part to enhanced LDH activity.

Glucokinase is a rate-limiting enzyme in beta -cell glycolysis and is thought to be the glucose sensor for glucose-induced insulin secretion (20), because it has a Km higher than the physiological concentration of glucose. Like normal beta -cells (17) and the original MIN6 cells (8), m9 cells express glucokinase predominantly, whereas m14 cells express hexokinase, a low-Km isozyme of the glucose-phosphorylating enzyme, in replacement of glucokinase (Fig. 4B). In accordance with the results of RNA blotting, glucokinase activity (glucose-phosphorylating activity in the presence of 50 mM glucose minus that in the presence of 0.5 mM glucose) in m14 cells is close to zero (Table 1), and this could account for the impaired glucose sensitivity in m14 cells (Fig. 2A).

The major isoform of glucose transporters expressed in rodent pancreatic beta -cells is GLUT-2 (30), and this high-Km glucose transporter has been a candidate glucose sensor for insulin secretion in beta -cells (34). However, it is now generally accepted that the most important beta -cell glucose sensor is not GLUT-2 but glucokinase (20). In support of this, the overexpression of the GLUT-1 isoform in MIN6 cells has been found not to affect glucose-induced insulin secretion, whereas overexpression of hexokinase does alter glucose sensitivity (8). Moreover, in the present study, we found that the GLUT-2 mRNA level is very low in both m9 and m14 cells (Fig. 4D); nevertheless, the glucose sensitivity of m9 cells is comparable to that of the original MIN6 cells predominantly expressing the GLUT-2 isoform (7, 19). These findings also suggest that GLUT-2 is not a major glucose sensor in pancreatic beta -cells and indicate that the loss of GLUT-2 expression cannot account for the impaired glucose-induced insulin secretion found in m14 cells.

Electrophysiological experiments show that the number of functional KATP channels of m14 cells is less than in m9 cells (Fig. 5A) and that the function of VDCCs also is impaired in m14 cells (Fig. 5D). In pancreatic beta -cells, KATP channels play a crucial role in glucose-induced insulin secretion by coupling cell metabolism to electrical activity. Closure of KATP channels depolarizes the plasma membrane and leads to activation of the VDCCs, triggering exocytosis of insulin-containing granules (25). Dysfunction of these channels, therefore, could be responsible for the impaired glucose-induced insulin secretion in m14 cells. The loss of KIC-induced insulin secretion and the loss of glucose-induced insulin secretion, despite the comparable amount of ATP production with m9 cells (even though the pathway of ATP synthesis is different), could be explained, at least in part, by abnormalities in KATP channels or VDCCs. Furthermore, it has recently been reported that mitochondria-derived glutamate potentiates glucose-induced insulin secretion distinct from that due to ATP (16). In m14 cells, because mitochondrial metabolism might be impaired due to enhanced LDH activity, sufficient glutamate may not be generated, and this could also reduce glucose-induced insulin secretion.

Interestingly, cAMP-mediated potentiation of glucose-induced insulin secretion appears to be retained in glucose-unresponsive m14 cells (Fig. 2D). As described above, the function of KATP channels and VDCCs is impaired in m14 cells, and [Ca2+]i remains unchanged after stimulation by high glucose (Fig. 5C). These findings suggest that the cAMP-mediated potentiation pathway is regulated differentially from KATP channel-mediated glucose-induced insulin secretion, consistent with previous findings with high KCl-exposed pancreatic islets (38).

Although pancreatic beta -cell lines derived from rodents have been extensively used to investigate the mechanism of glucose-induced insulin secretion, their phenotypic instability is a common problem (13). Thus attempts were made to isolate cell lines exhibiting stable responsiveness to glucose, and the cloning of beta TC and INS-1 cell-derived sublines have been reported (3, 13). These cell lines retain correct glucose responsiveness after prolonged propagation. cAMP-mediated potentiation of glucose-induced insulin secretion is observed in both clonal cell lines (3, 13), and glucokinase is the predominant glucose-phosphorylating enzyme in clonal beta TC cells (13). These observations are consistent with ours in m9 cells, although GLUT-2 mRNA expression is apparent in the beta TC cells (13). This further indicates that glucokinase, but not GLUT-2, is critical for glucose sensing in beta -cells.

As a model of type 2 diabetes, GK rats have been intensively investigated (10, 15, 33). Pancreatic islets of GK rats are poorly responsive to glucose, whereas high K+-stimulated insulin secretion is not reduced (10), and both glucose usage and lactate production are accelerated (15). Although these properties resemble those of m14 cells, KATP channel function itself is not altered in GK rat islets, and VDCC activity is rather enhanced (10, 33). Moreover, alterations in glucokinase have not been reported in GK rats, further suggesting that the physiological properties of m14 cells are different from those of GK rat islets.

Impaired glucose-induced insulin secretion may well be caused by alterations in the genes expressed in pancreatic beta -cells that are involved in insulin synthesis and/or insulin secretion. As an approach to identify genes associated with impaired glucose-induced insulin secretion, we performed mRNA differential display using m9 and m14 cells. An advantage of this strategy is that the number of differentially expressed genes between the glucose-responsive (m9) and -unresponsive (m14) insulin-secreting cell lines should be minimal because of their derivation from a single cell line. We found 10 genes to be quite differentially expressed between these cell lines. Among them, STC and DLK/Pref-1 are particularly interesting, because they should participate in the regulation of calcium mobilization (22) and beta -cell differentiation (21), respectively. STC is a calcium-regulating hormone originally discovered in bony fish (37), the high expression of which in differentiated brain neurons in humans and in mice was recently demonstrated (22, 41). Zhang et al. (41) have suggested that the molecule may regulate transmembrane calcium fluxes and contribute to protection against hypercalcemia in terminally differentiated neurons. The absence of STC gene expression, therefore, could be associated with dysfunction of calcium regulation in m14 cells. DLK is a transmembrane protein of the EGF-like family of homeotic proteins (14), and Pref-1 is a variant derived from the same gene (27). In the developing pancreas, fetal antigen-1 (FA-1), the cleaved form of Pref-1, is expressed in most epithelial cells at an early stage, but its expression later becomes restricted to the beta -cells (31). Moreover, it is reported that FA-1 is colocalized with insulin in the insulin secretory granules of beta -cells in the adult human pancreas (9). Pref-1/FA-1, therefore, may have a role as an autocrine growth factor in beta -cells and may regulate their differentiation and proliferation (9, 21). The reduced expression of the DLK/Pref-1 gene in m14 cells may thus affect their differentiation and proliferation. Among the five unknown clones, the expression of clones g-5 and p-4 is restricted to a few tissues, suggesting their unique roles. Whether or not alteration of these genes occurs independently in the development of impaired glucose-induced insulin secretion or whether there is a major gene responsible for the complex abnormalities remains to be determined.


    ACKNOWLEDGEMENTS

This study was supported by a Grant-in-Aid for Creative Basic Research (10NP0201) from the Ministry of Education, Science, Sports, and Culture, Japan, by a Scientific Research Grant from the Ministry of Health and Welfare, Japan, by a grant from Takeda Chemical Industries, by the Yamanouchi Foundation for Research on Metabolic Disorders, by the Suzuken Memorial Foundation, and by Mitsui Pharmaceuticals.


    FOOTNOTES

K. Minami is a visiting research fellow from the Institute of Biological Science, Mitsui Pharmaceuticals; C.-Z. Wang is supported by a Postdoctoral Fellowship for Foreign Researchers from the Japan Society for the Promotion of Science.

Address for reprint requests and other correspondence: S. Seino, Dept. of Molecular Medicine, Chiba Univ. Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan (E-mail: seino{at}molmed.m.chiba-u.ac.jp).

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.

Received 22 February 2000; accepted in final form 16 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   DeFronzo, RA. Lilly lecture 1987. The triumvirate: beta -cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37: 667-687, 1988[ISI][Medline].

2.   Gonoi, T, Mizuno N, Inagaki N, Kuromi H, Seino Y, Miyazaki J, and Seino S. Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line MIN6. J Biol Chem 269: 16989-16992, 1994[Abstract/Free Full Text].

3.   Hohmeier, HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, and Newgard CB. Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 49: 424-430, 2000[Abstract].

4.   Horikawa, Y, Iwasaki N, Hara M, Furuta H, Hinokio Y, Cockburn BN, Lindner T, Yamagata K, Ogata M, Tomonaga O, Kuroki H, Kasahara T, Iwamoto Y, and Bell GI. Mutation in hepatocyte nuclear factor-1beta gene (TCF2) associated with MODY. Nat Genet 17: 384-385, 1997[ISI][Medline].

5.   Ihara, Y, Yamada Y, Yasuda K, and Seino Y. Analysis of novel cDNAs in pancreatic islets of GK rats by fluorescent differential display procedure (Abstract). Diabetes 45, Suppl2: 312, 1996[ISI].

6.   Inagaki, N, Gonoi T, Clement JP, IV, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, and Bryan J. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270: 1166-1170, 1995[Abstract].

7.   Ishihara, H, Asano T, Tsukuda K, Katagiri H, Inukai K, Anai M, Kikuchi M, Yazaki Y, Miyazaki JI, and Oka Y. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia 36: 1139-1145, 1993[ISI][Medline].

8.   Ishihara, H, Asano T, Tsukuda K, Katagiri H, Inukai K, Anai M, Kikuchi M, Yazaki Y, Miyazaki J, and Oka Y. Overexpression of hexokinase I but not GLUT1 glucose transporter alters concentration dependence of glucose-stimulated insulin secretion in pancreatic beta -cell line MIN6. J Biol Chem 269: 3081-3087, 1994[Abstract/Free Full Text].

9.   Jensen, CH, Krogh TN, Hojrup P, Clausen PP, Skjodt K, Larsson LI, Enghild JJ, and Teisner B. Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2. Eur J Biochem 225: 83-92, 1994[Abstract].

10.   Kato, S, Ishida H, Tsuura Y, Tsuji K, Nishimura M, Horie M, Taminato T, Ikehara S, Odaka H, Ikeda H, Okada Y, and Seino Y. Alteration in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta  cells of non-insulin-dependent diabetes mellitus GK rat. J Clin Invest 97: 2417-2425, 1996[Abstract/Free Full Text].

11.   Kawaki, J, Nagashima K, Tanaka J, Miki T, Miyazaki M, Gonoi T, Mitsuhashi N, Nakajima N, Ieanaga T, Yano H, and Seino S. Unresponsiveness to glibenclamide curing chronic treatment induced by reduction of ATP-sensitive K+ channel activity. Diabetes 48: 2001-2006, 1999[Abstract].

12.   Kennedy, ED, Maechler P, and Wollheim CB. Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells. Diabetes 47: 374-380, 1998[Abstract].

13.   Knaack, D, Fiore DM, Surana M, Leiser M, Laurance M, Fusco-DeMane D, Hegre OD, Fleischer N, and Efrat S. Clonal insulinoma cell line that stably maintains correct glucose responsiveness. Diabetes 43: 1413-1417, 1994[Abstract].

14.   Lee, YL, Helman L, Hoffman T, and Laborda JJ. dlk, pG2 and Pref-1 mRNAs encode similar proteins belonging to the EGF-like superfamily. Identification of polymorphic variants of this RNA. Biochim Biophys Acta 1261: 223-232, 1995[ISI][Medline].

15.   Ling, ZC, Efendic S, Wibom R, Abel-Halim SM, Ostenson CG, Landau BR, and Kahn A. Glucose metabolism in Goto-Kakizaki rat islets. Endocrinology 139: 2670-2675, 1998[Abstract/Free Full Text].

16.   Maechler, P, and Wollheim CB. Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis. Nature 402: 685-689, 1999[ISI][Medline].

17.  Matschinsky FM. Glucokinase as glucose sensor and metabolic signal generator in pancreatic beta -cells and hepatocytes. Diabetes 39: 647-652.

18.   Miki, T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, Gonoi T, Iwanaga T, Miyazaki J, and Seino S. Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc Natl Acad Sci USA 95: 10402-10406, 1998[Abstract/Free Full Text].

19.   Miyazaki, J, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, and Yamamura K. Establishment of a pancreatic beta  cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127: 126-132, 1990[Abstract].

20.   Newgard, CB, and McGarry JD. Metabolic coupling factors in pancreatic beta -cell signal transduction. Annu Rev Biochem 64: 689-719, 1995[ISI][Medline].

21.   Nielsen, JH, Svensson C, Galsgaard ED, Moldrup A, and Billestrup N. Beta cell proliferation and growth factors. J Mol Med 77: 62-66, 1999[ISI][Medline].

22.   Olsen, HS, Cepeda MA, Zhang QQ, Rosen CA, and Vozzolo BL. Human stanniocalcin: a possible hormonal regulator of mineral metabolism. Proc Natl Acad Sci USA 93: 1792-1796, 1996[Abstract/Free Full Text].

23.   Polonsky Lilly Lecture 1994 KS. . The beta -cell in diabetes: from molecular genetics to clinical research. Diabetes 44: 705-717, 1995[Abstract].

24.   Schuit, F, De Vos A, Farfari S, Moens K, Pipeleers D, Brun T, and Prentki M. Metabolic fate of glucose in perifused islet cells. J Biol Chem 272: 18572-18579, 1997[Abstract/Free Full Text].

25.   Seino, S. ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61: 337-362, 1999[ISI][Medline].

26.   Sekine, N, Cirulli V, Regazzi R, Brown LJ, Gine E, Tamarit-Rodriguez J, Girotti M, Marie S, MacDonald MJ, Wollheim CB, and Rutter GA. Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta -cells. J Biol Chem 269: 4895-4902, 1994[Abstract/Free Full Text].

27.   Smas, CM, and Sul HS. Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell 73: 725-734, 1993[ISI][Medline].

28.   Soejima, A, Inoue K, Takai D, Kaneko M, Ishihara H, Oka Y, and Hayashi JI. Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic beta cell line, MIN6. J Biol Chem 271: 26194-26199, 1996[Abstract/Free Full Text].

29.   Stoffers, DA, Ferrer J, Clarke WL, and Habener JF. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 17: 138-139, 1997[ISI][Medline].

30.   Thorens, B, Sarkar HK, Kaback HR, and Lodish HF. Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney and beta -pancreatic islet cells. Cell 55: 281-290, 1988[ISI][Medline].

31.   Tornehave, D, Jansen P, Teisner B, Rasmussen HB, Chemnitz J, and Moscoso G. Fetal antigen 1 (FA1) in the human pancreas: cell type expression, topological and quantitative variations during development. Anat Embryol (Berl) 187: 335-341, 1993[ISI][Medline].

32.   Tsuruzoe, K, Araki E, Furukawa N, Shirotani T, Matsumoto K, Kaneko K, Motoshima H, Yoshizato K, Shirakami A, Kishikawa H, Miyazaki J, and Shichiri M. Creation and characterization of a mitochondrial DNA-depleted pancreatic beta -cell line: impaired insulin secretion induced by glucose, leucine, and sulfonylureas. Diabetes 47: 621-631, 1998[Abstract].

33.   Tsuura, Y, Ishida H, Okamoto Y, Kato S, Sakamoto K, Horie M, Ikeda H, Okada Y, and Seino Y. Glucose sensitivity of ATP-sensitive K+ channels is impaired in beta -cell of the GK rat. Diabetes 42: 1446-1453, 1993[Abstract].

34.   Unger, RH. Diabetic hypoglycemia: link to impaired glucose transport in pancreatic beta cells. Science 251: 1200-1205, 1991[ISI][Medline].

35.   Van den Ouweland, JM, Lemkes HH, Ruitenbeek W, Sandkuijl LA, de Vijlder MF, Struyvenberg PA, van de Kamp JJ, and Maassen JA. Mutation in mitochondrial tRNALeu (UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nat Genet 1: 368-371, 1992[ISI][Medline].

36.   Vionnet, N, Stoffel M, Takeda J, Yasuda K, Bell GI, Zouali H, Lesage S, Velho G, Iris F, Passa P, Froguel P, and Cohen D. Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. Nature 356: 721-722, 1992[ISI][Medline].

37.   Wagner, GF, Milliken C, Friesen HG, and Copp DH. Studies on the regulation and characterization of plasma stanniocalcin in rainbow trout. Mol Cell Endocrinol 79: 129-138, 1991[ISI][Medline].

38.   Yajima, H, Komatsu M, Schermerhorn T, Aizawa T, Kaneko T, Nagai M, Sharp GWG, and Hashizume K. cAMP enhanced insulin secretion by an action on the ATP-sensitive K+ channel-independent pathway of glucose signaling in rat pancreatic islets. Diabetes 48: 1006-1010, 1999[Abstract].

39.   Yamagata, K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, Signorini S, Stoffel M, and Bell GI. Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young. Nature 384: 458-460, 1996[ISI][Medline].

40.   Yamagata, K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire M, Southam L, Cox RD, Lathrop GM, Boriraj VV, Chen X, Cox NJ, Oda Y, Yano H, Le Beau MM, Yamada S, Nishigori H, Takeda J, Fajans SS, Hattersley AT, Iwasaki N, Hansen T, Pedersen O, Polonsky KS, Turner RC, Velho G, Chevre JC, Froguel P, and Bell GI. Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3). Nature 384: 455-458, 1996[ISI][Medline].

41.   Zhang, KZ, Westberg JA, Paetau A, von Boguslawsky K, Lindsberg P, Erlander M, Guo H, Su J, Olsen HS, and Andersson LC. High expression of stanniocalcin in differentiated brain neurons. Am J Pathol 153: 439-445, 1998[Abstract/Free Full Text].

42.   Zhao, C, and Rutter GA. Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 beta -cell lines. FEBS Lett 430: 213-216, 1998[ISI][Medline].


Am J Physiol Endocrinol Metab 279(4):E773-E781
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society