Interaction of glibenclamide and metformin at the level of translation in pancreatic β cells

in Journal of Endocrinology
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  • 1 Diabetes Research Center, Endocrine and Metabolic Division, Brussels Free University-VUB and JDRF Center for Beta Cell Therapy in Diabetes, Laarbeeklaan 103, B-1090 Brussels, Belgium

Sulfonylurea and metformin are used in the treatment of diabetes. Their chronic effects on β cells are not well known. We have shown that sustained exposure of rat β cells to glibenclamide increased their protein synthesis activity, while metformin caused an inhibition. The effect of glibenclamide was attributed to an activation of translation factors. This study examines whether both drugs interact at the level of protein translation in β cells. Purified rat β cells were cultured with and without glibenclamide and metformin before measurement of protein and insulin synthesis, abundance of (phosphorylated) translation factors, and cell viability. A 24 h exposure to metformin stimulated AMP-activated protein kinase (AMPK), suppressed activation of translation factors- both the mammalian target of rapamycin (mTOR; also known as mechanistic target of rapamycin, MTOR)-dependent ones (eukaryotic initiation factor 4E-binding protein 1 and ribosomal protein S6) and the mTOR-independent eukaryotic elongation factor 2-, and inhibited protein synthesis; a 72 h exposure resulted in 50% dead cells. These effects were counteracted by addition of glibenclamide, the action of which was blocked by the mTOR inhibitor rapamycin and the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs. In conclusion, metformin activates AMPK in β cells leading to suppression of protein translation through mTOR-dependent and -independent signaling. Glibenclamide antagonizes these metformin effects through activation of mTOR- and PKA-dependent signaling pathways.

Abstract

Sulfonylurea and metformin are used in the treatment of diabetes. Their chronic effects on β cells are not well known. We have shown that sustained exposure of rat β cells to glibenclamide increased their protein synthesis activity, while metformin caused an inhibition. The effect of glibenclamide was attributed to an activation of translation factors. This study examines whether both drugs interact at the level of protein translation in β cells. Purified rat β cells were cultured with and without glibenclamide and metformin before measurement of protein and insulin synthesis, abundance of (phosphorylated) translation factors, and cell viability. A 24 h exposure to metformin stimulated AMP-activated protein kinase (AMPK), suppressed activation of translation factors- both the mammalian target of rapamycin (mTOR; also known as mechanistic target of rapamycin, MTOR)-dependent ones (eukaryotic initiation factor 4E-binding protein 1 and ribosomal protein S6) and the mTOR-independent eukaryotic elongation factor 2-, and inhibited protein synthesis; a 72 h exposure resulted in 50% dead cells. These effects were counteracted by addition of glibenclamide, the action of which was blocked by the mTOR inhibitor rapamycin and the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs. In conclusion, metformin activates AMPK in β cells leading to suppression of protein translation through mTOR-dependent and -independent signaling. Glibenclamide antagonizes these metformin effects through activation of mTOR- and PKA-dependent signaling pathways.

Introduction

Sulfonylurea has been used in the treatment of diabetes for decades. Its hypoglycemic action is attributed to stimulation of insulin release that rapidly follows the drug's binding to the SUR receptor of β cells and the subsequent rise in cytoplasmic free calcium concentration (Sturgess et al. 1985, Henquin 1987, Proks et al. 2002). We have recently reported that glibenclamide, a second-generation sulfonylurea drug, also exerts stimulatory effects on β-cell protein and insulin synthesis when present over a prolonged period both in vivo and in vitro (Ling et al. 2006). This chronic effect was achieved through an activation of translation at the steps of initiation and elongation, as evidenced by phosphorylation of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and of ribosomal protein S6 (rpS6), and dephosphorylation of eIF2α and of eukaryotic elongation factor 2 (eEF2; Wang et al. 2008). It was shown to be calcium dependent and mediated through the mammalian target of rapamycin (mTOR; also known as mechanistic target of rapamycin, MTOR), protein kinase A (PKA), and MAPK/ERK kinases (MEKs; Wang et al. 2008). Thus, glibenclamide, which is used for its acute stimulatory effect on insulin release, exerts an mTOR-dependent stimulation of insulin synthesis when present for prolonged periods (Wang et al. 2008). mTOR is a serine and threonine protein kinase that regulates protein translation through a rapamycin-sensitive pathway involving the rpS6 kinase (S6K1) and the 4E-BP1 (Gingras et al. 2001, Raught et al. 2001, Arsham & Neufeld 2006). It is activated by nutrients and growth factors (McDaniel et al. 2002, Kwon et al. 2004) and inhibited by cellular energy deficiency through a mechanism that is dependent on AMP-activated protein kinase (AMPK; Inoki et al. 2003, Gleason et al. 2007). AMPK activation inhibits protein translation in many cell types, including pancreatic β cells (Bolster et al. 2002, Kefas et al. 2004, Williamson et al. 2006, Dowling et al. 2007, Cai et al. 2008). Sustained inhibition of protein synthesis is known to impair β-cell function and survival (Hoorens et al. 1996, Garcia-Barrado et al. 2001, Cai et al. 2008). It is unknown whether glibenclamide-induced mTOR activation could preserve protein translation and consequently β-cell survival in conditions of AMPK activation.

In pancreatic β cells, AMPK can be activated by the anti-diabetic drug metformin (Kefas et al. 2004, Leclerc et al. 2004). Metformin is used in the treatment of diabetes as a facilitator of insulin sensitivity in peripheral tissues (Cusi and Defronzo, 1998, Rutter et al. 2003). It is so far unknown whether metformin treatment in vivo exerts also direct effects on β cells. In vitro, metformin inhibits glucose oxidation, suppresses insulin secretion and biosynthesis, and subsequently induces apoptosis (Schatz et al. 1972, Kefas et al. 2004, Leclerc et al. 2004). As in peripheral tissues, this effect appears mediated, at least in part, through inhibition of mitochondrial respiratory complex I and activation of AMPK (El-Mir et al. 2000, Owen et al. 2000, Zou et al. 2004, Hinke et al. 2007). It is conceivable that a metformin-induced activation of AMPK also results in an inhibition of mTOR-dependent translation in β cells. If that is the case, the combination of metformin and glibenclamide may exert opposing effects on mTOR activity, and thus on β-cell function and survival. In this in vitro study, we therefore examined whether the earlier observed metformin inhibition of β-cell function and survival was associated with inactivation of mTOR-dependent translation factors, and whether these effects could be prevented by glibenclamide. Likewise, since glucose activates mTOR in pancreatic β cells (Kwon et al. 2004, 2006) and chronically elevated glucose results in sustained translational activation in β cells (Ling et al. 1996), we examined whether high levels of glucose could counter metformin inhibition of translation.

Materials and Methods

Preparation and culture of purified rat β cells

Pancreatic islets were isolated from adult male Wistar rats as described previously (Pipeleers et al. 1985). The animals were housed according to Belgian regulation of animal welfare. Islets were dissociated into single cells in calcium-free medium containing trypsin and DNase (Pipeleers et al. 1985). Single β cells were purified by autofluorescence-activated sorting, using cellular light-scatter and flavin adenine dinucleotide-autofluorescence as discriminating parameters (Pipeleers et al. 1985).

Purified β cells were suspended in Lux dishes (Miles, Naperville, IL, USA) containing 3 ml serum-free culture medium (Ling & Pipeleers 1994), reaggregated for 2 h in a rotatory shaking incubator (Braun, Melsugen, Germany), and then further cultured under static conditions in a CO2 incubator (37 °C, 95% air/5% CO2). Basal culture medium was Ham's F-10 containing 10 mmol/l glucose supplemented with 0.075 mg/ml penicillin, 0.1 mg/ml streptomycin, 2 mmol/l l-glutamine, 0.5% (weight/volume) BSA pretreated with charcoal (BSA, fraction V, Sigma Chemical Co.), and 1.7 mmol/l calcium (final concentration 2 mmol/l). After overnight culture, rat β cells were further cultured for 24–48 h with or without metformin (0.04–1 mmol/l, 1,1-dimethylbiguanide, Sigma–Aldrich). In a second series of experiments, the effect of metformin was tested in the absence or presence of glibenclamide (0.04–4 μmol/l, MP Biomedicals, Eschwege, Germany) or 20 mmol/l glucose instead of 10 mmol/l. In a third series, its effect was examined with or without glibenclamide, mTOR inhibitor rapamycin (25 nmol/l) (Cell Signaling Technology, Danvers, MA, USA), and/or PKA inhibitor Rp-8-Br-cAMPs (100 μmol/l) (Biolog, Bremen, Germany). At the end of culture, cells were analyzed for their protein synthesis and expression.

Purified β cells were also cultured as single cells in the above-mentioned test media for analysis of cell viability.

Protein synthesis and expression

The rate of protein synthesis was measured in β-cell aggregates following culture under the above conditions. Samples of 3×104 cells were incubated for an additional 1 h in Ham's F-10 medium at 0 or 10 mmol/l glucose without any of the tested compounds and with 1.85 MBq l-[3,5-3H]tyrosine (TRK200, Amersham) (Schuit et al. 1991). At the end of incubation, the cells were washed in Earle's-HEPES buffer containing 1 mmol/l unlabeled l-tyrosine and extracted in 2 M acetic acid containing 0.25% (weight/volume) BSA for analysis of total protein and insulin biosynthesis. Total protein synthesis was measured as precipitable radioactivity after mixing the acid extracts with trichloroacetic acid (Pipeleers et al. 1973, Schuit et al. 1988). Insulin biosynthesis was measured as precipitable radioactivity after incubation of the cell extracts with excess anti-insulin serum (Schuit et al. 1988). Fractions of cell extracts (50 μl) were dried and incubated at 37 °C for 1 h in insulin assay buffer (PBS with 0.5% BSA) containing guinea pig anti-insulin serum (kindly provided by Dr C Van Schravendijk). Thereafter, the samples were incubated for 15 min at room temperature with protein A-Sepharose CL 4B (5 mg/200 μl 0.9% NaCl) (Pharmacia), centrifuged, and extensively washed. The pellets were resuspended in 2 M acetic acid, and their soluble fraction was mixed with scintillation cocktail (Perkin Elmer, Shelton, CT, USA) and counted in a β counter (Tri-Carb model 1600 TR, Packard, Canberra Company, Meriden, CT, USA). Nonspecifically bound radioactivity was determined by incubating the samples with normal guinea pig serum instead of anti-insulin serum. Insulin synthesis was calculated as the difference between both counts (Schuit et al. 1988).

Protein expression in cultured β cells was analyzed immediately after drug treatment by western blot as described previously (Ling et al. 1998). Samples of 2–3×105 cells were run on 12% SDS-polyacrylamide before protein transfer on a nitrocellulose membrane and incubation with antibodies against 4E-BP1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), total eIF2α, phospho-eIF2α (Ser51), total rpS6, phospho-rpS6 (Ser235/236), total eEF2, phospho-eEF2 (Thr56), total AKT, phospho-AKT (Ser473), total AMPK and phospho-AMPK (Thr172) (all from Cell Signaling Technology), and actin (Santa Cruz Biotechnology, Inc.). HRP-linked anti-rabbit, anti-mouse, or anti-goat Igs (1:1000, Santa Cruz Biotechnology, Inc.) were used as secondary antibodies, and peroxidase activities were detected by enhanced chemiluminescence (Amersham). Intensities of bands were quantified by Scion Image for Windows (Scion Corporation, Frederick, MD, USA) expressed in arbitrary units of optical density and normalized for actin intensity in the same blot.

Viability assay

Viability assay consists of counting the percentage of dead cells and therefore requires the use of single cells. Purified single rat β cells (3×103 cells/well) were plated in polylysine-coated 96-well microtiter plates. After overnight culture in basal medium, cells were cultured for 72 h in test conditions. At the end of culture, cell viability was determined by fluorescence microscopy using propidium iodide (PI; Sigma) and Hoechst 3342 (HO342, Sigma; Hoorens et al. 1996). Viable cells were identified by their intact nuclei with blue fluorescence (HO342), and dead cells by nuclei with yellow–red fluorescence (HO342+PI). A cytotoxic test condition is detected by a lower cell survival than in control, and its toxicity index (TI) was calculated as TI=((% dead cells in test−% dead cells in control)/% living cells in control)×100%.

Statistical analysis

Results were expressed as mean±s.e.m. for the indicated number of independent experiments. Statistical significance of differences was calculated by Student's t-test or ANOVA with the Newman–Keuls test.

Results

Metformin suppresses protein translation and synthesis in rat β cells

After 24 h culture in the presence of 1 mmol/l metformin, protein synthesis by β cells was suppressed by 80% (Table 1). However, protein synthesis was almost completely restored after an additional 24 h culture without metformin (84–93% of protein synthesis activity in control cells). An inhibitory effect of metformin was also observed at lower concentrations (0.2–0.5 mmol/l), but this required a 48 h exposure (Fig. 1).

Table 1

Effects of glibenclamide and glucose on metformin-induced suppression of protein synthesis. Rat β cells were cultured for 24 h at 10 mmol/l glucose, with or without 1 mmol/l metformin (Metf), and with or without 4 μmol/l glibenclamide (Glib) or additional glucose (20 mmol/l glucose instead of 10 mmol/l=Gluc). By the end of culture, protein and insulin synthesis were measured during subsequent 1 h incubations at 0 or 10 mmol/l glucose in the absence of these compounds. Data represent means±s.e.m. of five to eight independent experiments

Addition to culture mediumTotal protein synthesis (dpm/cell per h)Insulin synthesis (dpm/cell per h)
StimuliMetf0 mmol/l glucose10 mmol/l glucose0 mmol/l glucose10 mmol/l glucose
2.7±0.38.5±0.71.9±0.46.8±0.8
+0.4±0.12.3±0.30.2±0.11.6±0.1
Glib3.4±0.48.4±0.81.9±0.36.0±0.9
Glib+1.7±0.35.1±0.5†,§0.9±0.23.3±0.4*
1.6±0.48.1±0.81.3±0.46.0±0.9
+0.3±0.05*1.4±0.30.1±0.02*1.4±0.1
Gluc3.1±0.7§8.2±1.42.4±0.66.8±1.3
Gluc+0.5±0.12.4±0.30.3±0.041.8±0.2

Statistical significance of differences with and without Metf at the same glucose concentration: *P<0.05, P<0.01, and P<0.001; with and without stimuli at the same glucose concentration: §P<0.05.

Figure 1
Figure 1

Effects of different concentrations of metformin on basal and glucose-induced protein synthesis. Rat β cells were cultured for 48 h at 10 mmol/l glucose, without or with 0.04, 0.2, and 0.5 mmol/l metformin. Total protein synthesis was measured during subsequent 1 h incubation at 0 mmol/l (a) or 10 mmol/l (b) glucose in the absence of metformin. Data are expressed as percentage of control (without metformin during 48 h culture) and represent means±s.e.m. of four independent experiments. The rates of protein synthesis in control cells were 1.6±0.2 dpm/cell per h in the absence of glucose and 7.0±0.6 dpm/cell per h at 10 mmol/l glucose. Statistical significance of differences with control: *P<0.01.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

A 24 h culture with 1 mmol/l metformin had decreased the phosphorylation state of translation initiation factors 4E-BP1 and rpS6 (Fig. 2a and b). The 4E-BP1 dephosphorylation was reflected in the shift of the highly phosphorylated γ-band to nonphosphorylated α-band and an intermediate β-band (Fig. 2a). Metformin did not significantly affect the phosphorylation of eIF2α (Fig. 2c), suggesting that the effect was initiation factor specific. Moreover, metformin inhibited translation elongation, as indicated by increased phosphorylation and thus inactivation of eEF2 (Fig. 2d).

Figure 2
Figure 2

Effects of metformin on expression and phosphorylation of translation factors. Rat β cells were cultured for 24 h at 10 mmol/l glucose, without (C) or with 1 mmol/l metformin (M). The expression of translation factors was analyzed by western blot using specific antibodies (see Materials and Methods). Band intensities of 4E-BP1γ, P-rpS6, P-eIF2α, P-eEF2, P-AMPK, and P-AKT were normalized for the corresponding actin intensity. Data represent means±s.e.m. of six independent experiments. Statistical significance of differences with control: *P<0.05, **P<0.01, and ***P<0.001.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

Metformin also increased the phosphorylation of AMPK (Fig. 2e), indicating its activation, while the activity of AKT (protein kinase B) was unaltered (Fig. 2f).

Glibenclamide, but not glucose, prevents metformin suppression of protein translation

Addition of glibenclamide (4 μmol/l) did not increase protein synthesis above the levels measured at 10 mmol/l glucose in the absence of metformin (Table 1) but counteracted the metformin suppression of protein synthesis (Table 1). This glibenclamide effect was also observed at lower concentrations (0.04 and 0.4 μmol/l, Fig. 3). Glibenclamide prevented the metformin-induced dephosphorylation of 4E-BP1 (γ-form) and rpS6 (Fig. 4a and b), whereas it did not alter the phosphorylation of eEF2 (Fig. 4c), suggesting that glibenclamide counteracted the metformin suppression of protein synthesis by increasing translation initiation. Glibenclamide did not prevent metformin-induced AMPK activation (Fig. 4d) but markedly increased the phosphorylation of AKT (Fig. 4e).

Figure 3
Figure 3

Effects of different concentrations of glibenclamide on metformin suppression of protein synthesis. Rat β cells were cultured for 24 h at 10 mmol/l glucose with 1 mmol/l metformin, in the absence or presence of different concentrations of glibenclamide (Glib, 0.04–4 μmol/l). Protein synthesis was measured during subsequent 1 h incubations at 10 mmol/l glucose in the absence of these compounds. Data represent means±s.e.m. of three independent experiments. Statistical significance of differences with and without Glib: *P<0.05.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

Figure 4
Figure 4

Effects of glibenclamide on metformin-induced suppression of translation factors. Rat β cells were cultured for 24 h at 10 mmol/l glucose, without or with 1 mmol/l metformin (Metf), and with or without 4 μmol/l glibenclamide (Glib). The expression of translation factors was analyzed as described in Fig. 2. Data represent means±s.e.m. of three or four independent experiments. Statistical significance of differences between conditions: *P<0.05 and **P<0.01.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

The protein synthesis stimulation by glibenclamide was inhibited by the mTOR inhibitor rapamycin and by the specific PKA inhibitor Rp-8-Br-cAMPs, and more strongly when both inhibitors were added together (Fig. 5a–c). This suggests that glibenclamide stimulated protein synthesis through both mTOR- and PKA-dependent pathways.

Figure 5
Figure 5

Effects of mTOR and PKA inhibitors on glibenclamide protection against metformin-induced suppression. Rat β cells were cultured for 24 h at 10 mmol/l glucose, without or with 1 mmol/l metformin (Metf), supplemented with 4 μmol/l glibenclamide (Glib) alone or in combination with the mTOR inhibitor rapamycin (25 nmol/l-Rapa), the PKA inhibitor Rp-8-Br-cAMPs (100 μmol/l-PKAi), or both. Total protein (a) and insulin synthesis (b) was measured during subsequent 1 h incubation at 10 mmol/l glucose. The expression of mTOR substrate p-rpS6 is shown in (c). Data represent means±s.e.m. of three to six independent experiments or are representative for two independent experiments (c: last condition with both inhibitors). Statistical significance of differences versus Glib/Metf condition in the absence of inhibitors: *P<0.05 and P<0.01.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

We then examined whether the metformin suppression of protein translation could be prevented by increasing the glucose concentration (20 mmol/l), a condition known to activate mTOR signaling and translation (Patel et al. 2001, McDaniel et al. 2002). Indeed, in the absence of metformin, 20 mmol/l glucose increased the basal protein and insulin synthesis of β cells (Table 1) and phosphorylation of 4E-BP1 and rpS6 (Fig. 6a and b). However, high glucose neither prevented the suppression of translational activity by metformin (Table 1 and Fig. 6a–c) nor the metformin-induced AMPK activation (Fig. 6d).

Figure 6
Figure 6

Effects of glucose on metformin-induced suppression of translation factors. Rat β cells were cultured for 24 h at 10 or 20 mmol/l glucose (Gluc) and without or with 1 mmol/l metformin (Metf). The expression of translation factors was analyzed as described in Fig. 2. Data represent means±s.e.m. of three or four independent experiments. Statistical significance of differences between conditions with and without metformin at the same glucose concentration: **P<0.01 and ***P<0.001; 10 vs 20 mmol/l glucose in the absence or presence of metformin: P<0.05 and ††P<0.01.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

Glibenclamide decreases β-cell toxicity of metformin

Culture of rat β cells for 24 h with 1 mmol/l metformin or for 48 h with ≤0.5 mmol/l metformin, which were the conditions used for analysis of protein translation (see above), did not induce β-cell death (results not shown), whereas a 72 h culture period at the highest dose (1 mmol/l) did cause death of 50% of the cells (Fig. 7). This confirms the previous finding that metformin-induced apoptosis requires extended periods of time (Kefas et al. 2004). The metformin-induced β-cell toxicity at 72 h was markedly reduced by addition of glibenclamide (Fig. 7). This protective effect of glibenclamide in β cells was less pronounced in the presence of the mTOR inhibitor rapamycin, or the PKA inhibitor Rp-8-Br-cAMPs, and disappeared completely in the presence of both inhibitors (Fig. 7). These data suggest that glibenclamide can protect β cells by stimulating mTOR and PKA.

Figure 7
Figure 7

Effects of mTOR and PKA inhibitors on glibenclamide protection against metformin-induced cell death. Rat β cells were cultured for 72 h in medium containing 10 mmol/l glucose with or without 1 mmol/l metformin (Metf) and supplemented with 4 μmol/l glibenclamide (Glib), alone or in combination with the mTOR inhibitor rapamycin (25 nmol/l-Rapa), the PKA inhibitor Rp-8-Br-cAMPs (100 μmol/l-PKAi), or both. The toxicity index was calculated as described in Materials and Methods. Percentage of dead cells in control condition (without metformin, glibenclamide, rapamycin, and PKAi) was 32±1. Data represent means±s.e.m. of six independent experiments. Statistical significance of differences with or without Glib, in the absence of inhibitors: *P<0.001; with or without inhibitor, in the presence of Metf/Glib: P<0.05 and P<0.01.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

Discussion

The hypoglycemic effect of glibenclamide, a second-generation sulfonylurea drug, is attributed to its acute stimulation of insulin release (Sturgess et al. 1985, Henquin 1987, Proks et al. 2002). We recently demonstrated that treatment with glibenclamide also stimulates insulin synthesis, at least in rats (Ling et al. 2006, Wang et al. 2008). On the other hand, metformin – an anti-diabetic drug that increases insulin sensitivity in peripheral tissues (Cusi and Defronzo, 1998) – was found to inhibit insulin synthesis in cultured rat β cells (Kefas et al. 2004). This study shows that both drugs exert opposing effects at the level of protein translation in β cells: glibenclamide counteracts the metformin-induced inhibition of protein synthesis.

The glibenclamide-induced elevation in insulin synthesis has been attributed to an activation of translation factors through calcium-dependent mTOR, PKA, and MEK signaling pathways (Wang et al. 2008). We now show that metformin suppresses activation of proteins involved in translation initiation and elongation, as evidenced by the lower amount of phosphorylated 4E-BP1 and rpS6 and the higher amount of phosphorylated eEF2. Total rpS6 was also lower, which is indicative of an associated decrease in ribosome biogenesis. Since the 4E-BP1 and rpS6 translation initiators are well-known downstream targets of mTOR (Gingras et al. 2001, Raught et al. 2001, Arsham & Neufeld 2006), our data suggest that metformin suppression of translation results from an inhibition of mTOR. Interestingly, glibenclamide, but not glucose, counteracted the metformin suppression of protein synthesis, and of 4E-BP1 and rpS6 phosphorylation, and these glibenclamide effects were partially blocked by the mTOR inhibitor rapamycin. This suggests that glibenclamide prevents β-cell dysfunction, at least in part, through activation of mTOR. Metformin inhibition of growth of breast cancer cells was also found to suppress translation initiation through inhibition of mTOR (Dowling et al. 2007). In these cells, the action mechanism of metformin involved activation of AMPK (Dowling et al. 2007), as appeared also to be the case in our β-cell preparations (Fig. 2). This mechanism is further supported by our previous finding that sustained activation of AMPK by the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide riboside also suppresses protein translation in β cells (Kefas et al. 2004, Cai et al. 2008).

The AMPK suppression of translation in β cells can be mediated via two pathways. In the first, active AMPK represses mTOR activity through phosphorylation of mTOR inhibitor TSC2 (Inoki et al. 2005), mTOR binding partner raptor (Gwinn et al. 2008), or mTOR itself on threonine 2446 (Cheng et al. 2004). In the second, active AMPK inhibits, in an mTOR-independent manner, the elongation factor eEF2 through activation of eEF2 kinase (Browne & Proud 2002, Browne et al. 2004). In β cells in vitro, both AMPK effector pathways seem operational. Indeed, the counteractive effect of glibenclamide on metformin inhibition of translation involved mTOR, supporting the first pathway, while increased phosphorylation of eEF2 by metformin suggested that the second pathway was involved as well. Our finding that glibenclamide partially restored protein synthesis in the presence of metformin can be explained by the observation that glibenclamide mainly interfered with the first AMPK pathway.

Glibenclamide not only antagonized metformin suppression of insulin and noninsulin protein synthesis but also protected β cells from the metformin cytotoxicity that is observed after longer culture periods. The counteractive effects of glibenclamide on metformin suppression of protein synthesis as well as cell survival observed in this study involved activation of both mTOR and PKA, as the glibenclamide effects were completely lost when both an mTOR inhibitor and a PKA inhibitor were present. These observations are consistent with previous work in which glibenclamide was found to activate translation through both mTOR-dependent and PKA-dependent pathways (Wang et al. 2008). It is unlikely that glibenclamide abrogates the negative effects of metformin on β-cell function through inhibition of apoptosis, because the metformin inhibition of protein translation was reversible and preceded its effect on death. On the contrary, the protection of glibenclamide against β-cell apoptosis may occur through its stimulatory effect on protein synthesis, since sustained inhibition of protein synthesis may result in a deficit of anti-apoptotic proteins (Hoorens et al. 1996, Kefas et al. 2003).

In contrast to glibenclamide, high glucose could not counteract metformin inhibition of translation, although it activated mTOR and translation in the absence of metformin. These observations indicate that glibenclamide and glucose regulate different pathways upstream of mTOR. Glibenclamide-induced signals can activate mTOR when AMPK is active, suggesting that glibenclamide acts downstream of AMPK. In contrast, the glucose-induced mTOR activation is blocked by AMPK activation, suggesting that glucose acts upstream of AMPK.

Based on the present in vitro observations, we propose a model for the interaction of metformin and glibenclamide at the level of translation (Fig. 8). Metformin induces an activation of AMPK that can result in an inhibition of mTOR. This effect interferes with a nutrient or growth factor stimulation of mTOR signaling that activates protein translation through phosphorylation of rpS6 and 4E-BP1. In addition to interfering with mTOR signaling, activated AMPK also inhibits protein elongation through phosphorylation of eEF2. The resulting suppression of protein synthesis is responsible for a decreased hormone production and an increased cellular susceptibility to apoptosis. Combination of metformin and glibenclamide does not result in these detrimental effects, because glibenclamide stimulates mTOR- and PKA-dependent signaling pathways that activate translational proteins.

Figure 8
Figure 8

Interaction of glibenclamide and metformin at the level of translation in β cells. Metformin activates AMPK, which will interfere with activation of mTOR and subsequently with the phosphorylation of rpS6 and 4E-BP1. Activated AMPK will directly phosphorylate eEF2 and thus inhibits its elongation activity. The metformin suppression of translation results in inhibition of protein synthesis. Glibenclamide partially counteracts these metformin effects through its activation of mTOR- and PKA-dependent pathways.

Citation: Journal of Endocrinology 208, 2; 10.1677/JOE-10-0372

It is unknown to which extent our in vitro observations on rat β cells are relevant for in vivo conditions. Although the metformin concentrations used in this study (0.04–1 mmol/l) were generally higher than steady-state plasma levels of metformin in patients (0.01–0.04 mmol/l; Cusi and Defronzo, 1998, Wiernsperger 1999), studies in rats have shown that tissue levels are several fold higher than in plasma (Wilcock & Bailey 1991). In an earlier report, we have shown that glibenclamide exerted the same effects in isolated rat β cells and in β cells in the rat pancreas (Ling et al. 2006). Adequate regulation of protein translation and synthesis in β cells is crucial for maintaining their survival and their insulin stores (Hoorens et al. 1996). Studies can thus be planned to investigate whether glibenclamide treatment of rats can help to maintain the functional β-cell mass through effects on β-cell protein synthesis and survival.

In conclusion, mTOR, PKA, and AMPK represent three protein kinases that are important targets of existing anti-diabetic pharmacologicals (Leibowitz et al. 2008, Rutter & Leclerc 2009) and that are expressed in β cells. Glibenclamide is now shown to act through stimulation of mTOR and PKA, while preventing the decline in protein synthesis and β-cell viability that are associated with AMPK activation.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by the Research Foundation Flanders (Fonds Voor Wetenschappelijk Onderzoek-Vlaanderen Grant FWO-G.0183.05, G.0400.07), the Inter-University Poles of Attraction Program (IUAP P5/17) from the Belgian Science Policy, the Onderzoeksraad Vrije Universiteit Brussel (OZR. 1153), the European Commission Sixth Framework Programme (FP6. 512145), and the E-Institutes of Shanghai Universities.

Author contribution statement

Q W, D P, and Z L designed the research; Q W and Y C performed the research; Q W and Z L analyzed the data; Q W, M V C, D P, and Z L wrote the manuscript.

Acknowledgements

We thank R De Proft and the staff of the Diabetes Research Center for their technical assistance.

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  • Cai Y, Wang Q, Ling Z, Pipeleers D, McDermott P, Pende M, Heimberg H & Van de Casteele M 2008 Akt activation protects pancreatic beta cells from AMPK-mediated death through stimulation of mTOR. Biochemical Pharmacology 75 19811993 doi:10.1016/j.bcp.2008.02.019.

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  • Cheng SW, Fryer LG, Carling D & Shepherd PR 2004 Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status. Journal of Biological Chemistry 279 1571915722 doi:10.1074/jbc.C300534200.

    • Search Google Scholar
    • Export Citation
  • Cusi K & Defronzo RA 1998 Metformin: a review of its metabolic effects. Diabetes Reviews 6 89131.

  • Dowling RJ, Zakikhani M, Fantus IG, Pollak M & Sonenberg N 2007 Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Research 67 1080410812 doi:10.1158/0008-5472.CAN-07-2310.

    • Search Google Scholar
    • Export Citation
  • El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M & Leverve X 2000 Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. Journal of Biological Chemistry 275 223228 doi:10.1074/jbc.275.1.223.

    • Search Google Scholar
    • Export Citation
  • Garcia-Barrado MJ, Ravier MA, Rolland JF, Gilon P, Nenquin M & Henquin JC 2001 Inhibition of protein synthesis sequentially impairs distinct steps of stimulus-secretion coupling in pancreatic beta cells. Endocrinology 142 299307 doi:10.1210/en.142.1.299.

    • Search Google Scholar
    • Export Citation
  • Gingras AC, Raught B & Sonenberg N 2001 Regulation of translation initiation by FRAP/mTOR. Genes and Development 15 807826 doi:10.1101/gad.887201.

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    • Export Citation
  • Gleason CE, Lu D, Witters LA, Newgard CB & Birnbaum MJ 2007 The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells. Journal of Biological Chemistry 282 1034110351 doi:10.1074/jbc.M610631200.

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    • Export Citation
  • Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE & Shaw RJ 2008 AMPK phosphorylation of raptor mediates a metabolic checkpoint. Molecular Cell 30 214226 doi:10.1016/j.molcel.2008.03.003.

    • Search Google Scholar
    • Export Citation
  • Henquin JC 1987 Regulation of insulin release by ionic and electrical events in B cells. Hormone Research 27 168178 doi:10.1159/000180806.

  • Hinke SA, Martens GA, Cai Y, Finsi J, Heimberg H, Pipeleers D & Van de Casteele M 2007 Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic beta-cells through restoration of mitochondrial electron transfer. British Journal of Pharmacology 150 10311043 doi:10.1038/sj.bjp.0707189.

    • Search Google Scholar
    • Export Citation
  • Hoorens A, Van de Casteele M, Kloppel G & Pipeleers D 1996 Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program. Journal of Clinical Investigation 98 15681574 doi:10.1172/JCI118950.

    • Search Google Scholar
    • Export Citation
  • Inoki K, Zhu T & Guan KL 2003 TSC2 mediates cellular energy response to control cell growth and survival. Cell 115 577590 doi:10.1016/S0092-8674(03)00929-2.

    • Search Google Scholar
    • Export Citation
  • Inoki K, Ouyang H, Li Y & Guan KL 2005 Signaling by target of rapamycin proteins in cell growth control. Microbiology and Molecular Biology Reviews 69 79100 doi:10.1128/MMBR.69.1.79-100.2005.

    • Search Google Scholar
    • Export Citation
  • Kefas BA, Cai Y, Ling Z, Heimberg H, Hue L, Pipeleers D & Van de Casteele M 2003 AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase. Journal of Molecular Endocrinology 30 151161 doi:10.1677/jme.0.0300151.

    • Search Google Scholar
    • Export Citation
  • Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, Pipeleers D & Van de Casteele M 2004 Metformin-induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochemical Pharmacology 68 409416 doi:10.1016/j.bcp.2004.04.003.

    • Search Google Scholar
    • Export Citation
  • Kwon G, Marshall CA, Pappan KL, Remedi MS & McDaniel ML 2004 Signaling elements involved in the metabolic regulation of mTOR by nutrients, incretins, and growth factors in islets. Diabetes 53 (Supplement 3) S225S232 doi:10.2337/diabetes.53.suppl_3.S225.

    • Search Google Scholar
    • Export Citation
  • Kwon G, Marshall CA, Liu H, Pappan KL, Remedi MS & McDaniel ML 2006 Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets. Journal of Biological Chemistry 281 32613267 doi:10.1074/jbc.M508821200.

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    • Export Citation
  • Leclerc I, Woltersdorf WW, da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R & Rutter GA 2004 Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. American Journal of Physiology. Endocrinology and Metabolism 286 E1023E1031 doi:10.1152/ajpendo.00532.2003.

    • Search Google Scholar
    • Export Citation
  • Leibowitz G, Cerasi E & Ketzinel-Gilad M 2008 The role of mTOR in the adaptation and failure of beta-cells in type 2 diabetes. Diabetes, Obesity and Metabolism 10 (Supplement 4) 157169 doi:10.1111/j.1463-1326.2008.00952.x.

    • Search Google Scholar
    • Export Citation
  • Ling Z & Pipeleers DG 1994 Preservation of glucose-responsive islet beta-cells during serum-free culture. Endocrinology 134 26142621 doi:10.1210/en.134.6.2614.

    • Search Google Scholar
    • Export Citation
  • Ling Z, Kiekens R, Mahler T, Schuit FC, Pipeleers-Marichal M, Sener A, Kloppel G, Malaisse WJ & Pipeleers DG 1996 Effects of chronically elevated glucose levels on the functional properties of rat pancreatic beta-cells. Diabetes 45 17741782 doi:10.2337/diabetes.45.12.1774.

    • Search Google Scholar
    • Export Citation
  • Ling Z, Chen MC, Smismans A, Pavlovic D, Schuit F, Eizirik DL & Pipeleers DG 1998 Intercellular differences in interleukin 1beta-induced suppression of insulin synthesis and stimulation of noninsulin protein synthesis by rat pancreatic beta-cells. Endocrinology 139 15401545 doi:10.1210/en.139.4.1540.

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  • Ling Z, Wang Q, Stange G, In't Veld P & Pipeleers D 2006 Glibenclamide treatment recruits beta-cell subpopulation into elevated and sustained basal insulin synthetic activity. Diabetes 55 7885 doi:10.2337/diabetes.55.01.06.db05-0820.

    • Search Google Scholar
    • Export Citation
  • McDaniel ML, Marshall CA, Pappan KL & Kwon G 2002 Metabolic and autocrine regulation of the mammalian target of rapamycin by pancreatic beta-cells. Diabetes 51 28772885 doi:10.2337/diabetes.51.10.2877.

    • Search Google Scholar
    • Export Citation
  • Owen MR, Doran E & Halestrap AP 2000 Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochemical Journal 348 607614 doi:10.1042/0264-6021:3480607.

    • Search Google Scholar
    • Export Citation
  • Patel J, Wang X & Proud CG 2001 Glucose exerts a permissive effect on the regulation of the initiation factor 4E binding protein 4E-BP1. Biochemical Journal 358 497503 doi:10.1042/0264-6021:3580497.

    • Search Google Scholar
    • Export Citation
  • Pipeleers DG, Marichal M & Malaisse WJ 1973 The stimulus-secretion coupling of glucose-induced insulin release. XIV. Glucose regulation of insular biosynthetic activity. Endocrinology 93 10011011 doi:10.1210/endo-93-5-1001.

    • Search Google Scholar
    • Export Citation
  • Pipeleers DG, in't Veld PA, Van de Winkel M, Maes E, Schuit FC & Gepts W 1985 A new in vitro model for the study of pancreatic A and B cells. Endocrinology 117 806816 doi:10.1210/endo-117-3-806.

    • Search Google Scholar
    • Export Citation
  • Proks P, Reimann F, Green N, Gribble F & Ashcroft F 2002 Sulfonylurea stimulation of insulin secretion. Diabetes 51 (Supplement 3) S368S376 doi:10.2337/diabetes.51.2007.S368.

    • Search Google Scholar
    • Export Citation
  • Raught B, Gingras AC & Sonenberg N 2001 The target of rapamycin (TOR) proteins. PNAS 98 70377044 doi:10.1073/pnas.121145898.

  • Rutter GA & Leclerc I 2009 The AMP-regulated kinase family: enigmatic targets for diabetes therapy. Molecular and Cellular Endocrinology 297 4149 doi:10.1016/j.mce.2008.05.020.

    • Search Google Scholar
    • Export Citation
  • Rutter GA, Da Silva Xavier G & Leclerc I 2003 Roles of 5′-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochemical Journal 375 116 doi:10.1042/BJ20030048.

    • Search Google Scholar
    • Export Citation
  • Schatz H, Katsilambros N, Nierle C & Pfeiffer EE 1972 The effect of biguanides on secretion and biosynthesis of insulin in isolated pancreatic islets of rats. Diabetologia 8 402407 doi:10.1007/BF01212167.

    • Search Google Scholar
    • Export Citation
  • Schuit FC, In't Veld PA & Pipeleers DG 1988 Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells. PNAS 85 38653869 doi:10.1073/pnas.85.11.3865.

    • Search Google Scholar
    • Export Citation
  • Schuit FC, Kiekens R & Pipeleers DG 1991 Measuring the balance between insulin synthesis and insulin release. Biochemical and Biophysical Research Communications 178 11821187 doi:10.1016/0006-291X(91)91017-7.

    • Search Google Scholar
    • Export Citation
  • Sturgess NC, Ashford ML, Cook DL & Hales CN 1985 The sulphonylurea receptor may be an ATP-sensitive potassium channel. Lancet 2 474475 doi:10.1016/S0140-6736(85)90403-9.

    • Search Google Scholar
    • Export Citation
  • Wang Q, Heimberg H, Pipeleers D & Ling Z 2008 Glibenclamide activates translation in rat pancreatic beta cells through calcium-dependent mTOR, PKA and MEK signalling pathways. Diabetologia 51 12021212 doi:10.1007/s00125-008-1026-8.

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  • Wiernsperger NF 1999 Membrane physiology as a basis for the cellular effects of metformin in insulin resistance and diabetes. Diabetes and Metabolism 25 110127.

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  • Wilcock C & Bailey CJ 1991 Reconsideration of inhibitory effect of metformin on intestinal glucose absorption. Journal of Pharmacy and Pharmacology 43 120121.

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  • Williamson DL, Bolster DR, Kimball SR & Jefferson LS 2006 Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR. American Journal of Physiology. Endocrinology and Metabolism 291 E80E89 doi:10.1152/ajpendo.00566.2005.

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  • Zou MH, Kirkpatrick SS, Davis BJ, Nelson JS, Wiles WGt, Schlattner U, Neumann D, Brownlee M, Freeman MB & Goldman MH 2004 Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species. Journal of Biological Chemistry 279 4394043951 doi:10.1074/jbc.M404421200.

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    Effects of different concentrations of metformin on basal and glucose-induced protein synthesis. Rat β cells were cultured for 48 h at 10 mmol/l glucose, without or with 0.04, 0.2, and 0.5 mmol/l metformin. Total protein synthesis was measured during subsequent 1 h incubation at 0 mmol/l (a) or 10 mmol/l (b) glucose in the absence of metformin. Data are expressed as percentage of control (without metformin during 48 h culture) and represent means±s.e.m. of four independent experiments. The rates of protein synthesis in control cells were 1.6±0.2 dpm/cell per h in the absence of glucose and 7.0±0.6 dpm/cell per h at 10 mmol/l glucose. Statistical significance of differences with control: *P<0.01.

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    Effects of metformin on expression and phosphorylation of translation factors. Rat β cells were cultured for 24 h at 10 mmol/l glucose, without (C) or with 1 mmol/l metformin (M). The expression of translation factors was analyzed by western blot using specific antibodies (see Materials and Methods). Band intensities of 4E-BP1γ, P-rpS6, P-eIF2α, P-eEF2, P-AMPK, and P-AKT were normalized for the corresponding actin intensity. Data represent means±s.e.m. of six independent experiments. Statistical significance of differences with control: *P<0.05, **P<0.01, and ***P<0.001.

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    Effects of different concentrations of glibenclamide on metformin suppression of protein synthesis. Rat β cells were cultured for 24 h at 10 mmol/l glucose with 1 mmol/l metformin, in the absence or presence of different concentrations of glibenclamide (Glib, 0.04–4 μmol/l). Protein synthesis was measured during subsequent 1 h incubations at 10 mmol/l glucose in the absence of these compounds. Data represent means±s.e.m. of three independent experiments. Statistical significance of differences with and without Glib: *P<0.05.

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    Effects of glibenclamide on metformin-induced suppression of translation factors. Rat β cells were cultured for 24 h at 10 mmol/l glucose, without or with 1 mmol/l metformin (Metf), and with or without 4 μmol/l glibenclamide (Glib). The expression of translation factors was analyzed as described in Fig. 2. Data represent means±s.e.m. of three or four independent experiments. Statistical significance of differences between conditions: *P<0.05 and **P<0.01.

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    Effects of mTOR and PKA inhibitors on glibenclamide protection against metformin-induced suppression. Rat β cells were cultured for 24 h at 10 mmol/l glucose, without or with 1 mmol/l metformin (Metf), supplemented with 4 μmol/l glibenclamide (Glib) alone or in combination with the mTOR inhibitor rapamycin (25 nmol/l-Rapa), the PKA inhibitor Rp-8-Br-cAMPs (100 μmol/l-PKAi), or both. Total protein (a) and insulin synthesis (b) was measured during subsequent 1 h incubation at 10 mmol/l glucose. The expression of mTOR substrate p-rpS6 is shown in (c). Data represent means±s.e.m. of three to six independent experiments or are representative for two independent experiments (c: last condition with both inhibitors). Statistical significance of differences versus Glib/Metf condition in the absence of inhibitors: *P<0.05 and P<0.01.

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    Effects of glucose on metformin-induced suppression of translation factors. Rat β cells were cultured for 24 h at 10 or 20 mmol/l glucose (Gluc) and without or with 1 mmol/l metformin (Metf). The expression of translation factors was analyzed as described in Fig. 2. Data represent means±s.e.m. of three or four independent experiments. Statistical significance of differences between conditions with and without metformin at the same glucose concentration: **P<0.01 and ***P<0.001; 10 vs 20 mmol/l glucose in the absence or presence of metformin: P<0.05 and ††P<0.01.

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    Effects of mTOR and PKA inhibitors on glibenclamide protection against metformin-induced cell death. Rat β cells were cultured for 72 h in medium containing 10 mmol/l glucose with or without 1 mmol/l metformin (Metf) and supplemented with 4 μmol/l glibenclamide (Glib), alone or in combination with the mTOR inhibitor rapamycin (25 nmol/l-Rapa), the PKA inhibitor Rp-8-Br-cAMPs (100 μmol/l-PKAi), or both. The toxicity index was calculated as described in Materials and Methods. Percentage of dead cells in control condition (without metformin, glibenclamide, rapamycin, and PKAi) was 32±1. Data represent means±s.e.m. of six independent experiments. Statistical significance of differences with or without Glib, in the absence of inhibitors: *P<0.001; with or without inhibitor, in the presence of Metf/Glib: P<0.05 and P<0.01.

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    Interaction of glibenclamide and metformin at the level of translation in β cells. Metformin activates AMPK, which will interfere with activation of mTOR and subsequently with the phosphorylation of rpS6 and 4E-BP1. Activated AMPK will directly phosphorylate eEF2 and thus inhibits its elongation activity. The metformin suppression of translation results in inhibition of protein synthesis. Glibenclamide partially counteracts these metformin effects through its activation of mTOR- and PKA-dependent pathways.

  • Arsham AM & Neufeld TP 2006 Thinking globally and acting locally with TOR. Current Opinion in Cell Biology 18 589597 doi:10.1016/j.ceb.2006.09.005.

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  • Bolster DR, Crozier SJ, Kimball SR & Jefferson LS 2002 AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. Journal of Biological Chemistry 277 2397723980 doi:10.1074/jbc.C200171200.

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  • Browne GJ & Proud CG 2002 Regulation of peptide-chain elongation in mammalian cells. European Journal of Biochemistry 269 53605368 doi:10.1046/j.1432-1033.2002.03290.x.

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  • Browne GJ, Finn SG & Proud CG 2004 Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398. Journal of Biological Chemistry 279 1222012231 doi:10.1074/jbc.M309773200.

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  • Cai Y, Wang Q, Ling Z, Pipeleers D, McDermott P, Pende M, Heimberg H & Van de Casteele M 2008 Akt activation protects pancreatic beta cells from AMPK-mediated death through stimulation of mTOR. Biochemical Pharmacology 75 19811993 doi:10.1016/j.bcp.2008.02.019.

    • Search Google Scholar
    • Export Citation
  • Cheng SW, Fryer LG, Carling D & Shepherd PR 2004 Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status. Journal of Biological Chemistry 279 1571915722 doi:10.1074/jbc.C300534200.

    • Search Google Scholar
    • Export Citation
  • Cusi K & Defronzo RA 1998 Metformin: a review of its metabolic effects. Diabetes Reviews 6 89131.

  • Dowling RJ, Zakikhani M, Fantus IG, Pollak M & Sonenberg N 2007 Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Research 67 1080410812 doi:10.1158/0008-5472.CAN-07-2310.

    • Search Google Scholar
    • Export Citation
  • El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M & Leverve X 2000 Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. Journal of Biological Chemistry 275 223228 doi:10.1074/jbc.275.1.223.

    • Search Google Scholar
    • Export Citation
  • Garcia-Barrado MJ, Ravier MA, Rolland JF, Gilon P, Nenquin M & Henquin JC 2001 Inhibition of protein synthesis sequentially impairs distinct steps of stimulus-secretion coupling in pancreatic beta cells. Endocrinology 142 299307 doi:10.1210/en.142.1.299.

    • Search Google Scholar
    • Export Citation
  • Gingras AC, Raught B & Sonenberg N 2001 Regulation of translation initiation by FRAP/mTOR. Genes and Development 15 807826 doi:10.1101/gad.887201.

    • Search Google Scholar
    • Export Citation
  • Gleason CE, Lu D, Witters LA, Newgard CB & Birnbaum MJ 2007 The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells. Journal of Biological Chemistry 282 1034110351 doi:10.1074/jbc.M610631200.

    • Search Google Scholar
    • Export Citation
  • Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE & Shaw RJ 2008 AMPK phosphorylation of raptor mediates a metabolic checkpoint. Molecular Cell 30 214226 doi:10.1016/j.molcel.2008.03.003.

    • Search Google Scholar
    • Export Citation
  • Henquin JC 1987 Regulation of insulin release by ionic and electrical events in B cells. Hormone Research 27 168178 doi:10.1159/000180806.

  • Hinke SA, Martens GA, Cai Y, Finsi J, Heimberg H, Pipeleers D & Van de Casteele M 2007 Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic beta-cells through restoration of mitochondrial electron transfer. British Journal of Pharmacology 150 10311043 doi:10.1038/sj.bjp.0707189.

    • Search Google Scholar
    • Export Citation
  • Hoorens A, Van de Casteele M, Kloppel G & Pipeleers D 1996 Glucose promotes survival of rat pancreatic beta cells by activating synthesis of proteins which suppress a constitutive apoptotic program. Journal of Clinical Investigation 98 15681574 doi:10.1172/JCI118950.

    • Search Google Scholar
    • Export Citation
  • Inoki K, Zhu T & Guan KL 2003 TSC2 mediates cellular energy response to control cell growth and survival. Cell 115 577590 doi:10.1016/S0092-8674(03)00929-2.

    • Search Google Scholar
    • Export Citation
  • Inoki K, Ouyang H, Li Y & Guan KL 2005 Signaling by target of rapamycin proteins in cell growth control. Microbiology and Molecular Biology Reviews 69 79100 doi:10.1128/MMBR.69.1.79-100.2005.

    • Search Google Scholar
    • Export Citation
  • Kefas BA, Cai Y, Ling Z, Heimberg H, Hue L, Pipeleers D & Van de Casteele M 2003 AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase. Journal of Molecular Endocrinology 30 151161 doi:10.1677/jme.0.0300151.

    • Search Google Scholar
    • Export Citation
  • Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, Pipeleers D & Van de Casteele M 2004 Metformin-induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochemical Pharmacology 68 409416 doi:10.1016/j.bcp.2004.04.003.

    • Search Google Scholar
    • Export Citation
  • Kwon G, Marshall CA, Pappan KL, Remedi MS & McDaniel ML 2004 Signaling elements involved in the metabolic regulation of mTOR by nutrients, incretins, and growth factors in islets. Diabetes 53 (Supplement 3) S225S232 doi:10.2337/diabetes.53.suppl_3.S225.

    • Search Google Scholar
    • Export Citation
  • Kwon G, Marshall CA, Liu H, Pappan KL, Remedi MS & McDaniel ML 2006 Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets. Journal of Biological Chemistry 281 32613267 doi:10.1074/jbc.M508821200.

    • Search Google Scholar
    • Export Citation
  • Leclerc I, Woltersdorf WW, da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R & Rutter GA 2004 Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. American Journal of Physiology. Endocrinology and Metabolism 286 E1023E1031 doi:10.1152/ajpendo.00532.2003.

    • Search Google Scholar
    • Export Citation
  • Leibowitz G, Cerasi E & Ketzinel-Gilad M 2008 The role of mTOR in the adaptation and failure of beta-cells in type 2 diabetes. Diabetes, Obesity and Metabolism 10 (Supplement 4) 157169 doi:10.1111/j.1463-1326.2008.00952.x.

    • Search Google Scholar
    • Export Citation
  • Ling Z & Pipeleers DG 1994 Preservation of glucose-responsive islet beta-cells during serum-free culture. Endocrinology 134 26142621 doi:10.1210/en.134.6.2614.

    • Search Google Scholar
    • Export Citation
  • Ling Z, Kiekens R, Mahler T, Schuit FC, Pipeleers-Marichal M, Sener A, Kloppel G, Malaisse WJ & Pipeleers DG 1996 Effects of chronically elevated glucose levels on the functional properties of rat pancreatic beta-cells. Diabetes 45 17741782 doi:10.2337/diabetes.45.12.1774.

    • Search Google Scholar
    • Export Citation
  • Ling Z, Chen MC, Smismans A, Pavlovic D, Schuit F, Eizirik DL & Pipeleers DG 1998 Intercellular differences in interleukin 1beta-induced suppression of insulin synthesis and stimulation of noninsulin protein synthesis by rat pancreatic beta-cells. Endocrinology 139 15401545 doi:10.1210/en.139.4.1540.

    • Search Google Scholar
    • Export Citation
  • Ling Z, Wang Q, Stange G, In't Veld P & Pipeleers D 2006 Glibenclamide treatment recruits beta-cell subpopulation into elevated and sustained basal insulin synthetic activity. Diabetes 55 7885 doi:10.2337/diabetes.55.01.06.db05-0820.

    • Search Google Scholar
    • Export Citation
  • McDaniel ML, Marshall CA, Pappan KL & Kwon G 2002 Metabolic and autocrine regulation of the mammalian target of rapamycin by pancreatic beta-cells. Diabetes 51 28772885 doi:10.2337/diabetes.51.10.2877.

    • Search Google Scholar
    • Export Citation
  • Owen MR, Doran E & Halestrap AP 2000 Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochemical Journal 348 607614 doi:10.1042/0264-6021:3480607.

    • Search Google Scholar
    • Export Citation
  • Patel J, Wang X & Proud CG 2001 Glucose exerts a permissive effect on the regulation of the initiation factor 4E binding protein 4E-BP1. Biochemical Journal 358 497503 doi:10.1042/0264-6021:3580497.

    • Search Google Scholar
    • Export Citation
  • Pipeleers DG, Marichal M & Malaisse WJ 1973 The stimulus-secretion coupling of glucose-induced insulin release. XIV. Glucose regulation of insular biosynthetic activity. Endocrinology 93 10011011 doi:10.1210/endo-93-5-1001.

    • Search Google Scholar
    • Export Citation
  • Pipeleers DG, in't Veld PA, Van de Winkel M, Maes E, Schuit FC & Gepts W 1985 A new in vitro model for the study of pancreatic A and B cells. Endocrinology 117 806816 doi:10.1210/endo-117-3-806.

    • Search Google Scholar
    • Export Citation
  • Proks P, Reimann F, Green N, Gribble F & Ashcroft F 2002 Sulfonylurea stimulation of insulin secretion. Diabetes 51 (Supplement 3) S368S376 doi:10.2337/diabetes.51.2007.S368.

    • Search Google Scholar
    • Export Citation
  • Raught B, Gingras AC & Sonenberg N 2001 The target of rapamycin (TOR) proteins. PNAS 98 70377044 doi:10.1073/pnas.121145898.

  • Rutter GA & Leclerc I 2009 The AMP-regulated kinase family: enigmatic targets for diabetes therapy. Molecular and Cellular Endocrinology 297 4149 doi:10.1016/j.mce.2008.05.020.

    • Search Google Scholar
    • Export Citation
  • Rutter GA, Da Silva Xavier G & Leclerc I 2003 Roles of 5′-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochemical Journal 375 116 doi:10.1042/BJ20030048.

    • Search Google Scholar
    • Export Citation
  • Schatz H, Katsilambros N, Nierle C & Pfeiffer EE 1972 The effect of biguanides on secretion and biosynthesis of insulin in isolated pancreatic islets of rats. Diabetologia 8 402407 doi:10.1007/BF01212167.

    • Search Google Scholar
    • Export Citation
  • Schuit FC, In't Veld PA & Pipeleers DG 1988 Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells. PNAS 85 38653869 doi:10.1073/pnas.85.11.3865.

    • Search Google Scholar
    • Export Citation
  • Schuit FC, Kiekens R & Pipeleers DG 1991 Measuring the balance between insulin synthesis and insulin release. Biochemical and Biophysical Research Communications 178 11821187 doi:10.1016/0006-291X(91)91017-7.

    • Search Google Scholar
    • Export Citation
  • Sturgess NC, Ashford ML, Cook DL & Hales CN 1985 The sulphonylurea receptor may be an ATP-sensitive potassium channel. Lancet 2 474475 doi:10.1016/S0140-6736(85)90403-9.

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