Hepatocyte growth factor protects rat RINm5F cell line against free fatty acid-induced apoptosis by counteracting oxidative stress

in Journal of Molecular Endocrinology
View More View Less
  • 1 National Centre for Food Quality and Risk Assessment, Section of Nutrition, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
  • 2 1Department of Drug Research and Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanità, Rome, Italy
  • 3 2Department of Clinical Sciences University ‘La Sapienza’, Rome, Italy
  • 4 3Division of Endocrinology and Metabolism Cedars-Sinai Medical Center, University of California, Los Angeles, California, USA

Type 2 diabetes is characterized by peripheral insulin resistance, pancreatic β-cells dysfunction, and decreased β-cell mass with increased rate of apoptosis. Chronic exposure to high levels of free fatty acids (FFAs) has detrimental effects on β-cell function and survival. FFAs have adverse effects on mitochondrial function, with a consequent increase in the production of reactive oxygen species. Hepatocyte growth factor (HGF) plays a critical role in promoting β-cell survival. In the present study, we investigated whether HGF was capable of protecting β-cells from death induced by prolonged exposure to FFAs. RINm5F cell line was cultured in the presence of FFAs (oleate:palmitate 2:1) for 72 h in order to induce apoptosis. Simultaneous administration of HGF and FFAs significantly suppressed the impaired insulin secretion and FFA-induced apoptosis. Specifically, HGF exerted its protective effect by counteracting: (i) the overproduction of either hydrogen peroxide and superoxide anion, (ii) the reduction of intracellular γ-glutamylcysteinylglycine level, and (iii) the depolarization of mitochondrial membrane, induced by prolonged FFAs exposure. These effects appear to be mediated by bcl-2 and phosphatidylinositol 3 kinase (PI3K)/Akt pathways. Indeed, HGF increased mRNA and protein expression of bcl-2 downregulated by FFAs-treatment; moreover, pre-treatment with the specific PI3-kinase inhibitor LY294002, significantly abolished the protective effect of HGF. In conclusion, in rat insulin-producing RINm5F cells, HGF exerts its prosurvival effect by counteracting the increased intracellular oxidative stress and, consequently, by inhibiting apoptosis induced by chronic exposure to FFAs.

Abstract

Type 2 diabetes is characterized by peripheral insulin resistance, pancreatic β-cells dysfunction, and decreased β-cell mass with increased rate of apoptosis. Chronic exposure to high levels of free fatty acids (FFAs) has detrimental effects on β-cell function and survival. FFAs have adverse effects on mitochondrial function, with a consequent increase in the production of reactive oxygen species. Hepatocyte growth factor (HGF) plays a critical role in promoting β-cell survival. In the present study, we investigated whether HGF was capable of protecting β-cells from death induced by prolonged exposure to FFAs. RINm5F cell line was cultured in the presence of FFAs (oleate:palmitate 2:1) for 72 h in order to induce apoptosis. Simultaneous administration of HGF and FFAs significantly suppressed the impaired insulin secretion and FFA-induced apoptosis. Specifically, HGF exerted its protective effect by counteracting: (i) the overproduction of either hydrogen peroxide and superoxide anion, (ii) the reduction of intracellular γ-glutamylcysteinylglycine level, and (iii) the depolarization of mitochondrial membrane, induced by prolonged FFAs exposure. These effects appear to be mediated by bcl-2 and phosphatidylinositol 3 kinase (PI3K)/Akt pathways. Indeed, HGF increased mRNA and protein expression of bcl-2 downregulated by FFAs-treatment; moreover, pre-treatment with the specific PI3-kinase inhibitor LY294002, significantly abolished the protective effect of HGF. In conclusion, in rat insulin-producing RINm5F cells, HGF exerts its prosurvival effect by counteracting the increased intracellular oxidative stress and, consequently, by inhibiting apoptosis induced by chronic exposure to FFAs.

Introduction

The correct balance between apoptosis and cell proliferation is a crucial factor in maintaining an appropriate mass of completely functional β-cells within the pancreatic islets. Type 2 diabetes occurs when the insulin secretory activity no longer meets the increase in demand due to presence of insulin resistance. Both a dysfunction in the glucose-dependent modality of secreting insulin and an insufficient β-cell mass have been considered as potential pathogenetic mechanisms leading to cell failure (Dickson & Rhodes 2004, Rhodes 2005). In the most recent years, concurrent with the obesity epidemic, the incidence of type 2 diabetes has shown a dramatic increase. In the setting of obesity, chronic hyperglycemia and hyperlipidemia contribute to β-cells dysfunction and a decrease of β-cell mass (Dickson & Rhodes 2004).

Free fatty acids (FFAs), at physiological concentrations, modulate the process of basal and glucose-induced insulin secretion in pancreatic β-cells, and chronic elevation of plasma fatty acid levels has been shown to be associated with insulin resistance and β-cell dysfunction (Lee et al. 1994, Evans et al. 2003).

It has been demonstrated that prolonged exposure of cultured human islets to FFAs has detrimental effects for β-cell function, including impairment of glucose-induced insulin release, suppression of proinsulin biosynthesis, and β-cell loss by apoptosis (Zhou & Grill 1995, Carpentier et al. 1999, Lupi et al. 2002). These findings are consistent with studies on the Zucker diabetic fatty rat showing that lipid accumulation in β-cells causes a substantial reduction of β-cell mass through apoptosis, and is associated with the development of diabetes (Shimabukuro et al. 1998a). In vitro studies indicate that long-term exposure of β-cell to FFAs induces adverse effects on mitochondrial function, with elevated production of reactive oxygen species (ROS) and, consequently, increased oxidative stress leading to β-cell death (Carlsson et al. 1999, Lupi et al. 2002, Evans et al. 2003, Wang et al. 2004). Oxidative stress refers to the imbalance between the production of ROS and ability of the cells to defend against them. The concentration of ROS and the cellular microenvironment appear to be important in determining the mode of cell death. Recent reports suggest that bcl-2 might prevent apoptosis by regulating the cellular antioxidant defense mechanisms, thus acting as free radical scavenger (Jang & Surth 2003). Indeed, bcl-2-overexpressing cells exhibit elevated expression of antioxidant enzymes and γ-glutamylcysteinylglycine (GSH); conversely, downregulation of bcl-2 expression is associated with GSH depletion (Voehringer 1999). Therapeutic approaches designed to arrest β-cell apoptosis by altering the balance between offending and defending mechanism(s), may be of importance in the management of type 2 diabetes.

Hepatocyte growth factor (HGF) is a mesenchyme-derived multifunctional protein that plays a critical role in cell survival, proliferation, migration, and differentiation. Earlier studies demonstrated that HGF and its specific receptor, a transmembrane tyrosine kinase encoded by c-met protooncogene, are highly expressed during pancreas development. HGF has also been shown to have insulinotropic properties and to promote β-cell proliferation and differentiation (Otonkoski et al. 1994). HGF is also an important modulator of cell death; it protects cardiomyocytes, endothelial cells, and neurons from apoptosis (Zhang et al. 2000, Kitta et al. 2001, Nakagami et al. 2002). A potential role for HGF in the pancreatic β-cell has been recently explored; the authors have demonstrated that the administration of HGF in transgenic mouse induced an increase of pancreatic β-cell and islets number, islet size, and overall islet mass (Garcia-Ocana et al. 2003). Recently, the same authors have demonstrated that by using adenovirus, the over-expression of HGF in murine islets markedly improved the function and survival of islets transplanted into diabetic mice; the protective effects of HGF have been observed even in Lewis rat in which islets were delivered intraportally and accompanied by immunosuppressant therapy (Lopez-Talavera et al. 2004).

The importance of identifying antiapoptotic agents to protect β-cell from apoptosis and the ability of HGF to enhance β-cell survival in different experimental settings, led us to investigate, in insulin-producing RIN5mF cells, the effect of HGF on cell death induced by prolonged exposure to FFAs.

Materials and methods

Cell line culture: FFA exposure and HGF treatment

The insulin-producing cells, RINm5F, were cultured in 75 ml flasks in the presence of RPMI 1640 medium with 10% fetal calf serum (FCS; Gibco, BRL) and 100 μg/ml penicillin, and 50 μg/ml streptomycin, at 37 °C under a humidified condition of 95% air and 5% CO2. On reaching 80% confluence, the cultures were washed twice with RPMI 1640 (without FBS) and kept in serum-free medium for 6 h before the induction of cell apoptosis. This was obtained by culturing cells in the presence of high levels of FFAs for 72 h. FFAs (2 mmol/l long-chain fatty acid oleate:palmitate 2:1) were prepared as previously described (Lee et al. 1994, Piro et l. 2002). Briefly, we first prepared a 2% fatty acid-free BSA solution in RPMI 1640, and then mixed oleate and palmitate (2:1) to obtain a 22 mmol/l stock solution. At each experiment, aliquots of stock solution were dissolved in the culture medium RPMI (Roswell Park Memorial Institute) 1640. For control experiments, BSA in the absence of fatty acids was prepared as described above. The unbound FFA concentrations were 180.6 and 20.3 nmol/l respectively for oleic and palmitic acids. Unbound concentration was calculated using the FFA-albumin association constants for the first six binding sites of albumin (Richieri et al. 1993, Cnop et al. 2001). At the end of the incubation, after a washout of the cell layer with PBS, untreated and FFA-treated adherent cells were scraped off the culture dishes, collected together with detached cells floating in the medium, and spun at 12000g for 30 s. The protective effect of HGF was studied by incubating FFA-treated cells with 50 ng/ml HGF (R&D System, Minnoapolis MN, USA) for 72 h; fresh aliquots of HGF were added every 8 h. Control cultures were grown under the same conditions as treated cells but in the absence of the drugs (FFAs and/or HGF). LY294002 (Calbiochem Co, Darmstadt, Germany), a selective inhibitor of protein kinase activity PI3-K, was used at 50 μM concentration, previously shown to be effective in blocking PI3-K activity (Hui et al. 2003). Depending on the specific assay for which the cell culture was prepared, the pellets were either stored at −70 °C or immediately used for the experiment.

Morphological determination of apoptosis

For morphological studies, the cells were grown in chamber slides and treated with HGF and FFA as described above. They were then washed in PBS (pH 7.4) and fixed for 20 min in 2% paraformaldehyde in PBS (pH 7.4) at room temperature (RT). After a wash in PBS, the cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate, rinsed twice in PBS, and stained with the karyophilic dye Hoechst 33342 (10 μg/ml) for 5 min at RT. After a final wash in PBS, the cells were mounted in Fluoromount G and visualized under u.v. light with an Axiophoto microscope (Carl Zeiss, New York, NY, USA).

Analysis of insulin secretion

After incubation with FFAs, in the presence or absence of HGF, the insulin secretion was assessed as previously described (Anastasi et al. 2005). Cells were kept in 5.5 mM glucose until the day of the experiment, then challenged with either 20 mM glucose or re-exposed to 5.5 mM glucose for 60 min. The level of insulin in the culture medium was measured by RIA (Linco Research, Inc. St. Charles, MO, USA) and normalized for the total cellular protein content detected in the pellet of each individual culture according to Bradford method (Bio-Rad Laboratories, Inc.).

Annexin V assay

Quantitative evaluation of apoptosis was performed by flow cytometry after double staining with annexin-V-fluorescein isothyocyanate (FITC) apoptosis detection kit (Eppendorf s.r.l., Milan, Italy), which allows discrimination among early apoptotic (single annexin V positive), late apoptotic (double annexin V/propidium iodide (PI) positive), and necrotic cells (single PI positive) which enables to differentiate cells that have lost membrane integrity (necrotic cells) from living cells by means of red staining of their nuclei with PI.

Analysis of the redox balance

For ROS production evaluation, control and treated cells (5×105 cells) were incubated in 495 μl Hanks’ balanced salt solution (pH 7.4) containing 10 μM dihydrorhodamine 123 (Molecular Probes, Eugene, OR, USA) or 1 μM dihydroethidium (Molecular Probes) in polypropylene test tubes for 15 min at 37 °C. After this time, cells were washed in ice-cold PBS and immediately analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) equipped with a 488 argon laser. Monochlorobi-mane (Molecular Probes) staining was performed for GSH as previously described (Sahaf et al. 2003). Cells exposed to the GSH depleting drug l-buthionine-[S,R]-sulfoximine 7.5 mM (Sigma) for 16 h were considered as negative controls (data not shown). After washings, samples were analyzed by a LRS II cytometer (Becton and Dickinson, San Jose’, CA, USA) equipped with a UVB laser. The median values of fluorescence intensity histograms were used to provide semiquantitative assessment of GSH content and ROS production.

Mitochondrial membrane potential (MMP)

The MMP (Δψ) of control and treated cells was studied by flow cytometry using 10 μM 5-5′,6-6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Molecular Probes). JC-1 is a metachromatic probe able to selectively enter the mitochondria. It exists in a monomeric form (in the green channel, FL1) but, depending on the membrane potential, it can form J-aggregates associated with a large shift in the emission range (in the orange channel, FL2). Analysis by JC-1 can be both qualitative (considering shift from green to orange) and quantitative (considering the ‘pure’ fluorescence intensity). As a methodological control (not shown), cells were also treated with increasing concentrations (from 0.1 to 10 μg/ml) of K+ iono-phore valinomycin (Sigma) which dissipates MMP but not pH gradient (Cossarizza et al. 1993). One additional probe, tetramethylrhodamine ester (TMRM; 1 μM, Molecular Probes, red fluorescence), was also used to confirm the data obtained by JC-1.

RT-PCR analysis of Bax and bcl-2 mRNA expression

The expression of bcl-2 and Bax genes, in RINm5F cell lines, was evaluated by RT-PCR in all of the experimental conditions. Total RNA was extracted from each sample with Trizol (Invitrogen-Life Technologies) according to manufacturer’s instructions. First-strand cDNA synthesis was performed in a total volume of 20 μl using 2 μg of each RNA sample primed with random hexamers with 200 U Superscript II (Invitrogen-Life Technologies); cDNA aliquots corresponding to 200 ng RNA were subsequently amplified in 25 μl reaction volume containing 20 pmol of sense and antisense-specific primers, and 2.5 U Taq DNA polymerase (Invitrogen-Life Technologies). The following primers were used for bcl-2 (Accession no. U34964), sense: 5′ GGA GGA TTG TGG CCT TCT TTG AG 3′(270 bp product); antisense: 5′ TAT GCA CCC AGA GTG ATG CAG GC 3′; and Bax (Accession no. U32098), sense: 5′ TGA ACT GGA CAA CAA CAT GGA GC 3′; antisense: 5′ GGT CTT GGA TCC AGA CAA ACA GC 3′ (259 bp product). Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as RNA control was analyzed employing the following primers, sense: 5′ GGA GCC AAA AGG GTC ATC ATC 3′; and antisense: 5′ AGA GGC AGG GAT GAT GTT CTG 3′ (342 bp product). Reaction conditions were standardized in order to observe a linear amplification of PCR products. All PCR products were electrophoresed on 1.5% agarose gel and the bands visualized by ethidium bromide staining. Semiquantitative analysis was performed by densitometric gel scanning using the ‘Gel Doc 2000’ video image system (Bio-Rad Laboratories). Results have been expressed as the ratio between the molecule of interest and GAPDH in each sample analyzed.

Western blot analysis

For immunblotting determination of CuZnSOD, MnSOD (SOD; superoxide dismutase), catalase (CAT), bcl-2, and bax molecules, cells were washed twice with ice-cold PBS and whole cell extract was prepared by treating cells with 50 μl Triton X 1% (Sigma), 5 μl of a mixture of protease inhibitors (Sigma), and incubated on ice for 20 min. Protein concentrations were determined using the Biorad Protein Assay kit (Bio-Rad). The cell lysates (15–50 μg/sample) were separated by 12–15% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes, blocked with PBS containing 5% dry milk and 0.1% Tween 20, were treated with antibodies that recognize Cu/ZnSOD (Upstate Biotechnology, Lake Placid, NY, USA), MnSOD (Stressgen, Ann Arbor, MI, USA), CAT (Sigma), bax, bcl-2 (Santa Cruz Bio-technology Inc, Santa Cruz, CA, USA), and β-tubulin (Santa Cruz Biotechnology Inc). The blots were treated with appropriate secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology Inc) followed by ECL detection (Amersham Biosciences). Densitometric analysis was performed with a molecular imager FX (Bio-Rad).

Data and statistical analysis

Data

For the flow cytometry studies, all samples were analyzed with a FACScan flow cytometer (Becton Dickinson) equipped with a 488 argon laser. At least 20 000 events were acquired. Data were recorded and statistically analyzed by a Macintosh computer using CellQuest Software or PC computer using DIVA Software (for GSH analysis; both by Becton and Dickinson). Data are reported as mean values of at least four separate experiments±s.d..

Statistical analysis

Statistical analysis of apoptosis data was performed by non-parametric ANOVA test. Statistical significance of flow cytometry studies was calculated by using the parametric Kolmogorov–Smirnov (K/S) test. As a general rule, only P values of less than 0.01 were considered as significant. As for RT-PCR and western blotting analysis, data are expressed as means±s.d. Statistical analysis was performed by ANOVA test. Differences were considered statistically significant when P<0.05.

Results

HGF protects insulin-secreting RINm5F cells from FFA-induced apoptosis

To examine whether HGF could protect against FFA-induced cell death, we cultured RINm5F cells, in vitro, for 72 h with or without 2 mmol/l long-chain fatty acid mixture (2:1 oleate to palmitate) in the presence or absence of 50 ng/ml of HGF. Our results showed that prolonged exposure to FFAs induced apoptotic cell death in rat RINm5F cells, and more importantly that the administration of HGF prevented the effect of FFAs on cell viability. Cytotoxicity was assayed by the morphological features of cell nuclei, demonstrating that, whereas the exposure of cells to FFAs promoted changes in the nuclear morphology characteristic, condensed and fragmented of apoptotic cells, the treatment with HGF in association with FFAs was capable of preventing nuclear fragmentation and inhibiting cell apoptosis (Fig. 1A).

To assess functional modifications, glucose-dependent secretion of insulin was evaluated in RINm5F cells exposed to FFA mixture in the presence or absence of HGF. Insulin release from cells exposed only to FFAs mixture was markedly impaired in absence of HGF (FFAs versus control; *P<0.01). The presence of HGF did not modify basal insulin release (at 5.5 mmol/l glucose) versus HGF untreated cells. When challenged with 20.0 mmol/l glucose, HGF+FFAs-treated cells showed a significant increase of insulin release when compared with FFA-treated cells. (*P<0.01; Table 1). To quantify the degree of cell apoptosis in the various culture conditions, we performed a biparametric cytofluorimetric analysis by using double staining with annexin V-FITC/PI. The annexin V/propidium iodide double staining allows discrimination among three different cell populations: (i) cells in the early phases of apoptosis (single annexin V positive), (ii) cells in the late apoptotic phases (double annexin V/PI positive), and (iii) necrotic cells (single PI positive) We found that exposure to FFAs was able to induce apoptosis in a significant percentage of cells (about 40%, *P<0.01 versus control; Fig. 1B). To note, in our experimental condition, only a small percentage of cells (<10%) underwent secondary necrosis after FFAs administration Importantly, HGF (50 ng/ml), which did not influence per se apoptosis in β-cells (*P>0.05 versus control), counteracted the effects of FFAs. Indeed, simultaneous administration of HGF and FFAs significantly reduced the occurrence of apoptosis (about 69%; *P<0.01 versus FFAs; Fig. 1B).

HGF inhibits mitochondrial damage and ROS production

To evaluate the protective activity of HGF against FFA-induced apoptosis, we analyzed ROS production in the experimental conditions, above described, by a semi-quantitative flow cytometry method. Our results confirmed that long-term FFAs exposure resulted in a significant overproduction of either hydrogen peroxide, H2O2, (Fig. 2A) or superoxide anion, O2, (Fig. 2B) with respect to cells cultured in the absence of FFAs. Importantly, HGF treatment was capable of preventing FFA-induced oxidative stress by significantly decreasing ROS production (*P<0.01 versus FFA).

We next investigated the cellular antioxidant defenses by analyzing GSH content by semiquantitative flow cytometry method and protein expression of MnSOD, CuZnSOD, and CAT enzymes by western blotting. We observed a significant decrease of intracellular GSH (Fig. 2C) content in FFA-treated cells whereas levels of MnSOD, CuZnSOD, and CAT proteins were unchanged (data not shown). When HGF was added to FFA-treated cells, the decrease of reduced GSH was strongly attenuated (*P<0.01 versus FFA; Fig. 2C) and no differences were observed in the expression of antioxidant enzymes tested (data not shown)

To further investigate the FFA-induced cell death, we analyzed the MMP in living cells by using JC-1 probe. This experiment demonstrated an involvement of mitochondrial transition pore (Fig. 2D), indicating that the treatment with FFAs induced the depolarization of the mitochondrial membrane in a significant percentage of insulin-producing cells (≥45%) when compared with control cells (about 5%; *P<0.01). On the other hand, the administration of HGF was able to significantly (*P<0.01) prevent this depolarization of mitochondrial membrane.

FFA and HGF modulate bcl-2 expression

Our study found that FFAs induced a significant reduction of bcl-2 expression, both at the mRNA (Fig. 3A and B; P<0.01) and protein level (Fig. 3C and D; P<0.01) in RINm5Fcell line. On the other hand, no significant change in bax expression was detected both at the mRNA (Fig. 3A and B) and at the protein (Fig. 3E and F) level. However, HGF treatment alone did not affect the expression of bcl-2 molecule in untreated control cells, but it was able to prevent the decrease of bcl-2 mRNA (*P<0.05) and FFA-induced protein (*P<0.01) (Fig. 3A–D).

HGF acts via PI3-K

The involvement of PI3-kinase in the survival effect of HGF was investigated by pre-treatment of RIN cells with LY294002 (50 μM), a synthetic specific inhibitor of PI-3-kinase, 30 min before FFAs+HGF stimulation. Data obtained clearly show that LY294002 abolish the protective effect of HGF (Fig. 4). Inhibition of PI-3-kinase led to an increase in the number of apoptotic cells, similarly to that observed in FFA-treated cells (Fig. 4A). Accordingly, pre-treatment with LY294002 also abrogated the protective effect exerted by HGF on the mitochondrial-membrane polarization state (Fig. 4B). Furthermore, the production of H2O2 (Fig. 4C) and O 2 (Fig. 4D) induced by FFAs was not prevented by HGF when LY294002 was present in the culture medium. Moreover, the pre-treatment with LY294002 completely prevented the increase of reduced GSH induced by HGF (Fig. 4E). Finally, the decrease of bcl-2 protein prevented by HGF in the presence of FFA, was completely counteracted by LY294002 (Fig. 4F and G).

Discussion

The present study was undertaken to investigate whether HGF exerted protective activity against cell death induced by a prolonged exposure to FFAs in insulin-producing RINm5F cell line. In addition, we provided an early elucidation of the mechanism by which HGF improves function and viability of insulin secreting cells. We demonstrated that HGF was able to inhibit, in RINm5F cells, the apoptosis and to prevent metabolic dysfunction induced by the exposure to FFAs. Furthermore, HGF inhibited oxidative stress induced by FFAs, thus exhibiting a ROS scavenging property. Finally, we showed that HGF might regulate the expression of antiapoptotic protein bcl-2 likely via PI3-K/Akt-dependent pathway. Previous reports have demonstrated that HGF is a survival factor with antioxidant properties for various cell types. For example, HGF protects adult cardiac myocytes against apoptosis induced by hydrogen peroxide, serum deprivation, and chemotherapeutic agents inducing oxidative stress (Kitta et al. 2001). In addition, HGF was found to protect cerebellar granule neurons against apoptotic death induced by low concentration of potassium (Zhang et al. 2000), and to rescue SAEC cell line from apoptosis induced by the treatment with tumour necrosis factor-Δor hydrogen peroxide (Okada et al. 2004). More importantly, it has been shown that HGF is a useful tool to preserve and enhance islet function and survival (Garcia-Ocana et al. 2003, Lopez-Talavera et al. 2004). Our results showed that HGF is able to improve function and viability of RIN5mF cells in accordance with a recent report indicating that HGF/ c-met has an essential role in the maintenance of normal glucose-dependent insulin secretion, and that genetic alterations in the HGF/c-met receptor/system, combined with environmental factors, may be implicated in the predisposition to develop type 2 diabetes (Roccisana et al. 2005). Oxidative stress is considered an important mediator of cellular damage following a prolonged exposure of pancreatic β-cell to elevated levels of FFAs (Lupi et al. 2002, Evans et al. 2003). Our observations, consistent with previous reports (Tiedge et al. 1997, Carlsson et al. 1999), demonstrated that long-term exposure to FFAs resulted in a significant overproduction of either hydrogen peroxide (H2O2) or superoxide anion O2 in RINm5F cells when compared with untreated cells. Moreover, these phenomena were accompanied by depolarization of mitochondrial membrane in a significant percentage of FFAs-treated cells.

It was noteworthy that by reducing H2O2 and O2production, HGF prevented FFA-induced oxidative stress, thus protecting mitochondrial function and rescuing ability to glucose response. It has been established that mitochondrial function is essential for glucose-stimulated secretion, and glucose-sensing defect observed in type 2 diabetes is caused by β-cell mitochondrial dysfunction (Lowell & Shulman 2005). Long-term exposure to FFAs determines an increase in energy flux through the electron transport chain, which leads to an increased ROS production and a proton leak that impairs glucose-stimulated insulin secretion, resulting in β-cell dysfunction. (Barbu et al. 2002, Lowell & Shulman 2005). In addition, the detrimental effect of increased oxidative stress might be due, in part, to the low levels of antioxidant enzyme defense system (SOD, CAT, glutathion peroxidase) present in β-cells when compared with other tissues (Lenzen et al. 1996, Tiedge et al. 1997). We showed that HGF was able to strongly attenuate depletion of reduced GSH levels, induced by FFA treatment in RIN5mF cells, whereas it did not modify protein expression of MnSOD, CuZnSOD, and CAT antioxidant enzymes.

Our data suggest that HGF exerts its protective effect by strengthening the intrinsic antioxidant defenses of RINmF5 cells through modulation of GSH intracellular and thus protecting them by acting on the redox status. This hypothesis is supported by recent findings showing that in type 2 diabetes, the human β-cell has functional defects associated with multiple alterations and increased oxidative stress and that 24-h exposure to glutathione improves glucose-stimulated insulin release, suggesting that it should be possible to reverse the functional impairment of type 2 diabetic islets by reducing oxidative stress in islet cells (Del Guerra et al. 2005).

Cells susceptibility to apoptosis is regulated by a balanced expression between apoptosis-suppressing and -inducing proteins. In humans and rodents islets, FFAs-induced apoptosis is associated with the downregulation of bcl-2/bax ratio (Shimabukuro et al. 1998b, Lupi et al. 2002). It has been reported that overexpression of bcl-2 proteins enhances islet viability (Rabinovitch et al. 1999). Our results demonstrated that HGF was capable of maintaining, in RINm5F cells, the bcl-2/bax ratio observed in control untreated cells, by counteracting the downregulation of the expression of bcl-2 induced by FFA-incubation. Several lines of evidence suggest that bcl-2 family members contribute to redox regulation in mammalian cells; bcl-2 deficiency mice show a chronic oxidative stress phenotype (Veis et al. 1993) and bcl-2 raises intracellular level of glutathione and other thiols in fibroblasts and other cells types (Papadopulos et al. 1998, Rudin et al. 2003). Moreover, bcl-2 appears to be a potent antioxidant and survival factor able to decrease ROS levels through regulation of cellular antioxidant enzymes (i.e., SOD and CAT; Deng et al. 2003). The role of bcl-2 in regulation of ROS levels is consistent with its mitochondrial localization, since the majority of ROS is produced in this organelle. However, the mechanism by which bcl-2 prevents ROS-induced apoptosis is unknown. HGF has multifunctional activities and exerts its prosurvival action by different mechanisms depending on the apoptotic stimuli. In renal epithelial cells, apoptosis induced by serum withdrawal is prevented by HGF overexpression through rapid phosphorylation of Akt, followed by phosphorylation/inactivation of Bad and simultaneously induced a dramatic expression of bcl-xL (Liu 1999). On the other hand, in human endothelial cells, hypoxia-induced apoptosis was attenuated by HGF and it was accompanied by a significant increase of bcl-2 molecule (Yamamoto et al. 2001). The survival effects of HGF inβ-cells appearto involvethe PI3-K and Aktpathway (Holst et al. 1998, Garcia-Ocana et al. 2003). Moreover, adenoviral-mediated expression of constitutively active Akt completely protected mouse INS-1 β-cell line (insulinonce β-cell line) from FFA-induced apoptosis (Wrede et al. 2002). Our data demonstrated that the rescue effect of HGF on the FFAs-induced apoptosis involves the PI3-kinase/Akt pathway. Indeed, pre-treatment with LY294002, a synthetic specific inhibitor of PI-3-kinase, abolished the protective effect of HGF, resulting in the overproduction of H2O2 and O2, in the decrease of reduced GSH as well as in the decrease of bcl-2 molecule. The above results provide evidences that PI3-K is an essential mediator through which HGF inhibits FFA-induced oxidative stress and apoptosis in RIN5mF cells possibly bcl-2 regulated. Because of its strong prosurvival and antioxidant action, HGF treatment appears to be a useful approach to preserve β-cell function and survival in the lipotoxicity setting.

Table 1

Insulin secretion from RINm5F cells in response to glucose (5.5 and 20 mmol/l) concentration after a 72-h incubation with control medium (control), medium containing free fatty acids (FFAs), plus HGF (FFAs+HGF), and medium containing HGF alone (HGF). Data are means±s.d. of three separate experiments

5.5 mmol/l20 mmol/l
*P<0.01 control and FFAs+HGF versus FFAs.
Culture conditions
Control35.2±2.068±1.6*
FFAs34.2±2.530±1.8
FFAs+HGF36±2.262±1.8*
HGF32±1.670±1.5
Figure 1
Figure 1

HGF protects RINm5F cells from FFA-induced apoptosis. (A) Cells were exposed to FFAs (oleate:palmitate 2:1, 2 mmol/l) for 72 h in the presence or absence of HGF (50 ng/ml). After fixing, cells were stained for the nuclear marker Hoechst 33342. Apoptotic cells were identified by condensed and fragmented nuclear appearance. Similar results were obtained in three independent experiments. Pictures were taken at 20× magnification. In the inserts are represented nuclei at greater magnification. (B) FACS analysis after double staining with annexin V/propidium iodide. RINm5F cells were cultured in regular medium (control; CTRL), exposed to free fatty acids mixture (oleate/palmitate 2:1) (FFA), cultured in medium containing 50 ng/ml HGF alone (HGF), treated with HGF in addition with FFA (HGF+FFA) for 72 h. Dot plots from a representative FACS experiment are shown. Numbers represent the percentage of annexin V single positive (early apoptosis, bottom quadrant) or annexin V/PI double positive (late apoptosis, lower quadrant) cells. In the histogram, the results obtained from four independent experiments are reported as means±s.d. *P<0.01.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02133

Figure 2
Figure 2

HGF inhibits mitochondrial damage and ROS production. Semiquantitative cytofluorimetric analysis of: (A) hydrogen peroxide production, H2O2; (B) superoxide anion production, O2 ; and (C) intracellular GSH content after long-term exposure of RINm5F cells to FFAs. In the upper panels results obtained in a representative FACS experiment are shown. In the lower panels the mean of data obtained from four independent experiments±s.d. is reported. P<0.01. (D) Biparametric flow cytometry analysis after staining of living cells with JC-1. In the area under the dashed line, the numbers indicate the percentage of cells with depolarized mitochondria. Results obtained in a representative experiment (upper panel) and the average of dataobtainedinfour independent experiments(lower panel, mean±s.d.;*P<0.01)are reported. CTRL, control.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02133

Figure 3
Figure 3

HGF modulates bcl-2 expression. Total RNA was isolated from RINm5F cells incubated for 72 h without (control; CTRL) or with free fatty acids (FFA) and in the presence of HGF with (FFA+HGF) or without FFA (HGF). (A) Expression of bcl-2, bax, and endogenous control GAPDH was evaluated by RT-PCR. PCR products were elecrophoresed on 1.5% agarose gel and analyzed by densitometric gel scanning. A representative experiment of three is shown. (B) Densitometric analysis of the bands is shown and results are expressed as means±s.d. of the different experiments. *P<0.01 versus control; P<0.05 versus FFAs. (C) Bcl-2 and bax expression evaluated by western blot analysis. A representative experiment of three is shown. (D) Densitometric analyses are reported as means±s.d. of the three different experiments. *P<0.01 versus FFAs.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02133

Figure 4
Figure 4

The antiapoptotic action of HGF requires the activation of a PI3K-dependent signaling pathway. HGF antiapototic activity was tested in the presence of the PI3K inhibitor LY294002. (A) Quantitative flow cytometry analysis of apoptosis after staining with annexin V and (B) mitochondrial membrane potential after staining of living cells with JC-1. Results are expressed as the percentage of annexin V-positive cells and percentage of cells with depolarized mitochondria membrane respectively. (C) Semi-quantitative flow cytometry analysis of hydrogen peroxide production H2O2, (D) superoxide anion production O2, and (E) intracellular GSH content. (F) Bcl-2 and bax protein expression evaluated by western blot and (G) densitometric analyses are reported as means±s.d. of the four different experiments conducted in the following experimental conditions: LY294002, FFA, FFA+HGF and FFA+HGF+LY294002. *P<0.01 versus FFAs. Dashed lines in the histograms represent the values found in control untreated cells for each specific parameter. Results reported in (A–G) are the means±s.d. from four independent experiments conducted in the following experimental conditions: LY294002, FFA, FFA+HGF, and FFA+HGF+ LY294002. *P<0.01.

Citation: Journal of Molecular Endocrinology 38, 1; 10.1677/jme.1.02133

We thank to Fabiola Masciotti and Gioacchino Romani for their technical assistance. This work has been supported by grants from Ministero della Salute and Ministero dell’Università e della Ricerca. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Anastasi E, Santangelo C, Bulotta A, Dotta F, Argenti B, Mincione C, Gulino A, Maroder M, Perfetti R & Di Mario U 2005 The acquisitation of an insulin-secreting phenotype by HGF-treated rat pancreatic ductal cells (ARIP) is associated with the development of susceptibility to cytokine-induced apoptosis. Journal of Molecular Endocrinology 34 367–376.

    • Search Google Scholar
    • Export Citation
  • Barbu A, Welsh N & Saldeen J 2002 Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential Deltapsi (m) in pancreatic RINm5F cells: prevention by Bcl-2. Molecular and Cellular Endocrinology 190 75–82.

    • Search Google Scholar
    • Export Citation
  • Carlsson C, Borg HLA & Welsh N 1999 Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro. Endocrinology 140 3422–3428.

    • Search Google Scholar
    • Export Citation
  • Carpentier A, Mittelman SD, Lamarche B, Bergman RN, Giacca A & Lewis GF 1999 Acute enhancement of insulin secretion by FFA in humans is lost with prolonged FFA elevation. American Journal of Physiology. Endocrinology and Metabolism 276 E1055–E1066.

    • Search Google Scholar
    • Export Citation
  • Cnop M, Hannaert JC, Hoorens A, Eizirik DL & Pipeleers DG 2001 Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50 1771–1777.

    • Search Google Scholar
    • Export Citation
  • Cossarizza A, Baccarani-Contri M, Kalashnikova G & Franceschi C 1993 A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochemical and Biophysical Research Communications 197 40–45.

    • Search Google Scholar
    • Export Citation
  • Del Guerra S, Lupi R, Marselli L, Masini M, Bugliani M, Sbrana S, Torri S, Pollera M, Boggi U, Mosca F et al. 2005 Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes 54 727–735.

    • Search Google Scholar
    • Export Citation
  • Deng X, Gao F & May WS Jr 2003 Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS. Blood 102 3179–3185.

  • Dickson LM & Rhodes CJ 2004 Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt? American Journal of Physiology. Endocrinology and Metabolism 287 E192–E198.

    • Search Google Scholar
    • Export Citation
  • Evans JL, Goldfine ID, Maddux BA & Grodsky GM 2003 Are oxidative stress-activated signaling pathways mediators of insulin resistance and β-cell dysfunction? Diabetes 52 1–8.

    • Search Google Scholar
    • Export Citation
  • Garcia-Ocana A, Takane KK, Reddy VT, Lopez-Talavera JC, Vasavada RC & Stevart AF 2003 Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death. Jounal of Biological Chemistry 278 343–351.

    • Search Google Scholar
    • Export Citation
  • Holst LS, Mulder H, Manganiello V, Sundler F, Ahren B, Holm C & Degerman E 1998 Protein kinase B is expressed in pancreatic beta cells and activated upon stimulation with insulin-like growth factor I. Biochemical and Biophysical Research Communications 250 181–186.

    • Search Google Scholar
    • Export Citation
  • Hui H, Nourpervar A, Zhao X & Perfetti R 2003 Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5′-adenosine monophosphate-dependent protein kinase A and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology 144 1444–1455.

    • Search Google Scholar
    • Export Citation
  • Jang JH & Surth YJ 2003 Potentiation of cellular antioxidant capacity by Bcl-2: implication for its antiapoptotic function. Biochemical Pharmacology 66 1371–1379.

    • Search Google Scholar
    • Export Citation
  • Kitta K, Day RM, Ikeda T & Suzuki YJ 2001 Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radical Biology and Medicine 31 902–910.

    • Search Google Scholar
    • Export Citation
  • Lee Y, Hirose H, Ohneda M, Johnson JH, McGarry JD & Unger RH 1994 Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships. PNAS 23 10878–10882.

    • Search Google Scholar
    • Export Citation
  • Lenzen S, Drinkgern J & Tiedge M 1996 Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radical Biology and Medicine 20 463–466.

    • Search Google Scholar
    • Export Citation
  • Liu Y 1999 Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms. American Journal of Physiology 277 F624–F633.

    • Search Google Scholar
    • Export Citation
  • Lopez-Talavera JC, Garcia-Ocana A, Sipula, Takane KK, Cozarcastellano I & Stewart AF 2004 Hepatocyte growth factor gene therapy for pancreatic islets in diabetes: reducing the minimal islet transplant mass required in a glucocorticoid-free rat model of allogenic portal vein islet transplantation. Endocrinoloy 145 467–474.

    • Search Google Scholar
    • Export Citation
  • Lowell BB & Shulman GI 2005 Mitochondrial dysfunction and type 2 diabetes. Science 307 384–387.

  • Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patanè G, Boggi U, Piro S, Anello M et al. 2002 Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets. Diabetes 51 1437–1442.

    • Search Google Scholar
    • Export Citation
  • Nakagami H, Morishita R, Yamamoto K, Taniyama Y, Aoki M, Yamasaki K, Matsumoto K, Nakamura T, Kaneda Y & Ogihara T 2002 Hepatocyte growth factor prevents endothelial cell death through inhibition of bax translocation from cytosol to mitochondrial membrane. Diabetes 51 2604–2611.

    • Search Google Scholar
    • Export Citation
  • Okada M, Sugita K, Inukai T, Goi K, Kagami K, Kawasaki K & Nakazawa S 2004 Hepatocyte growth factor protects small airway epithelial cells from apoptosis induced by tumor necrosis factor-Δor oxidative stress. Pediatric Research 56 336–344.

    • Search Google Scholar
    • Export Citation
  • Otonkoski T, Beattie GM, Rubin JS, Lopez AD, Baird A & Hayek A 1994 Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells. Diabetes 43 947–953.

    • Search Google Scholar
    • Export Citation
  • Papadopulos MC, Koumenis IL, Xu L & Giffard RG 1998 Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels. European Journal of Neuroscience 10 1252–1260.

    • Search Google Scholar
    • Export Citation
  • Piro S, Anello M, Di Pietro C, Lizzio MN, Patanè G, Rabuazzo AM, Vigneri R, Purrello M & Purrello F 2002 Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism 51 1340–1347.

    • Search Google Scholar
    • Export Citation
  • Rabinovitch A, Suarez-Pinzon W, Strynadka K, Ju Q, Edelstein D, Brownlee M, Korbutt GS & Rajotte RV 1999 Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes 48 1223–1229.

    • Search Google Scholar
    • Export Citation
  • Rhodes CJ 2005 Type 2 diabetes - a matter of beta-cell life and death? Science 307 380–384.

  • Richieri GV, Anel A & Kleinfeld AM 1993 Interaction of long-chain fatty acids and albumin: determination of free acid levels using the fluorescent probe ADIFAB. Biochemistry 32 7574–7580.

    • Search Google Scholar
    • Export Citation
  • Roccisana J, Reddy V, Vasavada RC, Gonzales-Pertusa JA, Magnuson MA & Garcia-Ocana A 2005 Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass. Diabetes 54 2090–2102.

    • Search Google Scholar
    • Export Citation
  • Rudin CM, Yang Z, Schumaker LM, Vander Weele DJ, Newkirk K, Egorin MJ, Zuhowski EG & Cullen JK 2003 Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Research 63 312–318.

    • Search Google Scholar
    • Export Citation
  • Sahaf B, Heydari K, Herzenberg LA & Herzenberg LA 2003 Lymphocyte surface thiol levels. PNAS 100 4001–4005.

  • Shimabukuro M, Zhou YT, Levi M & Unger RH 1998a Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. PNAS 95 2498–2502.

    • Search Google Scholar
    • Export Citation
  • Shimabukuro M, Wang MY, Zhou YT, Newgard CB & Unger RH 1998b Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression. PNAS 95 9558–9561.

    • Search Google Scholar
    • Export Citation
  • Tiedge M, Lortz S, Drinkgern J & Lenzen S 1997 Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46 1733–1742.

    • Search Google Scholar
    • Export Citation
  • Veis DJ, Sorensen CM, Shutter JR & Korsmayer SJ 1993 Bcl2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75 229–240.

    • Search Google Scholar
    • Export Citation
  • Voehringer DW 1999 Bcl-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity. Free Radical Biology and Medicine 27 945–950.

    • Search Google Scholar
    • Export Citation
  • Wang X, Li H, De Leo D, Guo W, Koshkin V, Fantus IG, Giacca A, Chan CB, Der S & Wheeler MB 2004 Gene and protein kinase expression profiling of reactive oxygen species-associated lipotoxicity in the pancreatic β-cell line MIN6. Diabetes 53 129–140.

    • Search Google Scholar
    • Export Citation
  • Wrede CE, Dickson LM, Lingohr MK, Briaud I & Rhodes CJ 2002 Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1). Journal of Biological Chemistry 277 49676–49684.

    • Search Google Scholar
    • Export Citation
  • Yamamoto K, Morishita R, Hayashi S, Matsushita H, Nakagami H, Moriguchi A, Matsumoto K, Nakamura T, Kaneda Y & Ogihara T 2001 Contribution of Bcl-2, but not Bcl-xL and Bax, to antiapoptotic actions of hepatocyte growth factor in hypoxia-conditioned human endothelial cells. Hypertension 37 1341–1348.

    • Search Google Scholar
    • Export Citation
  • Zhang L, Himi T, Morita I & Murota S 2000 Hepatocyte growth factor protects cultured rat cerebellar granule neurons from apoptosis via the phosphatidylinositol-3 kinase/Akt pathway. Journal of Neuroscience Research 59 489–496.

    • Search Google Scholar
    • Export Citation
  • Zhou YP & Grill V 1995 Long-term exposure to fatty acids and ketones inhibits beta-cell functions in human pancreatic islets of Langerhans. Journal of Clinical Endocrinology and Metabolism 80 1584–1590.

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

      Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 562 65 3
PDF Downloads 377 62 1
  • View in gallery

    HGF protects RINm5F cells from FFA-induced apoptosis. (A) Cells were exposed to FFAs (oleate:palmitate 2:1, 2 mmol/l) for 72 h in the presence or absence of HGF (50 ng/ml). After fixing, cells were stained for the nuclear marker Hoechst 33342. Apoptotic cells were identified by condensed and fragmented nuclear appearance. Similar results were obtained in three independent experiments. Pictures were taken at 20× magnification. In the inserts are represented nuclei at greater magnification. (B) FACS analysis after double staining with annexin V/propidium iodide. RINm5F cells were cultured in regular medium (control; CTRL), exposed to free fatty acids mixture (oleate/palmitate 2:1) (FFA), cultured in medium containing 50 ng/ml HGF alone (HGF), treated with HGF in addition with FFA (HGF+FFA) for 72 h. Dot plots from a representative FACS experiment are shown. Numbers represent the percentage of annexin V single positive (early apoptosis, bottom quadrant) or annexin V/PI double positive (late apoptosis, lower quadrant) cells. In the histogram, the results obtained from four independent experiments are reported as means±s.d. *P<0.01.

  • View in gallery

    HGF inhibits mitochondrial damage and ROS production. Semiquantitative cytofluorimetric analysis of: (A) hydrogen peroxide production, H2O2; (B) superoxide anion production, O2 ; and (C) intracellular GSH content after long-term exposure of RINm5F cells to FFAs. In the upper panels results obtained in a representative FACS experiment are shown. In the lower panels the mean of data obtained from four independent experiments±s.d. is reported. P<0.01. (D) Biparametric flow cytometry analysis after staining of living cells with JC-1. In the area under the dashed line, the numbers indicate the percentage of cells with depolarized mitochondria. Results obtained in a representative experiment (upper panel) and the average of dataobtainedinfour independent experiments(lower panel, mean±s.d.;*P<0.01)are reported. CTRL, control.

  • View in gallery

    HGF modulates bcl-2 expression. Total RNA was isolated from RINm5F cells incubated for 72 h without (control; CTRL) or with free fatty acids (FFA) and in the presence of HGF with (FFA+HGF) or without FFA (HGF). (A) Expression of bcl-2, bax, and endogenous control GAPDH was evaluated by RT-PCR. PCR products were elecrophoresed on 1.5% agarose gel and analyzed by densitometric gel scanning. A representative experiment of three is shown. (B) Densitometric analysis of the bands is shown and results are expressed as means±s.d. of the different experiments. *P<0.01 versus control; P<0.05 versus FFAs. (C) Bcl-2 and bax expression evaluated by western blot analysis. A representative experiment of three is shown. (D) Densitometric analyses are reported as means±s.d. of the three different experiments. *P<0.01 versus FFAs.

  • View in gallery

    The antiapoptotic action of HGF requires the activation of a PI3K-dependent signaling pathway. HGF antiapototic activity was tested in the presence of the PI3K inhibitor LY294002. (A) Quantitative flow cytometry analysis of apoptosis after staining with annexin V and (B) mitochondrial membrane potential after staining of living cells with JC-1. Results are expressed as the percentage of annexin V-positive cells and percentage of cells with depolarized mitochondria membrane respectively. (C) Semi-quantitative flow cytometry analysis of hydrogen peroxide production H2O2, (D) superoxide anion production O2, and (E) intracellular GSH content. (F) Bcl-2 and bax protein expression evaluated by western blot and (G) densitometric analyses are reported as means±s.d. of the four different experiments conducted in the following experimental conditions: LY294002, FFA, FFA+HGF and FFA+HGF+LY294002. *P<0.01 versus FFAs. Dashed lines in the histograms represent the values found in control untreated cells for each specific parameter. Results reported in (A–G) are the means±s.d. from four independent experiments conducted in the following experimental conditions: LY294002, FFA, FFA+HGF, and FFA+HGF+ LY294002. *P<0.01.

  • Anastasi E, Santangelo C, Bulotta A, Dotta F, Argenti B, Mincione C, Gulino A, Maroder M, Perfetti R & Di Mario U 2005 The acquisitation of an insulin-secreting phenotype by HGF-treated rat pancreatic ductal cells (ARIP) is associated with the development of susceptibility to cytokine-induced apoptosis. Journal of Molecular Endocrinology 34 367–376.

    • Search Google Scholar
    • Export Citation
  • Barbu A, Welsh N & Saldeen J 2002 Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential Deltapsi (m) in pancreatic RINm5F cells: prevention by Bcl-2. Molecular and Cellular Endocrinology 190 75–82.

    • Search Google Scholar
    • Export Citation
  • Carlsson C, Borg HLA & Welsh N 1999 Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro. Endocrinology 140 3422–3428.

    • Search Google Scholar
    • Export Citation
  • Carpentier A, Mittelman SD, Lamarche B, Bergman RN, Giacca A & Lewis GF 1999 Acute enhancement of insulin secretion by FFA in humans is lost with prolonged FFA elevation. American Journal of Physiology. Endocrinology and Metabolism 276 E1055–E1066.

    • Search Google Scholar
    • Export Citation
  • Cnop M, Hannaert JC, Hoorens A, Eizirik DL & Pipeleers DG 2001 Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50 1771–1777.

    • Search Google Scholar
    • Export Citation
  • Cossarizza A, Baccarani-Contri M, Kalashnikova G & Franceschi C 1993 A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochemical and Biophysical Research Communications 197 40–45.

    • Search Google Scholar
    • Export Citation
  • Del Guerra S, Lupi R, Marselli L, Masini M, Bugliani M, Sbrana S, Torri S, Pollera M, Boggi U, Mosca F et al. 2005 Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes 54 727–735.

    • Search Google Scholar
    • Export Citation
  • Deng X, Gao F & May WS Jr 2003 Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS. Blood 102 3179–3185.

  • Dickson LM & Rhodes CJ 2004 Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt? American Journal of Physiology. Endocrinology and Metabolism 287 E192–E198.

    • Search Google Scholar
    • Export Citation
  • Evans JL, Goldfine ID, Maddux BA & Grodsky GM 2003 Are oxidative stress-activated signaling pathways mediators of insulin resistance and β-cell dysfunction? Diabetes 52 1–8.

    • Search Google Scholar
    • Export Citation
  • Garcia-Ocana A, Takane KK, Reddy VT, Lopez-Talavera JC, Vasavada RC & Stevart AF 2003 Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death. Jounal of Biological Chemistry 278 343–351.

    • Search Google Scholar
    • Export Citation
  • Holst LS, Mulder H, Manganiello V, Sundler F, Ahren B, Holm C & Degerman E 1998 Protein kinase B is expressed in pancreatic beta cells and activated upon stimulation with insulin-like growth factor I. Biochemical and Biophysical Research Communications 250 181–186.

    • Search Google Scholar
    • Export Citation
  • Hui H, Nourpervar A, Zhao X & Perfetti R 2003 Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5′-adenosine monophosphate-dependent protein kinase A and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology 144 1444–1455.

    • Search Google Scholar
    • Export Citation
  • Jang JH & Surth YJ 2003 Potentiation of cellular antioxidant capacity by Bcl-2: implication for its antiapoptotic function. Biochemical Pharmacology 66 1371–1379.

    • Search Google Scholar
    • Export Citation
  • Kitta K, Day RM, Ikeda T & Suzuki YJ 2001 Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radical Biology and Medicine 31 902–910.

    • Search Google Scholar
    • Export Citation
  • Lee Y, Hirose H, Ohneda M, Johnson JH, McGarry JD & Unger RH 1994 Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships. PNAS 23 10878–10882.

    • Search Google Scholar
    • Export Citation
  • Lenzen S, Drinkgern J & Tiedge M 1996 Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radical Biology and Medicine 20 463–466.

    • Search Google Scholar
    • Export Citation
  • Liu Y 1999 Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms. American Journal of Physiology 277 F624–F633.

    • Search Google Scholar
    • Export Citation
  • Lopez-Talavera JC, Garcia-Ocana A, Sipula, Takane KK, Cozarcastellano I & Stewart AF 2004 Hepatocyte growth factor gene therapy for pancreatic islets in diabetes: reducing the minimal islet transplant mass required in a glucocorticoid-free rat model of allogenic portal vein islet transplantation. Endocrinoloy 145 467–474.

    • Search Google Scholar
    • Export Citation
  • Lowell BB & Shulman GI 2005 Mitochondrial dysfunction and type 2 diabetes. Science 307 384–387.

  • Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patanè G, Boggi U, Piro S, Anello M et al. 2002 Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets. Diabetes 51 1437–1442.

    • Search Google Scholar
    • Export Citation
  • Nakagami H, Morishita R, Yamamoto K, Taniyama Y, Aoki M, Yamasaki K, Matsumoto K, Nakamura T, Kaneda Y & Ogihara T 2002 Hepatocyte growth factor prevents endothelial cell death through inhibition of bax translocation from cytosol to mitochondrial membrane. Diabetes 51 2604–2611.

    • Search Google Scholar
    • Export Citation
  • Okada M, Sugita K, Inukai T, Goi K, Kagami K, Kawasaki K & Nakazawa S 2004 Hepatocyte growth factor protects small airway epithelial cells from apoptosis induced by tumor necrosis factor-Δor oxidative stress. Pediatric Research 56 336–344.

    • Search Google Scholar
    • Export Citation
  • Otonkoski T, Beattie GM, Rubin JS, Lopez AD, Baird A & Hayek A 1994 Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells. Diabetes 43 947–953.

    • Search Google Scholar
    • Export Citation
  • Papadopulos MC, Koumenis IL, Xu L & Giffard RG 1998 Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels. European Journal of Neuroscience 10 1252–1260.

    • Search Google Scholar
    • Export Citation
  • Piro S, Anello M, Di Pietro C, Lizzio MN, Patanè G, Rabuazzo AM, Vigneri R, Purrello M & Purrello F 2002 Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism 51 1340–1347.

    • Search Google Scholar
    • Export Citation
  • Rabinovitch A, Suarez-Pinzon W, Strynadka K, Ju Q, Edelstein D, Brownlee M, Korbutt GS & Rajotte RV 1999 Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes 48 1223–1229.

    • Search Google Scholar
    • Export Citation
  • Rhodes CJ 2005 Type 2 diabetes - a matter of beta-cell life and death? Science 307 380–384.

  • Richieri GV, Anel A & Kleinfeld AM 1993 Interaction of long-chain fatty acids and albumin: determination of free acid levels using the fluorescent probe ADIFAB. Biochemistry 32 7574–7580.

    • Search Google Scholar
    • Export Citation
  • Roccisana J, Reddy V, Vasavada RC, Gonzales-Pertusa JA, Magnuson MA & Garcia-Ocana A 2005 Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass. Diabetes 54 2090–2102.

    • Search Google Scholar
    • Export Citation
  • Rudin CM, Yang Z, Schumaker LM, Vander Weele DJ, Newkirk K, Egorin MJ, Zuhowski EG & Cullen JK 2003 Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Research 63 312–318.

    • Search Google Scholar
    • Export Citation
  • Sahaf B, Heydari K, Herzenberg LA & Herzenberg LA 2003 Lymphocyte surface thiol levels. PNAS 100 4001–4005.

  • Shimabukuro M, Zhou YT, Levi M & Unger RH 1998a Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. PNAS 95 2498–2502.

    • Search Google Scholar
    • Export Citation
  • Shimabukuro M, Wang MY, Zhou YT, Newgard CB & Unger RH 1998b Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression. PNAS 95 9558–9561.

    • Search Google Scholar
    • Export Citation
  • Tiedge M, Lortz S, Drinkgern J & Lenzen S 1997 Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46 1733–1742.

    • Search Google Scholar
    • Export Citation
  • Veis DJ, Sorensen CM, Shutter JR & Korsmayer SJ 1993 Bcl2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75 229–240.

    • Search Google Scholar
    • Export Citation
  • Voehringer DW 1999 Bcl-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity. Free Radical Biology and Medicine 27 945–950.

    • Search Google Scholar
    • Export Citation
  • Wang X, Li H, De Leo D, Guo W, Koshkin V, Fantus IG, Giacca A, Chan CB, Der S & Wheeler MB 2004 Gene and protein kinase expression profiling of reactive oxygen species-associated lipotoxicity in the pancreatic β-cell line MIN6. Diabetes 53 129–140.

    • Search Google Scholar
    • Export Citation
  • Wrede CE, Dickson LM, Lingohr MK, Briaud I & Rhodes CJ 2002 Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1). Journal of Biological Chemistry 277 49676–49684.

    • Search Google Scholar
    • Export Citation
  • Yamamoto K, Morishita R, Hayashi S, Matsushita H, Nakagami H, Moriguchi A, Matsumoto K, Nakamura T, Kaneda Y & Ogihara T 2001 Contribution of Bcl-2, but not Bcl-xL and Bax, to antiapoptotic actions of hepatocyte growth factor in hypoxia-conditioned human endothelial cells. Hypertension 37 1341–1348.

    • Search Google Scholar
    • Export Citation
  • Zhang L, Himi T, Morita I & Murota S 2000 Hepatocyte growth factor protects cultured rat cerebellar granule neurons from apoptosis via the phosphatidylinositol-3 kinase/Akt pathway. Journal of Neuroscience Research 59 489–496.

    • Search Google Scholar
    • Export Citation
  • Zhou YP & Grill V 1995 Long-term exposure to fatty acids and ketones inhibits beta-cell functions in human pancreatic islets of Langerhans. Journal of Clinical Endocrinology and Metabolism 80 1584–1590.

    • Search Google Scholar
    • Export Citation