Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2010 Feb 22;151(4):1487–1498. doi: 10.1210/en.2009-0975

Novel Proapoptotic Effect of Hepatocyte Growth Factor: Synergy with Palmitate to Cause Pancreatic β-Cell Apoptosis

José A González-Pertusa 1, John Dubé 1, Shelley R Valle 1, Taylor C Rosa 1, Karen K Takane 1, José M Mellado-Gil 1, Germán Perdomo 1, Rupangi C Vasavada 1, Adolfo García-Ocaña 1
PMCID: PMC2850223  PMID: 20176723

Abstract

Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to β-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect β-cells against streptozotocin and during islet engraftment. However, whether HGF is a β-cell prosurvival factor in situations of excessive lipid supply has not been deciphered. Mice overexpressing HGF in the β-cell [rat insulin type II promoter (RIP)-HGF transgenic mice] fed with standard chow display improved glucose homeostasis and increased β-cell mass and proliferation compared with normal littermates. However, after 15 wk of high-fat feeding, glucose homeostasis and β-cell expansion and proliferation are indistinguishable between normal and transgenic mice. Interestingly, RIP-HGF transgenic mouse β-cells and normal β-cells treated with HGF display increased sensitivity to palmitate-mediated apoptosis in vitro. Palmitate completely eliminates Akt and Bad phosphorylation in RIP-HGF transgenic mouse islets. HGF-overexpressing islets also show significantly decreased AMP-activated protein kinase-α and acetyl-coenzyme A carboxylase phosphorylation, diminished fatty acid oxidation, increased serine palmitoyltransferase expression, and enhanced ceramide formation compared with normal islets. Importantly, human islets overexpressing HGF also display increased β-cell apoptosis in the presence of palmitate. Treatment of both mouse and human islet cells with the de novo ceramide synthesis inhibitors myriocin and fumonisin B1 abrogates β-cell apoptosis induced by HGF and palmitate. Collectively, these studies indicate that HGF can be detrimental for β-cell survival in an environment with excessive fatty acid supply.

HGF is a novel pro-apoptotic factor for the murine, and more importantly human, β-cell in an environment rich in saturated free-fatty acids through the increase of ceramide formation.

It is becoming increasingly evident that chronic high levels of circulating free fatty acids (FFA) and triglycerides play a role in β-cell failure present in obesity-related type 2 diabetes. Sustained high concentrations of FFA induce apoptosis in β-cells (1,2,3), an effect amplified by high glucose concentrations (3) and reverted by increased fatty acid oxidation (FAO) (3,4). Interestingly, circulating hepatocyte growth factor (HGF) levels are highly increased in humans with metabolic syndrome and obesity (5,6). However, whether HGF plays a role in β-cell survival in situations of lipid oversupply remains unknown.

HGF, originally identified as a hepatic regeneration factor (7), is a potent β-cell mitogen and an insulinotropic agent in vivo and in vitro (8,9,10,11). HGF is also a β-cell prosurvival factor against the cytotoxic and diabetogenic agent streptozotocin (9,12,13) and in the hypoxic and nutrient deprivation environment present in the early hours after islet transplantation (10,13,14,15,16). Recently, Santangelo et al. (17) have shown that HGF also protects rat insulinoma RINm5F cells from FFA (2 mm 2:1 oleate:palmitate)-induced apoptosis.

To assess whether HGF improves glucose homeostasis and further enhances β-cell mass in vivo in a situation of obesity-mediated insulin resistance, we analyzed the phenotype of rat insulin type II promoter (RIP)-HGF transgenic mice after high-fat diet (HFD) feeding for 15 wk. Surprisingly, the superior glucose homeostasis and the increased rate in β-cell proliferation and mass observed in RIP-HGF transgenic mice on standard diet (SD) disappear when these mice are fed with HFD. Furthermore, HGF exacerbates palmitate-induced apoptosis in rodent and human β-cells in vitro. These proapoptotic effects are mediated by up-regulation of serine palmitoyltransferase (SPT) and increased ceramide production. Contrary to our expectations, these results suggest that HGF may participate in the events leading to β-cell insufficiency in humans with obesity/type 2 diabetes.

Materials and Methods

Materials

The [9,10-3H]-palmitic acid and [3H]-H2O were from PerkinElmer Life Sciences (Shelton, CT). Recombinant human HGF (hHGF) was from Research Diagnostics, Inc. (Flanders, NJ); and palmitate, oleate, fumonisin B1, Hoechst 33258, propidium iodide (PI), and fatty acid-free BSA were from Sigma (St. Louis, MO). Antibodies against acetyl-coenzyme A (CoA) carboxylase (ACC), phospho-ACC-(Ser79), phospho-Akt-(Ser473), phospho-Bad-(Ser136), phospho-AMP-activated protein kinase (AMPK)α-(Thr 172), AMPKα, Bad, and Akt were from Cell Signaling (Beverly, MA); carnitine palmitoyltransferase 1 (CPT-1) and hHGF from Santa Cruz Biotechnology (Santa Cruz, CA); actin from Sigma; tubulin from Calbiochem (La Jolla, CA); insulin from Abcam (Cambridge, MA); and SPT-1 from BD Biosciences (San Jose, CA) and SPT-2 from Cayman Chemical (Ann Arbor, MI). Myriocin was from Enzo Life Sciences (Plymouth Meeting, PA).

Generation of transgenic mice

The generation of transgenic mice with HGF overexpression in the β-cell driven by the RIP has been previously described (9). Two-month-old RIP-HGF transgenic mice on a CD-1 background were used for these studies. For the fat-feeding experiments, male littermate mice were fed ad libitum either a SD or a HFD with 60% kcal from fat (Research Diets, New Brunswick, NJ) for 15 wk. All studies were performed with the approval of, and in accordance with, guidelines established by the University of Pittsburgh Institutional Animal Care and Use Committee.

Glucose homeostasis measurements

Blood obtained by retroorbital bleed was analyzed for glucose, as previously described (9). Intraperitoneal glucose tolerance test was performed in 16–18 h fasted mice injected ip with 1 g d-glucose/kg body weight (wt) and insulin tolerance test (ITT) was performed in random-fed mice injected ip with 0.75 U (SD) or 7.5 U (HFD) of bovine insulin/kg body wt, as previously reported (10,18).

β-Cell proliferation and death in vivo and pancreas immunohistochemistry and histomorphometry

In vivo β-cell replication was determined by 5-bromo-2′-deoxyuridine (BrdU) incorporation in mice injected ip with BrdU (10 μl/g body wt) (Cell Proliferation kit; Amersham Pharmacia Biotech, Piscataway, NJ) and killed 6 h later, as previously reported (9,13). Islets were photographed at ×400, and the number of BrdU-positive β-cells was manually counted. PI staining was performed as previously described (9,13), islets photographed at ×400, and the number of β-cells with condensed nuclei (dead β-cells) was manually counted. At least 1000 β-cells/mouse were counted, β-cell mass was measured in three insulin-stained pancreas sections from each mouse as previously reported (18).

Islet cell cultures

Islets from normal and RIP-HGF transgenic mice were isolated after injection of collagenase P (Roche Molecular Biochemicals, Indianapolis, IN) through the pancreatic duct, as previously described (9). Islets were separated by density gradient in Histopaque (Sigma) and hand-picked under a microscope. Human islets were provided by the Islet Cell Resource Center and Juvenile Diabetes Research Foundation Basic Science Islet Distribution Programs. Mouse and human islets were trypsinized, and islet cells were plated and incubated for 48 h in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5 mm glucose, as previously reported (11). Mouse islet cells were incubated for 24 h in RPMI medium with 5 mm glucose and with 0.5% BSA or different concentrations of palmitate (0.25 and 0.5 mm) or oleate (0.5 mm), with or without recombinant hHGF (0–25 ng/ml), and with 50 nm myriocin or 15 μm fumonisin B1, two known inhibitors of the enzymes SPT and ceramide synthase, respectively (19). In another set of experiments, mouse islet cells were incubated in 25 mm glucose and treated with or without 0.5 mm palmitate and HGF for 24 h. After adenovirus transduction (see below), human islets cells were incubated for 24 h with 5 mm glucose and with 0.5% BSA or 0.5 mm palmitate and with or without myriocin or fumonisin B1.

FFA/BSA solution preparation

Fatty acids (palmitate or oleate) were preconjugated with fatty acid-free BSA to generate a solution of 5% (wt/vol) BSA, 5 mm fatty acid in serum-free RPMI medium, as previously described (20). Briefly, fatty acid stock solution prepared in 0.1 m NaOH by heating at 70 C and 5% BSA solution prepared in RPMI medium at 48 C were mixed, vortexed, cooled down to room temperature, and sterile filtered (0.22-μm pore size membrane filter) to generate the 5% (wt/vol) BSA, 5 mm palmitate, or oleate stock solution.

β-Cell proliferation in mouse islet cell cultures

β-Cell proliferation rates were assessed by BrdU incorpation into β-cells in primary cultures of RIP-HGF transgenic and normal mouse islet cells, as recently performed (11). Mouse islet cells were incubated in RPMI medium containing 5 mm glucose, BrdU (1:1000 dilution), and 0.25 mm palmitate or 2.5% BSA, with or without 50 μm of the myristolated PKCζ peptide inhibitor (Biomol International, Plymouth Meeting, PA) for 18 h.

Detection of β-cell apoptosis in vitro

Primary islet cells were fixed with 2% paraformaldehyde for 30 min and apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method (Promega Corp., Madison, WI), according to the manufacturer’s protocol. Subsequent to TUNEL, cells were stained with the antiinsulin antibody as described above. Visualization was performed by using a tetramethylrhodamine isothiocyanate-conjugated rabbit antiguinea pig IgG secondary antibody. Nuclei were detected with Hoechst 33258.

Generation of recombinant adenoviruses and human islet cell transduction

Adenovirus containing hHGF or ß-galactosidase (Adv-LacZ) cDNAs were prepared according to methods previously described in detail (13), originally reported by Becker et al. (21). Multiplicity of infection (MOI) calculation assumes 1000 cells per islet equivalent (IE; 125-μm diameter). At the time of plating, human islet cells were transduced with purified adenovirus at a MOI of 100 plaque-forming units per cell or left uninfected. Subsequently, complete medium was added, and cells were incubated for 48 h before treatment as indicated above.

Immunoblot analysis

Mouse islets were hand-picked from RPMI medium containing 5 mm glucose immediately after isolation, and islet protein extracts were prepared as previously described (18). In some experiments, mouse islets were incubated for 24 h in RPMI containing 5 mm glucose and 0.5% BSA or 0.5 mm palmitate before protein extraction. Human islets were hand-picked 48 h after adenoviral transduction, and protein extracts were prepared as previously reported (18). Islet protein extracts were separated using a 10% SDS-PAGE, and Western blot analysis was performed as previously described (18).

FAO in isolated islets

FAO was measured as described previously with some modifications (22,23). Briefly, RIP-HGF and normal mouse islets (60 IE/well) were placed in plate inserts (12-μm diameter pore size; Millipore, Cork, UK) RPMI medium containing 5 mm glucose, 0.25% fatty acid-free BSA, 0.1 mm palmitate, and 74 kBq/ml [9,10(n)-3H]palmitate and cultured for 40 h at 37 C. Islets were subsequently washed in Krebs-Ringer bicarbonate buffer (KRBB) supplemented with 10 mm HEPES, 0.25% fatty acid-free BSA, 3 mm glucose, and 0.1 mm palmitate. Then, islets were incubated in KRBB containing 0.25% fatty acid-free BSA, 0.1 mm palmitate, 1 mm carnitine, 74 kBq/ml [9,10(n)-3H]palmitate, and 3 or 16 mm glucose for 2 h. Medium was harvested, centrifuged at 3000 rpm for 5 min, and FAO was determined by measuring the [3H]H2O content in the conditioned medium by vapor-phase equilibration. Islets were washed with KRBB and digested in 0.1 n NaOH at 37 C. Protein was measured by the Bradford method.

Ceramide content in islets

Quantitation of ceramide content in lipid extracts from isolated islets was performed as previously reported by Dubé et al. (24) with some modifications. Briefly, lipids were extracted from normal and RIP-HGF transgenic mouse islets (200-350 IE) and ceramides phosphorylated in the lipid extract by the addition of diacylglycerol-kinase and 5 μCi [32P]ATP. Ceramides were subsequently separated by thin-layer chromatography. The silica plates were then exposed to autoradiograph film, ceramides located and scraped into separate scintillation vials. Ceramide concentrations were calculated as nanomoles/IE based on ceramide standards run in the same plate. This method is quantitatively similar to HPLC-fluorescence spectrometry for total ceramide content (25).

Statistical analysis

Data are expressed as means ± sem. Intergroup comparisons were performed by unpaired two-tailed Student’s t test, Mann-Whitney U test or ANOVA with Bonferroni post hoc test where appropriate. P < 0.05 was considered significant.

Results

Adaptive responses to a HFD in RIP-HGF transgenic mice

HGF overexpression in mouse β-cells in vivo results in increased β-cell mass and improved glucose homeostasis in basal conditions (9,10). To determine whether HGF overexpression in β-cells can further expand β-cell mass and improve glucose homeostasis in mice fed with a HFD, we compared the phenotype of RIP-HGF and normal mouse littermates fed with SD or HFD for 15 wk. As shown in Fig. 1A, RIP-HGF mice gained wt to the same extent as normal littermates when fed a HFD. As previously observed in 2-month-old mice, RIP-HGF transgenic mice on SD continued to display significantly decreased blood glucose, mild inappropriate hyperinsulinemia, and increased insulin/glucose ratios at 6 months of age (Fig. 1, B–D) (9). However, after high-fat feeding, RIP-HGF transgenic mice did not show any improvement in these metabolic parameters compared with normal mice (Fig. 1, B–D). As previously observed in 2-month-old mice, RIP-HGF transgenic mice on a SD displayed tighter control of glucose tolerance than normal mice at 6 months of age (Fig. 1, E and F). In contrast, RIP-HGF transgenic mice on HFD showed a small but nonsignificant improvement in glucose tolerance compared with normal littermates fed HFD (Fig. 1, E and F). Insulin sensitivity was not significantly different between RIP-HGF transgenic mice and normal littermates fed a HFD for 15 wk (Fig. 1G). Equally, ITT expressed as either absolute blood glucose values or as percentage of blood glucose at time 0 was not significantly different between both types of mice fed with a SD (Supplemental Fig. 1, A and B, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Collectively, these results indicate that the improvement in glucose homeostasis conferred by HGF overexpression in the β-cell in mice disappears in a situation of insulin resistance induced by high-fat feeding.

Figure 1.

Figure 1

Open in a new tab

Body weight and glucose homeostasis in adult male RIP-HGF transgenic mice and normal (NL) littermates fed with a SD or HFD. The 8-wk-old mice were fed with these diets for 15 wk and (A) body wt, (B) nonfasting blood glucose, and (C) plasma insulin were measured. D, Insulin/glucose ratios were calculated from these results. One week before killing, (E) ip glucose tolerance tests were performed and (F) areas under the curve calculated from these results. G, ITTs were measured in nonfasting mice fed for 14 wk and 3 d with their corresponding diets. Values are means ± sem of five to nine mice per group. *, P < 0.05 vs. same type of mice in SD; #, P < 0.05 vs. NL on the same diet.

β-cell mass was significantly increased in RIP-HGF transgenic mice compared with normal littermates when fed with a SD (Fig. 2, A, B, and E), as previously observed (9). As a result of the HFD feeding, both types of mice showed a significant increase in β-cell mass compared with their littermates fed with a SD (Fig. 2, A–E). Interestingly, β-cell mass was not significantly different between RIP-HGF transgenic mice and normal littermates after 15 wk of HFD feeding (Fig. 2, C–E), suggesting that the compensatory increase in β-cell mass mediated by HFD-induced insulin resistance was not further enhanced by the presence of HGF overexpression in the β-cell. This could be the result of alterations in the mitogenic and/or prosurvival actions of HGF in the β-cell in HFD-mediated insulin resistance states. To determine whether this is the case, we measured β-cell proliferation and death in both types of mice fed with SD or HFD. As previously reported (9), β-cell proliferation was significantly increased in RIP-HGF transgenic mice compared with normal littermates fed a SD (Fig. 2F). HFD for 15 wk significantly increased β-cell proliferation in normal mice. However, β-cell proliferation was not further enhanced in RIP-HGF transgenic mice fed a HFD when compared with normal littermates fed with HFD or RIP-HGF transgenic mice fed with a SD (Fig. 2F).

Figure 2.

Figure 2

Open in a new tab

β-Cell mass, proliferation and death in adult male RIP-HGF transgenic mice and normal (NL) littermates fed with a SD or HFD. Eight-week-old mice were fed with these diets for 15 wk, injected ip with BrdU and 6 h later pancreta harvested. Representative microphotographs of insulin-stained (brown) pancreas from (A) NL and (B) RIP-HGF transgenic mice fed with a SD, and (C) NL and (D) RIP-HGF transgenic mice fed with a HFD. E, β-Cell mass in NL and RIP-HGF transgenic mice fed with SD or HFD. F, BrdU incorporation in NL and RIP-HGF transgenic mice fed with SD or HFD. *, P < 0.05 vs. same type of mice in SD; #, P < 0.05 vs. NL on the same diet. G, Effect of 50 μm/liter cell-permeable PKCζ inhibitor and 0.25 mm palmitate (Palm) or 2.5% BSA in normal (NL) and RIP-HGF transgenic mouse β-cell proliferation measured by BrdU incorporation in primary islet cells cultures incubated at 5 mm glucose. Values are means ± se of four experiments in duplicate. *, P < 0.05 vs. BSA-NL; #, P < 0.05 vs. Palm+PKCζ inhibitor. H, PI condensed nuclei in β-cells of NL and RIP-HGF transgenic mice fed with HFD. In the in vivo experiments, values are means ± sem of five to nine mice per group.

We next examined the effect of FFAs, in particular palmitate, on β-cell proliferation in RIP-HGF transgenic mouse β-cells in vitro. As shown in Fig. 2G, 0.25 mm palmitate increased BrdU incorporation in normal mouse β-cells. As previously observed (11), RIP-HGF transgenic mouse β-cells also displayed increased β-cell proliferation in vitro; however, there was no additive effect of HGF and palmitate on β-cell proliferation. (Fig. 2G). Based on our earlier observation that HGF induces β-cell proliferation through the protein kinase C (PKC)ζ intracellular pathway, we determined its role in palmitate-mediated β-cell proliferation. Interestingly, the cell-permeable PKCζ inhibitor completely abolished the mitogenic effect of palmitate in both normal and HGF-overexpressing β-cells (Fig. 2G). These results indicate that the PKCζ signaling pathway is downstream of both HGF and palmitate on β-cell proliferation.

As expected, the number of condensed β-cell nuclei, a marker of β-cell death, was significantly increased in both types of mice fed a HFD (Fig. 2H) compared with the number in animals fed a SD (undetectable and data not shown). Interestingly, the number of condensed β-cell nuclei in RIP-HGF mice on HFD was increased compared with the number in normal littermates fed HFD, but this increase was not significant (Fig. 2H). Taken together, these results indicate that HFD suppresses the beneficial effects that HGF overexpression has on β-cell proliferation and mass and suggest a potential negative effect of HGF overexpression on β-cell survival in this setting.

HGF increases the sensitivity of primary β-cells to palmitate-induced apoptosis

To analyze in detail whether HGF alters β-cell survival in a lipotoxic environment potentially representing obesity situations, we treated islet cells from RIP-HGF transgenic mice and normal littermates with the saturated FFA palmitate. As shown in Fig. 3, A, B, and E, normal mouse primary β-cells cultured in 5 mm glucose were resistant to apoptosis-induced by 24-h treatment with 0.25–0.5 mm palmitate. To our surprise, however, β-cells from RIP-HGF transgenic mouse littermates displayed a significantly increased number of TUNEL-positive nuclei (Fig. 3, C and E). Importantly, this effect was also observed in normal β-cells treated with 25 ng/ml HGF and palmitate (Fig. 3, D and E). On the other hand, HGF was not capable of increasing β-cell apoptosis in the presence of the monounsaturated FFA oleate (0.5 mm) (data not shown).

Figure 3.

Figure 3

Open in a new tab

β-Cell apoptosis in primary islet cell cultures from RIP-HGF transgenic mice and normal (NL) littermates incubated at 5 mm glucose and treated for 24 h with palmitate. Representative microphotographs of (A) untreated NL, (B) 0.5 mm palmitate-treated NL, (C) 0.5 mm palmitate-treated RIP-HGF, and (D) 0.5 mm palmitate and 25 ng/ml HGF-treated NL mouse primary islet cell cultures stained for insulin (red), TUNEL (green), and Hoechst (blue). Arrows indicate TUNEL-positive β-cell nuclei. E, Quantitation of the number of TUNEL-positive β-cells in these primary islet cell cultures. F, Dose-dependent effect of HGF on the number of TUNEL-positive β-cells in primary islet cell cultures from NL mice treated with 0.5 mm palmitate for 24 h. An open circle represents untreated cells. Values are means ± sem of at least five experiments in duplicate. *, P < 0.05 vs. NL; #, P < 0.05 vs. not treated with HGF. There was no significant difference between the apoptosis levels found with 6 and 25 ng/ml HGF. G, Quantitation of the number of TUNEL-positive β-cells in primary islet cell cultures from normal mice incubated at 5 and 25 mm glucose and treated for 24 h with 0.5 mm palmitate (Palm) and/or 25 ng/ml HGF. *, P < 0.05 vs. untreated (UT); #, P < 0.05 vs. Palm+25 mm glucose; ^, P < 0.05 vs. Palm+5 mm glucose+HGF. H, Representative Western blot analyses of Akt phosphorylation (Ser473), Bad phosphorylation (Ser136) in protein extracts from RIP-HGF transgenic and NL mouse islets after treatment with or without 0.5 mm palmitate for 24 h. Samples from different parts of the same gel were used for this figure. Densitometric quantitation of several Western blottings analyzing (I) Akt phosphorylation (J) and Bad phosphorylation. The y-axis represents the ratio of phosphorylated vs. total in arbitrary units. Values are means ± sem of four different islet extracts from RIP-HGF transgenic and NL mice. *, P < 0.05 vs. NL untreated; #, P < 0.05 vs. RIP-HGF treated with palmitate.

We next analyzed whether HGF-induced increase in β-cell apoptosis was dose-dependent. These experiments revealed that 6 ng/ml HGF, a dose close to that present in the circulation of obese subjects (5), significantly increased β-cell apoptosis in the presence of 0.5 mm palmitate (Fig. 3F). This effect was not significantly enhanced by a higher HGF concentration (25 ng/ml) (Fig. 3F). In addition, HGF significantly amplified β-cell apoptosis induced by 0.5 mm palmitate in the presence of high glucose (25 mm) (Fig. 3G). Taken together, these results indicate that HGF, in a concentration range present in the circulation in obese subjects (5), enhances the sensitivity of primary β-cells to the cytotoxic effects of palmitate in vitro.

HGF activates Akt in islets and Akt is known to be a prosurvival signal for the β-cell (13). To analyze whether palmitate decreases Akt activation induced by HGF, we treated islets from RIP-HGF transgenic and normal littermate mice with 0.5 mm palmitate for 24 h. As shown in Fig. 3, H–J, RIP-HGF transgenic mouse islets displayed increased phosphorylation of Akt, and the known target of Akt, Bad. Importantly, palmitate significantly inhibited phosphorylation of both intracellular targets in RIP-HGF transgenic mouse islets. These studies suggest that the lack of a protective effect of HGF in β-cells treated with palmitate could be in part due to its inability to activate Akt.

HGF decreases FAO in isolated islets

To determine whether HGF has any effect on fatty acid metabolism in islets, we analyzed FAO rates in isolated islets from RIP-HGF transgenic and normal mouse littermates. As shown in Fig. 4A, both RIP-HGF transgenic and normal mouse islets displayed significantly decreased FAO at 16 mm compared with 3 mm glucose. Importantly, RIP-HGF islets displayed markedly and significantly decreased FAO compared with normal mouse islets at 16 mm glucose. Analysis of the expression and phosphorylation levels of AMPK and its target ACC in freshly isolated RIP-HGF transgenic mouse islets showed a significant decrease in the phosphorylated levels of both enzymes with no alteration of CPT-1 levels (Fig. 4, B–F). Because phosphorylation of ACC inhibits its enzymatic activity, these results suggest that FAO is diminished in RIP-HGF transgenic mouse islets through decreased ACC phosphorylation and potentially enhanced malonyl-CoA levels.

Figure 4.

Figure 4

Open in a new tab

RIP-HGF transgenic islets display decreased FAO at high glucose concentrations. A, Oxidation of palmitate in RIP-HGF transgenic and normal (NL) mouse islets incubated with 3 and 16 mm glucose. Values are means ± sem of five different experiments in duplicate. *, P < 0.05 vs. 3 mm glucose; #, P < 0.05 vs. NL at 16 mm glucose. Representative images and densitometric quantitation of several Western blottings analyzing (B) AMPKα phosphorylation (Thr172), (C) ACC phosphorylation (Ser79), and (D) CPT-1 expression in protein extracts from RIP-HGF transgenic and NL mouse islets. The y-axis represents the ratio of phosphorylated vs. total or total vs. tubulin in arbitrary units. Values are means ± sem of four different islet extracts from RIP-HGF transgenic and NL mice. *, P < 0.05 vs. NL.

HGF increases ceramide formation in islets

Saturated fatty acids, such as palmitate, promote accumulation of ceramide in islets, which has been reported as a possible inducer of FFA-mediated β-cell apoptosis (1). We next analyzed ceramide levels in islets from normal and RIP-HGF transgenic mouse littermates incubated for 24 h with 0.5 mm palmitate and 5 mm glucose As shown in Fig. 5A, ceramide content increased, although not significantly (P = 0.08), in normal islets after treatment with 0.5 mm palmitate (Fig. 5A). Importantly, however, ceramide accumulation in HGF-overexpressing islets was markedly and significantly amplified compared with normal islets after treatment with palmitate (Fig. 5A).

Figure 5.

Figure 5

Open in a new tab

Ceramide content and SPT expression in islets from RIP-HGF transgenic mice and normal (NL) littermates. A, Ceramide content was measured in RIP-HGF transgenic and NL mouse islets incubated in 5 mm glucose and treated with 0.5 mm palmitate for 24 h or left untreated. Values are means ± sem of experiments with islets from four to six different mice. *, P < 0.05 vs. RIP-HGF transgenic islets untreated; #, P < 0.05 vs. NL islets treated with palmitate. Representative images and densitometric quantitation of Western blot analyses performed with four different islet extracts from RIP-HGF transgenic and NL mice to determine (B) SPT-1 and (C) SPT-2 expression levels. The y-axis represents the ratio of SPT vs. actin in arbitrary units. Values are means ± sem. *, P < 0.05; **, P < 0.01 vs. NL.

SPT is a key enzyme regulating the de novo pathway of ceramide synthesis. Two gene products (SPT1 and SPT2) are necessary for its activity (27). Analysis of the expression levels of SPT1 and SPT2 in HGF-overexpressing islets revealed a marked up-regulation of both SPT enzymatic components in basal conditions (Fig. 5, B and C). Taken together, these results suggest that HGF overexpression enhances the formation of ceramide in islets treated with palmitate potentially by inhibiting FAO and activating the de novo ceramide synthesis pathway.

Inhibition of SPT and de novo ceramide synthesis blocks palmitate-mediated β-cell apoptosis in RIP-HGF transgenic mouse islets

We next explored whether inhibition of SPT and de novo ceramide synthesis could affect palmitate-mediated β-cell apoptosis in RIP-HGF transgenic mouse islets. As shown in Fig. 6, A and B, myriocin, a potent inhibitor of SPT, decreased the number of TUNEL-positive β-cell nuclei in RIP-HGF islet-cell cultures treated with palmitate. Quantitation of several experiments revealed that myriocin completely and significantly eliminated β-cell apoptosis induced by palmitate in HGF-overexpressing β-cells (Fig. 6C). Furthermore, similar results were found when RIP-HGF transgenic islet cell cultures were treated with fumonisin B1, an inhibitor of ceramide synthase (Fig. 6D). Collectively, these results indicate that HGF sensitizes β-cells to palmitate-induced apoptosis by a ceramide-mediated mechanism and confirms the functional role of SPT in this process.

Figure 6.

Figure 6

Open in a new tab

β-cell apoptosis in primary islet cell cultures from RIP-HGF transgenic mice and normal (NL) littermates incubated at 5 mm glucose and treated for 24 h with palmitate, myriocin, or fumonisin B1. Representative microphotographs of RIP-HGF primary islet cells treated with (A) 0.5 mm palmitate or (B) 0.5 mm palmitate plus 50 nm myriocin and stained for insulin (red), TUNEL (green), and Hoechst (blue). Arrows indicate TUNEL-positive β-cell nuclei. Quantitation of the number of TUNEL-positive β-cells in primary islet cell cultures treated with (C) 50 nm myriocin or (D) with 15 μm fumonisin B1. Values are means ± sem of at least four to six experiments in duplicate. *, P < 0.05 vs. NL treated with palmitate; #, P < 0.05 vs. RIP-HGF treated with palmitate and myriocin or fumonisin B1.

HGF enhances palmitate-mediated apoptosis in human β-cells: abrogation with myriocin and fumonisin B1

To determine whether HGF could also have a proapoptotic effect in human β-cells in the presence of palmitate, we overexpressed hHGF in primary cultures of human islet cells as previously reported (13). Transduction of human islets with Adv-hHGF resulted in marked overexpression of this growth factor compared with untransduced or Adv-LacZ transduced human islets (Fig. 7A). Interestingly, human islet cell cultures overexpressing HGF and treated with 0.5 mm palmitate displayed increased number of TUNEL-positive β-cell nuclei compared with Adv-LacZ-transduced human β-cells (Fig. 7, B–E). The increase in β-cell apoptosis induced by HGF overexpression in the presence of palmitate was significantly reduced after treatment with myriocin (Fig. 7D) or fumonisin B1 (Fig. 7E). Taken together, these results indicate that HGF is proapoptotic in both primary mouse and human β-cells in the presence of palmitate, potentially through increased expression of SPT and excessive de novo ceramide synthesis.

Figure 7.

Figure 7

Open in a new tab

β-cell apoptosis in primary human islet cell cultures transduced with Adv-LacZ as control or Adv-hHGF and treated with or without palmitate, myriocin, or fumonisin B1 for 24 h. A, Representative Western blot analysis of two different human islet preparations transduced with 500 MOI of Adv-LacZ, Adv-hHGF, or left untransduced (UT). In the right lane, 10 ng of recombinant hHGF peptide were used as positive control. Three different experiments with three different human islet preparations were performed with similar results. Representative microphotographs of primary human islet cell cultures transduced with (B) Adv-LacZ or (C) Adv-HGF and both treated with 0.5 mm palmitate and stained for insulin (red), TUNEL (green), and Hoechst (blue). Arrows indicate TUNEL-positive β-cell nuclei. Quantitation of the number of TUNEL-positive β-cells in human primary islet cell cultures transduced with Adv-LacZ or Adv-HGF and treated with or without 0.5 mm palmitate and (D) 50 nm myriocin or (E) 15 μm fumonisin B1. Values are means ± sem of four to six different islet cell cultures from four to six human islet preparations in duplicate. *, P < 0.05 vs. Adv-LacZ or Adv-HGF untreated; #, P < 0.05 vs. Adv-LacZ treated with palmitate; +, P < 0.05 vs. Adv-LacZ treated with palmitate and myriocin or fumonisin B1; ^, P < 0.05 vs. Adv-HGF treated with palmitate and myriocin or fumonisin B1.

Discussion

Present studies show that the in vivo beneficial effects of HGF overexpression in the β-cell in terms of glucose homeostasis, β-cell proliferation, and β-cell expansion in basal conditions disappear in a situation of obesity and insulin resistance induced by high-fat feeding. Furthermore, these studies clearly demonstrate a novel role for HGF in decreasing FFA oxidation, enhancing ceramide accumulation, and exacerbating apoptosis in rodent and human β-cells in a lipotoxic environment in vitro. Because circulating HGF levels are increased in obese subjects (5), it might be possible that HGF participates in the β-cell failure that leads to type 2 diabetes in obesity conditions.

Acute obesity-mediated insulin resistance is associated with increased β-cell proliferation and β-cell mass expansion to counter the decrease in peripheral insulin sensitivity (28). Chronic persistence of obesity-associated insulin resistance is thought to induce β-cell decompensation ending in β-cell failure and type 2 diabetes (29). Several genetic mouse models have shown that further enhancement of β-cell proliferation, survival, and concomitant β-cell expansion may improve glucose homeostasis in obesity-related and insulin resistant conditions (30,31). We have previously shown that 2- to 3-month-old RIP-HGF transgenic mice fed a SD, and now in approximately 6-month-old RIP-HGF transgenic mice on the same diet, display improved glucose homeostasis, increased β-cell proliferation, and augmented β-cell mass (9,10). We and others have also shown that HGF actions in the β-cell can improve β-cell survival after islet transplantation and increase β-cell mass and circulating plasma insulin in situations of diminished β-cell mass induced by streptozotocin administration (9,10,12,13,14,15,16). With this in mind, we sought to determine whether RIP-HGF transgenic mice would display improved glucose and β-cell homeostasis in a situation of obesity-related insulin resistance. Against our expectations, RIP-HGF transgenic mice fed with a HFD display similar blood glucose, plasma insulin, glucose tolerance, β-cell proliferation, and β-cell mass to normal littermates when fed a HFD. These data indicate that HGF overexpression in the β-cell does not confer any advantage in glucose and β-cell homeostasis in obesity-related insulin resistance. Nevertheless, it cannot be ruled out that HGF overexpression in the β-cell could confer an advantage in glucose and β-cell homeostasis when RIP-HGF transgenic mice are fed with HFD containing lower amounts of fat.

There are at least two possibilities that could potentially explain why HGF overexpression does not induce a further increase in β-cell proliferation under a HFD. First, it has been reported that FFA activate PKCζ in vitro and we have recently shown that HGF requires the activation of the phosphatidylinositol-3 kinase-PKCζ signaling pathway for increasing β-cell proliferation (11,20). Therefore, it could be possible that HGF and HFD-induced obesity and insulin resistance use this common intracellular signaling pathway to increase β-cell proliferation. Our in vitro experiments in RIP-HGF transgenic and normal mouse primary β-cells indicate that palmitate does not further enhance HGF-mediated β-cell proliferation and that PKCζ activation is required for palmitate- and HGF-induced β-cell proliferation. These studies suggest that the lack of enhanced β-cell proliferation in RIP-HGF transgenic mice fed a HFD could be the result of common activation of the PKCζ signaling pathway. Second, it is possible that lipids negatively regulate HGF signaling. It has been shown that FFAs interfere with the mitogenic signals of growth factors in β-cells via novel PKC activation (32). Novel PKCs are known to associate with the HGF receptor, c-met, to induce its phosphorylation in Ser-985 and to abrogate the cellular responsiveness to HGF (33). Indeed, we have recently shown that inhibition of novel PKCs increases HGF-mediated β-cell proliferation (11). Therefore, it could be possible that the potential activation of PKCs by the lipid oversupply after high-fat feeding may be responsible for the absence of further increase in β-cell proliferation in RIP-HGF transgenic mice on HFD.

We have previously shown that 2- to 3-month-old RIP-HGF transgenic mice display a 2- to 3-fold increase in β-cell mass when fed a SD (9). However, β-cell mass in RIP-HGF transgenic mice is not significantly increased after a 15-wk HFD compared with normal littermates. Because β-cell proliferation is similar in both types of mice on HFD, these data suggest a potential increase in β-cell death in transgenic mice that could mediate the disappearance of the 2- to 3-fold increase in β-cell mass present at the time of starting the high-fat feeding. Analysis of the number of condensed β-cell nuclei in mice fed with HFD shows an increase in β-cell death in HGF-overexpressing islets, although this increase is not significant possibly due to the small number of mice measured, the relative short duration of HFD administration, and the efficient elimination of dead β-cells from the islets that makes its detection difficult.

HGF has been shown to efficiently induce survival of multiple cell types, including β-cells, in the presence of various cell death inducers through the activation of signaling pathways mainly involving phosphatidylinositol-3 kinase/Akt activation (9,10,12,13,14,15,16,34). However, contrary to our expectation, in the current in vivo study, HGF-overexpression in the islet potentially facilitates β-cell death when the transgenic mice are on HFD. To analyze in more detail whether this is the case, we performed in vitro experiments in mouse and human primary β-cells. Indeed, the results included here clearly show that HGF exacerbates the apoptotic effect of palmitate in β-cells. Although this proapoptotic effect of HGF for the β-cell may look paradoxical, HGF has been reported to induce or facilitate apoptosis in other cell types (35). In fact, HGF was first described as a cytotoxic factor for different tumor cell lines through apoptotic and cytostatic mechanisms, although the signaling targets involved still remain unknown (35). Furthermore, c-met activation by HGF was found to liberate Fas from its interaction with c-met in liver and endothelial cells, which sensitizes these cells to Fas-mediated apoptosis (36,37). The current study adds new knowledge to the cellular responses to HGF because this growth factor can act as an antiapoptotic or proapoptotic agent in the same cell, i.e. the β-cell, depending on the cellular environment.

A recent report has shown that HGF can protect rat insulinoma RINm5F cells against chronic treatment with FFA (17). The differences between that other report and our studies may lie in the type of β-cell (insulinoma vs. normal primary β-cell) and the type of FFA (oleate + palmitate vs. palmitate alone) used in the experiments. Rodent insulinoma cells are normally grown in high glucose concentrations and display abnormal glucose metabolism compared with normal β-cells (38,39). This may lead to differences in FFA oxidation and lipid metabolism regulation when incubated with FFA. These potential differences in lipid metabolism between clonal cells and primary β-cells may explain their different sensitivity to the cytotoxic effects to FFA and HGF. Furthermore, it is known that saturated fatty acids induce β-cell apoptosis in vitro, (2,40,41), whereas unsaturated fatty acids are usually protective (2,40,41,42). It could be possible that the combination of HGF and oleate further protect RINm5F cells against the proapoptotic effects of palmitate as reported in the study by Santangelo et al. (17). In our hands, we did not find any change in cell viability when rat insulinoma INS-1 cells or mouse insulinoma MIN6 cells were incubated with HGF and palmitate (data not shown), indicating the importance of performing these studies in primary β-cells.

Elevations of plasma fatty acids frequently observed in obese/insulin-resistant conditions and the alteration of lipid partitioning and accumulation of ceramide have been proposed as events involved in the diminution of β-cell mass in type 2 diabetes (1,40). Thus, we wondered whether HGF might have any impact on FAO and ceramide formation in β-cells. HGF-overexpressing islets displayed markedly and significantly decreased FAO, similar to the effect recently observed in skeletal muscle cells (22). HGF potently increases glucose uptake, glucose transporters expression, and glucose utilization in various cell types, including pancreatic β-cells (10,22,43). AMPK is a metabolic sensor that balances energy production with the metabolic demands in eukaryotic cells. Among its functions, AMPK controls a number of ATP generating catabolic pathways including FAO (44). Malonyl-CoA is an allosteric inhibitor of CPT-1, the enzyme that controls the transfer of long-chain fatty acyl-CoAs into the mitochondria where they are oxidized. The formation of malonyl-CoA is regulated by changes in the activity of ACC and AMPK phosphorylates ACC and inhibits both its basal activity and activation by citrate. Interestingly, HGF-overexpressing islets display decreased levels of phosphorylated AMPK and its downstream target ACC. Because CPT-1 levels are normal in HGF-overexpressing islets, one potential explanation for the decreased FAO is that malonyl-CoA levels are increased in transgenic cells inhibiting CPT-1 activity (45). As FAO inhibition by HGF is observed only at 16 mm glucose, it is possible that the decrease in ACC phosphorylation in RIP-HGF transgenic islets is not enough to generate sufficient malonyl-CoA at low glucose concentrations to significantly inhibit FAO. Alterations in FAO could lead to changes in lipid partitioning in the RIP-HGF transgenic β-cells.

The two components of SPT, the enzyme that catalyzes the first step of the de novo synthesis of ceramide, are significantly up-regulated in HGF-overexpressing islets. The decrease in FAO and the increase in the expression of SPT in HGF-overexpressing islets could lead to the increase in intracellular accumulation of ceramide, a known mediator of β-cell death (1). Furthermore, palmitate has been shown to decrease Akt activation and Bad phosphorylation, two prosurvival signals for the β-cell (46,47). Importantly, palmitate markedly and significantly eliminated the activation of Akt signaling pathway induced by HGF. This decrease in prosurvival signals could make β-cells more vulnerable to apoptosis mediated by the accumulation of ceramide induced by HGF in these cells. Indeed this is the case. Inhibition of the de novo pathway of ceramide synthesis with myriocin or fumonisin B1 blocked the apoptosis induced by palmitate in HGF-overexpressing islets, suggesting that the impaired Akt activation, the dysregulation in lipid partitioning, and the increase in ceramide formation in these islets can lead to the hypersensitivity to lipotoxicity (Fig. 8).

Figure 8.

Figure 8

Open in a new tab

Schematic representation depicting the effects of HGF/c-met actions on glycolysis (10), phosphorylation of Akt, Bad, AMPK and ACC, FFA oxidation, SPT up-regulation, ceramide accumulation, and apoptosis in the β-cell in the presence of an excess of palmitate in the extracellular milieu. Glut-2, Glucose transporter-2; CL, citrate lyase; TG, triglycerides; DAG, diacylglycerols.

The results included in the current manuscript indicate that human β-cells are more sensitive to palmitate-induced apoptosis than mouse islets, because 0.5 mm palmitate induced β-cell apoptosis in human, but not in mouse islet cell cultures when both were incubated at 5 mm glucose. This phenomena has been previously observed, the effect in human β-cells being partially-dependent on ceramide synthesis (2,3). Importantly, human β-cell apoptosis induced by palmitate and 5 mm glucose was enhanced by HGF and decreased by myriocin and fumonisin B1. These results indicate that the proapoptotic effect of HGF is not species specific and requires increased ceramide synthesis in human β-cells as well. These studies suggest that the increased circulating levels of HGF found in human subjects with metabolic syndrome and obesity might participate in the deterioration of the β-cell mass that could potentially emerge in these situations (5,6).

Collectively, these studies indicate that HGF is a novel proapoptotic factor for the murine, and more importantly human, β-cell in an environment rich in saturated FFA through the alteration of FAO and the increase of ceramide formation. These results suggest a potential regulatory effect of HGF in β-cell survival in situations of human obesity.

Supplementary Material

[Supplemental Data]
en.2009-0975_index.html (795B, html)

Acknowledgments

We thank Dr. Andrew F. Stewart and Laura C. Alonso for thoughtful discussions of the ideas in this article, Jennifer Roccisana for superb technical assistance, and the Islet Cell Resource Center and Juvenile Diabetes Research Foundation Basic Science Islet Distribution Programs for providing us with human islets.

Footnotes

This work was supported by National Institutes of Health Grants DK067351 and DK077096 (to A.G.-O.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online February 22, 2010

Abbreviations: ACC, Acetyl-CoA carboxylase; Adv-LacZ, adenovirus containing ß-galactosidase; AMPK, AMP-activated protein kinase; BrdU, 5-bromo-2′-deoxyuridine; CoA, coenzyme A; CPT-1, carnitine palmitoyltransferase 1; FAO, fatty acid oxidation; FFA, free fatty acids; HFD, high-fat diet; HGF, hepatocyte growth factor; hHGF, human HGF; IE, islet equivalent; ITT, insulin tolerance test; KRBB, Krebs-Ringer bicarbonate buffer; MOI, multiplicity of infection; PI, propidium iodide; PKC, protein kinase C; RIP, rat insulin type II promoter; SD, standard diet; SPT, serine palmitoyltransferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; wt, weight.

References

  1. Shimabukuro M, Zhou YT, Levi M, Unger RH 1998 Fatty acid-induced β cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci USA 95:2498–2502 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, Santangelo C, Patané G, Boggi U, Piro S, Anello M, Bergamini E, Mosca F, Di Mario U, Del Prato S, Marchetti P 2002 Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that β-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 51:1437–1442 [DOI] [PubMed] [Google Scholar]
  3. El-Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L, Prentki M 2003 Saturated fatty acids synergize with elevated glucose to cause pancreatic β-cell death. Endocrinology 144:4154–4163 [DOI] [PubMed] [Google Scholar]
  4. Sol EM, Sargsyan E, Akusjärvi G, Bergsten P 2008 Glucolipotoxicity in INS-1E cells is counteracted by carnitine palmitoyltransferase 1 over-expression. Biochem Biophys Res Commun 375:517–521 [DOI] [PubMed] [Google Scholar]
  5. Rehman J, Considine RV, Bovenkerk JE, Li J, Slavens CA, Jones RM, March KL 2003 Obesity is associated with increased levels of circulating hepatocyte growth factor. J Am Coll Cardiol 41:1408–1413 [DOI] [PubMed] [Google Scholar]
  6. Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW 2004 Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 145:2273–2282 [DOI] [PubMed] [Google Scholar]
  7. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S 1989 Molecular cloning and expression of human hepatocyte growth factor. Nature 342:440–443 [DOI] [PubMed] [Google Scholar]
  8. 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 [DOI] [PubMed] [Google Scholar]
  9. Garcia-Ocaña A, Takane KK, Syed MA, Philbrick WM, Vasavada RC, Stewart AF 2000 Hepatocyte growth factor overexpression in the islet of transgenic mice increases β cell proliferation, enhances islet mass, and induces mild hypoglycemia. J Biol Chem 275:1226–1232 [DOI] [PubMed] [Google Scholar]
  10. García-Ocaña A, Vasavada RC, Cebrian A, Reddy V, Takane KK, López-Talavera JC, Stewart AF 2001 Transgenic overexpression of hepatocyte growth factor in the β-cell markedly improves islet function and islet transplant outcomes in mice. Diabetes 50:2752–2762 [DOI] [PubMed] [Google Scholar]
  11. Vasavada RC, Wang L, Fujinaka Y, Takane KK, Rosa TC, Mellado-Gil JM, Friedman PA, Garcia-Ocaña A 2007 Protein kinase C-ζ activation markedly enhances β-cell proliferation: an essential role in growth factor mediated β-cell mitogenesis. Diabetes 56:2732–2743 [DOI] [PubMed] [Google Scholar]
  12. Dai C, Li Y, Yang J, Liu Y 2003 Hepatocyte growth factor preserves β cell mass and mitigates hyperglycemia in streptozotocin-induced diabetic mice. J Biol Chem 278:27080–27087 [DOI] [PubMed] [Google Scholar]
  13. Garcia-Ocana A, Takane KK, Reddy VT, Lopez-Talavera JC, Vasavada RC, Stewart AF 2003 Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces β cell death. J Biol Chem 278:343–351 [DOI] [PubMed] [Google Scholar]
  14. Nakano M, Yasunami Y, Maki T, Kodama S, Ikehara Y, Nakamura T, Tanaka M, Ikeda S 2000 Hepatocyte growth factor is essential for amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving a marginal mass of intrahepatic islet grafts. Transplantation 69:214–221 [DOI] [PubMed] [Google Scholar]
  15. Lopez-Talavera JC, Garcia-Ocaña A, Sipula I, Takane KK, Cozar-Castellano 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 allogeneic portal vein islet transplantation. Endocrinology 145:467–474 [DOI] [PubMed] [Google Scholar]
  16. Fiaschi-Taesch NM, Berman DM, Sicari BM, Takane KK, Garcia-Ocaña A, Ricordi C, Kenyon NS, Stewart AF 2008 Hepatocyte growth factor enhances engraftment and function of nonhuman primate islets. Diabetes 57:2745–2754 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Santangelo C, Matarrese P, Masella R, Di Carlo MC, Di Lillo A, Scazzocchio B, Vecci E, Malorni W, Perfetti R, Anastasi E 2007 Hepatocyte growth factor protects rat RINm5F cell line against free fatty acid-induced apoptosis by counteracting oxidative stress. J Mol Endocrinol 38:147–158 [DOI] [PubMed] [Google Scholar]
  18. Roccisana J, Reddy V, Vasavada RC, Gonzalez-Pertusa JA, Magnuson MA, Garcia-Ocaña A 2005 Targeted inactivation of hepatocyte growth factor receptor c-met in β-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of β-cell mass. Diabetes 54:2090–2102 [DOI] [PubMed] [Google Scholar]
  19. Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ, Poitout V 2003 Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis. J Biol Chem 278:30015–30021 [DOI] [PubMed] [Google Scholar]
  20. Cousin SP, Hügl SR, Wrede CE, Kajio H, Myers Jr MG, Rhodes CJ 2001 Free fatty acid-induced inhibition of glucose and insulin-like growth factor I-induced deoxyribonucleic acid synthesis in the pancreatic β-cell line INS-1. Endocrinology 142:229–240 [DOI] [PubMed] [Google Scholar]
  21. Becker TC, Noel RJ, Coats WS, Gómez-Foix AM, Alam T, Gerard RD, Newgard CB 1994 Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 43:161–189 [DOI] [PubMed] [Google Scholar]
  22. Perdomo G, Martinez-Brocca MA, Bhatt BA, Brown NF, O'Doherty RM, Garcia-Ocaña A 2008 Hepatocyte growth factor is a novel stimulator of glucose uptake and metabolism in skeletal muscle cells. J Biol Chem 283:13700–13706 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nolan CJ, Leahy JL, Delghingaro-Augusto V, Moibi J, Soni K, Peyot ML, Fortier M, Guay C, Lamontagne J, Barbeau A, Przybytkowski E, Joly E, Masiello P, Wang S, Mitchell GA, Prentki M 2006 β Cell compensation for insulin resistance in Zucker fatty rats: increased lipolysis and fatty acid signaling. Diabetologia 49:2120–2130 [DOI] [PubMed] [Google Scholar]
  24. Dube JJ, Bhatt BA, Dedousis N, Bonen A, O'Doherty RM 2007 Leptin, skeletal muscle lipids, and lipid-induced insulin resistance. Am J Physiol Regul Integr Comp Physiol 293:R642–R650 [DOI] [PubMed] [Google Scholar]
  25. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, Bergeron R, Kim JK, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, Shulman GI 2002 Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 277:50230–50236 [DOI] [PubMed] [Google Scholar]
  26. Arner P 2002 Insulin resistance in type 2 diabetes: role of fatty acids. Diabetes Metab Res Rev 18(Suppl 2):S5–S9 [DOI] [PubMed] [Google Scholar]
  27. Linn SC, Kim HS, Keane EM, Andras LM, Wang E, Merrill AH Jr 2001 Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption. Biochem Soc Trans 29:831–835 [DOI] [PubMed] [Google Scholar]
  28. Unger RH 1995 Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Genetic and clinical implications Diabetes 44:863–870 [DOI] [PubMed] [Google Scholar]
  29. Chang-Chen KJ, Mullur R, Bernal-Mizrachi E 2008 β-Cell failure as a complication of diabetes. Rev Endocr Metab Disord 9:329–343 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Uchida T, Nakamura T, Hashimoto N, Matsuda T, Kotani K, Sakaue H, Kido Y, Hayashi Y, Nakayama KI, White MF, Kasuga M 2005 Deletion of Cdkn1b ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia in diabetic mice. Nat Med 11:175–182 [DOI] [PubMed] [Google Scholar]
  31. Tanabe K, Liu Z, Patel S, Doble BW, Li L, Cras-Méneur C, Martinez SC, Welling CM, White MF, Bernal-Mizrachi E, Woodgett JR, Permutt MA 2008 Genetic deficiency of glycogen synthase kinase-3β corrects diabetes in mouse models of insulin resistance. PLoS Biol 6:e37 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wrede CE, Dickson LM, Lingohr MK, Briaud I, Rhodes CJ 2003 Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic β-cells (INS-1). J Mol Endocrinol 30:271–286 [DOI] [PubMed] [Google Scholar]
  33. Hashigasako A, Machide M, Nakamura T, Matsumoto K, Nakamura T 2004 Bi-directional regulation of Ser-985 phosphorylation of c-met via protein kinase C and protein phosphatase 2A involves c-Met activation and cellular responsiveness to hepatocyte growth factor. J Biol Chem 279:26445–26452 [DOI] [PubMed] [Google Scholar]
  34. Tulasne D, Foveau B 2008 The shadow of death on the MET tyrosine kinase receptor. Cell Death Differ 15:427–434 [DOI] [PubMed] [Google Scholar]
  35. Higashio K, Shima N, Goto M, Itagaki Y, Nagao M, Yasuda H, Morinaga T 1990 Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor. Biochem Biophys Res Commun 170:397–404 [DOI] [PubMed] [Google Scholar]
  36. Wang X, DeFrances MC, Dai Y, Pediaditakis P, Johnson C, Bell A, Michalopoulos GK, Zarnegar R 2002 A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 9:411–421 [DOI] [PubMed] [Google Scholar]
  37. Smyth LA, Brady HJ 2005 cMet and Fas receptor interaction inhibits death-inducing signaling complex formation in endothelial cells. Hypertension 46:100–106 [DOI] [PubMed] [Google Scholar]
  38. Trautmann ME, Wollheim CB 1987 Characterization of glucose transport in an insulin-secreting cell line. Biochem J 242:625–630 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vischer U, Blondel B, Wollheim CB, Höppner W, Seitz HJ, Iynedjian PB 1987 Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells. Biochem J 241:249–255 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Poitout V, Robertson RP 2008 Glucolipotoxicity: fuel excess and β-cell dysfunction. Endocr Rev 29:351–366 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY 2003 Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic β-cell turnover and function. Diabetes 52:726–733 [DOI] [PubMed] [Google Scholar]
  42. Maedler K, Spinas GA, Dyntar D, Moritz W, Kaiser N, Donath MY 2001 Distinct effects of saturated and monounsaturated fatty acids on β-cell turnover and function. Diabetes 50:69–76 [DOI] [PubMed] [Google Scholar]
  43. Bertola A, Bonnafous S, Cormont M, Anty R, Tanti JF, Tran A, Le Marchand-Brustel Y, Gual P 2007 Hepatocyte growth factor induces glucose uptake in 3T3-L1 adipocytes through A Gab1/phosphatidylinositol 3-kinase/Glut4 pathway. J Biol Chem 282:10325–10332 [DOI] [PubMed] [Google Scholar]
  44. Winder WW, Hardie DG 1999 AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 277:E1–E10 [DOI] [PubMed] [Google Scholar]
  45. McGarry JD, Brown NF 1997 The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem 244:1–14 [DOI] [PubMed] [Google Scholar]
  46. Martinez SC, Tanabe K, Cras-Méneur C, Abumrad NA, Bernal-Mizrachi E, Permutt MA 2008 Inhibition of Foxo1 protects pancreatic islet β-cells against fatty acid and endoplasmic reticulum stress-induced apoptosis. Diabetes 57:846–859 [DOI] [PubMed] [Google Scholar]
  47. Choi SE, Kim HE, Shin HC, Jang HJ, Lee KW, Kim Y, Kang SS, Chun J, Kang Y 2007 Involvement of Ca2+-mediated apoptotic signals in palmitate-induced MIN6N8a β cell death. Mol Cell Endocrinol 272:50–62 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]
en.2009-0975_index.html (795B, html)
en.2009-0975_1.pdf (45.8KB, pdf)

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES