ORIGINAL

RESEARCH

Hepatocyte Growth Factor Ameliorates Hyperglycemia and Corrects ␤-Cell Mass in IRS2-Deficient Mice Juan C. Alvarez-Perez, Taylor C. Rosa, Gabriella P. Casinelli, Shelley R. Valle, Jayalakshmi Lakshmipathi, Carolina Rosselot, Francisco Rausell-Palamos, Rupangi C. Vasavada, and Adolfo García-Ocaña Diabetes, Obesity and Metabolism Institute (J.C.A.-P., J.L., C.R., F.R.-P., R.C.V., A.G.-O.), Division of Endocrinology, Diabetes and Bone Diseases, The Mindich Child Health and Development Institute, Icahn School of Medicine at Mt Sinai, New York, New York 10029; and Department of Medicine (T.C.R., G.P.C., S.R.V.), Division of Endocrinology and Metabolism, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Insulin resistance, when combined with decreased ␤-cell mass and relative insufficient insulin secretion, leads to type 2 diabetes. Mice lacking the IRS2 gene (IRS2⫺/⫺ mice) develop diabetes due to uncompensated insulin resistance and ␤-cell failure. Hepatocyte growth factor (HGF) activates the phosphatidylinositol 3-kinase/Akt signaling pathway in ␤-cells without recruitment of IRS1 or IRS2 and increases ␤-cell proliferation, survival, mass, and function when overexpressed in ␤-cells of transgenic (TG) mice. We therefore hypothesized that HGF may protect against ␤-cell failure in IRS2 deficiency. For that purpose, we cross-bred TG mice overexpressing HGF in ␤-cells with IRS2 knockout (KO) mice. Glucose homeostasis analysis revealed significantly reduced hyperglycemia, compensatory hyperinsulinemia, and improved glucose tolerance in TG/KO mice compared with those in KO mice in the context of similar insulin resistance. HGF overexpression also increased glucose-stimulated insulin secretion in IRS2⫺/⫺ islets. To determine whether this glucose homeostasis improvement correlated with alterations in ␤-cells, we measured ␤-cell mass, proliferation, and death in these mice. ␤-Cell proliferation was increased and death was decreased in TG/KO mice compared with those in KO mice. As a result, ␤-cell mass was significantly increased in TG/KO mice compared with that in KO mice, reaching levels similar to those in wild-type mice. Analysis of the intracellular targets involved in ␤-cell failure in IRS2 deficiency showed Pdx-1 up-regulation, Akt/FoxO1 phosphorylation, and p27 down-regulation in TG/KO mouse islets. Taken together, these results indicate that HGF can compensate for IRS2 deficiency and subsequent insulin resistance by normalizing ␤-cell mass and increasing circulating insulin. HGF may be of value as a therapeutic agent against ␤-cell failure. (Molecular Endocrinology 28: 2038 –2048, 2014)

ype 2 diabetes (T2D) results from combined defects in insulin action and secretion. Although the search for agents that can increase ␤-cell function is of great importance for treating T2D, the relentless decline in ␤-cell mass highlights the need for therapies that can also protect and expand ␤-cells in this disease. Mouse genetic

T

models have shown that the insulin receptor substrates (IRSs) participate through distinct biological actions in the response of the ␤-cell to insulin resistance (1). IRS1 participates in somatic growth and mediates insulin action in skeletal muscle, but IRS1 knockout (KO) mice do not develop diabetes because of a remarkable compensa-

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received July 8, 2014. Accepted October 22, 2014. First Published Online October 31, 2014

Abbreviations: Adv, adenovirus; BrdU, 5-bromo-2-deoxyuridine; DAPI, 4⬘,6-diamidino-2phenylindole; FoxO1, forkhead box protein O1; GFP, green fluorescent protein; GSIS, glucose-stimulated insulin secretion; GSK3, glycogen synthase kinase 3; HGF, hepatocyte growth factor; IRS, insulin receptor substrate; ITT, insulin tolerance test; KO, knockout; Pdx-1, pancreatic and duodenal homeobox 1; PI3K, phosphatidylinositol 3-kinase; PKC␨, protein kinase C␨; RIP, rat insulin type II promoter; T2D, type 2 diabetes; TG, transgenic; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling.

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doi: 10.1210/me.2014-1207

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tory ␤-cell mass expansion and hyperinsulinemia (2). On the other hand, IRS2 regulates hepatic gluconeogenesis and lipid metabolism, and its absence leads to hepatic insulin resistance (3). However, in IRS2 KO mice, there is no compensatory ␤-cell response and IRS2 deficiency leads to ␤-cell failure, further contributing to diabetes development in these mice. The cellular mechanisms involved in the demise of ␤-cells in IRS2 KO mice include increased apoptosis and decreased proliferation (3–7). At a molecular level, dysregulation of AKT/glycogen synthase kinase 3 (GSK3)/forkhead box protein O1 (FoxO1) signaling, down-regulation of pancreatic and duodenal homeobox 1 (Pdx-1), and up-regulation of p27 participate in the ␤-cell failure observed in these mice (4 –7). Hepatocyte growth factor (HGF) is a ␤-cell mitogen and prosurvival factor involved in ␤-cell expansion in physiologic and pathologic situations (8 –13). HGF is essential for ␤-cell expansion during pregnancy, with obesity, and after partial pancreatectomy (12–14). HGF is also required for ␤-cell survival during pregnancy and in a surrogate model of autoimmune type 1 diabetes in mice (11, 12). In addition, overexpression of HGF in the ␤-cells of transgenic (TG) mice by means of the rat insulin type II promoter (RIP) increases ␤-cell survival, proliferation, and mass (8 –10, 15–18). Upon binding of HGF, c-Met activates phosphatidylinositol 3-kinase (PI3K) in ␤-cells, but the participation of IRS proteins in this activation is unclear (19). HGF induces its proliferative and prosurvival effects in ␤-cells through the activation of the PI3K/ atypical protein kinase C␨ (PKC␨)/Akt (10, 18). Taken together, these studies highlight the potential therapeutic effects of HGF for the treatment of diabetes. However, whether IRS2 is involved in the beneficial effects of HGF in ␤-cells is unknown. Furthermore, whether HGF can ameliorate hyperglycemia and ␤-cell failure in insulinresistant states is uncertain. To address these points, we combined RIP-HGF transgenic (TG) mice and IRS2-deficient mice and analyzed their phenotype. HGF overexpression remarkably decreased blood glucose levels, enhanced plasma insulin, and normalized ␤-cell mass in the absence of IRS2. Akt and FoxO1 phosphorylation and Pdx-1 and p27 levels were normalized in islets overexpressing HGF and deficient in IRS2, indicating that HGF might be of therapeutic use against ␤-cell failure.

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crossed with IRS2⫹/⫺ mice maintained on a mixed background (C57BL6 ⫻ 129Sv) (The Jackson Laboratory) (3). Mice that were RIP-HGF-IRS2⫹/⫺ in F1 were crossed again with IRS2⫹/⫺ mice to obtain RIP-HGF-IRS2⫺/⫺ mice (named TG/KO mice) in F2. Only mice from F2 were used for these studies. Mouse progeny was obtained at the expected Mendelian frequency. The other genotypes from F2 used in this study were wild-type mice for both genes (WT), IRS2⫺/⫺ mice that did not carry the RIPHGF transgene (KO), and RIP-HGF TG mice that were WT for IRS2 (TG). IRS2⫹/⫺ mice that were TG or WT were not used in this study. Male mice at 6 to 12 weeks of age were phenotypically analyzed with the approval of and in accordance with the guidelines established by the Icahn School of Medicine at Mount Sinai and the University of Pittsburgh institutional animal care and use committees.

Glucose homeostasis analysis Blood glucose and plasma insulin were measured as described previously (8). For the intraperitoneal glucose tolerance tests, mice were fasted overnight, blood glucose was measured, and then mice were injected ip with glucose (2 g/kg) (9). Blood glucose was measured at 15, 30, 60, and 120 minutes after injection. Insulin tolerance tests (ITTs) were performed in nonfasted mice injected ip with human insulin (1.5 U/kg) (20). Blood glucose was measured at 0, 15, 30, and 60 minutes postinjection.

Immunohistochemistry, pancreas histomorphometry, and ␤-cell proliferation and death measurements ␤-Cell mass and islet number were measured in 3 insulinstained pancreas sections per mouse using ImageJ (National Institutes of Health) as described previously (13). 5-Bromo-2deoxyuridine (BrdU) incorporation in ␤-cells was measured in pancreatic sections from mice injected ip with BrdU (Amersham, Piscataway, NJ), killed 6 hours later, and stained for insulin and BrdU (13). Sections were also stained for Ki67 and insulin as an additional method to detect ␤-cell proliferation (13). ␤-Cell death was determined in pancreas sections stained for insulin using the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method (Promega) (12). Immunostainings with antibodies for Pdx-1 and IRS2 (Santa Cruz Biotechnology), HGF (R&D Systems), p27 (BD Pharmingen), Ki67 (Thermo Fisher Scientific), and insulin (DAKO) were performed using methods described previously (12, 13).

Islet isolation and Western blot analysis

Materials and Methods

Mouse islets were isolated after collagenase P injection through the pancreatic duct (8). Mouse islet extracts were analyzed by Western blotting with antibodies against IRS2, Pdx-1, and p85-PI3K (Santa Cruz Biotechnology), phospho-Ser473Akt, phospho-Ser256-FoxO1, and p27 (Cell Signaling, Danvers, MA), and actin (Sigma-Aldrich). After several washes, blots were incubated with peroxidase-conjugated secondary antibodies followed by chemiluminescence detection (13).

Generation of mice

Glucose-stimulated insulin secretion (GSIS)

RIP-HGF TG mice were generated and maintained on a CD-1 background as described previously (8). TG mice were

Insulin release from 20 islets of similar sizes per condition was measured as described previously (9). Islets were obtained

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from female WT and IRS2 KO mice and transduced with 250 multiplicity of infection of adenovirus (Adv) control containing green fluorescent protein (Adv.GFP) or HGF (Adv.HGF), as reported previously (10). Twenty-four hours after infection, islets were incubated in 5 or 20 mM glucose for 30 minutes, buffer was removed and frozen at ⫺20°C until insulin determination, islets were washed, and insulin content was extracted in acidethanol as described previously (8). Results are expressed as the percentage of insulin secreted per islet insulin content by WT islets transduced with Adv.GFP at 5 mM glucose.

INS-1 cell culture, real-time PCR, and immunoprecipitation assay INS-1 cells (107 cells/plate) were cultured in RPMI medium (18) with 10% fetal bovine serum overnight, and then serum was depleted for 6 h followed by incubation with 25 ng/mL recombinant human HGF (Research Diagnostics, Inc) or 10⫺7 M recombinant human IGF-1 (Sigma-Aldrich) for 10 minutes. Proteins were extracted in radioimmunoprecipitation assay buffer, and immunoprecipitation experiments were performed with 0.5 to 1 mg of protein incubated overnight at 4°C with the antibody against p85-PI3K (Millipore) and then with protein G-agarose (Thermo Fisher) for 4 hours before resuspension in Laemmli buffer and separation by SDS-PAGE (21). In a different set of experiments, INS-1 cells were incubated in serum-free RPMI medium with 25 ng/mL HGF for 24 h, and protein extracts and total RNA were obtained following previously described protocols (13, 22). Then, Western blot and real-time PCR analyses were performed to determine p27 protein and mRNA expression levels, respectively (13, 22).

Statistical analysis The data are presented as means ⫾ SE. Multiple comparisons were tested by one-way ANOVA followed by the Tukey post hoc test (http://statistica.mooo.com/). For comparison between 2 groups, the unpaired, two-tailed Student t test was used (indicated in the figure legends). A value of P ⱕ .05 was considered statistically significant.

Results HGF activates AKT in islets with IRS2 deficiency HGF has been shown to modulate ␤-cell proliferation and survival in vivo and in vitro through activation of the PI3K/atypical PKC␨/Akt pathway (10, 18). However, whether activation of IRS2 is involved in these effects is unknown. As shown in Figure 1A, HGF at a dose that increases PI3K activity in INS-1 cells (10) did not significantly induce the binding of PI3K to IRS2, suggesting that HGF activates PI3K without IRS2 binding in these cells. This result was in striking contrast with the effect of IGF-1 (Figure 1A). To further address whether HGF can stimulate the PI3K/AKT signaling pathway without the presence of IRS2, we incubated islets from IRS2-deficient mice with HGF protein. HGF was able to increase

Figure 1. HGF activates Akt in the absence of IRS2. A, Coimmunoprecipitation of PI3K and IRS2 in INS-1 cells after stimulation with 25 ng/mL HGF or 0.1 ␮M IGF-1 or left untreated. INS1 cells were serum depleted for 6 hours and then were incubated with HGF or IGF-1 for 10 minutes. PI3K was immunoprecipitated as indicated in the Materials and Methods, and immunoblotting (IB) was performed against IRS2 and PI3K. Densitometric analysis was performed using ImageJ and quantitation appears in the bottom graph. Results are means ⫾ SEM of 4 experiments. *, P ⬍ .05 vs. untreated (Unt) or HGF-treated cells. Notice the increase in IRS2/PI3K coimmunoprecipitation with IGF-1 but not with HGF. B, Western blot analysis of protein extracts from WT and IRS2 KO islets treated with 25 ng/mL HGF for 10 minutes or left untreated. Islets were serum depleted for 6 hours and then were treated with HGF. Immunoblots were performed against phospho-Ser473-AKT and actin as a housekeeping protein to control for loading. Densitometry of 3 different blots was performed using ImageJ, and ratios of the phospho-AKT against actin are represented on the bottom graph. Results are means ⫾ SEM. *, P ⬍ .05 vs control (C) untreated.

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AKT phosphorylation similarly in the absence or presence of IRS2 (Figure 1B).

Figure 2. Glucose homeostasis in RIP-HGF TG mice with IRS2 deficiency. TG/KO mice displayed significantly increased body weight (A), significantly decreased nonfasting blood glucose (B), and significantly increased plasma insulin (C) at 12 weeks of age compared with those of KO mice. Results are means ⫾ SEM and were obtained from 5 to 7 mice per genotype. *, P ⬍ .05 vs the other 3 groups of mice; #, P ⬍ .05 vs WT and TG. D–F, intraperitoneal glucose tolerance test (D), area under the curve (AUC) calculated from these experiments (E), and ITT represented as percentage of the blood glucose value at time 0 minutes (F). Blood glucose values at time 0 minutes in the ITT experiment were as follows: WT mice, 151 ⫾ 4; KO mice, 490 ⫾ 18; TG mice, 134 ⫾ 8; and TG/KO mice, 236 ⫾ 16 mg/dL. Results are means ⫾ SEM for WT (n ⫽ 7), TG (n ⫽ 6), KO (n ⫽ 6), and TG/KO (n ⫽ 5) mice at 12 weeks of age. *, P ⬍ .05 vs the other 3 groups of mice; #, P ⬍ .05 vs WT and TG; ∧, P ⬍ .05 vs WT. G, GSIS performed in groups of 20 islets of similar sizes obtained from female WT (n ⫽ 4) and IRS2 KO (n ⫽ 4) mice, transduced with Adv.GFP or Adv.HGF and incubated for 30 minutes with 5 or 20 mM glucose. Experiments were performed in triplicate. Results are means ⫾ SEM. *, P ⬍ .05 vs 5 mM glucose; #, P ⬍ .05 vs the same genotype-GFP at 20 mM glucose.

Generation of IRS2 KO mice with overexpression of HGF in the ␤-cell IRS2-deficient mice display decreased PI3K/Akt signaling pathway activation, insulin resistance, and ␤-cell failure (3–7). Because HGF is capable of activating Akt in the absence of IRS2 (Figure 1B), we wondered whether overexpression of HGF in the ␤-cell might have beneficial effects on preserving ␤-cell mass and improving glucose homeostasis in IRS2-deficient mice. For this purpose, we generated TG mice overexpressing HGF in ␤-cells that lack IRS2 expression. The combination of RIP-HGF TG mice with IRS2 KO mice led to the generation of 4 genotypes after 2 consecutive breedings: wild-type mice (WT), IRS2 knockout mice (KO), RIP-HGF transgenic mice (TG), and RIP-HGF transgenic mice with IRS2 deficiency in ␤-cells (TG/KO) (Supplemental Figure 1A). Western blot and immunohistochemical analyses performed in isolated islets and pancreatic sections from these 4 genotypes clearly indicate the absence of IRS2 expression in KO mice and TG/KO mice (Supplemental Figure 1, B and C). In addition, immunohistochemical analysis of pancreatic sections of these mice at 12 weeks of age reveal marked overexpression of HGF in TG and TG/KO mice (Supplemental Figure 1C). Taken together these results indicate that we have generated mice with overexpression of HGF in ␤-cells that lack IRS2. HGF overexpression in ␤-cells improves glucose homeostasis in IRS2-deficient mice It has previously been shown that IRS2 deficiency leads to a more consistent and rapid development of

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mice (Figure 2, B–C). Glucose tolerance was also improved in TG/KO mice compared with that in KO mice (Figure 2, D–E). The improvement in glucose tolerance in TG/KO mice might be underestimated because 4 of 6 KO mice displayed blood glucose values of 500 mg/dL at every single time point after glucose injection, the maximal glucose value the glucometer could register in these experiments. On the other hand, ITTs in these mice revealed that TG/KO and KO mice display insulin resistance similar to that in WT or TG mice (Figure 2F), suggesting that the improvement in blood glucose in TG/KO mice is not due to improved insulin sensitivity. Glucose homeostasis analysis in TG/KO mice suggests that HGF might be increasing GSIS in ␤-cells Figure 3. ␤-Cell homeostasis in RIP-HGF TG mice with IRS2 deficiency. A, Representative that lack IRS2. However, this could photomicrographs of mouse pancreatic sections stained for insulin (brown) and counterstained also be the result of potential differwith hematoxylin from WT, TG, KO, and TG/KO mice at 12 weeks of age. B–D, Quantification of ences in ␤-cell mass between TG/KO ␤-cell mass (B), islet number per pancreatic area (C), and singlet, doublet, and ductal insulinpositive cells per pancreatic area (D). Results are means ⫾ SEM and were obtained from 5 to 7 and KO mice (see below). Therefore, mice per genotype. *, P ⬍ .05 vs the other 3 groups of mice; #, P ⬍ .05 vs WT and TG/KO mice; to analyze whether HGF is capable ∧, P ⬍ .05 vs WT and TG mice. of acutely increasing GSIS in the absence of IRS2, we performed insulin frank diabetes and ␤-cell failure in male mice than in secretion analysis in static incubations of islets from fefemale mice (3, 23). For this reason, we decided to study male IRS2 KO mice transduced with Adv.HGF. We chose the glucose and ␤-cell phenotype of 6- and 12-week-old female IRS2 mouse islets because it has previously been male mice with IRS2 deficiency and HGF overexpression shown that these islets display normal GSIS responses in ␤-cells. Adult IRS2 KO mice have been reported to (23). Overexpression of HGF in WT islets led to an indisplay lower body weight that is associated with severe crease in GSIS (Figure 2G) as reported previously (9). prolonged hyperglycemia (3, 7). In our study, we ob- Interestingly, the absence of IRS2 did not significantly served that 12-week-old KO mice displayed significantly alter the increased GSIS response induced by HGF overlower body weight than TG and WT mice (Figure 2A). In expression (Figure 2G), suggesting that TG/KO islets can contrast with the body weight of KO mice, the body respond to insulin resistance by increasing insulin secreweight of TG/KO mice was indistinguishable from that of tion and plasma insulin levels (Figure 2, B and C). TG and WT mice (Figure 2A). TG mice displayed decreased nonfasting blood glu- ␤-Cell mass is preserved in IRS2-deficient mice cose, increased plasma insulin, and significantly im- with HGF overexpression in ␤-cells Representative areas of pancreatic sections immunoproved glucose tolerance compared with those of WT mice (Figure 2, B–E), as previously observed in these mice stained for insulin from these 4 types of mice at 12 weeks (8, 9). IRS2 KO mice displayed severe hyperglycemia, of age are shown in Figure 3A. Clearly, insulin-stained hypoinsulinemia, and highly impaired glucose tolerance islets were difficult to detect in KO mice (Figure 3A). In compared with those of WT mice (Figure 2, B–E), con- contrast, small and large islets were observed in TG/KO firming previous observations (3). In contrast to the KO mice, with numbers similar to those in WT mice (Figure mice, blood glucose was significantly reduced and plasma 3A). ␤-Cell histomorphometric analysis of several slides insulin was highly increased in TG/KO mice, suggesting per pancreas indicated that ␤-cell mass was severely dipotential effects on ␤-cells or insulin sensitivity in these minished in KO mice, whereas the mass in TG/KO mice

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did not differ from that of WT mice (Figure 3B), suggesting that overexpression of HGF corrects ␤-cell mass in the absence of IRS2. As reported previously, TG mice displayed significantly increased ␤-cell mass compared with that of WT mice (Figure 3B) (8). Islet numbers were markedly and significantly decreased in KO mice, but this number was similar in TG/KO and WT mice (Figure 3C). TG mice displayed significantly increased islet numbers and a trend toward an increased number of singlet/doublet insulin-positive cells in the acinar and ductal areas of the pancreas, a potential indication of increased islet neogenesis (24, 25) (Figure 3D). However, the number of singlet/ doublet insulin-positive cells was markedly decreased in KO mice (Figure 3D). Interestingly, this parameter was significantly increased in TG/KO mice compared with that in KO mice but was markedly decreased compared with that on WT mice or TG mice, suggesting decreased islet neogenesis with overexpression of HGF in the absence of IRS2 (Figure 3D).

␤-Cell death is reduced and proliferation is increased in islets of IRS2-deficient mice with HGF overexpression in ␤-cells The loss of ␤-cell mass in IRS2⫺/⫺ mice is associated with reduced ␤-cell proliferation and increased apoptosis (3, 26, 27). We therefore measured ␤-cell proliferation by Ki67 detection and BrdU incorporation in the nuclei of ␤-cells from 6-week-old mice. Representative images of islets immunostained for insulin, Ki67, and 4⬘,6-diamidino-2-phenylindole (DAPI) of the 4 types of mice are shown in Figure 4A. The percentage of ␤-cell proliferation was clearly decreased in KO mouse islets assessed by either Ki67 staining or BrdU incorporation in ␤-cells (Figure 4, B and C). In contrast, ␤-cell proliferation was increased in TG/KO mice compared with that in WT and KO mice (Figure 4, B and C). The percentage of ␤-cell proliferation in TG/KO mice was not significantly different from the value in TG mice (Figure 4, B and C). Next, we measured ␤-cell death by TUNEL and insulin staining in pancreatic sections of these mice. As shown in Figure 4D, the number of TUNEL-positive ␤-cells was significantly increased in IRS2⫺/⫺ mice compared with that of WT mice. Interestingly, ␤-cell death was significantly decreased in TG/KO mice compared with that in KO mice, and the values were similar to the values in WT and TG mice (Figure 4D). These results indicate that the preservation of ␤-cell mass in TG/KO mice is associated with increased ␤-cell proliferation and decreased ␤-cell death.

Figure 4. ␤-Cell proliferation and survival in RIP-HGF TG mice with IRS2 deficiency. A, Representative photomicrographs of mouse pancreatic sections stained for insulin (green), DAPI (blue), and Ki67 (red) from WT, TG, KO, and TG/KO mice at 6 weeks of age. Arrows indicate Ki67-positive ␤-cell nuclei. B and C, Quantification of the percentage of Ki67-positive (B) and BrdU-positive (C) ␤-cells. D, TUNELpositive ␤-cells to detect cell death in WT (n ⫽ 7), TG (n ⫽ 6), KO (n ⫽ 7), and TG/KO (n ⫽ 9) mice. Results are means ⫾ SEM. *, P ⬍ .05 vs the other 3 groups of mice; #, P ⬍ .05 vs WT.

Akt activation and Pdx-1 levels are increased in IRS2-deficient mice expressing HGF in ␤-cells. Dysregulation of AKT/GSK3/FoxO1 signaling and down-regulation of Pdx-1 are key molecular events that

participate in the ␤-cell failure observed in IRS2⫺/⫺ mice (4, 6 –7). Therefore, we performed Western blot analysis of these intracellular targets and confirmed that Akt and

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Figure 5. Western blot analysis of multiple intracellular molecules in RIP-HGF TG mouse islets with IRS2 deficiency that are involved in ␤-cell proliferation and survival. A–C, Analysis of AKT Ser473 phosphorylation (A), FoxO1 Ser256 phosphorylation (B), and Pdx-1 levels (C) in islet extracts from WT, TG, KO, and TG/KO mice (n ⫽ 4/group). A representative Western blot is shown at the top of the graph with the calculation of densitometry of 4 different blots. Actin was used as a housekeeping protein. Results are means ⫾ SEM. *, P ⬍ .05 vs the other 3 groups of mice; ∧, P ⬍ .05 vs WT; #, P ⬍ .05 vs WT and KO mice. D, Representative photomicrographs of pancreatic sections from WT, TG, KO, and TG/KO mice stained for Pdx-1 (red), insulin (green), and DAPI (blue). Inset: higher magnification image of specific islet areas stained for Pdx-1. Notice the Pdx-1 nuclear staining in WT, TG, and TG/KO mice and the more diffused cytoplasmic staining in KO mice.

FoxO1 phosphorylation were markedly decreased in KO mouse islets compared with that in islets from WT mice (Figure 5, A and B), as reported previously (3, 6, 7). In contrast, phosphorylation of AKT and FoxO1 was increased in TG/KO mice compared with that in KO mouse islets and similar to the levels found in TG mouse islets (Figure 5, A and B).

A decrease in the expression together with increased cytoplasmic localization of Pdx1 in IRS2⫺/⫺ mouse islets has been reported to correlate with decreased ␤-cell mass (4). In the current study, we observed a mild decrease in Pdx-1 in KO mouse islets that did not reach statistical significance (Figure 5C). However, Pdx-1 levels were significantly increased in TG/KO islets compared with those

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proliferation in TG and TG/KO mice (Figure 4, A and B) correlated with a decrease in p27kip1 in islet ␤-cells (Figure 6, A and B). To determine whether this was a direct effect of HGF in ␤-cells, we treated INS-1 cells with HGF for 24 hours and measured p27kip1 protein and mRNA levels in these cells. As shown in Figure 6C, HGF significantly decreased p27kip1 protein levels but did not alter mRNA levels, suggesting that HGF does not affect p27kip1 transcriptionally but rather at a translational or posttranslational level.

Discussion The current study indicates that HGF can overcome the deleterious effects of ␤-cell failure and insulin resistance in IRS-2-deficient mice by Figure 6. p27 expression levels in RIP-HGF TG mouse islets with IRS2 deficiency and in INS-1 cells treated with HGF. A, Analysis of p27 levels in islet extracts from WT, TG, KO, and TG/KO reducing ␤-cell apoptosis, increasing mice (n ⫽ 4/group). A representative Western blot is shown, and the graph with the ␤-cell proliferation, and normalizdensitometric calculation of the different samples tested appears on the right of the image. Actin ing ␤-cell mass, resulting in compenwas used as a housekeeping protein. Results are means ⫾ SEM. *, P ⬍ .05 vs the other 3 groups of mice. B, Representative photomicrographs of pancreatic sections from WT, TG, KO, and TG/ satory hyperinsulinemia and deKO mice stained for p27 (red), insulin (green), and DAPI (blue). Notice the increased p27 staining creased blood glucose levels. HGF in KO mice compared with that in WT, TG, and TG/KO mice. C and D, Analysis of p27 protein (C) also increases GSIS in islets lacking and mRNA expression levels (D) in INS-1 cells treated with 25 ng/mL HGF. A representative Western blot using actin as a housekeeping protein is shown to the left, next to the densitometry IRS2. These improvements in ␤-cell quantitation of 3 different blots. Real-time PCR quantitation of p27 mRNA expression in 3 function and glucose and ␤-cell hoexperiments in triplicate is shown to the right using actin as a housekeeping gene. Results are meostasis correlate with normalizameans ⫾ SEM. *, P ⬍ .05 vs vehicle-treated cells by the Student t test. tion or activation of a profile of signaling molecules known to be in KO and WT islets (Figure 5C). Staining for Pdx-1 involved in T2D development in the context of IRS2 deshows diffused cytoplasmic and reduced nuclear staining in KO mouse islets with increased nuclear staining in ficiency: HGF leads to phosphorylation of Akt and FoxO1, up-regulation of Pdx-1, and down-regulation of TG/KO mice (Figure 5D). p27kip1 in the absence of IRS2. Therefore, these studies truly highlight the HGF/c-Met pathway as a potential p27kip1 levels are reduced in IRS2-deficient mice therapeutic target for ␤-cell failure in insulin resistance expressing HGF in ␤-cells Up-regulation of the cyclin-dependent kinase inhibitor and diabetes. HGF increases ␤-cell proliferation and survival 1B p27kip1 has been observed in IRS2⫺/⫺ mouse islets (5), kip1⫺/⫺ and breeding with p27 mice corrects diabetes and through activation of PI3K, PKC␨ and Akt (10, 18). loss of ␤-cell mass in IRS2 haploinsufficient mice (5). Whether activation of PI3K by HGF requires the particiMice deficient in the HGF receptor, c-Met, in pancreas pation of IRS proteins in ␤-cells has not been clearly demdisplay a marked increase in p27kip1 levels in ␤-cells dur- onstrated. HGF receptor c-Met associates with Gab1, ing pregnancy, suggesting that HGF might be a physio- which in turn associates with PI3K, inducing its activation logic modulator of p27kip1 levels in ␤-cells (12). In the (28). Gab1 is a member of the IRS1-like family of multicurrent study, we found that KO mouse islets have in- substrate docking proteins and has been shown to be escreased p27kip1 and that p27kip1 localizes mostly in the sential for epithelial morphogenesis downstream of c-Met nuclei of ␤-cells (Figure 6, A and B). The increase in ␤-cell (28, 29). On the other hand, HGF-mediated hepatocyte

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growth after partial hepatectomy has been suggested to require Grb2 and IRS1 activation (30). In ␤-cells, Gahr et al (19) have shown that HGF can increase the association of PI3K to IRS4 in the absence of glucose, but there is no clear association of PI3K with IRS1 or IRS2. Araujo et al (14), however, have recently shown that rat islets incubated with HGF show increased phosphorylation of IRS2. Whether this increase is associated with c-Met or with insulin secreted from these islets is unknown. In our study, we found that incubation of INS-1 cells with HGF does not lead to the association of PI3K with IRS2 compared with the effect of IGF-1. Furthermore, IRS2-deficient islets respond to HGF in terms of Akt activation similar to WT islets. This finding suggests that IRS2 is not required for HGF-mediated PI3K-Akt activation. HGF increases ␤-cell proliferation and survival in basal conditions as well as during pregnancy, during islet inflammation, and after partial pancreatectomy (8 –13, 15, 16). These studies have highlighted the physiologic importance of HGF and its potential for regenerative therapies in diabetes. However, whether HGF can be of use in situations of ␤-cell failure and insulin resistance has not been addressed to our knowledge. Because IRS2 deficiency leads to insulin resistance and ␤-cell failure, we wondered whether HGF actions in ␤-cells could bypass the negative effects of IRS2 absence and normalize ␤-cell mass and improve glucose homeostasis in the context of insulin resistance. For that purpose, we crossed TG mice overexpressing HGF in the ␤-cell (RIP-HGF TG mice)

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with IRS2-deficient mice (IRS2 KO mice). The original backgrounds of RIP-HGF TG (CD-1 background) and IRS2 KO (C57BL6 ⫻ 129Sv background) mice were different from one another, and it is well known than the phenotype of a mouse with a genetic alteration can vary based on its genetic background (31). Importantly, however, the phenotypes of IRS2 KO and RIP-HGF TG mice on this new, mixed background are very similar to their phenotypes on the original backgrounds. IRS2 KO mice display hyperglycemia, hypoinsulinemia, insulin resistance, glucose intolerance, increased ␤-cell apoptosis, and diminished ␤-cell mass (3–7). RIP-HGF TG mice display increased ␤-cell mass, enhanced ␤-cell proliferation, and improved glucose tolerance (8 –9). This is significant because it demonstrates that these phenotypes previously described on one genetic background remain present on a second, mixed genetic background and provides confidence on the phenotypic results of the TG/KO mice. Importantly, overexpression of HGF in ␤-cells of TG mice deficient in IRS-2 resulted in increased ␤-cell proliferation and survival similar to the levels in TG mice, suggesting that the absence of IRS-2 does not impair the proliferative and prosurvival actions of HGF in the ␤-cell. To our surprise, ␤-cell mass in TG/KO mice was similar to that in WT mice, suggesting correction of the ␤-cell mass allotment in the absence of IRS-2. However, ␤-cell mass was lower in TG/KO mice than in TG mice alone, suggesting that IRS-2 deficiency takes a toll on ␤-cell mass in the context of HGF overexpression that is not related to

Figure 7. Schematic representation of HGF action in ␤-cells in the absence or presence of IRS2. HGF action in an adult ␤-cell under basal conditions (A), in the absence of IRS2 (B), and when overexpressed in the absence of IRS2 (C). Phosphorylation of PI3K/AKT/FoxO1 by overexpressed HGF in the absence of IRS2 leads to an increased nuclear presence of Pdx-1, p27 down-regulation, increased ␤-cell replication, and survival. An increase in the size of the font/boxes indicates up-regulation/activation of that protein.

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doi: 10.1210/me.2014-1207

␤-cell proliferation and survival. Indeed, analysis of islet number and singlet or doublet insulin-positive cells in the pancreas, markers thought to reflect ␤-cell neogenesis (24, 25) show that these parameters are decreased in IRS2-deficient mice with HGF overexpression. Analysis of the molecular and cellular mechanisms involved in the incapability of HGF to increase islet numbers in the absence of IRS2 warrants further future studies. Pdx-1 is a homeodomain transcription factor required for pancreas development and ␤-cell differentiation and function (32). Pdx-1 haploinsufficiency accelerated the onset of diabetes in IRS-2-deficient mice, whereas transgenic overexpression through the use of Pdx-1 promoter restored ␤-cell mass and improved glucose homeostasis in these mice (4). These studies indicated that Pdx-1 is vital for ␤-cell proliferation and survival in the absence of IRS-2 (4). Importantly, ␤-cell mass was normalized but not increased in the Pdx-1 TG/IRS-2 KO mice as we observed in our TG/KO mice, and the number of islets was decreased in Pdx-1 TG/IRS-2 KO mice compared with that in TG mice as we see in TG/KO mice in our study, indicating similar patterns of action in these 2 studies. Likewise, blood glucose levels were highly reduced but not completely normalized as we observed in the TG/KO mice in our study. Importantly, TG/KO mice in our study display increased Pdx-1 levels in the islet, similar to those of TG mice, and the pattern of staining is more nuclear than that in KO mice where the staining is more cytoplasmic and diffused. Therefore, the current studies highlight the important novel finding that HGF can increase Pdx-1 expression and nuclear localization and potentially thereby contribute to overcome ␤-cell failure in the context of IRS-2 deficiency (Figure 7). The cell cycle inhibitor p27Kip1 has been shown to play an important role in restricting the ability of ␤-cells to proliferate (33, 34). Indeed, deletion of the p27Kip1 gene ameliorated hyperglycemia in IRS-2-deficient mice by increasing ␤-cell proliferation and mass and maintaining compensatory hyperinsulinemia, suggesting that p27Kip1 contributes to ␤-cell failure during the development of T2D in IRS2⫺/⫺ mice (5). p27Kip1 expression is up-regulated in islets from pregnant mice with pancreatic c-Met deletion (12), suggesting that HGF might control directly or indirectly the expression of p27Kip1 during pregnancy. Overexpression of HGF in ␤-cells of IRS-2-deficient mice led to a decrease in the expression of p27Kip1 that could explain the increase in ␤-cell proliferation observed in these mice (Figure 7). The effect of HGF appears to be a direct translational/posttranslational effect because HGF is capable of down-regulating p27Kip1 protein in INS-1 cells without altering p27 mRNA levels. Further studies

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are warranted to determine how HGF regulates p27Kip1 at a molecular level in ␤-cells. In summary, HGF is capable of modulating Akt and FoxO1 activity, increasing Pdx-1 expression, and reducing p27Kip1 levels in ␤-cells in the absence of IRS2, alterations that contribute to enhanced ␤-cell proliferation and survival. These beneficial effects lead to ␤-cell mass correction and amelioration of hyperglycemia by maintaining compensatory hyperinsulinemia in IRS2⫺/⫺ mice. Therefore, HGF can be of therapeutic use for overcoming ␤-cell failure during the development of T2D.

Acknowledgments Address all correspondence and requests for reprints to: Adolfo Garcia-Ocaña, PhD, Diabetes, Obesity and Metabolism Institute, Division of Endocrinology, Diabetes and Bone Diseases, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1152, New York, NY 10029. E-mail: adolfo.g. [email protected]. This work was supported in part by the National Institutes of Health (Grants DK-067351 and DK-077096 to A.G.-O.). Disclosure Summary: The authors have nothing to disclose.

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Hepatocyte growth factor ameliorates hyperglycemia and corrects β-cell mass in IRS2-deficient mice.

Insulin resistance, when combined with decreased β-cell mass and relative insufficient insulin secretion, leads to type 2 diabetes. Mice lacking the I...
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