Global lack of mesencephalic astrocyte-derived neurotropic factor (MANF) leads to progressive postnatal loss of β-cell mass and insulin-dependent diabetes in mice. Similar to Manf−/− mice, embryonic ablation of MANF specifically from the pancreas results in diabetes. In this study, we assessed the importance of MANF for the postnatal expansion of pancreatic β-cell mass and for adult β-cell maintenance in mice. Detailed analysis of Pdx-1Cre+/−::Manffl/fl mice revealed mosaic MANF expression in postnatal pancreata and a significant correlation between the number of MANF-positive β-cells and β-cell mass in individual mice. In vitro, recombinant MANF induced β-cell proliferation in islets from aged mice and protected from hyperglycemia-induced endoplasmic reticulum (ER) stress. Consequently, excision of MANF from β-cells of adult MIP-1CreERT::Manffl/fl mice resulted in reduced β-cell mass and diabetes caused largely by β-cell ER stress and apoptosis, possibly accompanied by β-cell dedifferentiation and reduced rates of β-cell proliferation. Thus, MANF expression in adult mouse β-cells is needed for their maintenance in vivo. We also revealed a mechanistic link between ER stress and inflammatory signaling pathways leading to β-cell death in the absence of MANF. Hence, MANF might be a potential target for regenerative therapy in diabetes.

Insulin deficiency caused by autoimmune destruction of β-cells leads to type 1 diabetes, and β-cell death is involved in the failure of insulin secretion that leads to type 2 diabetes (1,2). Current diabetes therapies are based on treating hyperglycemia, but they are not targeting basic disease mechanisms. Because the prevalence of diabetes is increasing rapidly worldwide, the development of new therapies to protect and restore functional β-cell mass is important (3).

Increasing evidence suggests that endoplasmic reticulum (ER) stress followed by prolonged activation of the unfolded protein response (UPR) is one of the pathogenic mechanisms leading to β-cell death in both type 1 and type 2 diabetes (46). Pancreatic β-cells are especially vulnerable to ER stress–mediated cell death because of their heavy demand of insulin production, depending of proper folding and secretion (7). ER stress is triggered by the accumulation of unfolded and misfolded proteins in the ER lumen, leading to activation of UPR signaling cascades that are mediated by three ER transmembrane protein sensors: protein kinase R-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1α) (8). Mutations inactivating the PERK gene lead to ER dysfunction, neonatal diabetes, and growth defects in patients with Wolcott-Rallison syndrome (9). Similarly, a homozygous mutation in the Perk gene leads to retarded growth and neonatal diabetes in mice (10,11). In addition, inactivating mutations in several UPR genes result in β-cell death and diabetes in mice (12,13).

Mesencephalic astrocyte-derived neurotropic factor (MANF) originally was described as a secreted trophic factor for dopamine neurons in vitro with protective roles for neurons and cardiomyocytes in rodent disease models (1417). The protective effect of MANF has been suggested to depend on its ability to dampen ER stress (15,16,18,19). The mechanisms whereby MANF acts and enters cells are not fully understood. It was suggested to bind with its COOH-terminal KDEL motif (RTDL) to a KDEL receptor (KDELR) at the cell surface and thereby enter the cell (20). Recently, MANF was shown to bind to lipid sulfatides on the cell surface followed by endocytosis (21). We recently discovered that MANF deficiency in mice (Manf−/− mice) leads to postnatal reduction of pancreatic β-cell mass and severe insulin-deficient diabetes as a result of decreased β-cell proliferation and increased β-cell death (22). ER stress and chronic activation of the UPR in β-cells was found to precede the loss of β-cells in Manf−/− mice (22). Of note, adeno-associated virus vector–mediated overexpression of MANF was able to regenerate β-cells in vivo, and recombinant MANF was able to increase proliferation rates of β-cells in vitro, indicating that MANF protein is a potential new drug candidate for the treatment of type 1 diabetes (22).

In the current study, we examined the importance of MANF in vivo in mice by conditional removal of MANF specifically from pancreas and β-cells in embryos. Our results show that MANF expression in β-cells positively correlates with β-cell mass and negatively correlates with β-cell apoptosis, further revealing the importance of MANF expression for β-cell survival. We also reveal the role of MANF for adult β-cells by removal of MANF specifically from β-cells using MIP-1CreERT::Manffl/fl mice, which led to the development of diabetes caused by sustained ER stress, resulting in increased β-cell death. Finally, we show that MANF protein increased proliferation rates of even aged and MANF-deficient mouse β-cells in vitro. Thus, our work demonstrates that MANF is essential for the maintenance of adult β-cells in mice.

Animals and In Vivo Physiology

All experimental procedures involving mice were approved by the National Animal Experiment Board, Administrative Agency of Southern Finland. All mice were kept on a 12-h dark/light cycle with free access to chow diet and water. The generation of Manf−/− and conditional Manffl/fl mice has been described previously (22) (Supplementary Data). To delete MANF embryonically from the pancreas, we used a Pdx-1CreTUV transgenic mouse line (Stock 014647, C57BL/6J background; The Jackson Laboratory) (23) crossed with Manffl/fl mice [Hsd:ICR(CD-1) background). To specifically remove MANF from the β-cells in adult mice, we used tamoxifen (TMX)-inducible MIP-1CreERT1Lphi mice (24) (C57BL/6JRccHsd background) crossed with Manffl/fl mice [Hsd:ICR(CD-1) background]. Eight-week-old MIP-1CreERT::Manffl/fl and Manffl/fl mice were injected with either 33 mg/kg TMX [MIP-1CreERT::Manffl/fl(TMX)] or corn oil [MIP-1CreERT::Manffl/fl(OIL)] for 5 consecutive days. Four weeks after injection, mice were sacrificed and tissues and sera analyzed. All experimental animals were of mixed background. Analysis of blood samples were performed as described previously (22) and in Supplementary Data.

Histological Analysis

Immunohistochemistry (IHC), quantification of β-cell mass, and analysis of β-cell death were performed as described previously (22). For IHC, mouse pancreatic paraffin sections were stained with primary antibodies (Supplementary Table 1). For immunofluorescence staining, appropriate secondary antibodies conjugated with Alexa Fluor 488 or 568 (1:400; Molecular Probes, OR) and DAPI (VECTASHIELD; Vector Laboratories, Burlingame, CA) were used to visualize the labels.

In Vitro β-Cell Proliferation

In vitro β-cell proliferation experiments were performed as previously described and assessed by Click-iT EdU Alexa Fluor 647 Imaging Kit (C10340; Thermo Fisher Scientific, Waltham, MA) (22). Isolated islets from 1.3-year-old or 4–5-week-old Manf−/− female mice were stimulated with recombinant human (rh) placental lactogen (500 ng/mL; Affiland) or rhMANF (100 ng/mL; Icosagen) for 5 consecutive days in RPMI media containing 11 mmol/L glucose supplemented with 10% FBS and antibiotics. 5-Ethynyl-2'-deoxyuridine EdU (10 nmol/L) was added to the culture medium for the last 3 days of stimulation.

To study cell signaling pathways, islets from Manf−/− and wild-type (control) MANF mice (Manf+/+ mice) were kept overnight in RPMI medium supplemented with 10% FBS. The next day, islets were starved in 0.5% BSA in RPMI medium for 3 h and stimulated with 100 ng/mL rhMANF for 1 h followed by islet homogenization in 1× Laemmli buffer and Western blot analysis.

Serial Block-Face Scanning Electron Microscopy

Mice were perfused with 4% formaldehyde followed by the cutting of pancreata in pieces. The pieces were postfixed, stained, and embedded on the basis of a previously published protocol (25). The collected images were processed, aligned, and analyzed using Microscopy Image Browser (26).

Additional Materials and Methods

The methods for Western blot and RNA analysis and those concerning treatment of murine islets, serial block-face scanning electron microscopy analysis, and subcellular localization analysis are provided in the Supplementary Data.

Statistical Analysis

All data are presented as mean ± SEM. Statistical significance was assessed using Student t test between two groups or one-way ANOVA followed by the Tukey test among four groups as mentioned in the figure legends using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA).

MANF Is Specifically Expressed in β-Cells in the Islets of Langerhans

MANF is widely expressed in mouse tissues throughout embryonic development and in adulthood (27). Detailed examination of MANF expression in developing and adult mouse pancreas and islets revealed MANF expression in developing pancreatic primordium in embryonic day 13.5 (E13.5) embryos by IHC (Fig. 1A), when major differentiation of pancreas-specific endocrine cells begins (28). Consistent with our previous studies, we observed wide expression of MANF in adult mouse pancreas with positive signal in both exocrine acinar cells and endocrine islets of Langerhans (22,29,30) (Fig. 1B). Of note, no positive signal was detected in the pancreas of Manf−/− mice, confirming the specificity of MANF antibody used throughout this study (Fig. 1C and D and Supplementary Fig. 1.1D). Quantitative RT-PCR and Western blot analysis confirmed expression of Manf mRNA in pancreatic exocrine tissue and in isolated islets from adult mice (Fig. 1E and F). Thus, MANF protein is highly expressed in the mouse pancreas from early embryonic development until adulthood.

Figure 1

MANF is specifically expressed in the β-cells of the islets of Langerhans. AD: IHC localization of MANF protein in pancreatic primordium at E13.5 in Manf+/+ embryo (A) and in adult Manf +/+ mouse pancreas (B), using sections from E13.5 Manf−/− embryo (C) and adult Manf −/− mouse pancreas (D) as specificity controls for anti-MANF antibody (Icosagen). Scale bar = 50 μm. E: Quantitative RT-PCR analysis for Manf mRNA levels in islets of Langerhans isolated from Manf+/+ mice (n = 6) and exocrine tissue (n = 3) from Manf+/+ mice at P56. F: Western blot analysis of lysates from isolated islets confirms that MANF protein is not expressed in islets from Manf−/− mice. rhMANF protein (15 ng MANF) was used as positive control, and anti-GAPDH antibody was used for normalization of total protein content. G–V: Double IHC analysis with anti-MANF antibody (H, L, P, and T) and antibodies against other islet hormones, insulin (G), somatostatin (K), glucagon (O), and PP (S). MANF was coexpressed with insulin in β-cells and weakly with somatostatin in δ-cells. Almost no MANF signal was detected in α- or PP cells. Very weak or no MANF-positive staining was observed in Manf−/− pancreatic islets (J, N, R, and V). Cell nuclei were labeled with DAPI (blue). Scale bar = 10 μm. Arrowheads in K and L show overlapping expression of somatostatin and MANF in some δ-cells. Arrowheads in O and P and S and T confirm that MANF (P, T) is not expressed in glucagon-producing α-cells (O) or in PP-producing cells (S). WZ: Representative confocal laser scanning microscopy images of primary mouse islet β-cells double stained with anti-MANF and anti-protein disulphide isomerase (PDI) (ER marker), anti-GM130 (Golgi marker), anti-insulin, or anti-GRP78 antibodies. Quantitative colocalization analysis on the basis of Pearson correlation and Mander coefficients (shown in the Supplementary Table 2) revealed high colocalization of MANF with ER marker PDI and insulin, moderate colocalization with GRP78, and no colocalization of MANF with Golgi marker GM130. Data are mean ± SEM. ns, not significant.

Figure 1

MANF is specifically expressed in the β-cells of the islets of Langerhans. AD: IHC localization of MANF protein in pancreatic primordium at E13.5 in Manf+/+ embryo (A) and in adult Manf +/+ mouse pancreas (B), using sections from E13.5 Manf−/− embryo (C) and adult Manf −/− mouse pancreas (D) as specificity controls for anti-MANF antibody (Icosagen). Scale bar = 50 μm. E: Quantitative RT-PCR analysis for Manf mRNA levels in islets of Langerhans isolated from Manf+/+ mice (n = 6) and exocrine tissue (n = 3) from Manf+/+ mice at P56. F: Western blot analysis of lysates from isolated islets confirms that MANF protein is not expressed in islets from Manf−/− mice. rhMANF protein (15 ng MANF) was used as positive control, and anti-GAPDH antibody was used for normalization of total protein content. G–V: Double IHC analysis with anti-MANF antibody (H, L, P, and T) and antibodies against other islet hormones, insulin (G), somatostatin (K), glucagon (O), and PP (S). MANF was coexpressed with insulin in β-cells and weakly with somatostatin in δ-cells. Almost no MANF signal was detected in α- or PP cells. Very weak or no MANF-positive staining was observed in Manf−/− pancreatic islets (J, N, R, and V). Cell nuclei were labeled with DAPI (blue). Scale bar = 10 μm. Arrowheads in K and L show overlapping expression of somatostatin and MANF in some δ-cells. Arrowheads in O and P and S and T confirm that MANF (P, T) is not expressed in glucagon-producing α-cells (O) or in PP-producing cells (S). WZ: Representative confocal laser scanning microscopy images of primary mouse islet β-cells double stained with anti-MANF and anti-protein disulphide isomerase (PDI) (ER marker), anti-GM130 (Golgi marker), anti-insulin, or anti-GRP78 antibodies. Quantitative colocalization analysis on the basis of Pearson correlation and Mander coefficients (shown in the Supplementary Table 2) revealed high colocalization of MANF with ER marker PDI and insulin, moderate colocalization with GRP78, and no colocalization of MANF with Golgi marker GM130. Data are mean ± SEM. ns, not significant.

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Next, we studied MANF expression in detail in various islet endocrine cells by IHC. Although the majority of MANF-positive cells were insulin-positive β-cells (Fig. 1G–I), weak MANF expression also was observed in δ-cells (Fig. 1K–M), whereas almost no MANF signal was detected in α-cells (Fig. 1O–Q) or pancreatic polypeptide (PP) cells (Fig. 1S–U). MANF also was coexpressed with cells expressing GLUT2 (Supplementary Fig. 1.1E–G) and pancreatic and duodenal homeobox 1 (PDX1) (Supplementary Fig. 1.1H–J), further confirming that MANF is predominantly expressed in insulin expressing β-cells of the islets.

Subcellular localization analysis revealed strong colocalization of MANF with the ER and insulin, a partial colocalization of MANF with GRP78, and a complete segregation of MANF from the Golgi apparatus in the primary mouse islet cells on the basis of Mander and Pearson correlation coefficients (Fig. 1W–Z, Supplementary Fig. 1.1K–V, and Supplementary Table 2).

Of note, in pancreas tissue from diabetic mice, we found increased MANF expression in some β-cells located near lymphocytic cells invading the islets of 12-week-old prediabetic NOD mice (Supplementary Fig. 1.2A–L). Barely no MANF expression was detected in the lymphocytic cells characteristic for insulitis. In addition, increased expression of MANF was found in some β-cells of 8-week-old db/db mice (Supplementary Fig. 1.2M–Y).

MANF Expression in β-Cells Is Important for Maintaining β-Cell Mass in Pdx-1Cre+/−::Manffl/fl Mice Postnatally

Detailed characterization of the diabetic phenotype in Pdx-1Cre+/−::Manffl/fl mice revealed that embryonic deletion of MANF specifically from the pancreas results in postnatal β-cell failure (22) (Supplementary Fig. 2.1 and Supplementary Data). However, the decreased β-cell mass and diabetic phenotype appeared in Pdx-1Cre+/−::Manffl/fl mice later than in the Manf−/− mice (22) (Supplementary Fig. 2.1 and Supplementary Data). No ectopic Cre activity affected MANF expression in the brain of Pdx-1Cre+/−::Manffl/fl mice compared with Manffl/fl mice (Supplementary Fig. 2.2A–J). Analysis of double transgenic Pdx-1Cre+/−::dtTomato reporter mice revealed that Pdx-1Cre was not expressed in all MANF expressing pancreatic cells during development or in adult islet endocrine and exocrine cells (Supplementary Fig. 2.2K–R and Supplementary Data). Consequently, we observed incomplete recombination in Pdx-1Cre+/−::Manffl/fl mice leading to mosaic expression of MANF both in exocrine tissue and in endocrine islets in the postnatal and adult pancreas (Supplementary Fig. 2.3A–I and K). Strong mosaic MANF expression was detected in postnatal day 14 (P14) and adult MANF conditional pancreata with individual degrees of variation (Fig. 2F and Supplementary Fig. 2.3D–I). Some adult mice totally lacked MANF expression in the islets (Fig. 2C and Supplementary Fig. 2.3I). As expected, Manf mRNA levels in isolated islets from Pdx-1Cre+/−::Manffl/fl mice were significantly decreased at various postnatal stages compared with Manffl/fl islets, and the expression of Manf mRNA in conditional islets increased with age (Supplementary Fig. 2.3J). Moreover, we observed loss of islet architecture and decreased intensity of insulin immunoreactivity in the pancreata lacking MANF (Fig. 2A–C, K, M, and O), whereas islet structure remained intact when MANF expression was present in most of the β-cells (Fig. 2D–F, J, L, and N).

Figure 2

MANF expression in β-cells is important for restoring β-cell mass. A–I: Insulin (A, D, and G), glucagon (B, E, and H), and MANF (C, F, and I) IHC on pancreas sections from P56 Pdx-1Cre+/−::Manffl/fl mice. Scale bar = 50 μm. JO: Double IHC with anti-insulin (J and K) and anti-MANF antibodies (L and M) show mosaic (L) or no (M) MANF expression in P56 Pdx-1Cre+/−::Manffl/fl mice. Cell nuclei in the merged pictures (N and O) labeled with DAPI (blue). Scale bar = 20 μm. P: β-Cell mass in P56 Pdx-1Cre+/−::Manffl/fl mice positively correlates with the number of MANF-positive insulin-positive cells (n = 15). Q: The number of MANF-positive insulin-positive cells negatively correlates with blood glucose levels in P56 Pdx-1Cre+/−::Manffl/fl mice (n = 18). R: The fraction of MANF-positive insulin-positive β-cells negatively correlates with the number of TUNEL-positive β-cells in P56 Pdx-1Cre+/−::Manffl/fl mice (n = 11).

Figure 2

MANF expression in β-cells is important for restoring β-cell mass. A–I: Insulin (A, D, and G), glucagon (B, E, and H), and MANF (C, F, and I) IHC on pancreas sections from P56 Pdx-1Cre+/−::Manffl/fl mice. Scale bar = 50 μm. JO: Double IHC with anti-insulin (J and K) and anti-MANF antibodies (L and M) show mosaic (L) or no (M) MANF expression in P56 Pdx-1Cre+/−::Manffl/fl mice. Cell nuclei in the merged pictures (N and O) labeled with DAPI (blue). Scale bar = 20 μm. P: β-Cell mass in P56 Pdx-1Cre+/−::Manffl/fl mice positively correlates with the number of MANF-positive insulin-positive cells (n = 15). Q: The number of MANF-positive insulin-positive cells negatively correlates with blood glucose levels in P56 Pdx-1Cre+/−::Manffl/fl mice (n = 18). R: The fraction of MANF-positive insulin-positive β-cells negatively correlates with the number of TUNEL-positive β-cells in P56 Pdx-1Cre+/−::Manffl/fl mice (n = 11).

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Furthermore, the number of double MANF- and insulin-positive cells correlated with β-cell mass (Fig. 2P) and, consequently, with blood glucose levels in individual mice (Fig. 2Q). Quantification of TUNEL-positive insulin-positive β-cells revealed increased apoptosis in islets with an increased number of β-cells lacking MANF (Fig. 2R).

MANF Expression Is Required for Maintaining β-Cell Phenotype in Pdx-1Cre+/−::Manffl/fl Mice

Reduced β-cell mass in P14 and P56 conditional animals was accompanied by a significant reduction of β-cell markers Glut2, insulin1/insulin2 (Ins1/2), Pdx1, MafA, and glucokinase (Gck) mRNA levels in islets of Pdx-1Cre+/−::Manffl/fl mice (Fig. 3B and C and Supplementary Fig. 3A–C). This was expected because the proportion of β-cells compared with other islet cells, was decreased in the Pdx-1Cre+/−::Manffl/fl islets. No reduced expression levels of β-cell markers was observed in P1 islets (Fig. 3A) in contrast to what was observed in the conventional Manf−/− islets (22). Moreover, GLUT2 and MANF double IHC confirmed that the absence of MANF in β-cells was associated with loss of GLUT2 membrane localization (Fig. 3F–N).

Figure 3

MANF expression is required for maintaining β-cell mass. AC: Quantitative RT-PCR for mRNA levels of β-cell–specific genes Glut2, Ins1/2, Pdx-1, MafA, and Gck in islets from P1, P14, and P56 pancreata (n = 6–11 per group). D and E: Quantitative RT-PCR analysis of UPR genes Atf4, Grp78, Chop, spXbp1, tXbp, and Atf6α and Atf6β in P1 and P14 islets from Pdx-1Cre+/−::Manffl/fl and Manffl/fl mice (n = 6–11 per group). FN: Double IHC of MANF (IK) and GLUT2 (FH) in pancreatic sections from Manffl/fl (F, I, and L) and Pdx-1Cre+/−::Manffl/fl (G, H, J, K, M, and N) mice at P56. Cell nuclei were labeled with DAPI (blue). Scale bar = 10 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Figure 3

MANF expression is required for maintaining β-cell mass. AC: Quantitative RT-PCR for mRNA levels of β-cell–specific genes Glut2, Ins1/2, Pdx-1, MafA, and Gck in islets from P1, P14, and P56 pancreata (n = 6–11 per group). D and E: Quantitative RT-PCR analysis of UPR genes Atf4, Grp78, Chop, spXbp1, tXbp, and Atf6α and Atf6β in P1 and P14 islets from Pdx-1Cre+/−::Manffl/fl and Manffl/fl mice (n = 6–11 per group). FN: Double IHC of MANF (IK) and GLUT2 (FH) in pancreatic sections from Manffl/fl (F, I, and L) and Pdx-1Cre+/−::Manffl/fl (G, H, J, K, M, and N) mice at P56. Cell nuclei were labeled with DAPI (blue). Scale bar = 10 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

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UPR pathways already are activated in isolated islets and pancreata from conventional Manf−/− mice before birth at E18.5 (20). In addition, electron microscopy analysis of Manf−/− β-cells revealed an increased and expanded ER network with stacked and occasionally dilated sheets as a sign of ER stress in 4-week-old mice (Supplementary Fig. 3D–G and Supplementary Videos 1 and 2) (3133).

Consistent with this, we observed significant upregulation of Atf4, Grp78, Chop, spliced (sp) Xbp1, and total (t) Xbp1 but not Atf6 mRNA levels in P1 conditional islets (Fig. 3D). At P14, we demonstrated a continuous significant increase in mRNA levels of UPR markers (Fig. 3E). Altogether, activation of the main UPR sensors IRE1α, PERK, and later ATF6 and their downstream targets seem to precede the loss of β-cell phenotype in Pdx1-Cre+/−::Manffl/fl conditional islets.

Effects of Recombinant MANF Protein In Vitro

We have previously demonstrated that rhMANF protein stimulates proliferation of β-cells from young adult mice both in vitro and in vivo (22). Under normal conditions, the primary mechanism for maintaining postnatal β-cell mass is self-renewal of β-cells (34). Basal β-cell proliferation and response to mitogenic triggers decline markedly with age in both rodents and humans (3537). To realistically model the effect of MANF on adult human β-cell dynamics, we used islets from aged adult mice, wherein β-cell proliferation is extremely low. We found that rhMANF seems to increase β-cell proliferation rates in vitro, even in islets isolated from aged, 15-month-old mice (Fig. 4A–J).

Figure 4

Effects of MANF in vitro. A: Recombinant MANF protein increases proliferation of β-cells from aged, 15-month-old female C57BL/6JRccHsd mice after 5 days in culture. n quantified from 4–6 wells/group. The proliferative effect of MANF in a second experiment using 15-month-old female Hsd:ICR(CD-1) mice was 0.44 ± 0.067% compared with 0.25 ± 0.034% in control islets showing a similar significant increase in β-cell proliferation in the MANF-treated islets (data not shown). BJ: Double IHC of insulin (B, E, and H) and EdU (C, F, and I). Cell nuclei were labeled with DAPI (blue). Scale bar = 50 μm. KN: Quantitative RT-PCR analysis of UPR genes Manf, Grp78, spXbp1, and tXbp1 in primary mouse pancreatic islets treated (+) or nontreated (–) with 30 mmol/L glucose (Gls) or treated (+) or nontreated (−) with rhMANF overnight in RPMI medium supplemented with 0.5% BSA. (n = 4–5 wells/group). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Figure 4

Effects of MANF in vitro. A: Recombinant MANF protein increases proliferation of β-cells from aged, 15-month-old female C57BL/6JRccHsd mice after 5 days in culture. n quantified from 4–6 wells/group. The proliferative effect of MANF in a second experiment using 15-month-old female Hsd:ICR(CD-1) mice was 0.44 ± 0.067% compared with 0.25 ± 0.034% in control islets showing a similar significant increase in β-cell proliferation in the MANF-treated islets (data not shown). BJ: Double IHC of insulin (B, E, and H) and EdU (C, F, and I). Cell nuclei were labeled with DAPI (blue). Scale bar = 50 μm. KN: Quantitative RT-PCR analysis of UPR genes Manf, Grp78, spXbp1, and tXbp1 in primary mouse pancreatic islets treated (+) or nontreated (–) with 30 mmol/L glucose (Gls) or treated (+) or nontreated (−) with rhMANF overnight in RPMI medium supplemented with 0.5% BSA. (n = 4–5 wells/group). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

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We previously demonstrated that MANF partially protects mouse β-cells from thapsigargin-induced cell death in vitro (38). Herein, we analyzed whether MANF protects isolated mouse islet β-cells from glucotoxicity. Manf mRNA levels were significantly upregulated in islets after exposure to hyperglycemia (Fig. 4K). In addition, we found increased expression of spXbp1 mRNA in the islets treated with high glucose consistent with previous results (39,40) (Fig. 4M). Addition of rhMANF to the medium containing high glucose led to a significant decrease in the expression levels of spXbp1 and tXpb1 mRNA (Fig. 4L–N). Furthermore, MANF protein induced proliferation of Manf−/− β-cells in vitro as well as significantly reduced the expression of UPR markers in isolated islets of Manf−/− mice (Supplementary Fig. 4A–C).

Deletion of MANF Exclusively From Adult Pancreatic β-Cells Results in Diabetes in MIP-1CreERT::Manffl/fl Mice

To address whether MANF is required for adult β-cell survival and proliferation in vivo, we specifically removed MANF from β-cells in adult mice. MIP-1CreERT::Manffl/fl(TMX) mice did not differ in weight, but random-fed TMX-injected conditional mice had increased blood glucose levels and reduced serum insulin levels accompanied by impaired glucose clearance compared with control mice after a 4-week washout period of TMX injections (Fig. 5A–D and Supplementary Fig. 5.1A and B). Bolus injection of insulin normalized blood glucose levels in MIP-1CreERT::Manffl/fl(TMX) animals, suggesting intact insulin sensitivity (Supplementary Fig. 5.1C). Lower insulin levels after intraperitoneal glucose injection confirmed decreased insulin secretion in MIP-1CreERT::Manffl/fl(TMX) animals (Supplementary Fig. 5.1D).

Figure 5

Deletion of MANF exclusively from adult pancreatic β-cells results in diabetes in MIP-1CreERT::Manffl/fl mice. A: Body weights of MIP-1CreERT::Manffl/fl and Manffl/fl animals 4 weeks after last TMX injection (n = 11–12 mice/group, both sexes). B: Ad libitum fed blood glucose levels measured 4 weeks after last TMX injection (n = 11–12 mice/group, both sexes). C: Serum insulin levels measured 4 weeks after TMX injection from ad libitum fed mice (n = 11–12 mice/group, both sexes). D: Blood glucose levels measured after intraperitoneal glucose (2 g/kg) injection (n = 5–12 mice/group). EG: In vitro insulin release from islets in response to low glucose (1.67 mmol/L), high glucose (16.7 mmol/L), and high glucose with IBMX (1 mmol/L) normalized to islet DNA content after 1 h (E); total insulin content normalized to islet DNA content (F); and in vitro glucose-stimulated insulin release compared with total islet insulin content (G) (n = islets from 6 pancreata/group). HM: Recombination efficiency varies in individual mice showing different amounts of MANF expressing β-cells by double fluorescence IHC in pancreatic sections of MIP-1CreERT::Manffl/fl(TMX) mice stained with anti-insulin (K) and anti-MANF antibodies (L). Cell nuclei were labeled with DAPI (blue). Scale bar = 50 μm. NQ: IHC analysis of insulin-positive (N and O) and glucagon-positive (P and Q) cells in pancreatic sections reveals morphological changes in the islets of MIP-1CreERT::Manffl/fl(TMX) mice compared with MIP-1CreERT::Manffl/fl(OIL) mice. Scale bar = 50 μm. R: β-Cell mass is significantly reduced in P90 MIP-1CreERT::Manffl/fl(TMX) mice compared with that in MIP-1CreERT::Manffl/fl(OIL) mice (n = 4–5 mice/group, both sexes). S: β-Cell proliferation assessed by Ki67 and insulin staining (n = 6 mice/group). T: β-Cell apoptosis assessed by TUNEL and insulin staining (n = 6 mice/group). Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the corresponding control. GLS, glucose; ns, not significant.

Figure 5

Deletion of MANF exclusively from adult pancreatic β-cells results in diabetes in MIP-1CreERT::Manffl/fl mice. A: Body weights of MIP-1CreERT::Manffl/fl and Manffl/fl animals 4 weeks after last TMX injection (n = 11–12 mice/group, both sexes). B: Ad libitum fed blood glucose levels measured 4 weeks after last TMX injection (n = 11–12 mice/group, both sexes). C: Serum insulin levels measured 4 weeks after TMX injection from ad libitum fed mice (n = 11–12 mice/group, both sexes). D: Blood glucose levels measured after intraperitoneal glucose (2 g/kg) injection (n = 5–12 mice/group). EG: In vitro insulin release from islets in response to low glucose (1.67 mmol/L), high glucose (16.7 mmol/L), and high glucose with IBMX (1 mmol/L) normalized to islet DNA content after 1 h (E); total insulin content normalized to islet DNA content (F); and in vitro glucose-stimulated insulin release compared with total islet insulin content (G) (n = islets from 6 pancreata/group). HM: Recombination efficiency varies in individual mice showing different amounts of MANF expressing β-cells by double fluorescence IHC in pancreatic sections of MIP-1CreERT::Manffl/fl(TMX) mice stained with anti-insulin (K) and anti-MANF antibodies (L). Cell nuclei were labeled with DAPI (blue). Scale bar = 50 μm. NQ: IHC analysis of insulin-positive (N and O) and glucagon-positive (P and Q) cells in pancreatic sections reveals morphological changes in the islets of MIP-1CreERT::Manffl/fl(TMX) mice compared with MIP-1CreERT::Manffl/fl(OIL) mice. Scale bar = 50 μm. R: β-Cell mass is significantly reduced in P90 MIP-1CreERT::Manffl/fl(TMX) mice compared with that in MIP-1CreERT::Manffl/fl(OIL) mice (n = 4–5 mice/group, both sexes). S: β-Cell proliferation assessed by Ki67 and insulin staining (n = 6 mice/group). T: β-Cell apoptosis assessed by TUNEL and insulin staining (n = 6 mice/group). Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the corresponding control. GLS, glucose; ns, not significant.

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Basal and glucose-stimulated insulin release from islets isolated from MIP-1CreERT::Manffl/fl(TMX) mice was significantly lower compared with controls (Fig. 5E). Accordingly, islet insulin content also was reduced (Fig. 5F). However, as shown for Manf−/− islets, their capacity to secrete insulin relative to cellular insulin content did not differ from controls, and the response to 3-isobutyl-1-methylxanthine (IBMX) was even increased (Fig. 5G).

Immunofluorescence staining revealed MANF expression in some β-cells 4 weeks after the TMX injection in MIP-1CreERT::Manffl/fl mice (Fig. 5K–M). The relative proportion of double MANF- and insulin-positive β-cells observed in islets of TMX-injected mice was 15.22 ± 3.27% compared with islets of MIP-1CreERT::Manffl/fl(OIL) controls, suggesting incomplete recombination with a degree of β-cell regeneration during the 4-week washout period after TMX injections. We also observed some loss of MANF expression (14.8 ± 3.6%) in the insulin-positive β-cells of MIP-1CreERT::Manffl/fl(OIL) islets (Fig. 5H–J and Supplementary Fig. 5.1F–W). This finding is in line with quantitative RT-PCR showing significantly reduced Manf mRNA levels in isolated islets from MIP-1CreERT::Manffl/fl(OIL) mice compared with islets from oil-injected Manffl/fl and TMX-injected Manffl/fl mice, suggesting some degree of leakiness resulting in Cre translocation to the nucleus even in the absence of TMX (Supplementary Fig. 5.1E). Compared with islets from MIP-1CreERT::Manffl/fl(OIL) mice, Manf mRNA levels were significantly reduced in MIP-1CreERT::Manffl/fl(TMX) islets (Supplementary Fig. 5.1E). Of note, TMX seemed to repress Manf mRNA expression by itself in Manffl/fl islets (Supplementary Fig. 5.1E). MANF expression in the brain did not differ between MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (Supplementary Fig. 5.2A–J).

Immunoperoxidase staining of pancreatic sections 4 weeks after TMX injection revealed that lack of MANF in the β-cells of adult MIP-1CreERT::Manffl/fl(TMX) mice resulted in β-cell loss, decreased insulin expression, and loss of islet architecture by redistribution of glucagon-stained α-cells into the core of islets (Fig. 5N–Q and Supplementary Fig. 5.1F–W). Significant reduction of the β-cell mass and islet cell mass and a slight, but nonsignificant increase in α-cell mass were observed in MIP-1CreERT::Manffl/fl(TMX) pancreata compared with the control groups (Fig. 5R and Supplementary Fig. 5.1X–Z). Whereas the proliferation rate of insulin-positive β-cells in pancreata of MIP-1CreERT::Manffl/fl(OIL) mice was 0.87 ± 0.08%, quantification of Ki67-positive insulin-positive β-cells revealed a 50% reduction in proliferative β-cells in the MIP-1CreERT::Manffl/fl(TMX) pancreata (0.45 ± 0.03%) (Fig. 5S). In addition, the number of TUNEL-positive insulin-positive β-cells were significantly increased in pancreata of MIP-1CreERT::Manffl/fl(TMX) animals (4.4 ± 0.23%) compared with MIP-1CreERT::Manffl/fl(OIL) (1.77 ± 0.11%), demonstrating increased β-cell apoptosis in pancreata of adult mutant mice (Fig. 5T).

Consistent with decreased β-cell mass in adult Manf-deleted mice, we found a significant decrease in mRNA levels for Glut2, Ins1/2, Pdx1, and MafA but not Gck in islets of MIP-1CreERT::Manffl/fl(TMX) mice compared with control islets (Fig. 6A). In line with reduced expression of Glut2 and Pdx1 in islets, we observed loss of GLUT2 membrane expression in β-cells where MANF was lacking (Fig. 6C–H). Reduced nuclear PDX1 staining also was detected in islets deficient for MANF at adult age (Fig. 6I–N). Thus, expression of MANF in β-cells is essential to maintain β-cell phenotype and mass also in adult mice.

Figure 6

MANF expression in β-cells is essential for maintaining adult β-cell mass. A: Quantitative RT-PCR for mRNA levels of β-cell–specific genes Glut2, Ins1/2, Pdx-1, Gck, and MafA in islets from P90 MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (n = islets from 7–9 mice/group). B: Quantitative RT-PCR analysis of UPR genes Grp78, Chop, Atf4, spXbp1, tXbp, Atf6α, and Atf6β in islets from MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (n = islets from 7–9 mice/group). CH: Double IHC of MANF (D and G) and GLUT2 (C and F) in pancreatic sections from P90 MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice. Scale bar = 10 μm. IN: Double IHC of PDX1 (J and M) and insulin (I and L) in pancreatic sections from MIP-1CreERT::Manffl/fl(OIL) or MIP-1CreERT::Manffl/fl(TMX) mice. Scale bar = 10 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the corresponding control. ns, not significant.

Figure 6

MANF expression in β-cells is essential for maintaining adult β-cell mass. A: Quantitative RT-PCR for mRNA levels of β-cell–specific genes Glut2, Ins1/2, Pdx-1, Gck, and MafA in islets from P90 MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (n = islets from 7–9 mice/group). B: Quantitative RT-PCR analysis of UPR genes Grp78, Chop, Atf4, spXbp1, tXbp, Atf6α, and Atf6β in islets from MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (n = islets from 7–9 mice/group). CH: Double IHC of MANF (D and G) and GLUT2 (C and F) in pancreatic sections from P90 MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice. Scale bar = 10 μm. IN: Double IHC of PDX1 (J and M) and insulin (I and L) in pancreatic sections from MIP-1CreERT::Manffl/fl(OIL) or MIP-1CreERT::Manffl/fl(TMX) mice. Scale bar = 10 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the corresponding control. ns, not significant.

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MANF deficiency in β-cells during development leads to increased and unresolved ER stress and chronic activation of the UPR response. In line with previous data, high levels of mRNA for Grp78, Chop, Atf4, Atf6α, and Atf6β were detected in islets from adult MIP-1CreERT::Manffl/fl(TMX) mice (Fig. 6B). The level of spXbp1 and tXbp1 mRNA remained the same in both groups (Fig. 6B). Hence, increased and chronic activation of the UPR seem to be one of the mechanisms behind β-cell failure after MANF-deletion also in mature adult β-cells.

Insights in MANF Mechanism of Action

Differences in cell signaling pathways between Manf+/+ and Manf−/− islets were studied in the presence or absence of rhMANF in vitro. Previous studies have suggested that MANF exerts its protective effect on neurons through activation of the PI3K/AKT signaling pathway (41). We found a trend for an increase in AKT (Ser473) phosphorylation in Manf+/+ islets after MANF addition (Fig. 7A and B) besides a detectable, but nonsignificant upregulation of pan-AKT levels in MANF-treated islets (Supplementary Fig. 6C). A slight decrease in phosphorylated (P) AKT was found in Manf−/− islets, which was clearly, but not significantly upregulated by the addition of rhMANF (Fig. 7A and B and Supplementary Fig. 6C). Furthermore, Tribbles homolog 3 (Trib3) mRNA levels were significantly increased in Manf–/– islets (Fig. 7C and D and Supplementary Fig. 6A). Because Trib3 mRNA expression is modulated by ATF4 in the PERK-UPR pathway and TRIB3 is a negative regulator of AKT, the reduced AKT phosphorylation in Manf−/− islets might be caused by the chronic activation of ATF4 followed by increased TRIB3 expression (5,42,43). We also found a trend for a slight decrease in the level of P-ERK1 (44 kDa) in Manf−/− islets compared with Manf+/+ islets (Fig. 7E and F and Supplementary Fig. 6D). MANF has been shown to relieve nuclear factor (NF)-κB activation and to downregulate B-cell lymphoma/leukemia 10 (BCL10) expression in human islets exposed to cytokines in vitro (30). Because BCL10 is a known inducer of apoptosis and an upstream regulator of NF-κB signaling (44,45), we next tested whether NF-κB was differently regulated in β-cells in vitro. We found a trend for increased NF-κB phosphorylation in Manf−/− islets compared with Manf+/+ islets (Fig. 7G and H and Supplementary Fig. 6E). Furthermore, BCL10, a binding partner of tumor necrosis factor receptor–associated factor 2 (TRAF2) and a regulator of NF-κB (46), was found to be significantly upregulated in Pdx-1Cre+/−::Manffl/fl islets at P1 (Fig. 7I and J and Supplementary Fig. 6B). Because IRE1α in prolonged ER stress forms a complex with apoptosis signal-regulating kinase-1 (ASK1) and TRAF2 triggering c-jun terminal kinase (JNK) activation (47), we tested c-Jun activation in the Manf−/− islets. We observed a trend for increased phosphorylation of c-Jun (Ser63) in Manf−/− islets compared with Manf+/+ islets (Fig. 7K and L and Supplementary Fig. 6G). In addition to JNK, IRE1α stimulates the activation of p38 mitogen-activated protein kinase that promotes apoptosis through phosphorylation of CHOP (48). A significant increase in levels of P-p38 also was detected in Manf−/− islets compared with Manf+/+ islets (Fig. 7M and N and Supplementary Fig. 6F). In rat β-cells, NF-κB induces expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production (47). We found a trend for increased iNOS expression in the Manf−/− islets (Fig. 7O and P), suggesting NO production, inhibition of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2b) pump, and depletion of Ca2+ from the ER, increasing the amplitude of ER stress (47).

Figure 7

Insights in MANF mechanism of action. AP: Cell signaling pathways in islet β-cells from 4–5-week-old Manf−/− and Manf+/+ mice in the presence or absence of recombinant MANF (A, E, G, K, M, and O). Western blot analysis with indicated antibodies on islet lysates from Manf−/− and Manf+/+ mice, nontreated (−) or treated (+) with recombinant MANF. Quantified intensities of Western blot bands of P-AKT (Ser 473) compared with the total amount of AKT (B), P-ERK1/2 to total ERK1/2 (F), P-NF-κB p65 (Ser536) to total NF-κB p65 (H), P-c-Jun (Ser63) to total c-Jun (L), P-p38 to total p38 (N), and iNOS compared with GAPDH (P) (n = 2–3 wells/group). Quantitative real-time PCR analysis of Trib3 mRNA expression in islets isolated from Manffl/fl and Pdx-1Cre+/−::Manffl/fl mice at P1 and P14 (C) and MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (D) (n = islets from 4–6 mice/group). Quantitative RT-PCR analysis of Bcl10 mRNA expression in islets isolated from Manffl/fl and Pdx-1Cre+/−::Manffl/fl mice at P1 and P14 (I) and from MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (J) (n = islets from 4–6 mice/group). Q: A link between ER stress and inflammation in Manf–/– mouse pancreatic β-cells. MANF deficiency in β-cells leads to ER stress and activation of all three UPR branches: ATF6, PERK, and IRE1α. Transcription factor ATF4, which escapes the translational inhibition induced by P-eIF2α in the PERK pathway, increases Trib3 expression. TRIB3 is known to block AKT phosphorylation and thereby inhibits insulin signaling and β-cell cycle progression (62). Expression of CHOP, a proapoptotic transcription factor, is induced by both cleaved ATF6 and ATF4. IRE1α activation results in activation of JNK, NF-κB, and p38 MAPK inflammatory signaling cascades, contributing to the increased expression of several proapoptotic genes, including Bcl10, which is a known inducer of apoptosis and regulator of NF-κB. NF-κB activation leads to increase iNOS production, which contributes to increased NO production. NO has been found to inhibit the SERCA2b pump and thereby contributes to depletion of Ca2+ from the ER, leading to increased ER stress (46). Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. the corresponding control. ERAD, ER-associated protein degradation; IΚB, inhibitor of κB kinases; ns, not significant.

Figure 7

Insights in MANF mechanism of action. AP: Cell signaling pathways in islet β-cells from 4–5-week-old Manf−/− and Manf+/+ mice in the presence or absence of recombinant MANF (A, E, G, K, M, and O). Western blot analysis with indicated antibodies on islet lysates from Manf−/− and Manf+/+ mice, nontreated (−) or treated (+) with recombinant MANF. Quantified intensities of Western blot bands of P-AKT (Ser 473) compared with the total amount of AKT (B), P-ERK1/2 to total ERK1/2 (F), P-NF-κB p65 (Ser536) to total NF-κB p65 (H), P-c-Jun (Ser63) to total c-Jun (L), P-p38 to total p38 (N), and iNOS compared with GAPDH (P) (n = 2–3 wells/group). Quantitative real-time PCR analysis of Trib3 mRNA expression in islets isolated from Manffl/fl and Pdx-1Cre+/−::Manffl/fl mice at P1 and P14 (C) and MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (D) (n = islets from 4–6 mice/group). Quantitative RT-PCR analysis of Bcl10 mRNA expression in islets isolated from Manffl/fl and Pdx-1Cre+/−::Manffl/fl mice at P1 and P14 (I) and from MIP-1CreERT::Manffl/fl(OIL) and MIP-1CreERT::Manffl/fl(TMX) mice (J) (n = islets from 4–6 mice/group). Q: A link between ER stress and inflammation in Manf–/– mouse pancreatic β-cells. MANF deficiency in β-cells leads to ER stress and activation of all three UPR branches: ATF6, PERK, and IRE1α. Transcription factor ATF4, which escapes the translational inhibition induced by P-eIF2α in the PERK pathway, increases Trib3 expression. TRIB3 is known to block AKT phosphorylation and thereby inhibits insulin signaling and β-cell cycle progression (62). Expression of CHOP, a proapoptotic transcription factor, is induced by both cleaved ATF6 and ATF4. IRE1α activation results in activation of JNK, NF-κB, and p38 MAPK inflammatory signaling cascades, contributing to the increased expression of several proapoptotic genes, including Bcl10, which is a known inducer of apoptosis and regulator of NF-κB. NF-κB activation leads to increase iNOS production, which contributes to increased NO production. NO has been found to inhibit the SERCA2b pump and thereby contributes to depletion of Ca2+ from the ER, leading to increased ER stress (46). Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. the corresponding control. ERAD, ER-associated protein degradation; IΚB, inhibitor of κB kinases; ns, not significant.

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We recently reported that global absence of MANF in mouse results in insulin-deficient diabetes and reduced viability (22). Herein, we provide new insights for the role of MANF during pancreas development and in mature β-cells.

MANF expression in the developing and adult mouse was studied in detail, revealing expression of MANF already in the endocrine pancreatic primordium during mouse embryonic development. Adult mouse pancreas exhibited MANF protein and Manf mRNA expression in both islets of Langerhans and pancreatic exocrine acinar cells. Of note, in endocrine islets, MANF was predominantly expressed in β-cells with some expression in δ-cells, but not in α- or PP cells, comparable to results obtained in human islets (30). MANF protein levels are highly upregulated in pancreatic β-cells of Ins2Akita mice, where UPR is triggered by the accumulation of misfolded proinsulin in the ER, leading to β-cell death (29,49,50). Here, we provide evidence that MANF expression is increased in β-cells near infiltrated lymphocytes in prediabetic NOD islets, in agreement with data showing increased Manf mRNA expression and UPR activation preceding β-cell death in islets from prediabetic NOD mice (51,52). Increased MANF expression was detected in some β-cells in the islets of db/db mice in line with recently published data (53). Conversely, MANF was downregulated as a consequence of reduced Glis3 expression in β-cells of diabetes-susceptible NOD mice, where chronic ER stress led to β-cell death (54). This study further supports our results that MANF expression in β-cells is needed for restoring cellular homeostasis by preventing long-term ER stress leading to β-cell apoptosis, senescence, and diabetes.

Similarly to Manf−/− mice, Pdx-1Cre+/−::Manffl/fl mice developed hyperglycemia and hypoinsulinemia as a result of postnatal reduced β-cell mass caused by reduced β-cell proliferation and increased β-cell death. However, because of mosaic Cre recombination, some Pdx-1Cre+/−::Manffl/fl mice demonstrated patchy expression of MANF in both β-cells and exocrine acinar cells at P14 and onward, suggesting clonal expansion of MANF-expressing cells in these pancreata. Of note, ectopic Cre expression and recombination in the hypothalamic region of the brain of Pdx-1Cre+/− mice has been reported (55). However, MANF was not removed from hypothalamic neurons in the Pdx-1Cre+/−::Manffl/fl brain, suggesting that the diabetic phenotype is caused solely by the pancreatic deletion of MANF.

We previously demonstrated that β-cell mass is unaffected in E18.5 Manf−/− mice but decreased by 50% in newborn Manf−/− mice (22), suggesting that MANF is not needed for the development of β-cell mass before birth but, rather, for the postnatal expansion of β-cell mass. Here, we demonstrated for the first time in our knowledge that the number of MANF-positive β-cells positively correlates with β-cell mass as well as with blood glucose levels and TUNEL-positive β-cells in individual Pdx-1Cre+/−::Manffl/fl mice. The delayed onset of diabetes in Pdx-1Cre+/−::Manffl/fl mice compared with conventional Manf−/− mice likely was due to an incomplete removal of MANF from the pancreas.

To directly address the role of MANF in mature β-cells, we specifically excised MANF from β-cells in adult mice. Although MANF was not completely deleted from all pancreatic β-cells in MIP-1CreERT::Manffl/fl(TMX) mice, we demonstrate that lack of MANF in the adult β-cells results in reduced expression of β-cell–specific genes and poor β-cell survival and proliferation, leading to hyperglycemia and diabetes. The above results strongly argue against a systemic effect of circulating MANF in rescuing β-cells and clearly demonstrates that local expression of MANF is required to maintain the β-cell phenotype in mice (56,57). Furthermore, MANF expression in pancreatic exocrine acinar cells did not protect MANF-deficient adult β-cells.

The ability to stimulate adult β-cell proliferation and survival, inhibit β-cell apoptosis, and induce β-cell neogenesis would significantly progress the prospects of therapy for diabetes. Currently, a number of different factors and hormones have been identified that induce β-cell proliferation in vitro and/or in vivo (58,59). However, not many can enhance the rates of β-cell proliferation and restrain β-cell apoptosis at the same time. In this study, we demonstrated that the MANF protein intensifies islet β-cell proliferation from aged mice, suggesting that MANF could partly reverse age-dependent repression of β-cell replication. Previously, we found that recombinant MANF protein partially rescues mouse and human β-cells from cytokine- and ER stress–induced cell death in culture (30,38) and induces human β-cell proliferation (30). Here, we show that Manf mRNA is upregulated in mouse β-cells in hyperglycemic conditions in vitro and that MANF protein reduces ER stress–induced glucotoxicity in mouse β-cells. We show that MANF protein induces β-cell proliferation and reduces ER stress in Manf–/– islets. Previously, we showed that inflammatory cytokines increase MANF secretion from human β-cells in vitro (30). These results strongly indicate that exogenous MANF acts in a paracrine manner by dampening ER stress, thus favoring β-cell protection and proliferation.

Still unknown is how and where MANF exerts its beneficial effects. We show that MANF resides in the ER where it modulates ER stress and is crucial for β-cell survival. Upon ER stress, MANF is secreted from β-cells (30), and rhMANF has protective and restorative effects when applied exogenously to human and mouse β-cells (30,38). Thus, MANF seems to have a dual mode of action: 1) cotranslationally where it inserts in the ER, thereby modulating ER stress, and 2) extracellularly (possibly through autocrine/paracrine signaling) by activating a signal transduction pathway and/or being endocytosed and transported to the ER.

The identification of signaling receptors for MANF has been challenging. With its suboptimal ER retention signal in its COOH-terminal domain, MANF was found to bind weakly to the KDELR in the ER (20). The same study suggested that MANF also binds KDELRs at the cell membrane of cell lines overexpressing KDELRs (20). However, direct binding of MANF to the KDELR was not confirmed. Recently, MANF was reported to bind to lipid sulfatides (e.g., 3-O-sulfogalactosylceramide) located at the outer leaflet of cell membranes in Caenorhabditis elegans and in mammalian cells after MANF uptake by endocytosis into the cells (21). This report suggested that sulfatides are important for MANF cell surface binding, transport, and secretion (21). The N-terminal domain of MANF contains a saposin-like domain known to be able to bind lipids (60). In addition, in the pancreas, sulfatides are selectively synthesized in β-cells but not in pancreatic exocrine tissue (61). Thus, exogenous MANF might be taken up by β-cells through lipid sulfatide binding and endocytosed back to the ER where it relieves ER stress, leading to restored proliferation and/or protection from ER stress–induced apoptosis. However, the increased mitogenic effect of MANF on β-cells still might depend on as-yet unidentified signaling receptors and signaling cascades.

Previously, we demonstrated the activation of all three UPR branches in pancreatic islets in Manf −/− mice (22). Herein, we confirm that chronic UPR activation and persistent ER stress in Manf −/− islets is one of the mechanisms behind the reduced β-cell proliferation and increased β-cell death in islets from both Pdx-1Cre+/−::Manffl/fl mice and MIP-1CreERT::Manffl/fl(TMX) mice. In addition, we show a trend to decreased AKT phosphorylation and increased activation of inflammatory signaling in Manf−/− islets probably caused by chronically activated UPR pathways. In agreement, we recently reported that MANF relieves cytokine-induced β-cell death through NF-κB inhibition in human β-cells (30). In addition, MANF has been implicated as a negative regulator of inflammation by inhibiting expression of NF-κB target genes in the nucleus of fibroblast-like synoviocytes (62). Of note, platelet-derived growth factor released from injured retina promoted MANF expression in innate immune cells and biased cells toward an anti-inflammatory phenotype, thereby promoting retinal tissue repair in mouse and fruit fly (63). Thus, roles for MANF in inflammation are emerging. In this study, we provide evidence for a mechanistic link between chronic ER stress and increased inflammation leading to β-cell death in Manf−/− islets (Fig. 7Q). A modest activation of the UPR has been suggested to adapt β-cells to the high demand of insulin production (10,64). Therefore, reduced insulin production by knockdown of Ins2 gene in Ins1−/− background in adult mouse β-cells results in decreased PERK/eIF2α activation (65). The decrease in UPR leads to an increase in proliferation through upregulation of the AKT/cyclin D1 axis caused by a decrease in Trib3 expression, suggesting that increased UPR signaling through P-eIF2α reduces β-cell proliferation. Similarly, addition of MANF to ER-stressed β-cells might reduce ER stress and lead to increased β-cell proliferation. A recent study revealed that genetically induced MANF overexpression in the hypothalamus leads to increased feeding behavior and obesity in mice caused by impaired insulin signaling and insulin resistance in the hypothalamus (66). In contrast, decreased MANF expression in the hypothalamus results in hypophagia and reduced body weight. Increased MANF at the ER has been suggested to recruit and activate PIP4k2b, thus reducing AKT phosphorylation downstream of insulin receptor signaling leading to the hyperphagia (66). This result suggests that cellular MANF levels are critical for insulin-regulated AKT phosphorylation. Thus, MANF deficiency seems to result in ER stress, increased PERK/P-eIF2α signaling, and selective ATF4 translation inducing Trib3 expression leading to reduced AKT phosphorylation and proliferation.

In conclusion, deletion of MANF from mouse β-cells both in early pancreatic development and in adulthood results in insulin-deficient diabetes. Consequently, endogenous MANF levels in β-cells correlates with β-cell survival in normal, diabetes-susceptible, and stressed β-cells, but the precise mechanisms of MANF action needs further investigation. The ability of MANF to induce proliferation and at the same time protect β-cells from death makes it a promising drug candidate for the treatment of diabetes.

Acknowledgments. The authors thank S. Tynkkynen, Mari Heikkinen, Antti Salminen, and Mervi Lindman (all from the Institute of Biotechnology, HiLIFE, University of Helsinki) for excellent technical assistance. The authors also acknowledge National Institutes of Health Knockout Mouse Phenotyping for the MANF-targeted embryonic stem cell clone used to develop the MANF knockout mice, FIMM High Content Imaging and Analysis unit services (University of Helsinki) for imaging and data analysis, and the scanning service of the Institute of Biotechnology and Research Programs Unit, Faculty of Medicine, University of Helsinki, Biocenter Finland, for fluorescence or light microscopy images captured with the Pannoramic 250 Flash II series digital scanner (3DHISTECH, Budapest, Hungary). Finally, the authors thank Mart Saarma (University of Helsinki) for financial support and valuable comments on the article.

Funding. This project was supported by JDRF grant 17-2013-410, the Commission of the European Union collaborative project MOLPARK (no. 400752), the Sigrid Juselius Foundation, the Academy of Finland grant no. 141122, Biocenter Finland (to I.B. and E.J.), and the Helsinki Institute of Life Science infrastructure. M.L. was supported by grants from the Finnish Diabetes Research Foundation and the Academy of Finland (no. 117044).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. T.D. designed and performed experiments and wrote the manuscript. T.D., I.B., H.L., E.P., E.J., T.O., and M.L. accepted the final version of the manuscript. I.B. and E.J. performed serial block-face scanning electron microscopy experiments and analyzed the data. H.L. and E.P. performed experiments. T.O. designed experiments and revised the manuscript. M.L. designed and performed experiments, provided funding, and wrote the manuscript. T.D. and M.L. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in poster form at the 2nd Joint Meeting of the European Association for the Study of Diabetes Islet Study Group and Βeta Cell Workshop, Dresden, Germany, 7–10 May 2017, and orally at the 53rd Annual Meeting of the European Association for the Study of Diabetes, Lisbon, Portugal, 10–15 September 2017.

1.
Eizirik
DL
,
Colli
ML
,
Ortis
F
.
The role of inflammation in insulitis and beta-cell loss in type 1 diabetes
.
Nat Rev Endocrinol
2009
;
5
:
219
226
[PubMed]
2.
Olokoba
AB
,
Obateru
OA
,
Olokoba
LB
.
Type 2 diabetes mellitus: a review of current trends
.
Oman Med J
2012
;
27
:
269
273
[PubMed]
3.
Donath
MY
,
Halban
PA
.
Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications
.
Diabetologia
2004
;
47
:
581
589
[PubMed]
4.
Eizirik
DL
,
Cnop
M
.
ER stress in pancreatic beta cells: the thin red line between adaptation and failure
.
Sci Signal
2010
;
3
:
pe7
[PubMed]
5.
Eizirik
DL
,
Miani
M
,
Cardozo
AK
.
Signalling danger: endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation
.
Diabetologia
2013
;
56
:
234
241
[PubMed]
6.
Cnop
M
,
Foufelle
F
,
Velloso
LA
.
Endoplasmic reticulum stress, obesity and diabetes
.
Trends Mol Med
2012
;
18
:
59
68
[PubMed]
7.
Kaufman
RJ
,
Back
SH
,
Song
B
,
Han
J
,
Hassler
J
.
The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in β-cells
.
Diabetes Obes Metab
2010
;
12
(
Suppl. 2
):
99
107
[PubMed]
8.
Ron
D
,
Walter
P
.
Signal integration in the endoplasmic reticulum unfolded protein response
.
Nat Rev Mol Cell Biol
2007
;
8
:
519
529
[PubMed]
9.
Delépine
M
,
Nicolino
M
,
Barrett
T
,
Golamaully
M
,
Lathrop
GM
,
Julier
C
.
EIF2AK3, encoding translation initiation factor 2-alpha kinase 3, is mutated in patients with Wolcott-Rallison syndrome
.
Nat Genet
2000
;
25
:
406
409
[PubMed]
10.
Harding
HP
,
Ron
D
.
Endoplasmic reticulum stress and the development of diabetes: a review
.
Diabetes
2002
;
51
(
Suppl. 3
):
S455
S461
[PubMed]
11.
Harding
HP
,
Zeng
H
,
Zhang
Y
, et al
.
Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival
.
Mol Cell
2001
;
7
:
1153
1163
[PubMed]
12.
Hetz
C
.
The unfolded protein response: controlling cell fate decisions under ER stress and beyond
.
Nat Rev Mol Cell Biol
2012
;
13
:
89
102
[PubMed]
13.
Scheuner
D
,
Kaufman
RJ
.
The unfolded protein response: a pathway that links insulin demand with beta-cell failure and diabetes
.
Endocr Rev
2008
;
29
:
317
333
[PubMed]
14.
Petrova
P
,
Raibekas
A
,
Pevsner
J
, et al
.
MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons
.
J Mol Neurosci
2003
;
20
:
173
188
[PubMed]
15.
Glembotski
CC
,
Thuerauf
DJ
,
Huang
C
,
Vekich
JA
,
Gottlieb
RA
,
Doroudgar
S
.
Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion
.
J Biol Chem
2012
;
287
:
25893
25904
[PubMed]
16.
Airavaara
M
,
Shen
H
,
Kuo
CC
, et al
.
Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats
.
J Comp Neurol
2009
;
515
:
116
124
[PubMed]
17.
Yang
S
,
Huang
S
,
Gaertig
MA
,
Li
XJ
,
Li
S
.
Age-dependent decrease in chaperone activity impairs MANF expression, leading to Purkinje cell degeneration in inducible SCA17 mice
.
Neuron
2014
;
81
:
349
365
[PubMed]
18.
Apostolou
A
,
Shen
Y
,
Liang
Y
,
Luo
J
,
Fang
S
.
Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death
.
Exp Cell Res
2008
;
314
:
2454
2467
[PubMed]
19.
Voutilainen
MH
,
Bäck
S
,
Pörsti
E
, et al
.
Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson’s disease
.
J Neurosci
2009
;
29
:
9651
9659
[PubMed]
20.
Henderson
MJ
,
Richie
CT
,
Airavaara
M
,
Wang
Y
,
Harvey
BK
.
Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors
.
J Biol Chem
2013
;
288
:
4209
4225
[PubMed]
21.
Bai
M
,
Vozdek
R
,
Hnízda
A
, et al
.
Conserved roles of C. elegans and human MANFs in sulfatide binding and cytoprotection
.
Nat Commun
2018
;
9
:
897
[PubMed]
22.
Lindahl
M
,
Danilova
T
,
Palm
E
, et al
.
MANF is indispensable for the proliferation and survival of pancreatic β cells
.
Cell Rep
2014
;
7
:
366
375
[PubMed]
23.
Hingorani
SR
,
Petricoin
EF
,
Maitra
A
, et al
.
Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse
.
Cancer Cell
2003
;
4
:
437
450
[PubMed]
24.
Tamarina
NA
,
Roe
MW
,
Philipson
L
.
Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic β-cells
.
Islets
2014
;
6
:
e27685
[PubMed]
25.
Deerinck
TJ
,
Bushong
E
,
Thor
A
,
Ellisman
MH
.
NCMIR methods for 3D EM: a new protocol for preparation of biological specimens for serial block-face SEM
[article online].
Microscopy
2010
;
6
8
. Available from https://ncmir.ucsd.edu/sbem-protocol
26.
Belevich
I
,
Joensuu
M
,
Kumar
D
,
Vihinen
H
,
Jokitalo
E
.
Microscopy image browser: a platform for segmentation and analysis of multidimensional datasets
.
PLoS Biol
2016
;
14
:
e1002340
[PubMed]
27.
Lindholm
P
,
Peränen
J
,
Andressoo
JO
, et al
.
MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain
.
Mol Cell Neurosci
2008
;
39
:
356
371
[PubMed]
28.
Seymour
PA
,
Sander
M
.
Historical perspective: beginnings of the beta-cell: current perspectives in beta-cell development
.
Diabetes
2011
;
60
:
364
376
[PubMed]
29.
Mizobuchi
N
,
Hoseki
J
,
Kubota
H
, et al
.
ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element
.
Cell Struct Funct
2007
;
32
:
41
50
[PubMed]
30.
Hakonen
E
,
Chandra
V
,
Fogarty
CL
, et al
.
MANF protects human pancreatic beta cells against stress-induced cell death
.
Diabetologia
2018
;
61
:
2202
2214
[PubMed]
31.
Akiyama
M
,
Hatanaka
M
,
Ohta
Y
, et al
.
Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice
.
Diabetologia
2009
;
52
:
653
663
[PubMed]
32.
Riggs
AC
,
Bernal-Mizrachi
E
,
Ohsugi
M
, et al
.
Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis
.
Diabetologia
2005
;
48
:
2313
2321
[PubMed]
33.
Wang
J
,
Takeuchi
T
,
Tanaka
S
, et al
.
A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse
.
J Clin Invest
1999
;
103
:
27
37
[PubMed]
34.
Dor
Y
,
Brown
J
,
Martinez
OI
,
Melton
DA
.
Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation
.
Nature
2004
;
429
:
41
46
[PubMed]
35.
Teta
M
,
Long
SY
,
Wartschow
LM
,
Rankin
MM
,
Kushner
JA
.
Very slow turnover of beta-cells in aged adult mice
.
Diabetes
2005
;
54
:
2557
2567
[PubMed]
36.
Kushner
JA
,
Weir
GC
,
Bonner-Weir
S
.
Ductal origin hypothesis of pancreatic regeneration under attack
.
Cell Metab
2010
;
11
:
2
3
[PubMed]
37.
Kushner
JA
.
The role of aging upon β cell turnover
.
J Clin Invest
2013
;
123
:
990
995
[PubMed]
38.
Cunha
DA
,
Cito
M
,
Grieco
FA
, et al
.
Pancreatic β-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF)
.
J Biol Chem
2017
;
292
:
14977
14988
[PubMed]
39.
Elouil
H
,
Bensellam
M
,
Guiot
Y
, et al
.
Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets
.
Diabetologia
2007
;
50
:
1442
1452
[PubMed]
40.
Lipson
KL
,
Fonseca
SG
,
Urano
F
.
Endoplasmic reticulum stress-induced apoptosis and auto-immunity in diabetes
.
Curr Mol Med
2006
;
6
:
71
77
[PubMed]
41.
Zhang
J
,
Tong
W
,
Sun
H
, et al
.
Nrf2-mediated neuroprotection by MANF against 6-OHDA-induced cell damage via PI3K/AKT/GSK3β pathway
.
Exp Gerontol
2017
;
100
:
77
86
[PubMed]
42.
Cunard
R
.
Mammalian tribbles homologs at the crossroads of endoplasmic reticulum stress and Mammalian target of rapamycin pathways
.
Scientifica (Cairo)
2013
;
2013
:
750871
[PubMed]
43.
Du
K
,
Herzig
S
,
Kulkarni
RN
,
Montminy
M
.
TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver
.
Science
2003
;
300
:
1574
1577
[PubMed]
44.
Mazzone
P
,
Scudiero
I
,
Ferravante
A
, et al
.
Functional characterization of zebrafish (Danio rerio) Bcl10
.
PLoS One
2015
;
10
:
e0122365
[PubMed]
45.
Ruland
J
,
Duncan
GS
,
Elia
A
, et al
.
Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure
.
Cell
2001
;
104
:
33
42
[PubMed]
46.
Yoneda
T
,
Imaizumi
K
,
Maeda
M
, et al
.
Regulatory mechanisms of TRAF2-mediated signal transduction by Bcl10, a MALT lymphoma-associated protein
.
J Biol Chem
2000
;
275
:
11114
11120
[PubMed]
47.
Brozzi
F
,
Eizirik
DL
.
ER stress and the decline and fall of pancreatic beta cells in type 1 diabetes
.
Ups J Med Sci
2016
;
121
:
133
139
[PubMed]
48.
Ron
D
,
Hubbard
SR
.
How IRE1 reacts to ER stress
.
Cell
2008
;
132
:
24
26
[PubMed]
49.
Oyadomari
S
,
Araki
E
,
Mori
M
.
Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells
.
Apoptosis
2002
;
7
:
335
345
[PubMed]
50.
Oyadomari
S
,
Koizumi
A
,
Takeda
K
, et al
.
Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes
.
J Clin Invest
2002
;
109
:
525
532
[PubMed]
51.
Morita S, Villalta SA, Feldman HC, et al. Targeting ABL-IRE1α signaling spares ER-stressed pancreatic β cells to reverse autoimmune diabetes [published correction appears in Cell Metab 2017;25:1207]. Cell Metab 2017;25:883–897.e8
52.
Tersey
SA
,
Nishiki
Y
,
Templin
AT
, et al
.
Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model
.
Diabetes
2012
;
61
:
818
827
[PubMed]
53.
Neelankal John
A
,
Ram
R
,
Jiang
FX
.
RNA-seq analysis of islets to characterise the dedifferentiation in type 2 diabetes model mice db/db
.
Endocr Pathol
2018
;
29
:
207
221
[PubMed]
54.
Sharma
RB
,
O’Donnell
AC
,
Stamateris
RE
, et al
.
Insulin demand regulates β cell number via the unfolded protein response
.
J Clin Invest
2015
;
125
:
3831
3846
[PubMed]
55.
Wicksteed
B
,
Brissova
M
,
Yan
W
, et al
.
Conditional gene targeting in mouse pancreatic β-cells: analysis of ectopic Cre transgene expression in the brain
.
Diabetes
2010
;
59
:
3090
3098
[PubMed]
56.
Galli
E
,
Härkönen
T
,
Sainio
MT
, et al
.
Increased circulating concentrations of mesencephalic astrocyte-derived neurotrophic factor in children with type 1 diabetes
.
Sci Rep
2016
;
6
:
29058
[PubMed]
57.
Wu
T
,
Zhang
F
,
Yang
Q
, et al
.
Circulating mesencephalic astrocyte-derived neurotrophic factor is increased in newly diagnosed prediabetic and diabetic patients, and is associated with insulin resistance
.
Endocr J
2017
;
64
:
403
410
[PubMed]
58.
Vasavada
RC
,
Gonzalez-Pertusa
JA
,
Fujinaka
Y
,
Fiaschi-Taesch
N
,
Cozar-Castellano
I
,
Garcia-Ocaña
A
.
Growth factors and beta cell replication
.
Int J Biochem Cell Biol
2006
;
38
:
931
950
[PubMed]
59.
Tarabra
E
,
Pelengaris
S
,
Khan
M
.
A simple matter of life and death-the trials of postnatal Beta-cell mass regulation
.
Int J Endocrinol
2012
;
2012
:
516718
[PubMed]
60.
Parkash
V
,
Lindholm
P
,
Peränen
J
, et al
.
The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional
.
Protein Eng Des Sel
2009
;
22
:
233
241
[PubMed]
61.
Boslem
E
,
Meikle
PJ
,
Biden
TJ
.
Roles of ceramide and sphingolipids in pancreatic β-cell function and dysfunction
.
Islets
2012
;
4
:
177
187
[PubMed]
62.
Chen
L
,
Feng
L
,
Wang
X
, et al
.
Mesencephalic astrocyte-derived neurotrophic factor is involved in inflammation by negatively regulating the NF-κB pathway
.
Sci Rep
2015
;
5
:
8133
[PubMed]
63.
Neves
J
,
Zhu
J
,
Sousa-Victor
P
, et al
.
Immune modulation by MANF promotes tissue repair and regenerative success in the retina
.
Science
2016
;
353
:
aaf3646
[PubMed]
64.
Lee
AH
,
Heidtman
K
,
Hotamisligil
GS
,
Glimcher
LH
.
Dual and opposing roles of the unfolded protein response regulated by IRE1alpha and XBP1 in proinsulin processing and insulin secretion
.
Proc Natl Acad Sci U S A
2011
;
108
:
8885
8890
[PubMed]
65.
Szabat
M
,
Page
MM
,
Panzhinskiy
E
, et al
.
Reduced insulin production relieves endoplasmic reticulum stress and induces β cell proliferation
.
Cell Metab
2016
;
23
:
179
193
[PubMed]
66.
Yang
S
,
Yang
H
,
Chang
R
, et al
.
MANF regulates hypothalamic control of food intake and body weight
.
Nat Commun
2017
;
8
:
579
[PubMed]
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