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 (4–6). 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 (14–17). 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.
Research Design and Methods
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.
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
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.
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.
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).
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).
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) (31–33).
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 (35–37). 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).
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).
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.
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).
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.