Dichotomous role of pancreatic HUWE1/MULE/ARF-BP1 in modulating beta cell apoptosis in mice under physiological and genotoxic conditions
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Diabetes mellitus represents a significant burden on the health of the global population. Both type 1 and type 2 diabetes share a common feature of a reduction in functional beta cell mass. A newly discovered ubiquitination molecule HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase (HUWE1 [also known as MULE or ARF-BP1]) is a critical regulator of p53-dependent apoptosis. However, its role in islet homeostasis is not entirely clear.
We generated mice with pancreas-specific deletion of Huwe1 using a Cre-loxP recombination system driven by the Pdx1 promoter (Pdx1cre + Huwe1 fl/fl) to assess the in vivo role of HUWE1 in the pancreas.
Targeted deletion of Huwe1 in the pancreas preferentially activated p53-mediated beta cell apoptosis, leading to reduced beta cell mass and diminished insulin exocytosis. These defects were aggravated by ageing, with progressive further decline in insulin secretion and glucose homeostasis in older mice. Intriguingly, Huwe1 deletion provided protection against genotoxicity, such that Pdx1cre + Huwe1 fl/fl mice were resistant to multiple-low-dose-streptozotocin-induced beta cell apoptosis and diabetes.
HUWE1 expression in the pancreas is essential in determining beta cell mass. Furthermore, HUWE1 demonstrated divergent roles in regulating beta cell apoptosis depending on physiological or genotoxic conditions.
KeywordsApoptosis ARF-BP1 Beta cell Diabetes HUWE1 MULE
V-myc avian myelocytomatosis viral oncogene homologue
Glucose-stimulated insulin secretion
HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase
Myeloid cell leukaemia sequence 1
Mouse double minute 2 homologue
Multiple low dose streptozotocin
Pancreatic and duodenal homeobox 1
Type 1 diabetes is characterised by the autoimmune destruction of insulin-producing beta cells and typically has an early onset, while type 2 diabetes is associated with obesity and insulin resistance [1, 2]. Although caused by distinct underlying mechanisms, one common feature is an inadequate number of beta cells, which are required to maintain a sufficient plasma insulin level for glucose homeostasis.
Increased apoptotic cells have been observed in islets of patients with both type 1 and type 2 diabetes, in association with dramatic reduction in beta cell mass [1, 3]. The well-known tumour suppressor p53, when activated, leads to cell cycle arrest and apoptosis. It can be induced by a variety of insults, including DNA damage, oncogenic expression and ribosomal stress [4, 5]. The p53-dependent apoptotic pathway plays a critical role in beta cell demise during diabetes pathogenesis. The expression of p53 is increased in both human amylin- and nitric oxide-induced beta cell apoptosis in culture [6, 7]. In addition, beta-cell-specific p53 stabilisation and upregulation has been implicated in beta cell mass reduction in humans with diabetes [8, 9]. On the other hand, deletion of p53 in beta cells in culture provides resistance from NEFA-induced apoptosis . Furthermore, p53-null mice are protected from streptozotocin (STZ)-induced beta cell death and type 1 diabetes , which suggests that p53 plays an important regulatory role in beta cell homeostasis under both physiological and pathological conditions. Thus, p53 and its regulators represent a viable target for diabetes.
For years, mouse double minute 2 homologue (MDM2) has been touted as the principle inhibitor of p53, via its specific E3 ubiquitin ligase activity leading to p53 degradation . However, MDM2-null cells continue to undergo p53 stabilisation and subsequent apoptosis , suggesting an MDM2-independent regulation of p53. Indeed, multiple studies have shown molecules that directly regulate p53 function without MDM2 [14, 15, 16]. One such molecule is myeloid cell leukaemia sequence 1 (MCL1) ubiquitin ligase E3 (MULE) or HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase (HUWE1 [also known as ARF-BP1 or LASU1]). The Huwe1 gene is encoded on the X-chromosome and its protein product contains multiple domains, including BH3, which allows for polyubiquitination and degradation of p53 independent of MDM2 . Thus, HUWE1 may have a regulatory role in p53-mediated apoptosis of beta cells that occurs during diabetogenesis.
Apart from p53, HUWE1 has multiple other polyubiquitination substrates [18, 19, 20, 21, 22]. Among them is MCL1, which belongs to the anti-apoptotic B cell CLL/lymphoma 2 (BCL-2) family of proteins . MCL1 is rapidly induced after exposure to cell damaging stimuli, thus offering acute protection against apoptosis . Therefore, from a functional point of view, the interaction of HUWE1 with MCL1 is pro-apoptotic in nature. This is in direct contrast to the anti-apoptotic role of HUWE1 in its polyubiquitination of p53. Therefore, it is very likely that the role of HUWE1 is cell-type- and context-specific , and the function of HUWE1 in beta cell homeostasis and diabetes pathogenesis remains unclear.
In this study, we examined the role of HUWE1 in beta cells by targeted deletion of Huwe1 in mouse pancreas. Under basal conditions, we observed an overall anti-apoptotic role of HUWE1, predominantly through its inhibition of p53. As such, HUWE1-deficient mice showed a decline in beta cell mass with ageing without essential involvement of other known substrates of HUWE1 under physiological conditions. Furthermore, we showed that HUWE1 was critical for modulating exocytosis of insulin granules in beta cells. Intriguingly, Huwe1 deletion demonstrated an opposite effect in response to genotoxic stress, whereby HUWE1-deficient beta cells were resistant to streptozotocin (STZ)-induced apoptosis and diabetes. Thus, HUWE1 is a critical modulator of beta cell integrity and function under both physiological and pathological conditions.
Heterozygote Huwe1 fl/y mice (Tak Mak, Toronto, ON, Canada; exons 76 and 77 of Huwe1 flanked by loxP sites) were bred with Pdx1cre mice (Jackson Laboratories, Bar Harbor, ME, USA) [25, 26] to generate male Pdx1cre + Huwe1 +/y, male Pdx1cre + Huwe1 fl/y, female Pdx1cre + Huwe1 +/+ and female Pdx1cre + Huwe1 fl/fl mice. Male mice were used for experiments and littermates as controls. Genotypes were determined as described previously . Mice were maintained on a mixed 129J-C57BL/6 background and housed in a pathogen-free facility on a 12 h light/dark cycle and fed ad libitum with standard irradiated rodent chow (5% fat; Harlan Tecklad, Indianapolis, IN, USA) without restriction of activity in accordance with University Health Network Animal Care Facility Protocol.
Serum insulin, blood glucose, glucose tolerance tests, insulin tolerance tests and glucose-stimulated insulin secretion (GSIS) tests were performed as previously described . Serum insulin was measured by enzyme-linked immunosorbent assay kit (Crystal Chem, Downers Grove, IL, USA).
Low doses of STZ were injected i.p. into mice (40 mg/kg body weight) for 5 days (consecutive) .
Immunohistochemistry and immunofluorescence
The pancreas was fixed, stained and islet area quantified as previously described . Antibodies against insulin (Dako, Glostrup, Denmark), glucagon (Cell Signaling Technology, Beverly, MA, USA), HUWE1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TUNEL (Cell Signaling Technology), GLUT2 (Cell Signaling Technology), PDX1 (Santa Cruz Biotechnology), p53 (Cell Signaling Technology), p-p53 (Cell Signaling Technology) and MCL1 (Cell Signaling Technology) were used.
Liver, muscle, visceral adipose tissue, islets and hypothalamus protein lysates were obtained as previously described . Antibodies against actin (Santa Cruz Biotechnology), AMPK (Cell Signaling Technology), p-AMPK (Cell Signaling Technology), Akt (Cell Signaling Technology), p-Akt (Cell Signaling Technology), cleaved caspase 3 (Cell Signaling Technology), DNA polymerase beta (Abcam, Cambridge, MA, USA), GLUT2 (Chemicon, Temecula, CA, USA), MCL1 (Cell Signaling Technology), HUWE1 (Cell Signaling Technology), p53 (Cell Signaling Technology), PDX1 (Chemicon) were used.
Recording pipettes were from 1.5 mm borosilicate glass capillary tubes using a programmable micropipette puller. Pipettes were heat-polished and tip resistance ranged from 2 to 3 mΩ when filled with intracellular solution. For measurement of membrane capacitance, the intracellular solution contained: 125 mmol/l caesium glutamate, 10 mmol/l CsCl, 10 mmol/l NaCl, 1 mmol/l MgCl2, 5 mmol/l HEPES, 0.05 mmol/l EGTA, 3 mmol/l MgATP, 0.1 mmol/l cAMP, pH to 7.2. The extracellular solution consisted of 118 mmol/l NaCl, 5.6 mmol/l KCl, 1.2 mmol/l MgCl2, 10 mmol/l CaCl2, 20 mmol/l tetraethylammonium chloride, 5 mmol/l HEPES and 5 mmol/l d-glucose, pH 7.4. Cell membrane capacitance (Cm) was estimated by the Lindau–Neher technique, implementing the ‘sine-DC’ feature of the Lock-in module (40 mV peak-to-peak and a frequency of 500 Hz) in the whole-cell configuration. Recordings were conducted using an EPC10 patch clamp amplifier and the Pulse and X-Chart software programs (HEKA Electronik, Lambrecht, Germany).
Data are presented as means ± SEM and were analysed by one-sample t test.
Generation of pancreas-specific Huwe1-knockout mice
The mouse Huwe1 gene is located on the X-chromosome. To generate mice with Huwe1 deletion specifically in pancreatic progenitor cells during early pancreatic development (E8.5-9), we employed the Cre-loxP system with cre expression regulated by the pancreas-specific promoter of pancreatic and duodenal homeobox 1. Both adult male Pdx1cre + Huwe1 fl/y mice and female Pdx1cre + Huwe1 fl/fl mice showed dramatic reduction in Huwe1 mRNA (Fig. 1c and ESM Fig. 1d) in isolated islets. This translated into a significant reduction in HUWE1 protein (Fig. 1a and ESM Fig. 1a) and immunohistochemistry (Fig. 1b and ESM Fig. 1c). Pdx1 promoter expression has also been reported in the central nervous system, especially the hypothalamus, the command centre for many endocrine functions such as glucose and energy homeostasis. However, in our model no observable reduction in HUWE1 protein level was detected in isolated hypothalami from Pdx1cre + Huwe1 fl/y or Pdx1cre + Huwe1 fl/fl mice compared with control littermates (Fig. 1a and ESM Fig. 1a). Accordingly, total body weight, a major hypothalamic regulatory target, remained similar to controls in both sexes (ESM Fig. 2a–d). The levels of HUWE1 protein was also comparable between genotypes in other peripheral tissues, including liver, muscle and visceral fat (Fig. 1a and ESM Fig. 1a).
Pdx1cre + Huwe1 fl/y mice demonstrated age-dependent glucose intolerance and beta cell dysfunction without changes in insulin sensitivity
Pdx1cre + Huwe1 fl/y islets demonstrated age-dependent reduction in beta cell mass and increase in apoptosis
Individual beta cells from 6 month old male Pdx1cre + Huwe1 fl/y mice demonstrated secretory dysfunction
Huwe1 deletion preferentially activated p53-dependent apoptotic machinery
Multiple polyubiquitination and degradation substrates of HUWE1 have been identified. Among them, p53, MCL1 and DNA polymerase beta are better characterised [16, 20, 33]. Intriguingly, p53 induces apoptosis while MCL-1 and DNA polymerase inhibit the apoptotic pathway. Thus, HUWE1 seemingly regulates two opposing pathways involved in cell survival. To identify the signalling alterations that may have resulted from Huwe1 deletion in the pancreas in vivo, we measured the activation of p53 and MCL1 pathways. We found that Huwe1 deletion in islets led to increased p53 in Pdx1cre + Huwe1 fl/y mice at 2 months of age (Fig. 5f), with an even more significant difference in older mice at 6 months of age (ESM Fig. 5a). In keeping with the well-known pro-apoptotic role of p53, there was an associated increase in cleaved caspase 3 (ESM Fig. 5a). In comparison, levels of MCL1 and DNA polymerase beta were not significantly different between Pdx1cre + Huwe1 fl/y and control islets (Fig. 5f and ESM Fig. 5a). Therefore, p53-dependent apoptosis in HUWE1-deficient islets likely contributed to reduced beta cell mass and glucose intolerance in Pdx1cre + Huwe1 fl/y mice, while Huwe1 deletion in islets had minimal effect on pro-survival pathways, such as p-Akt, p-AMPK, MCL1 and DNA polymerase beta (Fig. 5f and ESM Fig. 5a). Consistent with reduction in beta cell secretory function at 6 months of age, beta cell differentiation marker PDX1 also showed a reduction, while glucose-sensing receptor GLUT2 was similar in HUWE1-deficient islets compared with controls (Fig. 5f and ESM Fig. 5a). Coupled with reduced granule filling/mobilising rate we observed in individual beta cells, these findings suggest that glucose intolerance in HUWE1-deficient mice is due to both a defect in exocytosis and survival of pancreatic beta cells.
HUWE1 deletion paradoxically leads to protection against STZ-induced beta cell apoptosis
HUWE1 is a newly discovered apoptosis regulator that acts via its E3 ligase activity, which polyubiquitinates known players in apoptosis. Intriguingly, these HUWE1 targets consist of effectors of both pro-apoptotic and anti-apoptotic pathways, thus the precise function of HUWE1 in beta cells was unknown. This study showed that HUWE1 modulated beta cell homeostasis by exerting a tonic inhibition on p53-dependent apoptosis in beta cells, such that pancreas-specific Huwe1 deletion led to increased beta cell death and glucose intolerance in mice under basal conditions. In contrast, HUWE1 appeared to play an opposite role when beta cells were under genotoxic stress. As such, the same HUWE1-deficient mice were paradoxically protected against MLDS-induced beta cell apoptosis and diabetes. This protection was not associated with a significant difference in expression of GLUT2, which is required for STZ action. Rather, these findings suggest that HUWE1 can regulate substrates such as p53 differently in specific settings, similar to the recent report on the dual role of HUWE1 in regulation of B lymphocyte survival and proliferation . HUWE1 demonstrated similar dual function in pancreatic beta cells, depending on the experimental context in which the role of HUWE1 was examined.
In agreement with the physiological pro-survival role of HUWE1, Kon and colleagues reported that rat insulin promoter (RIP)-mediated Huwe1 deletion in pancreatic beta cells led to similar beta cell apoptosis and diabetes starting at 6 months of age . However, the possibility of RIP-mediated effects in the central nervous system shown by other groups [35, 36, 37] was not explored in their study. On the other hand, the hypothalamic expression pattern of PDX1 is much more restricted . This was further supported in our mice where we used Pdx1-promoter-driven deletion of Huwe1, which resulted in no change in hypothalamic HUWE1 expression or peripheral insulin sensitivity. Furthermore, the region of the Huwe1 gene that was targeted was different between the two groups (loxP sites flanked exon 11 in the report by Kon et al, whereas we used mice with loxP sites flanking exons 76 and 77) . This difference is reflected in the early embryonic lethality (E12.5) of the whole-body knockout using the targeting construct used in our study, whereas perinatal lethality was observed by Kon et al [17, 34]. These differences suggest a more complete genetic ablation in mice that we used for our study in comparison to the report by Kon et al [17, 34].
Multiple HUWE1 substrates have been identified; among them is v-myc avian myelocytomatosis viral oncogene homologue (c-Myc) [18, 39]. Huwe1 deletion leads to accumulation of c-Myc/zinc finger and BTB domain containing 17 (MIZ1) complex and subsequent downregulation of p21 in models of skin carcinogenesis . Interestingly, in pancreatic beta cells, the absence of HUWE1 does not appear to have an impact on cell proliferation. It is possible that the intrinsic properties of low mitotic activity in these endocrine cells render them resistant against uncontrolled proliferation and tumorigenesis. Alternatively, this difference in HUWE1 function is a reflection of its context- and cell-type-dependent specificity in its biological role, which is highlighted by our findings in this report. Similarly, while HUWE1 can facilitate degradation of anti-apoptotic MCL1 [21, 24, 40], deletion of HUWE1 in the pancreas did not change MCL1 protein expression. This is consistent with the only slight differences in MCL1 that were seen at steady state in B lymphocytes with Huwe1 deletion, suggesting MCL1 regulation is context-specific [18, 24]. In addition to the essential role of HUWE1 in determining beta cell survival, we also showed an age-dependent decline in secretory function. Thus, the role of HUWE1 may be more complex than previously thought. Indeed, HUWE1 has been shown to modulate histone ubiquitination and chromatin condensation in addition to its role in cellular survival . Therefore, HUWE1 deletion may have a wide range of effects on protein expression important in other areas of cellular function, including potentially the machinery required for insulin granule secretion as suggested by our data showing a defect in exocytosis in Pdx1cre + Huwe1 fl/y beta cells.
In summary, our study shows HUWE1 is an important physiological regulator of both beta cell function and survival, such that Huwe1 deletion activates p53-dependent apoptosis and impairs beta cell secretory function with ageing under basal conditions. In contrast, HUWE1 has an opposite role under genotoxic conditions such that Huwe1 deletion provides protection against further apoptosis. These complexities in the roles of molecules that determine beta cell fate illustrate the need for clearer understanding of their intricacies as we strive to find novel therapeutic targets that enhance beta cell survival and function for treatment of both type 1 and type 2 diabetes.
This work was supported by grants to MW from the Canadian Institutes of Health Research (CIHR) MOP-93707 and Canadian Diabetes Association (CDA), and to HG from CIHR MOP 86544. LW was supported by a Frederick Banting and Charles Best Canada Graduate Scholarship from CIHR, a Novo Nordisk graduate scholarship from the Banting and Best Diabetes Centre (BBDC), a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC) and a Comprehensive Research Experience for Medical Students (CREMS) scholarship from the Faculty of Medicine, University of Toronto. CTL is supported by the Eliot Phillipson Clinician Scientist Training Program and a BBDC Postdoctoral Fellowship. EPC is supported by the CDA Doctoral Student Research Award. MW is supported by a Canada Research Chair in Signal Transduction in Diabetes Pathogenesis.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors contributed to conception, data, revising the manuscript, and approved the final version of the manuscript. LW and CTL performed experiments, analysed and interpreted data, drafted the manuscript and contributed to the study design. SAS developed methods and animal models, performed experiments, analysed and interpreted data. AMS, XL and EPC performed experiments, analysed and interpreted data. HG and PEM contributed to study design and discussion. ZH and TWM developed animal models and contributed to study design. MW supervised the project and developed the study design. MW is the guarantor of this work.
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