Discussion
Intra-islet HS was previously reported to be involved in beta cell dysfunction in autoimmune diabetes (Simeonovic et al. , 2013), islet amyloid formation, and islet amyloid polypeptide-induced islet cell apoptosis (Oskarsson et al. , 2015). In addition, some important genes that participate in insulin secretion, like BETA2/NeuroD (Noguchi et al. , 2007) and pancreatic and duodenal homeobox factor 1 (PDX1) (Ueda et al. , 2008), could be regulated by cell surface HSPG. In addition, murine islets selectively lose HS to 11–27% of normal levels during primary isolation before islet transplantation (Choong et al. , 2015). These data suggest that the maintenance of intra-islet HS level is important to the function of islets.
However, the mechanism of the protective effect of HS on beta cells is still unclear. First, we detected the distribution of HS in the islets from normal and STZ-treated mice. Previous study suggested that perlecan, one important type of HSPG, is localized in the peri-islet and surrounding intra-islet capillaries and is destroyed in the islets of NOD mice (Irving-Rodgers et al. , 2008). Takahashi et al., using anti-HS antibody (3G10 site) and immunohistochemistry, found that HS is significantly expressed in the insulin-positive beta cells but not in glucagon-positive alpha cells of islets (Takahashi et al. , 2009). In the present study, we precisely showed that 10E4 epitope mainly exists in islet beta cells but less expresses in other type of islet cells, which is similar from that of a previous study using anti-10E4 antibody, where HS co-localized with insulin but not glucagon staining in the normal human islets (Simeonovicet al. , 2018). However, after use heparitinase-treatment to expose the 3G10 epitope of HS, we found that 3G10 staining was not only highly co-localized with beta cells, but also colocalized with other type of islet endocrine cells in greater amounts than peripheral acinar cells. This difference may indicate different sites of HS antigen have distinct patterns in pancreatic islets (Theodoraki et al. , 2015). Importantly, in STZ mice, 10E4 staining remains a similar pattern with insulin-positive cells, confirmed that 10E4 could be a beta cell-specific HS epitope. In contrast, 3G10 staining is lost in most beta cells of STZ mice, which exhibits a similar net shape as that in the peripheral acinar cells. Unlike 10E4 data, 3G10 result provided stronger mark of intra-islet HS loss. Although the detailed distribution pattern of HS in the islets should be further investigated in more species, like the data from the islets from NOD mice (Irving-Rodgerset al. , 2008) and from patients with type 1 diabetes (Simeonovicet al. , 2018), the intra-islet HS is obviously lost in STZ-treated diabetic mice, it seems clear that HS is highly expressed in the normal islets, where it performs critical biological roles, especially in pancreatic beta cells.
Since the loss of intra-islet HS could be a marker of type 1 diabetes progression in both patients and animal models, it is worthful to further investigate its underlying mechanism. As an endoglucuronidase, heparanase could specifically cleave the glycosidic bond of GlcA-GlcNS in HS, thereby promoting the degradation of HS (Mao et al. , 2014). In many cancers, high expression of heparanase could enhance the hydrolysis of HS in the extracellular matrix to promote tumor invasion, metastasis, and angiogenesis (Dai et al. , 2017). Thus, heparanase inhibitors, including sulfated polysaccharides/oligosaccharides (HS analogs), anti-heparanase antibodies, nucleic acid-based inhibitors, and small molecules, have been developed to treat cancer and other rare diseases (Rivara et al. , 2016). Ziolkowski et al. found that PI-88, an analog of HS, inhibited heparanase and improved intra-islet HS level to protect against islet dysfunction in NOD mice (Ziolkowskiet al. , 2012). In line with this evidence, our data showed that a small molecular heparanase inhibitor, OGT2115 (Courtney et al. , 2005), not only protects islet beta cells from STZ-induced apoptosis, but also improves glucose profiles and increases insulin secretion in STZ-induced diabetic mice by preventing the loss of intra-islet HS. It is known that the onset age and morbidity of NOD mice are uncertain (Acharjee et al. , 2013), this multiple injection of low dose STZ-induced diabetic model could be reliable in the pharmacological study of heparanase inhibitors as anti-diabetic drugs. Furthermore, morphological staining confirms a significant reduction in the infiltration of immunocytes in the islets of OGT2115-treated STZ mice. In view of the important role of immune inflammatory damage in the process of beta cell injury in type 1 diabetes (Lombardi et al. , 2018), the decrease in intra-islet immunocytes infiltration in this STZ-induced diabetic mouse model may be vital for improved glucose control in OGT2115-treated STZ mice.
Interestingly, OGT2115 does not reduce STZ-induced beta cell dysfunction in vitro, suggested that preventing HS loss in beta cells by OGT2115 could not directly prevent STZ-induced beta cell damage. In vitro , STZ is transported into beta cells via glucose transporter 2 (GLUT2), thereby directly damaging beta cells (Deshpande et al. , 2019). However, in the present in vivo experiment, infiltration of the recruited inflammatory cells in the islets is the main mechanism of induction of diabetes by multiple low-dose injections of STZ (Yanget al. , 2003). Therefore, we focused the in vivoinhibitory effect of OGT2115 on increased intra-islet heparanase level in STZ mice. Heparanase was previously reported to be upregulated by high glucose in renal epithelial cells (Maxhimer et al. , 2005) and retinal microvascular endothelial cells (Hu et al. , 2017), and urine and plasma heparanase level are associated with blood glucose levels in patients with diabetes (Shafat et al. , 2011). A reduction in heparanase activity prevents the high glucose-induced epithelial-mesenchymal transition (Masola et al. , 2017).We speculate that heparanase level is also increased in STZ-induced hyperglycemic mice and that heparanase subsequently hydrolyzes intra-islet HS to aggravate islet damage under an inflammatory microenvironment. In line with this hypothesis, higher intra-islet heparanase expression is found in STZ mice than in control mice. Previous study showed that heparanase is strongly expressed in monocytes in diabetes-onset NOD pancreas, while weak cell surface expression of heparanase could be also observed (Ziolkowski et al. , 2012). Using a commercial heparanase antibody that recognizes 65 kD precursor as well as 50 kD and 8 kD subunits of heparanase I, we found that although heparanase is highly expressed in not only F4/80-positive macrophages, but also obvious heparanase staining could be observed in murine islet beta cells. The expression of heparanase in beta cells was confirmed by bioinformatic analysis of single cell sequencing data of human and mice islets from GEO database (Xin et al. , 2016; Lawloret al. , 2017; Sharon et al. , 2019). Together, these results at least suggest that heparanase is also highly expressed in islet cells, and this beta cell-expressed heparanase may contribute to wide-spread loss of HS in STZ islets as 3G10 staining indicated.
How glucose regulates the expression of heparanase is not fully understood. ROS is reported to mediate high glucose-induced heparanase expression (Han et al. , 2007; Rao et al. , 2011). The promoter region of heparanase contains GC-rich sequences and lacks TATA or CCAAT box (Dong et al. , 2000). In addition, some factors, including nuclear factor-κB (NF-κB) (Hao et al. , 2015; Anet al. , 2018), forkhead box protein O4 (An et al. , 2011), early growth response gene 1 (EGR1) (de Mestre et al. , 2005), truncated glioma-associated oncogene homolog 1 (TGLI1) (Zhu et al. , 2014), and ETS1 (Lu et al. , 2003) can up-regulate heparanase expression in many cancers; while tumor suppressor p53 (Barazet al. , 2006), SMAD family member 4 (SMAD4) and lymphoid enhancer binding factor 1 (LEF1) (Qu et al. , 2016), are reported to down-regulate heparanase expression. Here, we verified that chronic high glucose has less effect on the expression of SMAD4, LEF1 and TGLI1 in cultured islets, while the expression of ETS1, EGR1, and p53 are increased. Notably, the expression of PPARγ is significantly reduced in high glucose-treated islets. In line with this result, our previous study has showed that high glucose-treated islets have an increased ETS1 expression to injury beta cell function (Chen et al. , 2016). Decreased PPARγ expression is also reported in high glucose-treated islets (So et al. , 2013). In addition, activation of PPARγ is reported to downregulate the expression of heparanase to inhibit hepatocellular carcinoma metastases (Shen et al. , 2012). We further confirmed that the direct binding of PPARγ to the promoter region negatively regulates the transcriptional activity of the heparanase gene. Thus, PPARγ may act as an important inhibitory transcription factor, together with other stimulating factors like ETS1 or EGR1, thereby involves in hyperglycemia-stimulated heparanase expression.
To summarize, STZ-induced hyperglycemia in mice decreases the expression of PPARγ in the islets, thereby eliminating the occupancy of the heparanase promoter region and stimulating the expression of heparanase. Increased heparanase hydrolyzes intra-islet HS, which promotes infiltration of immunocytes, damages beta cells and, subsequently aggravates hyperglycemia in type 1 diabetes. Although further experiments are needed to improve the knowledge of the underlying mechanisms, this study provided pharmacological evidence that the use of a heparanase inhibitor could prevent the loss of intra-islet HS, thereby providing a protective effect on islet function in the process of type 1 diabetes.