Localization of HS in islets of normal and STZ-treated diabetic mice
In this study, we used two HS antibodies to explore the detailed localization of HS in murine islets. The antibody reacts with 10E4 epitope of HS recognizes N-sulfated glucosamine residue of HS, while the 3G10 antibody reacts with a neo-epitope of HS, which is generated by heparitinase I digestion on sections (David et al. , 1992). The staining results of both 10E4 and 3G10 antibodies showed that HS was highly expressed in normal islets (Fig. 1A and 1B). However, minor differences were observed. The 10E4 antibody staining in control group only exists in intra-islets (Fig. 1A). The 3G10 antibody staining showed that HS was not only highly expressed in the islets, but also distributed in a net shape in the extracellular matrix around the pancreatic acinar cells (Fig. 1B). Double-fluorescence staining of HS with four islet endocrine hormones showed that 10E4 mainly expresses in insulin-positive beta cells but less in other types of islet endocrine cells, including glucagon-positive alpha cells, somatostatin-positive delta cells, and pancreatic polypeptide (PP)-positive PP cells (Fig. 1A), whereas 3G10 covers most islet endocrine cell types (Fig. 1B).
Obviously different expression patterns between 10E4 and 3G10 were found in STZ-treated mice. As in normal islets, 10E4 was co-localized with insulin, which is reduced with damage beta cells in STZ mice (Fig. 1A). Using 3G10 antibody, the amount of intra-islet HS was clearly decreased in the whole islets of STZ-treated mice, and HS exhibited a similar pattern of net shape to that in the peripheral acinar cells (Fig. 1B). It should be noticed that residual 3G10-stained HS could be still observed in alpha cells, delta cells, and PP cells in STZ mice (Fig. 1B).
Heparanase is highly expressed in pancreatic beta cells and monocytes in islets. Immunostaining showed that heparanase is expressed in islets rather than peripheral acinar cells (Fig. 2A and 2B). Remarkably, intra-islet content of heparanase is increased in STZ-treated mice, in which we noticed some cells had extremely higher expression of heparanase (Fig. 2A and 2B). We further analyzed the expression level of heparanase in different islet cell types from reported single cell sequencing data. Although the difference of heparanase expression between diabetic and normal samples was not significant due to limited sample number, it confirmed the expression of heparanase is high in the alpha and beta endocrine cells in the islets (Fig. 2C). To confirm this, double-immunofluorescence was used to investigate the detailed localization of heparanase in islets. In control mice, less F4/80-positive monocytes could be observed (Fig. 2D), heparanase was expressed in most insulin-positive beta cells (Fig. 2E). In contrast, heparanase was found to co-localized with F4/80-positive monocytes in STZ islets, and heparanase-positive cells was still present in islet without F4/80 (Fig. 2D). As shown in Fig. 2E, those F4/80-negative cells that expressed heparanase was co-localized with insulin.