Two Dipolar α-Helices within Hormone-encoding Regions of Proglucagon Are Sorting Signals to the Regulated Secretory Pathway

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229–240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting “signature.”     

Electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of the calf fundic glands

The electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of calf fundic glands was studied using specific antisera for prochymosin and pepsinogen with a protein A-gold method. Prochymosin and pepsinogen immunoreactivities were detected in the same secretory granules of the chief, mucous neck and transitional cells, simultaneously using small and large colloidal gold particles. In chief cells, both immunoreactivities were distributed uniformly over the same zymogen granules showing a round, large, homogeneous and electron-dense appearance. In mucous neck cells, both immunoreactivities were found exclusively on the same electron-dense core located eccentrically in the mucous granule showing light or moderate electron density. In transitional mucous neck/chief cells, electron-dense cores became larger in size and some granules were occupied by the electron-dense core without a halo between the core and the limiting membrane. Both immunoreactivities were found uniformly over the electron-dense core. The granules having no halo in the transitional cells could not be distinguished from the typical zymogen granules in the chief cells.     

Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C- peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.     

Compartmentalization of Pancreatic Secretory Zymogen Granules as Revealed by Low-Voltage Transmission Electron Microscopy

Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means. It corresponds to the segregation of the different secretory proteins inside the granule as demonstrated by high-resolution immunocytochemistry and reflects a well-organized aggregation of the secretory proteins at the time of granule formation in the trans-Golgi. Such partition of the granule matrix undergoes changes under experimental conditions known to alter the secretory process such as stimulation of secretion or diabetes.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Topology of chromogranins in secretory granules of endocrine cells

Summary Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions.Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities.Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide-and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Topology of chromogranins in secretory granules of endocrine cells.

Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions. Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities. Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide- and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Glucagon- and glicentin-immunoreactive cells in the human digestive tract

Summary The distribution and cellular location of substances reacting with anti-glucagon or anti-glicentin sera, i.e., glucagon-like and glicentin-like immunoreactivities, were studied in the human digestive tract using the immunofluorescence and immunoperoxidase methods. Both types of immunoreactivity were (1) absent in the antrum, (2) abundant in cells located at the periphery of pancreatic islets, (3) unevenly present in cells scattered in the epithelium of the small intestinal mucosa, the glicentin-immunoreactive cells being particularly abundant in the ileum. In the pancreas, and, when simultaneously present, in the intestine, both glucagon and glicentin immunoreactivities were located in the same cells.The precise ultrastructural location of each immunoreactivity was readily made using colloidal gold and ferritin tracers on ultrathin sections of glutaraldehyde-osmium fixed and epoxy resin-embedded tissues. In the pancreas, both glucagon and glicentin immunoreactivities were found in the granules of the A-type cells; the glucagon immunoreactivity was only present in the core of the granule, whereas the glicentin immunoreactivity was found either in the peripheral halo only, or throughout the entire granule. In the small intestine, both immunoreactivities were located inside the granules of the L-type cells.Quantitative specificity tests suggested that the glucagon- and the glicentin-like substances of the pancreas differ from those found in the intestine.Work supported by INSERM, A.T.P. number: 167539     

Formation and exocytosis of secretory granules in the endocrine cells of normal and transplanted pancreas: an electromicroscopic study

The mechanism of secretory granule formation and exocytosis in the endocrine cells of normal and transplanted rat pancreas was studied using electron microscopy. On the one hand, formation of secretory granules starts with the dilatation of the 2 ends or the vesicularization of the middle parts of rough endoplasmatic reticulum (RER). On the other hand, prohormone ribosomes condense into the vesicles of the GOLGI apparatus. This probably indicates that the GOLGI complex is not the only source of formation of secretory granules. Exocytosis occurs with the formation of an electron dense streak between the perigranular membrane and the apical cell membrane. This is followed by the rupture of the streak at this midpoint allowing the granule to extrude into the space between the cell membrane and the parenchymal basal membrane. This fusion-rupture-extrusion mechanism repeats itself at the parenchymal and capillary basal membranes and also at the endothelium until it gets into the capillary lumen, showing that hormones of pancreatic endocrine cells may be actively transported into circulation as intact secretory granules. There is no significant morphological difference between the mechanism of secretory granule formation in normal and transplanted pancreatic tissue.     

Human glucagon-like peptides 1 and 2 activate rat brain adenylate cyclase

Two human glucagon-like peptides, GLP-1 and GLP-2, which are coencoded with pancreatic glucagon in the preproglucagon gene, do not significantly inhibit [125I]monoiodoglucagon binding to rat liver and brain membranes and do not activate adenylate cyclase in liver plasma membranes. Nevertheless, GLP-1 and GLP-2 were each found to be potent stimulators of both rat hypothalamic and pituitary adenylate cyclase. Only 30-50 pM concentrations of each peptide elicited half-maximal adenylate cyclase stimulation. Our data suggest that GLP-1 and GLP-2 may be neurotransmitters and/or neuroendocrine effectors, which would account for their high degree of sequence conservation through vertebrate evolution.     

Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000- 15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p- chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.     

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP- or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas. Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide- and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide- or glucagon-immunoreactivity were also found. Glucagon- and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Summary Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP-or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas.Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide-and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide-or glucagon-immunoreactivity were also found. Glucagon-and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.