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Overexpression of a chaperone protein liberates functional insulin from a misfolded mutant partner to improve insulin secretion.
Copyright © 2017, American Association for the Advancement of Science.
Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.
We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with beta cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially beta cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of beta cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.
Mutations in PERK (EIF2AK3) result in permanent neonatal diabetes as well as several other anomalies that underlie the human Wolcott-Rallison syndrome, and these anomalies are mirrored in Perk knockout mice. To identify the cause of diabetes in PERK-deficient mice, we generated a series of tissue- and cell-specific knockouts of the Perk gene and performed a developmental analysis of the progression to overt diabetes. We discovered that PERK is specifically required in the insulin-secreting beta cells during the fetal and early neonatal period as a prerequisite for postnatal glucose homeostasis. However, PERK expression in beta cells is not required at the adult stage to maintain beta cell functions and glucose homeostasis. We show that PERK-deficient mice exhibit severe defects in fetal/neonatal beta cell proliferation and differentiation, resulting in low beta cell mass, defects in proinsulin trafficking, and abrogation of insulin secretion that culminate in permanent neonatal diabetes.
Insulin granule trafficking is a key step of glucose-stimulated insulin secretion from pancreatic beta cells. Using quantitative live cell imaging, we examined insulin granule movements within the reserve pool upon secretory stimulation in betaTC3 cells. For this study, we developed a custom image analysis program that permitted automatic tracking of the individual motions of over 20,000 granules. This analysis of a large sample size enabled us to study micro-populations of granules that were not quantifiable in previous studies. While over 90% of the granules depend on Ca2+ efflux from the endoplasmic reticulum for their mobilization, a small and fast-moving population of granules responds to extracellular Ca2+ influx after depolarization of the plasma membrane. We show that this differential regulation of the two granule populations is consistent with localized Ca2+ signals, and that the cytoskeletal network is involved in both types of granule movement. The fast-moving granules are correlated temporally and spatially to the replacement of the secreted insulin granules, which supports the hypothesis that these granules are responsible for replenishing the readily releasable pool. Our study provides a model by which glucose and other secretory stimuli can regulate the readily releasable pool through the same mechanisms that regulate insulin secretion.
We generated transgenic mice expressing firefly (Photinus pyralis) luciferase (luc) under the control of the mouse insulin I promoter (MIP). The mice have normal glucose tolerance and pancreatic islet architecture. The luciferase-expressing beta cells can be readily visualized in living mice using whole-body bioluminescent imaging. The MIP-luc transgenic mice may be useful for monitoring changes in beta cell function or mass in living animals with normal or altered metabolic states.
genesis 43:80-86, 2005. (c) 2005 Wiley-Liss, Inc.
In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.
Antigenic determinants recognized by human proinsulin (HPI)-specific monoclonal antibodies (Mabs) and Mabs crossreacting with free human C-peptide (HCP) were mapped by using various forms of purified, partially converted HPI intermediates. Two HPI-specific mouse Mabs (GS-4G9 and GS-9A8) reacted with the same antigenic determinant, GS, which was localized to the site of linkage of the B-chain to the C-peptide (Arg-Arg) at positions 31-32. These antibodies bind with equal efficiency to C65-A1 split proinsulin and to intact HPI. The binding of C32-C33 split proinsulin is markedly reduced. A rat Mab (GN-VIIB6), which crossreacts with free HCP in addition to HPI, reacted similarly with various HPI intermediates as it had with the corresponding synthetic HCP fragments, as previously reported (see ref. 9). This determinant (GN) is a three-dimensional structure composed of residues located in two separate regions in the C-peptide segment (positions 40-45 and 57-63). Reduced, carboxymethylated HPI retains the GN-determinant, whereas all insulin-like immunoreactivity identified with a conventional guinea pig insulin antiserum is completely lost. The binding of the two GS Mabs to the denatured HPI was reduced by 40-50% compared with intact HPI. It is concluded that the strong GN-determinant can readily form in the C-peptide segment of HPI, independently of the presence of ordered structure in the insulin moiety. A predicted beta-turn at position 47-50 may play an important role in bringing N- and C-terminal regions of the C-peptide segment into close proximity.(ABSTRACT TRUNCATED AT 250 WORDS)
The development of a simple microscale solid phase screening RIA and improved methods for cell cloning has lead to the establishment of 2 prohormone- and species-specific mouse monoclonal antibodies against human proinsulin (HPI). These antibodies react to determinant(s) only expressed on the HPI molecule. In addition, 11 rat monoclonal antibodies were generated which react with both human C-peptide (HCP) and HPI. All 11 rat antibodies recognize a very similar antigenic determinant in the C-peptide that appears to be made up of residues 40-45 and 57-63, which are probably brought into close proximity by a beta-turn near the center of the connecting segment. The identical behavior of both HPI-specific mouse antibodies in competition experiments indicates that the antigenic structure recognized in proinsulin might be the same for both antibodies. This structure could not be regenerated by mixing equimolar amounts of human insulin and C-peptide, including the chemically synthesized complete proinsulin connecting segment (Arg X Arg X HCP X Lys X Arg), which contains the entire sequence removed from proinsulin in the conversion to mature insulin. Indirect evidence is provided that a HPI molecule simultaneously can bind a C-peptide-directed rat antibody and a HPI-specific mouse antibody when the first antibody is presented to HPI in solution phase. However, when HPI is immobilized on a plastic surface, the binding of 1 type of antibody completely blocks the binding of the other. These antibodies provide useful new tools for studying the biosynthesis and 3-dimensional structure of HPI and HCP.