As a tool to explore proinsulin (PI) trafficking, a human PI cDNA has been constructed with GFP fused within the C peptide. In regulated secretory cells containing appropriate prohormone convertases, the hProCpepGFP construct undergoes endoproteolytic processing to CpepGFP and native human insulin, which are specifically detected and cosecreted in parallel with endogenous insulin. Expression of C(A7)Y mutant PI results in autosomal dominant diabetes in Akita mice. We directly identify the misfolded PI in Akita islets and also show that C(A7)Y mutant PI, either in the context of the hProCpepGFP chimera or not, engages directly in protein complexes with nonmutant PI, impairing the trafficking and recovery of nonmutant PI. This trapping mechanism decreases insulin production in  cells. Thereafter we observe a loss of  cell viability. The data imply that PI misfolding leading to impaired endoplasmic reticulum exit of nonmutant PI may be a key early step in a chain reaction of  cell dysfunction and demise leading to onset and progression of diabetes.diabetes mellitus ͉ endoplasmic reticulum storage disease ͉ insulin secretion ͉ proinsulin disulfide isomers
Type 1B diabetes (typically early onset; without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene-induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed proinsulin mutants exerting dominant-negative (gain of toxic function) behavior in pancreatic beta cells. Herein, we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.
It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with "hProCpepGFP" (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these "bystander" proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit -cellspecific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in -secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic -cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to -cell failure.During the progression of diabetes mellitus, the endocrine pancreas encounters difficulty in meeting insulin requirements (1); -cell dysfunction is recognized as a major contributor to the disease (2-5). One element of -cell dysfunction is ER 3 stress (6 -11) with ER accumulation of misfolded protein (12), especially proinsulin (13, 14). -Cells ordinarily maintain a high level of proinsulin production with finite additional capacity before the biosynthetic apparatus is taxed to the point of ER stress (15). Chronically increased secretory demand, either in animal models or in humans, results in morphological depletion of -secretory granules with a compensatory increase in apparent secretory pathway activity, including distention of the ER (16 -18). These conditions may favor additional proinsulin misfolding (19).The causality between misfolded proinsulin and -cell failure is unequivocally established in congenital diabetes caused by preproinsulin coding sequence mutations, in which diabetes is inherited in an autosomal dominant manner (20 -24). Insulin haploinsufficiency cannot account for the diabetes (25), yet despite three normal proinsulin alleles, both Akita and Munich mice each develop overt diabetes by expressing from a single allele a mutant proinsulin with replacement of one Cys residue that disrupts one of the three proinsulin disulfide bonds (26,27). In addition to being retained in the ER, it has been suggested that misfolded proinsulin may impair normal insulin production via physical interactions between mutant and wild-type proinsulin gene products (26). Indeed, we have direct...
Background: Insulin therapy is most effective if dosage titrations are done regularly and frequently, which is seldom possible for busy clinicians. The d-Nav® Insulin Guidance System was design to address the insulin titration gap in patients with type 2 diabetes. It relies on the d-Nav handheld device, which is used to measure glucose, determine the glucose patterns and automatically determine the appropriate next insulin dose. It closes the titration gap in a scalable way utilizing the support of dedicated health care professionals (HCP). This multicenter randomized controlled study tested whether the combination of the d-Nav system and HCP support (d-Nav+HCP-S) is superior to HCP support alone (HCP-S).Methods: 181 subjects using insulin with sub-optimally controlled type 2 diabetes were randomized 1:1 to either d-Nav+HCP-S or HCP-S alone. Both groups were contacted 7 times
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