OBJECTIVE—Activation of the cyclooxygenase (COX) pathway with secondary neurovascular deficits are implicated in the pathogenesis of experimental diabetic peripheral neuropathy (DPN). The aim of this study was to explore the interrelationships between hyperglycemia, activation of the COX-2 pathway, and oxidative stress and inflammation in mediating peripheral nerve dysfunction and whether COX-2 gene inactivation attenuates nerve fiber loss in long-term experimental diabetes. RESEARCH DESIGN AND METHODS—Motor and sensory digital nerve conduction velocities, sciatic nerve indexes of oxidative stress, prostaglandin content, markers of inflammation, and intraepidermal nerve fiber (IENF) density were measured after 6 months in control and diabetic COX-2–deficient (COX-2−/−) and littermate wild-type (COX-2+/+) mice. The effects of a selective COX-2 inhibitor, celecoxib, on these markers were also investigated in diabetic rats. RESULTS—Under normal conditions, there were no differences in blood glucose, peripheral nerve electrophysiology, markers of oxidative stress, inflammation, and IENF density between COX-2+/+ and COX-2−/− mice. After 6 months, diabetic COX-2+/+ mice experienced significant deterioration in nerve conduction velocities and IENF density and developed important signs of increased oxidative stress and inflammation compared with nondiabetic mice. Diabetic COX-2−/− mice were protected against functional and biochemical deficits of experimental DPN and against nerve fiber loss. In diabetic rats, selective COX-2 inhibition replicated this protection. CONCLUSIONS—These data suggest that selective COX-2 inhibition may be useful for preventing or delaying DPN.
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...
Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY.
Alterations in cyclooxygenase (COX) pathway activity have been implicated in the pathogenesis of experimental diabetic neuropathy (EDN).
The etiology of painful diabetic neuropathy is poorly understood, but may result from neuronal hyperexcitability secondary to alterations of Ca2+ signaling in sensory neurons. The naturally occurring amino acid taurine functions as an osmolyte, antioxidant, Ca2+ modulator, inhibitory neurotransmitter, and analgesic such that its depletion in diabetes may predispose one to neuronal hyperexcitability and pain. This study reports the effects of taurine replacement on hyperalgesia and sensory neuron Ca2+ homeostasis in streptozotocin-diabetic (STZ-D) rats. Nondiabetic and STZ-D rats were treated with a 2% taurine-supplemented diet for 6-12 wk. Thermal hyperalgesia and mechanical allodynia were determined by measuring hindpaw withdrawal latency to radiant heat and the withdrawal threshold to the von Frey anesthesiometer. Intracellular Ca2+ signaling was explored in neurons from L4-L6 dorsal root ganglia (DRG), using fura 2 fluorescence. Taurine replacement of diabetic rats attenuated deficits of nerve conduction and prevented reductions of mechanical and thermal withdrawal threshold and latency, respectively. In small DRG sensory neurons from diabetic rats, recovery of intracellular Ca2+ concentration ([Ca2+]i) in response to KCl was slowed and 73% corrected by taurine. The amplitudes of caffeine and ATP-induced [Ca2+]i transients were decreased by 47 and 27% (P < 0.05), respectively, in diabetic rat DRG sensory neurons and corrected by 74 and 93% (P < 0.05), respectively, by taurine replacement. These data indicate that taurine is important in the regulation of neuronal Ca2+ signaling and that taurine deficiency may predispose one to nerve hyperexcitability and pain, complicating diabetes.
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