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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 2, 2004 as doi:10.1096/fj.04-1894fje. |
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* Department of Pharmacology and Therapeutics and
Department of Surgery, McGill University, Montreal, Quebec, Canada
1Correspondence: Department of Pharmacology, 3655 Promenade Sir-William-Osler, Room 1314, Montreal, Quebec, Canada, H3G 1Y6. E-mail: dusica.maysinger{at}mcgill.ca
SPECIFIC AIMS
INGAP peptide comprises the core active sequence of islet neogenesis-associated protein (INGAP), a pancreatic cytokine able to induce new islet formation and correct hyperglycemia in rodents. The ability of INGAP peptide to enhance nerve outgrowth from sensory ganglia in vitro suggests its potential utility in nervous system disorders. The aims of this study were to 1) assess the effectiveness of INGAP peptide on measures of diabetes-associated peripheral neuropathy and 2) characterize some of the biochemical changes effected by INGAP peptide in the nervous system.
PRINCIPAL FINDINGS
1. INGAP peptide administration corrects nociceptive dysfunction in diabetic mice without inducing hyperalgesia and without additivity or synergism with insulin
Diabetes was induced in C57BL/6 mice by streptozotocin injection; after 3 months of diabetes, thermal hypoalgesia was observed in diabetic animals (Fig. 1
). Control and diabetic mice were then administered INGAP peptide (25 mg/kg, i.p.), insulin (0.1 U/day, s.c.), INGAP peptide plus insulin, or saline vehicle every second day for 2 wk. Sensory function was assessed at 2 or 4 day intervals during the treatment period, at the end of which thermal hypoalgesia was found to be ameliorated in diabetic mice receiving INGAP peptide. In clinical trials using various growth factors to treat diabetic neuropathy, the development of hyperalgesia has been a major problem. However, sensory hyperalgesia measured by response latencies to thermal stimuli did not develop in control mice receiving INGAP peptide. In our 2 wk dosing regimen, INGAP peptide did not decrease the hyperglycemia measured in the diabetic mice (although INGAP peptide can restore euglycemia in diabetic rodents when provided longer term and with more frequent administration). Insulin treatment alone in diabetic mice brought about a small but significant improvement of thermal hypoalgesia, and we considered whether the production of small amounts of insulin by INGAP peptide using our dosing regimen could contribute to its effect on nociception. However, diabetic mice receiving the combination of INGAP peptide and insulin did not show greater improvement of sensory dysfunction than that produced by INGAP peptide alone. We conclude that INGAP peptide can restore normal nociceptive function in diabetic mice with associated peripheral neuropathy and that this effect of INGAP peptide in vivo in the nervous system is not dependent on normalization of blood glucose levels.
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2. INGAP peptide administered in vivo enhances nerve regeneration examined in vitro
Our findings in the nociceptive assays prompted us to consider whether INGAP peptide improved overall regenerative capacity of the peripheral nerves. We used tissue explant cultures of dorsal root ganglia (DRG), which contain the sensory neuronal cell bodies of the peripheral nerves, as an in vitro model of axotomy. We found that the most pronounced outgrowth at the end of a 7 day culture was observed from the DRG of diabetic mice that received prior treatment with INGAP peptide. DRG from INGAP peptide-treated control mice also showed enhanced regenerative outgrowth in the in vitro paradigm. Densitometric analysis of total neuritic density correlated with our in vivo nociceptive findings; the effects of INGAP peptide on the sensory nerves was not additive or synergistic with insulin, since mean outgrowth in these in vitro regeneration assays was not significantly different between the DRG from INGAP peptide-treated mice and those from mice treated with both INGAP peptide and insulin.
3. INGAP peptide induces structural protein and signal transduction changes characteristic of nerve regeneration, and enhances mitochondrial metabolic activity
We sought to characterize some of the biochemical changes occurring in the dorsal root ganglia that underlie the effects of INGAP peptide on the peripheral nervous system in diabetes. One hallmark of diabetic neuropathy is the hyperphosphorylation of structural neurofilament proteins in DRG. We observed a marked reduction in the amount of phosphorylated neurofilament in the DRG of diabetic mice treated with INGAP peptide (Fig. 2
). Another typical change observed in regenerating nerves is up-regulation of ß-tubulin and actin proteins. Western blot analyses showed that the expression levels of these proteins were significantly increased in the DRG of INGAP peptide-treated diabetic mice relative to untreated diabetic mice. An increase in ß-tubulin levels in control mice treated with INGAP peptide was also noted. At the level of signal transduction, the transcription factor STAT3 is known to be a major effector of regenerative growth in peripheral nerves. The ratio of phosphorylated:nonphosphorylated STAT3 was up-regulated in the DRG of diabetic mice treated with INGAP peptide, although the most dramatic increase was observed in the INGAP peptide-treated control mice. These findings are consistent with the induction or priming of a nerve regenerative program by INGAP peptide administration in vivo (Fig. 3
).
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CONCLUSIONS AND SIGNIFICANCE
We have provided evidence that INGAP peptide can exert effects on the peripheral sensory nervous system in vivo, manifested as improvements in defects that characterize experimental diabetic neuropathy in mice, namely impaired thermal nociception and hyperphosphorylation of neurofilament proteins. In addition, we find that INGAP peptide induces an up-regulation of proteins involved in nerve regeneration (ß-tubulin, actin, STAT3) that seems to underlie an enhanced regenerative capacity in an in vitro model of axotomy. The beneficial effects on the nervous system of INGAP peptide, a candidate therapeutic for Type 1 diabetes, are of particular importance given the common clinical finding that diabetic neuropathy, once established, may be essentially irreversible despite correction of the hyperglycemia. Administration of INGAP peptide to diabetic patients may, in addition to restoring glycemic control, prove effective in preventing or delaying the development of diabetic neuropathy.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1894fje;
This article has been cited by other articles:
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  D. A. Taylor-Fishwick, A. Bowman, M. Korngiebel-Rosique, and A. I. Vinik Pancreatic Islet Immunoreactivity to the Reg Protein INGAP J. Histochem. Cytochem., February 1, 2008; 56(2): 183 - 191. [Abstract] [Full Text] [PDF]
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Significant difference at day 0 of diabetic groups vs. control groups; **significant differences of D vs. C; NS denotes nonsignificant differences of C vs. C + IG. b) 
