Schwann cells (SCs), the myelinating cells of the peripheral nervous system, are lost or damaged in patients suffering from diabetic neuropathy. In the current study, 2 model systems are used to study the mechanism of SC damage in diabetic neuropathy: the streptozotocin (STZ)-treated diabetic rat and cultures of purified SCs in vitro. Electron microscopy of dorsal root ganglia from STZ-treated rats reveals classic ultrastructural features of apoptosis in SCs, including chromatin clumping and prominent vacuolation. Bisbenzamide staining of SCs cultured in hyperglycemic defined media shows nuclear blebbing of apoptotic cells. Insulin-like growth factor-I (IGF-I) is protective. LY294002, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, blocks the effect of IGF-I. High glucose induces caspase cleavage in apoptotic SCs--an effect that is blocked by bok-asp-fmk (BAF), a caspase inhibitor. Although Bcl-xL expression remains unchanged in experimental conditions, over-expression of Bcl-xL protects SCs from apoptosis. In summary, hyperglycemia induces caspase activation and morphologic changes in SCs consistent with apoptotic death, both in vivo and in vitro. Over-expression of Bcl-xL, or IGF-I, signaling via PI 3-kinase, protects SCs from glucose-mediated apoptosis in vitro. IGF-I may be useful in preventing hyperglycemia-induced damage to SCs in patients suffering from diabetic neuropathy.

We have previously shown that insulin-like growth factor I (IGF-I) activation of the IGF-I receptor rescues SH-SY5Y human neuroblastoma cells from high glucosemediated programmed cell death (PCD). In the current study, we further explored the potential points in the cell death cascade where IGF-I receptor activation may afford neuroprotection. As an initial step, we examined the effects of the PCD stimulus, high glucose, on stressactivated protein kinases, specifically the two mitogenactivated protein kinases p38 kinase and c-Jun N-terminal kinase (JNK). High glucose treatment activated the tyrosine phosphorylation of both p38 kinase and JNK in a dose-and time-dependent fashion. We next examined the effects of IGF-I on JNK and p38 kinase under normoglycemic and hyperglycemic conditions. IGF-I activated p38 kinase alone and had additive effects on glucose-induced p38 kinase phosphorylation. In contrast, IGF-I inhibited glucose activation of JNK phosphorylation and JNK activity. IGF-I also inhibited the glucoseinduced nuclear translocation of JNK, but did not effect glucose-induced translocation of p38 kinase. Finally, IGF-I inhibition of JNK phosphorylation was blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Collectively, these data imply cross-talk between the mitogen-activated protein kinase pathway and JNK and suggest that IGF-I activation of mitogen-activated protein kinases interferes with JNK activation and protects cells from PCD.The mitogen-activated protein (MAP) 1 kinases are a family of serine-threonine protein kinases that are activated in response to a variety of extracellular stimuli (1). The first members identified in the family were p42 and p44 extracellular signal-regulated kinases (ERKs), now known as ERK1 and ERK2, respectively. In the Ras pathway, phosphorylation of c-Raf 1 activates the downstream protein kinase, MAP kinase kinase 1 (also known as MAPK/ERK (MEK) or MKK1) or MAP kinase kinase 2 (MKK2). MKK1 and 2 activate ERK1 and ERK2, which leads to activation and translocation of ERKs to the nucleus. In the nucleus, the ERKs phosphorylate transcription factors, including Elk-1 and ATF-2. This signaling cascade is activated by growth factors and is important for cellular growth and mitogenesis (2).Another class of MAP kinase family members, the stressactivated c-Jun N-terminal kinases (JNKs), are primarily responsive to stressful stimuli. There are 10 identified isoforms of JNK originating from three homologous genes (JNK1, JNK2, and JNK3) with molecular masses of 46 or 54 kDa due to alternative splicing (3, 4). The Thr and Tyr sites of active phosphorylation are conserved between ERK and JNK; however, these sites are located within distinct phosphorylation motifs: Thr-Pro-Tyr (JNKs) and Thr-Glu-Try (ERKs) (5-7). JNK activation induces the phosphorylation of transcription factors, including c-Jun, Elk-1, and ATF-2, which regulate immediate early gene expression (4,8). Ultraviolet radiation, cytokines, and environmental stressors all ...

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Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999

The insulin-like growth factors (IGFs) are trophic factors whose growth-promoting actions are mediated via the lGF-I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF-l and lGF-I receptor after rat sciatic nerve transection. Schwann cells (SOs) were the main source of IGF-l and IGFBP-5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF-l positive. In vitro, CF-I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF-l receptor and IGFBP-5. IGF-I treatment increased the intensity of IGFBP-5 without affecting gene expression. Des(1-3)ICF-l, an IGF-l analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP-5 with SC conditioned media revealed CF-I protection of IGFBP-5 from proteolysis, implying the presence of an ICFBP-5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF-l and SC express the IGF-I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete ICFBPs, which in turn may increase local IGF-l bioavailability.