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Early changes of LIFR and gp130 in sciatic nerve and muscle of diabetic mice
Acta Histochemica 114 (2012) 159–165
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Early changes of LIFR and gp130 in sciatic nerve and muscle of diabetic mice Claudia M. Toledo-Corral 1 , Lisa R. Banner ∗ Department of Biology, California State University, Northridge, 18111 Nordhoff St., Northridge, CA 91330-8330, USA
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Article history: Received 23 March 2011 Received in revised form 11 April 2011 Accepted 12 April 2011
Keywords: Diabetic neuropathy LIFR Gp130 Peripheral nerve Skeletal muscle Mice
a b s t r a c t Peripheral neuropathy is a common complication of diabetes mediated by alterations of growth factors. Members of the neuropoietic cytokine family, which include IL-6, LIF, and CNTF among others, have been shown to be important regulators of peripheral nerves and the muscles that they innervate. To investigate their potential role in diabetic nerve and muscle, we studied the expression of the shared receptor subunits, LIFR and gp130 in a mouse model of streptozotocin (STZ)-induced diabetes. The results of Western blotting and densitometric analysis showed that both LIFR and gp130 protein expression were increased in diabetic sciatic nerve compared to control mice at early time points following STZ injection. In diabetic gastrocnemius muscle, LIFR and gp130 were increased from 3 days to 24 weeks following STZ injection. In contrast, both LIFR and gp130 protein expression were decreased in diabetic soleus muscle at 3-days post-injection. Our results suggest that hyperglycemia results in changes to nerve and muscle soon after the onset of diabetes and that cytokines may play a role in this process. © 2011 Elsevier GmbH. All rights reserved.
Introduction According to the World Diabetes Foundation, 6.4% or 285 million people worldwide live with diabetes. The chronic nature of the disease increases the risk of complications, one of the most common of which involves damage to the peripheral nervous system. This diabetic neuropathy, which occurs in more than 50% of diabetic individuals within 25 years of diagnosis (Hounsom and Tomlinson, 1997; Sugimoto et al., 2000). While the precise mechanisms responsible for the development of neuropathy have yet to be elucidated, it is mostly likely mediated by a variety of metabolic responses. In diabetic patients, both peripheral nerves and skeletal muscle progressively degenerate. Symptoms in diabetic neuropathy originate in the extremities and research into its causation has focused on the theory proposing that neuropathy arises from the loss of proteins necessary to maintain normal nerve growth and function (Calcutt et al., 2008).
Abbreviations: BDNF, brain-derived neurotrophic factor; CT-1, cardiotropin1; CLC, cardiotrophin-like cytokine; CIDP, chronic inflammatory demyelinating polyneuropathy; CNTF, ciliary neurotrophic factor; GDNF, glial cell-line derived neurotrophic factor; IGF, insulin-like growth factor; IL-1, interleukin-1; IL6, interleukin-6; IL-11, interleukin-11; IL-1RA, IL-1 receptor antagonist; LIF, leukemia inhibitory factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; OSM, oncostatin-M; STZ, streptozotocin; VEGF, vascular endothelial growth factor. ∗ Corresponding author. E-mail address: [email protected] (L.R. Banner). 1 Present address: Department of Preventive Medicine, School of Medicine, University of Southern California, Los Angeles, CA, USA. 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.04.003
Members of the neuropoietic cytokine family mediate a variety of functions during development and adulthood including acute phase protein synthesis in liver cells to cell survival and gene expression in the nervous system (Gadient and Patterson, 1999; Kishimoto et al., 1995; Murphy et al., 1997; Patterson, 1994; Senaldi et al., 1999). This family includes interleukin-6 (IL-6), interleukin-11 (IL-11), cardiotropin-1 (CT-1), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-like cytokine (CLC) (Senaldi et al., 1999) and the neuropoietin (Derouet et al., 2004). The actions of these cytokines are mediated through a shared, signal transducing receptor subunit, gp130 (Kishimoto et al., 1995; Taga, 1994), to elicit their biological actions (Kishimoto et al., 1995; Senaldi et al., 1999). Several of the neuropoietic cytokines also share an additional receptor subunit, LIFR. This is the ligand-binding subunit for LIF, which together with gp130 constitutes a functional heterodimeric receptor complex. The receptor gp130 acts as the signal transducer that activates the JAK/STAT pathway, which ultimately leads to the activation of promoters of ligand-responsive genes (Ernst and Jenkins, 2004). In addition to the JAK/STAT pathway, the cytokines can trigger the Ras/MEK/MAPK pathway, which will lead to the activation of a number of transcription factors (Ernst and Jenkins, 2004). Evidence for the involvement of the neuropoietic cytokines in peripheral neuropathies is emerging. In experiments examining chronic inflammatory demyelinating polyneuropathy, mRNAs for LIF and IL-6 were up-regulated while CNTF was down-regulated (Yamamoto et al., 2002). Biopsy studies in humans with various types of peripheral neuropathies, have exhibited an increase in the ligand-binding receptor subunit, CNTFR, and LIFR in diseased neurons (Ito et al., 2001). LIF can positively affect nerve regeneration in
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two additional models of neuropathy: autoimmune neuritis and chemotherapy-induced peripheral neuropathy (Kilpatrick et al., 2001; Laura et al., 2002). Given their roles in peripheral nerve function, it has been postulated that members of the neuropoietic cytokine family might be involved in diabetic neuropathy (Skundric and Lisak, 2003). Reduced levels of CNTF-like activity were found in sciatic nerves of diabetic animals (Calcutt et al., 1992), while replacement of CNTF resulted in an improvement in diabetic nerve conduction velocities and sensory nerve regeneration following nerve crush (Mizisin et al., 2004). Interleukin-6 has been shown to regulate Na+ channel expression in hyperglycemic Schwann cells (Skundric et al., 2003) and the normal injury-induced increase in IL-6 expression in transected nerve was not seen in diabetic animals (Takagi et al., 2001). Recently, IL-6 treatment has been shown to attenuate the development of and correct nerve dysfunction in experimental diabetes (Andriambeloson et al., 2006; Cameron and Cotter, 2007). Given this background, the purpose of the present study was to examine the potential role of the cytokine family receptor subunits, LIFR and gp130, in sciatic nerve, gastrocnemius and soleus muscles of diabetic mice. Materials and methods Animals Inbred C57/BL6J mice (Jackson Laboratory, Bar Harbor, Maine) were purchased, housed and cared for in the CSUN biology vivarium according to the Institutional Animal Care and Use Committee (IACUC) animal care protocol. Food was given ad libitum and animals were maintained on a constant 12 h light/12 h dark cycle. Diabetes induction and blood glucose monitoring Male mice of eight weeks of age were used for diabetes induction using a single intraperitoneal injection of streptozotocin (Sigma–Aldrich, St. Louis, MO, USA) at 200 mg/kg diluted in a 0.1 M citrate buffer, pH 4.2. Control animals were injected with citrate buffer only. Both sexes were used for diabetic induction, but only males were used for the protein expression analysis. Male diabetic and control animals were divided into 6 time points: pretreatment, 3-days, 1-week, 4-weeks, 8-weeks, and 24-weeks post-STZ injection. Blood glucose levels were monitored on a weekly basis using a Fast Take glucose monitor (Lifescan, Milpitas, CA, USA). Tail vein blood was used for blood glucose analyses. Readings above 400 mg/dL were considered as hyperglycemic, whereas control mice had blood glucose readings below 200 mg/dL. Any mice with a reading between 200 and 400 mg/dL were not used for this study. Tissue extraction and storage Diabetic mice and respective controls from each time point were killed by CO2 inhalation. Various tissues were harvested, including whole sciatic nerve, gastrocnemius muscle, and soleus muscle. Tissues used for Western blotting were frozen in liquid nitrogen and stored at −80 ◦ C until ready for use. Protein extraction and Western blots Tissues were homogenized using a glass tissue grinder (Kontes Glass, Vineland, NJ, USA) on ice. The protein extraction solution consisted of RIPA (Tris–HCl, pH 7.4, 10% Triton X-100, 10% NaDOC, 100 mM EDTA) buffer, 1 mM Pefabloc (Roche, Indianapolis, IN, USA) and protease cocktail inhibitor (Sigma–Aldrich, St. Louis, MO, USA). Homogenized tissue was then centrifuged at 7500 × g (4 ◦ C) for
5 min. Resultant supernatant was removed and stored at −80 ◦ C until ready to use for gel electrophoresis. Protein was quantified using a Bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA) per manufacturer’s instructions. Double density (7% resolving gel on top and 4% stacking gel on bottom) SDSgels were used. Sciatic nerve protein extracts were prepared with sample buffer (50 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA). Protease inhibitors used were a 1 mM Pefabloc (Roche, Indianapolis, IN, USA) and 1 mg/20 g of protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) containing beta-mercaptoethanol in a 1:2 ratio and heated to 95 ◦ C for 4 min, then loaded at 3 g per well. Soleus and gastrocnemius muscle were loaded with 10 g and 20 g, respectively. Gels were run at 200 V for 45 min in a running buffer of pH = 8.3 (24 mM Tris, 192 mM glycine, 3.5 mM SDS). Proteins were then transferred to 0.45 m nitrocellulose (Osmonics, USA) at 100 V for 45 min in transfer buffer, pH 8.3 (25 mM Tris, 192 mM glycine, 20%, v/v, methanol) using a Mini Trans-Blot Electrophoretic Transfer Module (BioRad, Hercules, CA, USA). Overnight blocking was performed at 4 ◦ C in 5% dried non-fat milk and 1% bovine serum albumin (BSA). LIFR or gp130 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted at 1:300 in 5% BSA and blots incubated at 37 ◦ C for 1 h, followed by a 1 h incubation at 37 ◦ C in pre-absorbed, horseradish peroxidase (HRP) conjugated, donkeyanti rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:2500 in 5% dried non-fat milk and 1% BSA. Blots were vigorously washed three times with TBS, pH 8.3 (20 mM Tris, 15 mM NaCl, and 0.09 mM KCl), two times with TBST (TBS plus 0.025% Tween-20) and then one time with TBS. Visualization was achieved by X-ray film using West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) per manufacturer’s instructions. Exposure times are optimized to the linear range. No signal was detected when blots were probed with secondary antibody only. To control for loading differences, the same sample was loaded in each well and probed for LIFR and gp130. Band intensity was consistent in all wells of the gel. In addition samples from each animal were run in at least 3 and mostly 5 independent gels to ensure consistency in gel loading. Densitometry Images of the blots were captured with a BioRad densitometer and densitometric software (BioRad, Hercules, CA, USA) was used to analyze the data in linear intensity units. Each gel contained either all diabetic or control time points in order to do comparative ratios. Three to five replicates of each sample were performed. Local background subtraction was performed for each band on each gel. Pixel numbers were always within 5% of each other. All time points were normalized to the 0-day (pretreatment) value within each gel. Ratio values were taken for each gel and then averaged with data from replica gels of the same sample. The averaged ratio for each time point was then averaged with samples from other animals of that time point. Western blots containing dilution curves were performed using varying levels of gp130 and LIFR (96pg, 192pg, 384pg, 768pg, and 1538pg). A standard curve was plotted with density versus protein concentration to determine saturation levels for each protein. Any densities falling outside of linear range were omitted from statistical analysis. Statistical analysis All data are presented as ±SEM. Single-factor ANOVA was performed to compare diabetic to control at each time point. For all analyses, p < 0.05 was considered statistically significant.
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Time (post-injection) Fig. 1. Effects of streptozotocin on blood glucose and body weight of mice. Diabetes resulted in elevated blood glucose levels in STZ-injected mice (A). Error bars represent ±SEM. All diabetic time points are significantly different from their respective same-sexed controls (p < 0.001). Diabetes results in lower body weight in compared to controls (B). Error bars represent ±SEM. The means with * indicate a significant difference from control using Student’s t-test (p < 0.05). N = 8–18 for all control values except 24 weeks where N = 4 for each time point. N = 16–26 for all experimental values except for 24 weeks where N = 4.
Results
Fig. 2. LIFR and gp130 expression in sciatic nerve. (A) Western blot analysis of LIFR and gp130 in diabetic and control sciatic nerve. Quantitative analysis of LIFR (B) and gp130 (C) Western blots. Each time point represents 5 animals except for 24 weeks where N = 4. Significance between experimental and control values were determined using Single-factor ANOVA and * denotes significant (p < 0.05) or ** denotes (p < 0.01) differences between groups.
Blood glucose and body weight Streptozotocin injection significantly elevated blood glucose levels compared to control mice at each time point (Fig. 1). Diabetic mice blood glucose levels were 2–3-fold greater than were control mice. The blood glucose of control mice was consistently below 200 mg/dL throughout the time course of the experiments. Body weights of animals were assessed starting on the day of injection and followed thereafter at one-week intervals to observe changes in weights as correlated with duration/length of diabetic state (Fig. 1). Control mice showed an increase in body weight over the 8-week experimental period, but diabetic mice lost weight over the course of the experiments so that by 8-weeks post-injection diabetic mice weighed almost 5 g less than controls. Expression of LIFR and gp130 in sciatic nerve following STZ injection Since the actions of the neuropoietic cytokines are mediated through the common signal-transducing receptor subunit, gp130, and several members also utilize LIFR, levels of these proteins were determined by Western blot analysis. Quantitative analysis revealed that diabetic sciatic nerve displayed an increase of LIFR and gp130 as early as 3-days post-STZ injection and became significant by 1-week post-
injection (Fig. 2). This increase peaked at 4-weeks post-injection, where levels of both LIFR and gp130 were 2-fold higher in diabetic nerve, and returned to pretreatment values by 8weeks post-injection. There was a significant decrease in LIFR expression in diabetic nerve from 4 to 8 weeks post-injection. The levels of gp130 also approached significance at 3-days post-injection (diab = 1.82 ± 0.66, con = 1.23 ± 0.23, p = 0.09); oneweek post-injection (diab = 1.50 ± 0.41, con = 0.84 ± 0.09, p = 0.16); and four-week post-injection (diab = 1.88 ± 0.46, con = 1.00 ± 0.08, p = 0.09). At 24-week post-injection, diabetic nerve still displayed higher levels of both LIFR and gp130 compared to control. Overall, control nerve shows a steady decrease of LIFR and gp130 expression from 3-days to the 24-week post-injection time point. When analyzing LIFR and gp130 in diabetic nerve, we noted that the fold change in LIFR expression was slightly higher than gp130 at early time points of diabetes (3-days, 1-week, and 4-week post-STZ injection). In control nerve, we saw the same fold change in LIFR at all time points post-injection. Expression of LIFR and gp130 in diabetic gastrocnemius and soleus muscles Diabetic gastrocnemius muscle was also examined as it is the largest skeletal muscle innervated by the sciatic nerve. Our densito-
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Fig. 4. LIFR and gp130 expression in soleus muscle. A. Western blot analysis of LIFR and gp130. Quantitative analysis of LIFR (B) and gp130 (C) Western blots. N = 3 for all time points. Significance differences were determined as in Fig. 2. Fig. 3. LIFR and gp130 expression in gastrocnemius muscle. (A) Western blot analysis of LIFR and gp130. Quantitative analysis of LIFR (B) and gp130 (C) Western blots. Each time point represents 5 animals except for 24 weeks where N = 4. Significance differences were determined as in Fig. 2.
Discussion
metric analyses showed LIFR levels drastically increased in diabetic gastrocnemius muscle as early as 3-days post-STZ injection (Fig. 3). This increase was significant at 3-days and continued through 24-weeks post-STZ injection. We also found a dramatic and significant increase of gp130 in diabetic muscle beginning at 1-week post-STZ injection and continuing through 24-weeks. Only the 3day post-STZ injection time point displayed an increase in gp130 expression that was not statistically significant (diab = 1.96 ± 0.65, con = 0.99 ± 0.05, p = 0.17). We observed a significant age-associated decrease in expression of both LIFR and gp130 in control gastrocnemius muscle, comparing 3-days post-injection to the 24-week (1.05 ± 0.03 vs. 0.49 ± 0.11, p = 0.001) for LIFR and 3-days to 24-week (0.99 ± 0.05 vs. 0.56 ± 0.11, p = 0.007) for gp130. Diabetic gastrocnemius also displayed a slight drop in LIFR expression over the same time period (2.52 ± 0.15) at 3 days post-injection compared to 24-week post-injection (1.71 ± 0.66), which was not statistically significant (p = 0.71). Glycoprotein130 displayed a drop in expression over the time course of 3-days post-injection (1.96 ± 0.65) to 24-week postinjection (1.56 ± 0.26), which was also not statistically significant (p = 0.17). Diabetic soleus muscle, with differing functional responsibilities than the gastrocnemius, was selected for comparison. In contrast to the other tissues examined, diabetic soleus showed a significant decrease at 3-day post-STZ injection in LIFR and gp130 (Fig. 4). We did not see any other significant differences in either LIFR or gp130 at any of the other time points examined.
We found a rapid up-regulation of LIFR and gp130 in sciatic nerve that occurred as early as 3-days following the induction of diabetes. Levels of the receptor subunits continued to increase until 4-weeks post-STZ injection, where levels were elevated 2-fold in diabetic nerve over controls, followed by a return to control values by 8-weeks post-injection. Our results also show striking increases in LIFR and gp130 in diabetic gastrocnemius muscle. As early as 3-days post-injection, we saw a significant increase in LIFR that continued throughout the entire 24-week time course. Early increased levels of both LIFR and gp130 may point to a role for the neuropoietic cytokines in diabetic nerve and muscle, particularly those that use LIFR as a ligand-binding subunit. Several groups have addressed the role of growth factor receptors in diabetic neuropathy. Expression of the low-affinity NGF receptor, p75, increased at 2-week post-STZ injection (Conti et al., 2002) and then returned to baseline levels. Our studies confirmed a similar pattern of increased LIFR and gp130 at early time points from 3-days to 4-week post-STZ injection followed by a return to baseline by 8-weeks. Studies of neurotrophin receptors revealed a decrease in the expression of trkB and trkC in diabetic animals (Rodriguez-Pena et al., 1995) and humans (Sobue et al., 1998). The respective ligands, NGF, BDNF, and NT-3, respectively, also decreased in diabetic nerve (Cai et al., 1999; Tomlinson et al., 1997). Although, we were unable to do a direct comparison with the neurotrophin receptors at the same time points, our results demonstrate an increase in LIFR and gp130 receptor expression from 3-days to 4-weeks of diabetes.
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Increases in receptor levels can occur due to increased expression of the ligand or potentially to a diminishing supply of the ligand. While not examined as early as 3-days, Calcutt et al. (1992) found lower levels of CNTF following 1–2 months of diabetes. Treatment with CNTF following 4 weeks of STZ-induced diabetes resulted in significant improvement of nerve conduction velocities (Mizisin et al., 2004). Reports have shown treatment with IL-6 prevented the development (Andriambeloson et al., 2006) and corrected nerve abnormalities (Cameron and Cotter, 2007) in STZ-induced diabetic neuropathy. The fact that we found a significant decrease in expression of LIFR from 4 to 8 weeks implies that one or more of the neuropoietic cytokines plays a role earlier than 4 weeks. Human studies on diseased nerves suggest that the underlying pathology of neuropathies, rather than the specific diseased state, is regulated by neuropoietic cytokines (Ito et al., 2001). Expression of various cytokine receptor mRNAs was all increased in different types of neuropathies. In studies of specific neuropathies, such as chronic inflammatory demyelinating polyneuropathy (CIDP), evidence of NGF, GDNF, CNTF, and IL-6 and their receptors were cooperatively expressed suggesting their importance in CIDP (Yamamoto et al., 2002). Several members of the neuropoietic cytokine family are also crucial to the maintenance and regeneration of muscle. Previous studies of the role of cytokines in diseased states, such as muscular dystrophy and inflammatory myopathies, revealed that they play an important part in the loss of muscle mass (Fujita et al., 1996; Garcia-Martinez et al., 1997). Our results showed striking increases in LIFR and gp130 in diabetic gastrocnemius muscle. As early as 3-days post-injection, we saw a significant increase in LIFR that continued throughout the entire 24-week time course. Streptozotocin studies of diabetic neuropathy suggest skeletal muscle alterations as early as 2-weeks post-injection (Fahim et al., 2000), but our results would propose that alterations in cytokine levels occur prior to measurable changes in muscle. Studies showed that LIFR is expressed on myoblasts in vitro (Bower et al., 1995) and continued until fusion of myotubes is complete. In vivo, an upregulation of LIFR occurred following denervation (Helgren et al., 1994). Interestingly, in nerve we see a return of receptor expression to baseline levels between 4 and 8-weeks post-injection, while levels remain elevated in gastrocnemius muscle throughout the course of the experiments. Perhaps correlating with this, a report examining force loss in diabetic muscle, demonstrated that an early nerve deficit improved between 4 and 8-weeks, but that the muscle loss continued (Lesniewski et al., 2003). Expression studies following muscle contusion demonstrate that IL-6, LIF, and CNTF receptors are all important factors during the regeneration process. The mRNA for these receptors was detected on myonuclei and muscle precursor cells (satellite cells) at early stages of regeneration (3 h to 2 days), and was no longer detected once myotubes were formed (Kami et al., 2000). CNTFR in human denervated skeletal muscles is also shown to be up-regulated (Weis et al., 1998). Our findings of up-regulated expression of LIFR and gp130 expression in the gastrocnemius of diabetic mice suggest that members of this cytokine family might be involved in muscle damage and repair. Experiments involving mouse and human myoblasts in culture have implicated LIF as a regeneration factor, and its effects can be magnified when combined with other growth factors (Austin et al., 1992, 2000). Muscle injury studies with exogenous LIF treatment improve muscle mass and function, indicating the importance of LIF in muscle function (Austin et al., 2000; Barnard et al., 1994; Brown et al., 2002). Additionally, it has been shown that LIF therapy following myoblast transplantation can enhance the regeneration of skeletal muscle in mice (White et al., 2001). Administration of CNTF to denervated muscle can reduce atrophy by accelerating myotube differentiation (Marques and Neto, 1997) and regulates muscle strength
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during aging (Guillet et al., 1999). Cardiotropin-1 (CT-1), which utilizes LIFR (Arce et al., 1999), has been cited as an important muscle-derived cytokine that is required during the development of motoneurons, specifically for the survival of certain subpopulations of motoneurons (Arce et al., 1999; Oppenheim et al., 2001). A recent study has shown that following muscle damage, satellite cells will proliferate in response to IL-6-induced STAT3 signalling (Toth et al., 2011). In contrast to our results with the gastrocnemius muscle, receptor expression in the soleus muscle was significantly decreased 3-days post-STZ injection. This is a transient decrease as levels of both receptor subunits returned to control levels by 1-week post-injection. Contractile properties of the gastrocnemius and soleus muscle differ. The gastrocnemius is considered a fast twitch muscle, responsible for most of the weight-bearing and strenuous workloads. In contrast, the soleus muscle is considered to be a slow twitch muscle, mostly used for low-intensity work and quick movements. The mechanical demand on the gastrocnemius muscle is much greater than the soleus, therefore its degeneration may be more costly to normal muscle function. Our results of differing expression patterns between the two muscles might point to potential muscle-specific functions of these cytokines. The significant age associated decreases of both LIFR and gp130 in control gastrocnemius muscle may be attributed to a decline in skeletal muscle function in aged mice (Brooks and Faulkner, 1998). Deterioration of muscle over time and decrease in satellite cell numbers may contribute to the decrease of growth factor proteins and their receptors in aging mice. In spite of the fact that diabetic skeletal muscle loses strength (Lesniewski et al., 2003), levels of LIFR and gp130 in diabetic animals showed only a slight (non significant) decrease with age. Although we did not compare receptor expression with muscle mass, our results might suggest that the receptor levels would actually increase over time considering the muscle atrophy that occurs in diabetic muscle. Deficits to the peripheral nervous system and associated skeletal muscles due to hyperglycemia have been linked with sub-optimal support from neuropoietic cytokines and other trophic factors. Consequently, an up-regulation of receptor complexes for these respective proteins may be necessary for optimal binding of already decreased levels of neuropoietic cytokines, particularly those that use LIFR as a ligand-binding subunit. Alterations seen as early as 3days following STZ-injection suggest that hyperglycemic-induced damage to nerve and muscle begins with the onset of diabetes and that therapeutic measures need to be addressed prior to the advent of symptoms of diabetic neuropathy.
Acknowledgements This study was supported by 1R15NS060117-01A2 from the National Institute of Neurological Disorders and Stroke and SO6 GM48630-08 from The National Institute of General Medical Sciences to LRB and NIH MBRS-RISE and CSUN Thesis Support Grant to CMT.
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Early changes of LIFR and gp130 in sciatic nerve and muscle of diabetic mice Claudia M. Toledo-Corral 1 , Lisa R. Banner ∗ Department of Biology, California State University, Northridge, 18111 Nordhoff St., Northridge, CA 91330-8330, USA
a r t i c l e
i n f o
Article history: Received 23 March 2011 Received in revised form 11 April 2011 Accepted 12 April 2011
Keywords: Diabetic neuropathy LIFR Gp130 Peripheral nerve Skeletal muscle Mice
a b s t r a c t Peripheral neuropathy is a common complication of diabetes mediated by alterations of growth factors. Members of the neuropoietic cytokine family, which include IL-6, LIF, and CNTF among others, have been shown to be important regulators of peripheral nerves and the muscles that they innervate. To investigate their potential role in diabetic nerve and muscle, we studied the expression of the shared receptor subunits, LIFR and gp130 in a mouse model of streptozotocin (STZ)-induced diabetes. The results of Western blotting and densitometric analysis showed that both LIFR and gp130 protein expression were increased in diabetic sciatic nerve compared to control mice at early time points following STZ injection. In diabetic gastrocnemius muscle, LIFR and gp130 were increased from 3 days to 24 weeks following STZ injection. In contrast, both LIFR and gp130 protein expression were decreased in diabetic soleus muscle at 3-days post-injection. Our results suggest that hyperglycemia results in changes to nerve and muscle soon after the onset of diabetes and that cytokines may play a role in this process. © 2011 Elsevier GmbH. All rights reserved.
Introduction According to the World Diabetes Foundation, 6.4% or 285 million people worldwide live with diabetes. The chronic nature of the disease increases the risk of complications, one of the most common of which involves damage to the peripheral nervous system. This diabetic neuropathy, which occurs in more than 50% of diabetic individuals within 25 years of diagnosis (Hounsom and Tomlinson, 1997; Sugimoto et al., 2000). While the precise mechanisms responsible for the development of neuropathy have yet to be elucidated, it is mostly likely mediated by a variety of metabolic responses. In diabetic patients, both peripheral nerves and skeletal muscle progressively degenerate. Symptoms in diabetic neuropathy originate in the extremities and research into its causation has focused on the theory proposing that neuropathy arises from the loss of proteins necessary to maintain normal nerve growth and function (Calcutt et al., 2008).
Abbreviations: BDNF, brain-derived neurotrophic factor; CT-1, cardiotropin1; CLC, cardiotrophin-like cytokine; CIDP, chronic inflammatory demyelinating polyneuropathy; CNTF, ciliary neurotrophic factor; GDNF, glial cell-line derived neurotrophic factor; IGF, insulin-like growth factor; IL-1, interleukin-1; IL6, interleukin-6; IL-11, interleukin-11; IL-1RA, IL-1 receptor antagonist; LIF, leukemia inhibitory factor; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; OSM, oncostatin-M; STZ, streptozotocin; VEGF, vascular endothelial growth factor. ∗ Corresponding author. E-mail address: [email protected] (L.R. Banner). 1 Present address: Department of Preventive Medicine, School of Medicine, University of Southern California, Los Angeles, CA, USA. 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.04.003
Members of the neuropoietic cytokine family mediate a variety of functions during development and adulthood including acute phase protein synthesis in liver cells to cell survival and gene expression in the nervous system (Gadient and Patterson, 1999; Kishimoto et al., 1995; Murphy et al., 1997; Patterson, 1994; Senaldi et al., 1999). This family includes interleukin-6 (IL-6), interleukin-11 (IL-11), cardiotropin-1 (CT-1), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-like cytokine (CLC) (Senaldi et al., 1999) and the neuropoietin (Derouet et al., 2004). The actions of these cytokines are mediated through a shared, signal transducing receptor subunit, gp130 (Kishimoto et al., 1995; Taga, 1994), to elicit their biological actions (Kishimoto et al., 1995; Senaldi et al., 1999). Several of the neuropoietic cytokines also share an additional receptor subunit, LIFR. This is the ligand-binding subunit for LIF, which together with gp130 constitutes a functional heterodimeric receptor complex. The receptor gp130 acts as the signal transducer that activates the JAK/STAT pathway, which ultimately leads to the activation of promoters of ligand-responsive genes (Ernst and Jenkins, 2004). In addition to the JAK/STAT pathway, the cytokines can trigger the Ras/MEK/MAPK pathway, which will lead to the activation of a number of transcription factors (Ernst and Jenkins, 2004). Evidence for the involvement of the neuropoietic cytokines in peripheral neuropathies is emerging. In experiments examining chronic inflammatory demyelinating polyneuropathy, mRNAs for LIF and IL-6 were up-regulated while CNTF was down-regulated (Yamamoto et al., 2002). Biopsy studies in humans with various types of peripheral neuropathies, have exhibited an increase in the ligand-binding receptor subunit, CNTFR, and LIFR in diseased neurons (Ito et al., 2001). LIF can positively affect nerve regeneration in
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two additional models of neuropathy: autoimmune neuritis and chemotherapy-induced peripheral neuropathy (Kilpatrick et al., 2001; Laura et al., 2002). Given their roles in peripheral nerve function, it has been postulated that members of the neuropoietic cytokine family might be involved in diabetic neuropathy (Skundric and Lisak, 2003). Reduced levels of CNTF-like activity were found in sciatic nerves of diabetic animals (Calcutt et al., 1992), while replacement of CNTF resulted in an improvement in diabetic nerve conduction velocities and sensory nerve regeneration following nerve crush (Mizisin et al., 2004). Interleukin-6 has been shown to regulate Na+ channel expression in hyperglycemic Schwann cells (Skundric et al., 2003) and the normal injury-induced increase in IL-6 expression in transected nerve was not seen in diabetic animals (Takagi et al., 2001). Recently, IL-6 treatment has been shown to attenuate the development of and correct nerve dysfunction in experimental diabetes (Andriambeloson et al., 2006; Cameron and Cotter, 2007). Given this background, the purpose of the present study was to examine the potential role of the cytokine family receptor subunits, LIFR and gp130, in sciatic nerve, gastrocnemius and soleus muscles of diabetic mice. Materials and methods Animals Inbred C57/BL6J mice (Jackson Laboratory, Bar Harbor, Maine) were purchased, housed and cared for in the CSUN biology vivarium according to the Institutional Animal Care and Use Committee (IACUC) animal care protocol. Food was given ad libitum and animals were maintained on a constant 12 h light/12 h dark cycle. Diabetes induction and blood glucose monitoring Male mice of eight weeks of age were used for diabetes induction using a single intraperitoneal injection of streptozotocin (Sigma–Aldrich, St. Louis, MO, USA) at 200 mg/kg diluted in a 0.1 M citrate buffer, pH 4.2. Control animals were injected with citrate buffer only. Both sexes were used for diabetic induction, but only males were used for the protein expression analysis. Male diabetic and control animals were divided into 6 time points: pretreatment, 3-days, 1-week, 4-weeks, 8-weeks, and 24-weeks post-STZ injection. Blood glucose levels were monitored on a weekly basis using a Fast Take glucose monitor (Lifescan, Milpitas, CA, USA). Tail vein blood was used for blood glucose analyses. Readings above 400 mg/dL were considered as hyperglycemic, whereas control mice had blood glucose readings below 200 mg/dL. Any mice with a reading between 200 and 400 mg/dL were not used for this study. Tissue extraction and storage Diabetic mice and respective controls from each time point were killed by CO2 inhalation. Various tissues were harvested, including whole sciatic nerve, gastrocnemius muscle, and soleus muscle. Tissues used for Western blotting were frozen in liquid nitrogen and stored at −80 ◦ C until ready for use. Protein extraction and Western blots Tissues were homogenized using a glass tissue grinder (Kontes Glass, Vineland, NJ, USA) on ice. The protein extraction solution consisted of RIPA (Tris–HCl, pH 7.4, 10% Triton X-100, 10% NaDOC, 100 mM EDTA) buffer, 1 mM Pefabloc (Roche, Indianapolis, IN, USA) and protease cocktail inhibitor (Sigma–Aldrich, St. Louis, MO, USA). Homogenized tissue was then centrifuged at 7500 × g (4 ◦ C) for
5 min. Resultant supernatant was removed and stored at −80 ◦ C until ready to use for gel electrophoresis. Protein was quantified using a Bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA) per manufacturer’s instructions. Double density (7% resolving gel on top and 4% stacking gel on bottom) SDSgels were used. Sciatic nerve protein extracts were prepared with sample buffer (50 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA). Protease inhibitors used were a 1 mM Pefabloc (Roche, Indianapolis, IN, USA) and 1 mg/20 g of protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) containing beta-mercaptoethanol in a 1:2 ratio and heated to 95 ◦ C for 4 min, then loaded at 3 g per well. Soleus and gastrocnemius muscle were loaded with 10 g and 20 g, respectively. Gels were run at 200 V for 45 min in a running buffer of pH = 8.3 (24 mM Tris, 192 mM glycine, 3.5 mM SDS). Proteins were then transferred to 0.45 m nitrocellulose (Osmonics, USA) at 100 V for 45 min in transfer buffer, pH 8.3 (25 mM Tris, 192 mM glycine, 20%, v/v, methanol) using a Mini Trans-Blot Electrophoretic Transfer Module (BioRad, Hercules, CA, USA). Overnight blocking was performed at 4 ◦ C in 5% dried non-fat milk and 1% bovine serum albumin (BSA). LIFR or gp130 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted at 1:300 in 5% BSA and blots incubated at 37 ◦ C for 1 h, followed by a 1 h incubation at 37 ◦ C in pre-absorbed, horseradish peroxidase (HRP) conjugated, donkeyanti rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:2500 in 5% dried non-fat milk and 1% BSA. Blots were vigorously washed three times with TBS, pH 8.3 (20 mM Tris, 15 mM NaCl, and 0.09 mM KCl), two times with TBST (TBS plus 0.025% Tween-20) and then one time with TBS. Visualization was achieved by X-ray film using West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) per manufacturer’s instructions. Exposure times are optimized to the linear range. No signal was detected when blots were probed with secondary antibody only. To control for loading differences, the same sample was loaded in each well and probed for LIFR and gp130. Band intensity was consistent in all wells of the gel. In addition samples from each animal were run in at least 3 and mostly 5 independent gels to ensure consistency in gel loading. Densitometry Images of the blots were captured with a BioRad densitometer and densitometric software (BioRad, Hercules, CA, USA) was used to analyze the data in linear intensity units. Each gel contained either all diabetic or control time points in order to do comparative ratios. Three to five replicates of each sample were performed. Local background subtraction was performed for each band on each gel. Pixel numbers were always within 5% of each other. All time points were normalized to the 0-day (pretreatment) value within each gel. Ratio values were taken for each gel and then averaged with data from replica gels of the same sample. The averaged ratio for each time point was then averaged with samples from other animals of that time point. Western blots containing dilution curves were performed using varying levels of gp130 and LIFR (96pg, 192pg, 384pg, 768pg, and 1538pg). A standard curve was plotted with density versus protein concentration to determine saturation levels for each protein. Any densities falling outside of linear range were omitted from statistical analysis. Statistical analysis All data are presented as ±SEM. Single-factor ANOVA was performed to compare diabetic to control at each time point. For all analyses, p < 0.05 was considered statistically significant.
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Time (post-injection) Fig. 1. Effects of streptozotocin on blood glucose and body weight of mice. Diabetes resulted in elevated blood glucose levels in STZ-injected mice (A). Error bars represent ±SEM. All diabetic time points are significantly different from their respective same-sexed controls (p < 0.001). Diabetes results in lower body weight in compared to controls (B). Error bars represent ±SEM. The means with * indicate a significant difference from control using Student’s t-test (p < 0.05). N = 8–18 for all control values except 24 weeks where N = 4 for each time point. N = 16–26 for all experimental values except for 24 weeks where N = 4.
Results
Fig. 2. LIFR and gp130 expression in sciatic nerve. (A) Western blot analysis of LIFR and gp130 in diabetic and control sciatic nerve. Quantitative analysis of LIFR (B) and gp130 (C) Western blots. Each time point represents 5 animals except for 24 weeks where N = 4. Significance between experimental and control values were determined using Single-factor ANOVA and * denotes significant (p < 0.05) or ** denotes (p < 0.01) differences between groups.
Blood glucose and body weight Streptozotocin injection significantly elevated blood glucose levels compared to control mice at each time point (Fig. 1). Diabetic mice blood glucose levels were 2–3-fold greater than were control mice. The blood glucose of control mice was consistently below 200 mg/dL throughout the time course of the experiments. Body weights of animals were assessed starting on the day of injection and followed thereafter at one-week intervals to observe changes in weights as correlated with duration/length of diabetic state (Fig. 1). Control mice showed an increase in body weight over the 8-week experimental period, but diabetic mice lost weight over the course of the experiments so that by 8-weeks post-injection diabetic mice weighed almost 5 g less than controls. Expression of LIFR and gp130 in sciatic nerve following STZ injection Since the actions of the neuropoietic cytokines are mediated through the common signal-transducing receptor subunit, gp130, and several members also utilize LIFR, levels of these proteins were determined by Western blot analysis. Quantitative analysis revealed that diabetic sciatic nerve displayed an increase of LIFR and gp130 as early as 3-days post-STZ injection and became significant by 1-week post-
injection (Fig. 2). This increase peaked at 4-weeks post-injection, where levels of both LIFR and gp130 were 2-fold higher in diabetic nerve, and returned to pretreatment values by 8weeks post-injection. There was a significant decrease in LIFR expression in diabetic nerve from 4 to 8 weeks post-injection. The levels of gp130 also approached significance at 3-days post-injection (diab = 1.82 ± 0.66, con = 1.23 ± 0.23, p = 0.09); oneweek post-injection (diab = 1.50 ± 0.41, con = 0.84 ± 0.09, p = 0.16); and four-week post-injection (diab = 1.88 ± 0.46, con = 1.00 ± 0.08, p = 0.09). At 24-week post-injection, diabetic nerve still displayed higher levels of both LIFR and gp130 compared to control. Overall, control nerve shows a steady decrease of LIFR and gp130 expression from 3-days to the 24-week post-injection time point. When analyzing LIFR and gp130 in diabetic nerve, we noted that the fold change in LIFR expression was slightly higher than gp130 at early time points of diabetes (3-days, 1-week, and 4-week post-STZ injection). In control nerve, we saw the same fold change in LIFR at all time points post-injection. Expression of LIFR and gp130 in diabetic gastrocnemius and soleus muscles Diabetic gastrocnemius muscle was also examined as it is the largest skeletal muscle innervated by the sciatic nerve. Our densito-
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Fig. 4. LIFR and gp130 expression in soleus muscle. A. Western blot analysis of LIFR and gp130. Quantitative analysis of LIFR (B) and gp130 (C) Western blots. N = 3 for all time points. Significance differences were determined as in Fig. 2. Fig. 3. LIFR and gp130 expression in gastrocnemius muscle. (A) Western blot analysis of LIFR and gp130. Quantitative analysis of LIFR (B) and gp130 (C) Western blots. Each time point represents 5 animals except for 24 weeks where N = 4. Significance differences were determined as in Fig. 2.
Discussion
metric analyses showed LIFR levels drastically increased in diabetic gastrocnemius muscle as early as 3-days post-STZ injection (Fig. 3). This increase was significant at 3-days and continued through 24-weeks post-STZ injection. We also found a dramatic and significant increase of gp130 in diabetic muscle beginning at 1-week post-STZ injection and continuing through 24-weeks. Only the 3day post-STZ injection time point displayed an increase in gp130 expression that was not statistically significant (diab = 1.96 ± 0.65, con = 0.99 ± 0.05, p = 0.17). We observed a significant age-associated decrease in expression of both LIFR and gp130 in control gastrocnemius muscle, comparing 3-days post-injection to the 24-week (1.05 ± 0.03 vs. 0.49 ± 0.11, p = 0.001) for LIFR and 3-days to 24-week (0.99 ± 0.05 vs. 0.56 ± 0.11, p = 0.007) for gp130. Diabetic gastrocnemius also displayed a slight drop in LIFR expression over the same time period (2.52 ± 0.15) at 3 days post-injection compared to 24-week post-injection (1.71 ± 0.66), which was not statistically significant (p = 0.71). Glycoprotein130 displayed a drop in expression over the time course of 3-days post-injection (1.96 ± 0.65) to 24-week postinjection (1.56 ± 0.26), which was also not statistically significant (p = 0.17). Diabetic soleus muscle, with differing functional responsibilities than the gastrocnemius, was selected for comparison. In contrast to the other tissues examined, diabetic soleus showed a significant decrease at 3-day post-STZ injection in LIFR and gp130 (Fig. 4). We did not see any other significant differences in either LIFR or gp130 at any of the other time points examined.
We found a rapid up-regulation of LIFR and gp130 in sciatic nerve that occurred as early as 3-days following the induction of diabetes. Levels of the receptor subunits continued to increase until 4-weeks post-STZ injection, where levels were elevated 2-fold in diabetic nerve over controls, followed by a return to control values by 8-weeks post-injection. Our results also show striking increases in LIFR and gp130 in diabetic gastrocnemius muscle. As early as 3-days post-injection, we saw a significant increase in LIFR that continued throughout the entire 24-week time course. Early increased levels of both LIFR and gp130 may point to a role for the neuropoietic cytokines in diabetic nerve and muscle, particularly those that use LIFR as a ligand-binding subunit. Several groups have addressed the role of growth factor receptors in diabetic neuropathy. Expression of the low-affinity NGF receptor, p75, increased at 2-week post-STZ injection (Conti et al., 2002) and then returned to baseline levels. Our studies confirmed a similar pattern of increased LIFR and gp130 at early time points from 3-days to 4-week post-STZ injection followed by a return to baseline by 8-weeks. Studies of neurotrophin receptors revealed a decrease in the expression of trkB and trkC in diabetic animals (Rodriguez-Pena et al., 1995) and humans (Sobue et al., 1998). The respective ligands, NGF, BDNF, and NT-3, respectively, also decreased in diabetic nerve (Cai et al., 1999; Tomlinson et al., 1997). Although, we were unable to do a direct comparison with the neurotrophin receptors at the same time points, our results demonstrate an increase in LIFR and gp130 receptor expression from 3-days to 4-weeks of diabetes.
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Increases in receptor levels can occur due to increased expression of the ligand or potentially to a diminishing supply of the ligand. While not examined as early as 3-days, Calcutt et al. (1992) found lower levels of CNTF following 1–2 months of diabetes. Treatment with CNTF following 4 weeks of STZ-induced diabetes resulted in significant improvement of nerve conduction velocities (Mizisin et al., 2004). Reports have shown treatment with IL-6 prevented the development (Andriambeloson et al., 2006) and corrected nerve abnormalities (Cameron and Cotter, 2007) in STZ-induced diabetic neuropathy. The fact that we found a significant decrease in expression of LIFR from 4 to 8 weeks implies that one or more of the neuropoietic cytokines plays a role earlier than 4 weeks. Human studies on diseased nerves suggest that the underlying pathology of neuropathies, rather than the specific diseased state, is regulated by neuropoietic cytokines (Ito et al., 2001). Expression of various cytokine receptor mRNAs was all increased in different types of neuropathies. In studies of specific neuropathies, such as chronic inflammatory demyelinating polyneuropathy (CIDP), evidence of NGF, GDNF, CNTF, and IL-6 and their receptors were cooperatively expressed suggesting their importance in CIDP (Yamamoto et al., 2002). Several members of the neuropoietic cytokine family are also crucial to the maintenance and regeneration of muscle. Previous studies of the role of cytokines in diseased states, such as muscular dystrophy and inflammatory myopathies, revealed that they play an important part in the loss of muscle mass (Fujita et al., 1996; Garcia-Martinez et al., 1997). Our results showed striking increases in LIFR and gp130 in diabetic gastrocnemius muscle. As early as 3-days post-injection, we saw a significant increase in LIFR that continued throughout the entire 24-week time course. Streptozotocin studies of diabetic neuropathy suggest skeletal muscle alterations as early as 2-weeks post-injection (Fahim et al., 2000), but our results would propose that alterations in cytokine levels occur prior to measurable changes in muscle. Studies showed that LIFR is expressed on myoblasts in vitro (Bower et al., 1995) and continued until fusion of myotubes is complete. In vivo, an upregulation of LIFR occurred following denervation (Helgren et al., 1994). Interestingly, in nerve we see a return of receptor expression to baseline levels between 4 and 8-weeks post-injection, while levels remain elevated in gastrocnemius muscle throughout the course of the experiments. Perhaps correlating with this, a report examining force loss in diabetic muscle, demonstrated that an early nerve deficit improved between 4 and 8-weeks, but that the muscle loss continued (Lesniewski et al., 2003). Expression studies following muscle contusion demonstrate that IL-6, LIF, and CNTF receptors are all important factors during the regeneration process. The mRNA for these receptors was detected on myonuclei and muscle precursor cells (satellite cells) at early stages of regeneration (3 h to 2 days), and was no longer detected once myotubes were formed (Kami et al., 2000). CNTFR in human denervated skeletal muscles is also shown to be up-regulated (Weis et al., 1998). Our findings of up-regulated expression of LIFR and gp130 expression in the gastrocnemius of diabetic mice suggest that members of this cytokine family might be involved in muscle damage and repair. Experiments involving mouse and human myoblasts in culture have implicated LIF as a regeneration factor, and its effects can be magnified when combined with other growth factors (Austin et al., 1992, 2000). Muscle injury studies with exogenous LIF treatment improve muscle mass and function, indicating the importance of LIF in muscle function (Austin et al., 2000; Barnard et al., 1994; Brown et al., 2002). Additionally, it has been shown that LIF therapy following myoblast transplantation can enhance the regeneration of skeletal muscle in mice (White et al., 2001). Administration of CNTF to denervated muscle can reduce atrophy by accelerating myotube differentiation (Marques and Neto, 1997) and regulates muscle strength
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during aging (Guillet et al., 1999). Cardiotropin-1 (CT-1), which utilizes LIFR (Arce et al., 1999), has been cited as an important muscle-derived cytokine that is required during the development of motoneurons, specifically for the survival of certain subpopulations of motoneurons (Arce et al., 1999; Oppenheim et al., 2001). A recent study has shown that following muscle damage, satellite cells will proliferate in response to IL-6-induced STAT3 signalling (Toth et al., 2011). In contrast to our results with the gastrocnemius muscle, receptor expression in the soleus muscle was significantly decreased 3-days post-STZ injection. This is a transient decrease as levels of both receptor subunits returned to control levels by 1-week post-injection. Contractile properties of the gastrocnemius and soleus muscle differ. The gastrocnemius is considered a fast twitch muscle, responsible for most of the weight-bearing and strenuous workloads. In contrast, the soleus muscle is considered to be a slow twitch muscle, mostly used for low-intensity work and quick movements. The mechanical demand on the gastrocnemius muscle is much greater than the soleus, therefore its degeneration may be more costly to normal muscle function. Our results of differing expression patterns between the two muscles might point to potential muscle-specific functions of these cytokines. The significant age associated decreases of both LIFR and gp130 in control gastrocnemius muscle may be attributed to a decline in skeletal muscle function in aged mice (Brooks and Faulkner, 1998). Deterioration of muscle over time and decrease in satellite cell numbers may contribute to the decrease of growth factor proteins and their receptors in aging mice. In spite of the fact that diabetic skeletal muscle loses strength (Lesniewski et al., 2003), levels of LIFR and gp130 in diabetic animals showed only a slight (non significant) decrease with age. Although we did not compare receptor expression with muscle mass, our results might suggest that the receptor levels would actually increase over time considering the muscle atrophy that occurs in diabetic muscle. Deficits to the peripheral nervous system and associated skeletal muscles due to hyperglycemia have been linked with sub-optimal support from neuropoietic cytokines and other trophic factors. Consequently, an up-regulation of receptor complexes for these respective proteins may be necessary for optimal binding of already decreased levels of neuropoietic cytokines, particularly those that use LIFR as a ligand-binding subunit. Alterations seen as early as 3days following STZ-injection suggest that hyperglycemic-induced damage to nerve and muscle begins with the onset of diabetes and that therapeutic measures need to be addressed prior to the advent of symptoms of diabetic neuropathy.
Acknowledgements This study was supported by 1R15NS060117-01A2 from the National Institute of Neurological Disorders and Stroke and SO6 GM48630-08 from The National Institute of General Medical Sciences to LRB and NIH MBRS-RISE and CSUN Thesis Support Grant to CMT.
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