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Peripheral nervous system insulin resistance in ob/ob mice

Peripheral nervous system insulin resistance in ob/ob mice Background: A reduction in peripheral nervous system (PNS) insulin signaling is a proposed mechanism that may contribute to sensory neuron dysfunction and diabetic neuropathy. Neuronal insulin resistance is associated with several neurological disorders and recent evidence has indicated that dorsal root ganglion (DRG) neurons in primary culture display altered insulin signaling, yet in vivo results are lacking. Here, experiments were performed to test the hypothesis that the PNS of insulin-resistant mice displays altered insulin signal transduction in vivo. For these studies, nondiabetic control and type 2 diabetic ob/ob mice were challenged with an intrathecal injection of insulin or insulin-like growth factor 1 (IGF-1) and downstream signaling was evaluated in the DRG and sciatic nerve using Western blot analysis. Results: The results indicate that insulin signaling abnormalities documented in other “insulin sensitive” tissues (i.e. muscle, fat, liver) of ob/ob mice are also present in the PNS. A robust increase in Akt activation was observed with insulin and IGF-1 stimulation in nondiabetic mice in both the sciatic nerve and DRG; however this response was blunted in both tissues from ob/ob mice. The results also suggest that upregulated JNK activation and reduced insulin receptor expression could be contributory mechanisms of PNS insulin resistance within sensory neurons. Conclusions: These findings contribute to the growing body of evidence that alterations in insulin signaling occur in the PNS and may be a key factor in the pathogenesis of diabetic neuropathy. Keywords: Diabetic neuropathy, Neuronal insulin resistance, Neurotrophic support Background Furthermore, clinical evidence has reported that insulin Diabetes and metabolic syndrome are risk factors for resistance appears to be an independent risk factor for several neurological diseases, and emerging evidence has both autonomic and peripheral neuropathy [8]. indicated that neuronal insulin resistance may be in- Although neurons do not appear to rely on insulin volved in disease pathogenesis [1]. While altered insulin for glucose uptake [9], insulin does have an import- signaling is known to be the key factor in the develop- ant role in both the CNS and PNS. Insulin promotes ment of diabetes, the role that it plays in diabetic neur- in vivo nerve regeneration [4,10,11], induces neurite opathy (DN) is not well understood. However, it has outgrowth [12,13], maintains neuronal mitochondrial been demonstrated that neuronally-targeted insulin function [14,15], supports memory formation [16,17], treatment can improve signs of neuropathy without and regulates hypothalamic metabolic control [18,19]. altering blood glucose levels [2-4]. Recent evidence sug- While the exact mechanisms through which insulin pro- gests that cultured sensory neurons from insulin- motes these functions remain unclear, insulin is considered resistant mice display classic signs of insulin resistance a potent neurotrophic factor key to maintaining proper and that insulin resistance may be contributing to mito- neuronal function. chondrial dysfunction and increased ROS in DN [5-7]. Insulin and insulin-like growth factor 1 (IGF-1) signal- ing is propagated by phosphorylation events that begin with the intrinsic tyrosine kinase activity of the insulin * Correspondence: dwright@kumc.edu or IGF receptor (reviewed in [20,21]) and continue with Department of Anatomy and Cell Biology, the University of Kansas Medical subsequent activation of both the PI3K-Akt and MAPK Center, Kansas City, KS 66160, USA cascades. While these pathways are well defined in Full list of author information is available at the end of the article © 2013 Grote et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 2 of 11 http://www.actaneurocomms.org/content/1/1/15 muscle, adipose, and liver, insulin signaling and its ac- Furthermore, the PNS of ob/ob mice has alterations in tions in the PNS are poorly understood. cellular mechanisms of insulin resistance, including In an insulin-resistant state, the cellular effects of insu- decreased DRG insulin receptor expression and up- lin are blunted due to improper signal propagation regulation of JNK activity in the sciatic nerve. resulting from several different mechanisms, including 1) degradation of the insulin receptor [22-25], 2) removal Results of keytyrosinephosphorylation sitesbyoveractivationof Insulin resistance in ob/ob mice protein tyrosine phosphatases [26-29], and 3) increased To quantify the extent of systemic insulin resistance in phosphorylation at inhibitory IRS serine residues due to el- ob/ob mice, nondiabetic and diabetic ob/ob mice under- evated stress kinases, such as JNK [30-35]. However, the went an IPGTT at 9 weeks of age (Figure 1A). Blood glu- extent to which these mechanisms affect insulin signal cose levels of the ob/ob mice were significantly higher transduction in the PNS is not clear. than nondiabetic mice throughout the course of the ex- Growing evidence suggests that neurons may become periment and the area under the curve (AUC) was also insulin resistant similar to other tissues. However, no significantly elevated for ob/ob mice (Figure 1B). Results in vivo evidence of PNS insulin resistance has been from the ITT indicated that nondiabetic, insulin-injected presented, and the cellular mechanisms associated with mice exhibited an expected physiological decrease in PNS insulin resistance have not been thoroughly investi- blood glucose in response to insulin; however, ob/ob gated. Here, we demonstrate that the DRG and sciatic mice displayed a transient elevation of glucose levels nerve of ob/ob mice display reduced insulin signaling (Figure 1C). Statistical analysis of the data revealed in response to an intrathecal injection of insulin. that ob/ob mice maintained elevated glucose levels Figure 1 Ob/ob mice display classic signs of insulin resistance. A, B) An IPGTT showed significantly elevated blood glucose levels in ob/ob mice throughout the test. The blood glucose of ob/ob mice increased more than 10 mmol/L at its maximal level as opposed to nondiabetic mice that elevated less than 6 mmol/L after glucose injection, indicating severe glucose intolerance in ob/ob mice. C, D) Similar to the IPGTT, data from the ITT showed reduced insulin sensitivity in ob/ob mice. In fact, an insulin dose of 1.5 U/Kg did not decrease the blood glucose level of ob/ob mice, whereas this dose lowered the blood glucose of nondiabetic controls by approximately 3.6 mmol/L. E-G)At10weeks of age, ob/ob mice had significantly elevated blood glucose and serum insulin levels. Accordingly, the HOMA-IR measure of insulin resistance was significantly higher in ob/ob mice. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. IPGTT n = 7 nondiabetic mice, n = 6 ob/ob. ITT n = 4 nondiabetic mice, n = 4 ob/ob. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 3 of 11 http://www.actaneurocomms.org/content/1/1/15 compared to nondiabetic controls throughout most of Akt is a hallmark of insulin resistance [5,6,37,38]. Here, the study, and that the AUC was significantly higher for nondiabetic and diabetic ob/ob mice were administered diabetic ob/ob mice (Figure 1D). The HOMA-IR, a meas- either intrathecal PBS or insulin and the DRG and sci- ure of insulin resistance, was calculated using fasting atic nerve were harvested 30 minutes later for Western blood glucose and serum insulin levels from 10 week old blot analysis to assess Akt activation. Both nondiabetic mice. Ob/ob mice had significantly higher blood glucose control and ob/ob mice display significantly elevated levels (14.3 ± 2.1 mmol/L) compared to nondiabetic mice blood glucose levels following intrathecal injection of (8.2 ±0.5 mmol/L) (Figure 1E). Fasting insulin levels were PBS. Nondiabetic mice glucose levels increased from also significantly higher in diabetic ob/ob mice (6780 ± 6.6 ± 0.4 mmol/L to 8.6 ± 0.5 mmol/L, whereas ob/ob levels 1610 pmol/L) compared to nondiabetic mice (198 ± 25 increased from 12.1 ± 2.4 mmol/L to 22.3 ± 2.4 mmol/L. pmol/L, Figure 1F). As such, ob/ob mice had a signifi- Glucose levels in nondiabetic mice significantly decreased cantly elevated HOMA-IR as compared to nondiabetic from 7.0 ± 0.3 mmol/L to 3.3 ± 0.3 mmol/L after intra- mice (557 ± 130 compared to 10.1 ± 1.4, respectively, thecal insulin injection. Ob/ob mice glucose levels 30 mi- Figure 1G). These results demonstrate significant glu- nutes after insulin injection were not significantly different cose intolerance and insulin resistance in ob/ob mice at from baseline, starting at 12.7 ± 0.9 mmol/L and ending at this age. 12.3 ± 1.5 mmol/L after 30 minutes. In nondiabetic mice, insulin produced a strong eleva- Mechanical allodynia in ob/ob mice tion in levels of activated Akt (p(ser473)Akt/total Akt) in To quantify a known behavioral abnormality associated both the DRG and sciatic nerve (Figure 3A,B). However with neuropathy in mice, mechanical sensitivity was in ob/ob mice, Akt activation was significantly lower in assessed in nondiabetic and diabetic ob/ob mice at 8, 9, the DRG and sciatic nerve. In fact, insulin failed to sig- 10, and 11 weeks of age. There were no differences in nificantly increase Akt activation over baseline in the mechanical thresholds between nondiabetic and ob/ob DRG of ob/ob mice. For comparison, Akt activation in diabetic mice at 8, 9, or 10 weeks of age. However, at 11 the DRG was increased 3.1 fold in nondiabetic mice and weeks, there was a significant decrease in the mechanical 1.6 fold in ob/ob diabetic mice. In the sciatic nerve, insu- thresholds of diabetic ob/ob mice compared to nondiabetic lin produced 9.7 and 6.1 fold increase in Akt activation mice (Figure 2), consistent with sensory aberrations associ- in nondiabetic and ob/ob mice, respectively. ated with peripheral neuropathy as previously reported To confirm that these results were not dependent on in this genetic mouse strain [36]. the intrathecal route of delivery, a small number of mice were administered intraperitoneal insulin at a dose of Blunted insulin and IGF-1 Akt activation in ob/ob DRG 3.33 U/kg. PBS injections once again appeared to cause an and sciatic nerve increase in blood glucose levels from baseline, nondiabetic Insulin stimulation causes a robust activation of Akt in mice levels started at 7.0 ± 1.4 mmol/L and ended at 8.6 ± insulin-sensitive tissues like muscle and adipose, as well 1.4 mmol/L (p > 0.05), whereas ob/ob mice showed a as in neurons of both the peripheral and central nervous significant increase from 10.8 ± 0.8 mmol/L to 15.4 ± systems. Moreover, reduced insulin-induced activation of 1.0 mmol/L. IP insulin resulted in significantly lower blood glucose levels in nondiabetic mice after 30 minutes, 7.8 ± 0.6 versus 4.2 ± 0.5 mmol/L, respectively. Ob/ob mice blood glucose levels were not significantly altered by IP in- sulin injection 15.3 ± 3.0 versus 13.6 ± 3.9 mmol/L. Similar to the intrathecal delivery route, a significant increase in Akt activation was observed in the DRG and sciatic nerve of nondiabetic mice stimulated with insulin; however, no significant change was observed in either tissue from ob/ob mice. (Figure 4A,B). In the DRG, nondiabetic mice displayeda2.4foldchangeinAkt activation,comparedto a 1.5 fold change in ob/ob mice. IP insulin induced a 3.8 fold change in Akt in the sciatic nerve of nondiabetic mice, Figure 2 Ob/ob mice develop mechanical allodynia. Mechanical but only a 1.4 fold change in ob/ob in the sciatic nerve thresholds were tested using von Frey monofilaments at 8, 9, 10, from ob/ob mice. and 11 weeks of age. Ob/ob mice did not display significant differences from nondiabetic controls at 8, 9, or 10 weeks. However, IGF-1 and insulin activate many of the same intracellular at week 11, ob/ob mice had a significant decrease in their signaling pathways [21], and altered IGF-1 signaling has mechanical withdrawal thresholds. * = p < 0.05. n = 6 nondiabetic been demonstrated in states of insulin resistance [39]. Fur- mice, n = 6 ob/ob diabetic mice. thermore, IGF-1 resistance has recently been demonstrated Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 4 of 11 http://www.actaneurocomms.org/content/1/1/15 Figure 3 Intrathecal insulin-induced Akt activation is blunted in the PNS of ob/ob mice. DRG (A) and sciatic nerve (B) were harvested after an intrathecal injection of PBS (nondiabetic n = 10, ob/ob n = 7) or insulin (nondiabetic n = 10, ob/ob n = 9) was administered to nondiabetic control and ob/ob mice. Nondiabetic mice displayed a robust and significant increase in Akt activation with insulin stimulation; however insulin failed to significantly activate Akt in the DRG of ob/ob mice. Furthermore, the maximal increase in Akt activation with insulin stimulation was significantly lower in both the DRG and sciatic nerve of ob/ob mice. There were no differences in mice that received PBS in either the DRG or sciatic nerve. * = p < 0.05, *** = p < 0.001. to be associated with brain insulin resistance and cognitive versus 8.1 ± 0.3 mmol/L. Ob/ob mice that received IT IGF- decline in Alzheimer’s patients [40]. To investigate IGF-1 1 had similar blood glucose profiles to ob/ob mice that re- signal transduction in the PNS of ob/ob mice, a dose of ceived IT PBS, with a significant increase in blood glucose IGF-1 equimolar to 0.1U insulin was administered via an after 30 minutes, 16.3 ± 2.7 mmol/L as compared to 27.7 ± intrathecal injection. Blood glucose levels in both 1.2 mmol/L. Akt was significantly activated in the DRG nondiabetic control and ob/ob mice once again appeared from both nondiabetic (13.3 fold) and ob/ob diabetic mice to increase with intrathecal PBS injection, 7.7 ± 0.3 mmol/ (6.0 fold). However, Akt activation was significantly lower L at baseline as compared to 9.7 ± 0.8 mmol/L (p = 0.056) in the DRG from ob/ob mice compared to nondiabetic after 30 minutes for nondiabetic mice and 12.1 ± mice (Figure 5A). In the sciatic nerve of nondiabetic mice, 1.9 mmol/L at baseline to 23.7 ± 3.1 mmol/L after 30 mi- IGF stimulation produced a significant 2.8 fold increase in nutes for ob/ob mice. IT IGF-1 did not significantly alter Akt activation. In contrast, Akt was not significantly acti- blood glucose levels in nondiabetic mice, 9.3 ± 0.4 mmol/L vated in the sciatic nerve of ob/ob mice (Figure 5B). Figure 4 The PNS of ob/ob mice showed reduced insulin-induced Akt activation in response to intraperitoneally-delivered insulin. Nondiabetic and ob/ob diabetic mice were given intraperitoneal injections of PBS (nondiabetic n = 3, ob/ob n = 3) or insulin at a dose of 3.33 U/kg (nondiabetic n =3 and ob/ob n = 3). In both the DRG (A) and sciatic nerve (B) of nondiabetic mice, there was a significant increase in Akt activation in the insulin stimulated group as compared to mice that received PBS, yet no statistically significant changes were observed in the PNS from ob/ob mice. * = p < 0.05, ** = p < 0.01. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 5 of 11 http://www.actaneurocomms.org/content/1/1/15 Figure 5 The PNS of ob/ob mice displayed reduced Akt activation in response to intrathecal IGF-1. Similar to the results shown for intrathecal insulin, an intrathecal injection of IGF-1 produced a strong activation of Akt in both the DRG and sciatic nerve of nondiabetic mice, but the response was somewhat blunted in the PNS of ob/ob mice. In the DRG (A), there was a significant increase in Akt activation in both the nondiabetic and ob/ob mice; however, the activation level was significantly lower in the DRG from ob/ob mice. In the sciatic nerve (B), IGF-1 stimulation resulted in a significant Akt activation in nondiabetic mice, but not in the ob/ob mice. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n = 8 nondiabetic PBS, n = 9 nondiabetic IGF-1, n = 7 diabetic PBS, n = 10 diabetic IGF-1. Insulin signaling downstream of Akt in the DRG and The PNS of ob/ob mice display reduced insulin receptor sciatic nerve expression and increased JNK activation To assess whether diabetes-induced blunting of Akt To explore possible mechanisms responsible for reduced activation was maintained downstream, several other PNS insulin sensitivity, we investigated several pathways insulin-responsive proteins were investigated via known in other insulin-resistant tissues. One contributor Western blot analysis, including mTor (protein syn- to reduced insulin signaling is a downregulation of insu- thesis), p70S6K (protein synthesis), AS160 (glucose lin receptor expression induced by hyperinsulinemia uptake), and GSK3β (glycogen synthesis). At the in- [23]. As shown in Figure 6A, protein levels of the in- sulin dose (0.1U) and time point (30 minute stimula- sulin receptor subunit β were significantly lower in the tion) that were investigated, no statistical differences DRG of ob/ob mice compared to nondiabetic mice. How- (p > 0.05) were observed in the activation of these ever, there was no statistical difference in the expression proteins even in control mice (Table 1). Additionally, of insulin receptor between nondiabetic and ob/ob similar to Akt results, no differences were observed in mice in the sciatic nerve (Figure 6B). No significant baseline levels between all proteins investigated. However, differences between groups were observed in IGF-1 it is interesting to note that in both the DRG and sciatic receptor expression in either the DRG or sciatic nerve nerve from ob/ob mice, there is a consistent pattern of a (data not shown). reduced fold change in response to insulin as compared to Our previous studies in primary DRG cultures reported responses in nondiabetic mice. an upregulation of IRS2 serine phosphorylation [6], a Table 1 Downstream Akt pathway activation in the DRG and sciatic nerve after intrathecal insulin stimulation Protein of DRG Sciatic nerve interest Control nondiabetic ob/ob diabetic Control nondiabetic ob/ob diabetic Insulin-induced fold change Insulin-induced fold change Insulin-induced fold change Insulin-induced fold change mTor 1.52 1.00 1.13 0.98 AS160 1.47 1.28 2.13 1.22 p70S6K 1.00 1.04 1.09 0.93 GSK3β 1.26 1.17 1.56 0.88 Four proteins downstream of Akt that are known to be involved in the intracellular actions of insulin signaling were investigated in the PNS of nondiabetic and ob/ob mice. In both the DRG and sciatic nerve, there were no significant changes in the activation of mTor, p70S6K, or AS160 in either nondiabetic or ob/ob mice, nor was there a significant change in the inhibition of GSK3β. Data presented is the fold change of protein modification (measured with Western blot analysis) induced by insulin as compared to that observed in mice that received PBS. n = 7-10 for all groups. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 6 of 11 http://www.actaneurocomms.org/content/1/1/15 Figure 6 (See legend on next page.) Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 7 of 11 http://www.actaneurocomms.org/content/1/1/15 (See figure on previous page.) Figure 6 Possible mechanisms that may be contributing to insulin resistance in the PNS. A) In this study the expression of the beta subunit of the insulin receptor was significantly reduced in the DRG of ob/ob mice as compared to nondiabetic controls. B) No significant change in insulin receptor expression was observed in the sciatic nerve. C) The stress kinase JNK was not significantly activated in the DRG of ob/ob mice; however in the sciatic nerve (D) there was a significant upregulation of JNK in ob/ob mice. E, F) No differences in PTP1B expression profiles were observed in either the DRG or sciatic nerve between nondiabetic and diabetic groups. * = p < 0.05. n = 9 nondiabetic PBS, n = 9 nondiabetic insulin, n = 7 diabetic PBS, n = 9 diabetic insulin. recognized mechanism of insulin resistance in muscle and These experiments used an in vivo approach to sup- adipose. In the current study, we investigated both IRS1 port the mounting in vitro evidence pointing to PNS in- (muscle and adipose isoform) [41] and IRS2 (neural iso- sulin resistance in diabetes. Interestingly, Akt activation form) [6,42] serine phosphorylation. In contrast to neurons was very prominent in the DRG and sciatic nerve of in vitro, IRS serine phosphorylation does not appear to be nondiabetic mice, yet very few significant changes were significantly affected in the PNS in vivo within this model, seen in downstream signaling molecules. This may be (data not shown). Interestingly, there was significant activa- due to a temporal effect, as downstream mediators of tion of the stress kinase JNK (p(Thr183/Tyr185)JNK/total the Akt pathway may have not yet been activated during JNK) in the sciatic nerve of ob/ob mice compared to the 30-minute stimulation period used for this study. nondiabetic mice (Figure 6D) and a similar pattern of acti- However, it is also plausible that the downstream Akt vatedJNK wasobservedinthe DRGof ob/ob mice, how- signaling proteins explored in this study do not play a ever significance was not reached (nondiabetic vs. diabetic prominent role in insulin pathways within the DRG. In- p = 0.122) (Figure 6C). stead of driving protein synthesis through mTor and In addition to stress kinase activation and reduced in- p70S6K or regulation of GSK3β actions, insulin may be sulin receptor expression, insulin resistance can also be playing a more important role in lipid and glucose me- induced by over activation of tyrosine phosphatases [29]. tabolism, gene regulation, or mitochondrial maintenance However, PTP1B expression was not elevated in the in peripheral neurons. Further studies are underway to DRG or sciatic nerve of ob/ob mice, nor did insulin stimu- explore these other downstream components of insulin lation appear to alter its expression levels (Figure 6E,F, signaling and to define the temporal components of this respectively). signaling pathway. An additional caveat to this study is the use of leptin- Discussion deficient ob/ob mice. Leptin’s role in the nervous system Diabetic neuropathy is associated with profound loss of is receiving increasing attention, and it may have a distal limb sensation and/or pain, causing significant neuroprotective role [44]. It is not known how reduced decline in the quality of life and potential morbidity neuronal leptin may have contributed to our results. and mortality for patients. Currently, there are no clin- Thus, confirming these results in a high-fat diet model ical treatments that successfully improve neuropathic of obesity will be an important step to further investigat- damage to peripheral sensory nerve fibers, likely due to ing PNS insulin resistance. the multifactorial etiology of neuropathy development In experiments presented here, it appeared that in- and progression. Here, we have demonstrated in vivo sulin produced a stronger Akt activation in the sciatic PNS insulin resistance in ob/ob mice. These results are nerve compared to the DRG (Figure 3), whereas IGF- consistent with recent in vitro studies and support the 1 produced a stronger Akt activation in the DRG view that altered insulin signaling may contribute to compared to the sciatic nerve (Figure 5). These results DN [43]. A robust activation of insulin-sensitive path- point to an apparent separation in insulin/IGF-1 sig- naling support within the PNS. One plausible explan- ways was observed in the DRG and sciatic nerve of nondiabetic mice, with a blunted response in both tis- ation may be that insulin and IGF-1 have different sues from insulin-resistant ob/ob mice. While no one actions on the DRG soma and satellite cells compared to sensory axons, motor axons, and Schwann cells in mechanism of insulin resistance was clearly prevalent, significant changes were seen in two known pathways the peripheral nerve, leading to alternative signaling of insulin resistance, including increased JNK activity profiles. How this potential divergence in signaling may affect sensory neuron function is yet to be deter- and reduced insulin receptor expression. Although more research is needed to fully elucidate the path- mined and ongoing research is further delineating the ways leading to PNS insulin resistance, these results differential roles that insulin and IGF-1 may play in sensory nerve biology. suggest that cellular mechanisms of insulin resistance that have been defined in muscle may also play an im- In ob/ob mice, both the DRG and sciatic nerve displayed portant role in the PNS. reduced insulin-induced Akt activation, a classic indication Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 8 of 11 http://www.actaneurocomms.org/content/1/1/15 of insulin resistance. Several mechanisms of insulin resist- PTP1B knockout mice display increased insulin sensitiv- ance outlined in muscle also appear to be altered in the ity [48]. In the current study, we did not detect signifi- PNS, and may be contributing to the observed reduction cant upregulation of PTP1B in the DRG or sciatic nerve in insulin signal transduction. However, these results must of insulin resistant mice. While there was no change in be interpreted with caution as significant changes were not PTP1B expression, there still could be alterations in seen consistently across PNS tissues, and further research phosphatase activity and further studies are underway will need to be completed to fully establish a clear mechan- to explore this possibility. ism. Interestingly, no change in baseline Akt activation It will be important to put the current results in levels was observed between nondiabetic and ob/ob mice context with other contributory mechanisms of DN, as may be expected in states of insulin resistance. These re- including glucose and/or lipid mediated toxicity as sults are intriguing and suggest that future research focus- well as oxidative stress [49]. We postulate that the ing on pathways driving Akt signaling is warranted. metabolic dysfunction associated with hyperglycemia Hyperinsulinemia can promote insulin resistance and dyslipidemia in concert with reduced neuro- through downregulation of the insulin receptor [22]. This trophic support promotes deterioration and reduced effect was demonstrated in our data. The ob/ob mice in regeneration of the distal axon. Furthermore, the loss this cohort had serum insulin levels 34.3 fold higher than of appropriate insulin signaling could make neurons nondiabetic mice and the DRG of ob/ob mice displayed even more susceptible to these pathogenic cascades. It significantly lower insulin receptor expression. Thus, the should be noted that both intrathecal and intraperito- extreme hyperinsulinemia in the ob/ob mice may be neal insulin injections altered blood glucose levels. promoting insulin receptor downregulation and contrib- Thus, these results should be viewed in the context uting to PNS insulin resistance. This idea is supported that glucose levels were also transiently altered by in- by a recent study that reported a significant decrease in sulin administration. Further research into disrupted insulin receptor mRNA in cultured DRG neurons that PNS insulin signaling relative to other pathogenic displayed insulin resistance when treated with high levels mechanisms is needed, as this will be a key step in of insulin [7]. translating these basic science results into clinical An alternative mediator of insulin resistance is the applications. stress kinase JNK, which is activated in response to vari- ous cellular stressors, including low grade chronic in- flammation induced by obesity [33,45]. In fact, ob/ob Conclusions mice with a JNK null mutation have improved whole Insulin resistance is emerging as a potential mediator of body glucose tolerance and insulin sensitivity [31]. Add- several neurological syndromes (reviewed in [1]). This itionally, JNK activation has been implicated in altered study, along with recent data of in vitro DRG insulin re- neurofilament phosphorylation in the PNS of diabetic sistance, strongly supports altered insulin signaling as a rats [46]. JNK activation is proposed to promote insulin pathogenic mechanism in DN. While deficient insulin resistance through upregulation of IRS serine phosphor- signaling has been a proposed contributor to DN in type ylation, and IRS is a key common signaling component 1 models for some time [3,4,14,43,50], little has been of both the insulin and IGF-1 pathways. In the current known about insulin signaling effectiveness in type 2 study we observed increased JNK activation without a (hyperinsulinemic) models of DN. We observed re- significant elevation in either IRS1 or IRS2 serine phos- duced insulin signaling in vivo in the PNS of type 2 phorylation. Some controversy does exist as to which diabetic ob/ob mice and suggest possible mechanisms serine sites are most important in insulin resistance, thus that may be contributing to these changes. It is now the serine sites that we probed (p(ser731)IRS2 and becoming evident that decreased insulin neurotrophic (p(ser307)IRS1) may not be heavily involved in inhibiting supportinthe PNSis anintegralpartofDN and insulin signaling in the PNS. More powerful approaches, may be a congruent mechanism between type 1 and such as mass spectrometry, may be needed to establish a type 2 diabetic models of DN, as both have reduced global change in the IRS phosphorylation profile within insulin signaling either due to insulinopenia or neur- the PNS [47]. onal insulin resistance. Another possible component of the insulin receptor Future studies will focus on mechanisms through signaling pathway that could be affected in insulin which insulin supports proper PNS function, as reveal- resistance is PTP1B. PTP1B is the canonical member ing these pathways may provide insight into how de- of protein tyrosine phosphatases and serves an import- creased insulin support contributes to the pathogenesis ant role in insulin signaling regulation [29]. Over- of DN. Furthermore, delineating the details of PNS insu- expression of PTP1B has been linked to insulin lin signaling may open new avenues for therapeutic resistance in peripheral tissues of ob/ob mice [26] and intervention in patients with DN. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 9 of 11 http://www.actaneurocomms.org/content/1/1/15 Methods followed by an additional 4 more applications. The Animals withdrawal threshold was calculated using the formula All experiments were approved by the University of from the up-down method previously described [52]. Kansas Medical Center Institutional Animal Care and Use Committee. Male ob/ob leptin null mutant and age- Insulin and IGF-1 injections matched control mice (ob/+) were purchased from Jackson Sterile PBS (vehicle), 0.1U (~0.7 nmol) Humulin R insu- Laboratories (Bar Harbor, Maine) at 8 weeks of age. Mice lin, or recombinant IGF-1 equimolar to 0.1U insulin was were given access to food and water ad libitum and directly administered to both nondiabetic and ob/ob type housed on a 12-hour light/dark cycle. Weekly blood glu- 2 diabetic mice via a one-time intrathecal injection. Pre- cose (Glucose Diagnostic Assay Sigma-Aldrich, St. Louis, viously, intrathecal 0.1U insulin and equimolar IGF-1 MO), serum insulin (Insulin ELISA Alpco, Salem, NH) have been shown to have beneficial effects on the symp- and weights were monitored and mice were sacrificed at toms of DN [10]. All injections were 50 μL and adminis- 11 weeks of age. tered with a 1cc 28½ gauge insulin syringe between the L6 and S1 vertebrae. In an additional preliminary study, Glucose tolerance test sterile PBS or insulin was delivered through an intra- At 9 weeks of age, an intraperitoneal glucose tolerance peritoneal injection at a dose of 3.33 U/kg, such that test (IPGTT) was used to assess the response of mice to the total insulin administered was approximately 0.1 U a glucose challenge. After a 6-hour fast, mice were given for nondiabetic mice and 0.17U (~1.2 nmol) for ob/ob an intraperitoneal injection of glucose at 1g of glucose mice. The doses administered and stimulation time frames per kg body weight. Blood glucose levels were measured used were confirmed to be sufficient for Akt activation in via tail clip immediately prior to the glucose bolus and the PNS with dose curve and time course studies (Grote, then at 15, 30, 60, and 120 minutes after injection. unpublished observation). Insulin tolerance test Western blots At 10 weeks of age, mice underwent an insulin toler- After a 30 minute insulin stimulation period, the right ance test (ITT). Mice were fasted for 6 hours and then and left lumbar DRG and sciatic nerves were harvested administered IP insulin (Humulin R, Lilly, Indianapolis, for each sample from 11 week old mice and frozen Indiana) at a dosage of 1.5 U per kg body weight. Blood at −80°C. Tissues were sonicated in Cell Extraction Buffer glucose levels were monitored immediately prior to in- (Invitrogen, Carlsbad, CA) containing 55.55 μl/ml protease sulin injection and then at 15, 30, 60, and 120 minutes inhibitor cocktail, 200 mM Na VO , and 200 mM NaF. 3 4 thereafter. Following sonication, protein was extracted on ice for 60 minutes and vortexed every 10 minutes. After centrifuga- HOMA-IR tion, protein concentration of the supernatant was mea- Fasting insulin and fasting glucose levels were used to sured with a Bradford assay (Bio-Rad, Hercules, CA). calculate the homeostatic model assessment of insulin Samples were then boiled with Lane Marker Reducing resistance (HOMA-IR). Scores were calculated with the Sample Buffer (Thermo Scientific, Waltham, MA) for 3 following equation: (blood glucose (mg/dl) X (serum insulin minutes. Equal amounts of protein (30 μg) were loaded (uU/mL))/405) [51]. per lane and samples were separated on a 4-15% gradient tris-glycine gel (Bio-Rad), and then transferred to a nitro- Mechanical sensitivity cellulose membrane. Membranes were probed with the Mechanical behavioral responses to Semmes Weinstein-von following primary antibodies and all antibodies were Frey monofilaments (0.07 to 5.0 g) were assessed at 8, purchased from Cell Signaling (Danvers, MA) unless 9, 10, and 11 weeks of age. Mice underwent acclima- otherwise noted: total Akt (1:2000), p-(Ser473)Akt tion 2 days prior to the first day of behavioral testing. (1:500), total p70S6K (1:500), p-(Thr389)p70S6K (1:500), Mice were placed in individual clear plastic cages total GSK3β (1:1500), p-(Ser9)GSK3β (1:1000), total JNK (11×5×3.5 cm) on a wire mesh grid 55 cm above the (1:1000), p-(Thr183/Tyr185)JNK (1:500), total mTor table and were acclimated for 30 minutes prior to be- (1:500), p-(Ser2448)mTor (1:500), Insulin-like growth fac- havioral analysis. The filaments were applied perpen- tor 1 receptor β subunit (1:500), PTP1B (1:500) (Abcam, dicularly to the plantar surface of the hindpaw until Cambridge, MA), total AS160 (1:1000) (Millipore, Biller- the filament bent. Testing began with the 0.7 g fila- ica, MA), p-(Thr642)AS160 (1:500) (Millipore), Insulin ment, and in the presence of a response, the next Receptor β subunit (1:500) (Santa Cruz, Santa Cruz, CA), smaller filament was applied. If no response was ob- and α-tubulin (1:5000) (Abcam). Bands were visualized served, the next larger filament was used. Filaments with either anti-mouse or anti-rabbit HRP-conjugated sec- were applied until there was a change in response, ondary antibodies (Santa Cruz) and ECL with X-ray film. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 10 of 11 http://www.actaneurocomms.org/content/1/1/15 Densitometry with ImageJ (NIH) was then used to analyze Received: 20 March 2013 Accepted: 19 April 2013 Published: 10 May 2013 each lane. All samples from each tissue were run simul- taneously across multiple gels and each group was equally represented on each gel (approximately 3 nondiabetic References 1. Kim B, Feldman EL: Insulin resistance in the nervous system. Trends Endocrinol PBS, 3 nondiabetic insulin, 3 ob/ob PBS, and 3 ob/ob insu- Metab 2012, 23(3):133–141. lin per gel). Data is presented as the ratio of integrated 2. 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Schubert M, et al: Insulin receptor substrate-2 deficiency impairs brain growth and promotes tau phosphorylation. JNeurosci 2003, 23(18):7084–7092. 43. Ishii DN: Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy. Brain Res Brain Res Rev 1995, 20(1):47–67. 44. Dicou E, Attoub S, Gressens P: Neuroprotective effects of leptin in vivo and in vitro. Neuroreport 2001, 12(18):3947–3951. 45. Nguyen MT, et al: JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes. J Biol Chem 2005, Submit your next manuscript to BioMed Central 280(42):35361–35371. and take full advantage of: 46. Fernyhough P, et al: Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 1999, • Convenient online submission 48(4):881–889. • Thorough peer review 47. Langlais P, et al: Global IRS-1 phosphorylation analysis in insulin resistance. Diabetologia 2011, 54(11):2878–2889. • No space constraints or color figure charges 48. Elchebly M, et al: Increased insulin sensitivity and obesity resistance in • Immediate publication on acceptance mice lacking the protein tyrosine phosphatase-1B gene. Science 1999, • Inclusion in PubMed, CAS, Scopus and Google Scholar 283(5407):1544–1548. 49. Vincent AM, et al: Diabetic neuropathy: cellular mechanisms as • Research which is freely available for redistribution therapeutic targets. Nat Rev Neurol 2011, 7(10):573–583. Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Neuropathologica Communications Springer Journals

Peripheral nervous system insulin resistance in ob/ob mice

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Springer Journals
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Copyright © 2013 by Grote et al.; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Pathology; Neurology
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2051-5960
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10.1186/2051-5960-1-15
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24252636
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Abstract

Background: A reduction in peripheral nervous system (PNS) insulin signaling is a proposed mechanism that may contribute to sensory neuron dysfunction and diabetic neuropathy. Neuronal insulin resistance is associated with several neurological disorders and recent evidence has indicated that dorsal root ganglion (DRG) neurons in primary culture display altered insulin signaling, yet in vivo results are lacking. Here, experiments were performed to test the hypothesis that the PNS of insulin-resistant mice displays altered insulin signal transduction in vivo. For these studies, nondiabetic control and type 2 diabetic ob/ob mice were challenged with an intrathecal injection of insulin or insulin-like growth factor 1 (IGF-1) and downstream signaling was evaluated in the DRG and sciatic nerve using Western blot analysis. Results: The results indicate that insulin signaling abnormalities documented in other “insulin sensitive” tissues (i.e. muscle, fat, liver) of ob/ob mice are also present in the PNS. A robust increase in Akt activation was observed with insulin and IGF-1 stimulation in nondiabetic mice in both the sciatic nerve and DRG; however this response was blunted in both tissues from ob/ob mice. The results also suggest that upregulated JNK activation and reduced insulin receptor expression could be contributory mechanisms of PNS insulin resistance within sensory neurons. Conclusions: These findings contribute to the growing body of evidence that alterations in insulin signaling occur in the PNS and may be a key factor in the pathogenesis of diabetic neuropathy. Keywords: Diabetic neuropathy, Neuronal insulin resistance, Neurotrophic support Background Furthermore, clinical evidence has reported that insulin Diabetes and metabolic syndrome are risk factors for resistance appears to be an independent risk factor for several neurological diseases, and emerging evidence has both autonomic and peripheral neuropathy [8]. indicated that neuronal insulin resistance may be in- Although neurons do not appear to rely on insulin volved in disease pathogenesis [1]. While altered insulin for glucose uptake [9], insulin does have an import- signaling is known to be the key factor in the develop- ant role in both the CNS and PNS. Insulin promotes ment of diabetes, the role that it plays in diabetic neur- in vivo nerve regeneration [4,10,11], induces neurite opathy (DN) is not well understood. However, it has outgrowth [12,13], maintains neuronal mitochondrial been demonstrated that neuronally-targeted insulin function [14,15], supports memory formation [16,17], treatment can improve signs of neuropathy without and regulates hypothalamic metabolic control [18,19]. altering blood glucose levels [2-4]. Recent evidence sug- While the exact mechanisms through which insulin pro- gests that cultured sensory neurons from insulin- motes these functions remain unclear, insulin is considered resistant mice display classic signs of insulin resistance a potent neurotrophic factor key to maintaining proper and that insulin resistance may be contributing to mito- neuronal function. chondrial dysfunction and increased ROS in DN [5-7]. Insulin and insulin-like growth factor 1 (IGF-1) signal- ing is propagated by phosphorylation events that begin with the intrinsic tyrosine kinase activity of the insulin * Correspondence: dwright@kumc.edu or IGF receptor (reviewed in [20,21]) and continue with Department of Anatomy and Cell Biology, the University of Kansas Medical subsequent activation of both the PI3K-Akt and MAPK Center, Kansas City, KS 66160, USA cascades. While these pathways are well defined in Full list of author information is available at the end of the article © 2013 Grote et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 2 of 11 http://www.actaneurocomms.org/content/1/1/15 muscle, adipose, and liver, insulin signaling and its ac- Furthermore, the PNS of ob/ob mice has alterations in tions in the PNS are poorly understood. cellular mechanisms of insulin resistance, including In an insulin-resistant state, the cellular effects of insu- decreased DRG insulin receptor expression and up- lin are blunted due to improper signal propagation regulation of JNK activity in the sciatic nerve. resulting from several different mechanisms, including 1) degradation of the insulin receptor [22-25], 2) removal Results of keytyrosinephosphorylation sitesbyoveractivationof Insulin resistance in ob/ob mice protein tyrosine phosphatases [26-29], and 3) increased To quantify the extent of systemic insulin resistance in phosphorylation at inhibitory IRS serine residues due to el- ob/ob mice, nondiabetic and diabetic ob/ob mice under- evated stress kinases, such as JNK [30-35]. However, the went an IPGTT at 9 weeks of age (Figure 1A). Blood glu- extent to which these mechanisms affect insulin signal cose levels of the ob/ob mice were significantly higher transduction in the PNS is not clear. than nondiabetic mice throughout the course of the ex- Growing evidence suggests that neurons may become periment and the area under the curve (AUC) was also insulin resistant similar to other tissues. However, no significantly elevated for ob/ob mice (Figure 1B). Results in vivo evidence of PNS insulin resistance has been from the ITT indicated that nondiabetic, insulin-injected presented, and the cellular mechanisms associated with mice exhibited an expected physiological decrease in PNS insulin resistance have not been thoroughly investi- blood glucose in response to insulin; however, ob/ob gated. Here, we demonstrate that the DRG and sciatic mice displayed a transient elevation of glucose levels nerve of ob/ob mice display reduced insulin signaling (Figure 1C). Statistical analysis of the data revealed in response to an intrathecal injection of insulin. that ob/ob mice maintained elevated glucose levels Figure 1 Ob/ob mice display classic signs of insulin resistance. A, B) An IPGTT showed significantly elevated blood glucose levels in ob/ob mice throughout the test. The blood glucose of ob/ob mice increased more than 10 mmol/L at its maximal level as opposed to nondiabetic mice that elevated less than 6 mmol/L after glucose injection, indicating severe glucose intolerance in ob/ob mice. C, D) Similar to the IPGTT, data from the ITT showed reduced insulin sensitivity in ob/ob mice. In fact, an insulin dose of 1.5 U/Kg did not decrease the blood glucose level of ob/ob mice, whereas this dose lowered the blood glucose of nondiabetic controls by approximately 3.6 mmol/L. E-G)At10weeks of age, ob/ob mice had significantly elevated blood glucose and serum insulin levels. Accordingly, the HOMA-IR measure of insulin resistance was significantly higher in ob/ob mice. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. IPGTT n = 7 nondiabetic mice, n = 6 ob/ob. ITT n = 4 nondiabetic mice, n = 4 ob/ob. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 3 of 11 http://www.actaneurocomms.org/content/1/1/15 compared to nondiabetic controls throughout most of Akt is a hallmark of insulin resistance [5,6,37,38]. Here, the study, and that the AUC was significantly higher for nondiabetic and diabetic ob/ob mice were administered diabetic ob/ob mice (Figure 1D). The HOMA-IR, a meas- either intrathecal PBS or insulin and the DRG and sci- ure of insulin resistance, was calculated using fasting atic nerve were harvested 30 minutes later for Western blood glucose and serum insulin levels from 10 week old blot analysis to assess Akt activation. Both nondiabetic mice. Ob/ob mice had significantly higher blood glucose control and ob/ob mice display significantly elevated levels (14.3 ± 2.1 mmol/L) compared to nondiabetic mice blood glucose levels following intrathecal injection of (8.2 ±0.5 mmol/L) (Figure 1E). Fasting insulin levels were PBS. Nondiabetic mice glucose levels increased from also significantly higher in diabetic ob/ob mice (6780 ± 6.6 ± 0.4 mmol/L to 8.6 ± 0.5 mmol/L, whereas ob/ob levels 1610 pmol/L) compared to nondiabetic mice (198 ± 25 increased from 12.1 ± 2.4 mmol/L to 22.3 ± 2.4 mmol/L. pmol/L, Figure 1F). As such, ob/ob mice had a signifi- Glucose levels in nondiabetic mice significantly decreased cantly elevated HOMA-IR as compared to nondiabetic from 7.0 ± 0.3 mmol/L to 3.3 ± 0.3 mmol/L after intra- mice (557 ± 130 compared to 10.1 ± 1.4, respectively, thecal insulin injection. Ob/ob mice glucose levels 30 mi- Figure 1G). These results demonstrate significant glu- nutes after insulin injection were not significantly different cose intolerance and insulin resistance in ob/ob mice at from baseline, starting at 12.7 ± 0.9 mmol/L and ending at this age. 12.3 ± 1.5 mmol/L after 30 minutes. In nondiabetic mice, insulin produced a strong eleva- Mechanical allodynia in ob/ob mice tion in levels of activated Akt (p(ser473)Akt/total Akt) in To quantify a known behavioral abnormality associated both the DRG and sciatic nerve (Figure 3A,B). However with neuropathy in mice, mechanical sensitivity was in ob/ob mice, Akt activation was significantly lower in assessed in nondiabetic and diabetic ob/ob mice at 8, 9, the DRG and sciatic nerve. In fact, insulin failed to sig- 10, and 11 weeks of age. There were no differences in nificantly increase Akt activation over baseline in the mechanical thresholds between nondiabetic and ob/ob DRG of ob/ob mice. For comparison, Akt activation in diabetic mice at 8, 9, or 10 weeks of age. However, at 11 the DRG was increased 3.1 fold in nondiabetic mice and weeks, there was a significant decrease in the mechanical 1.6 fold in ob/ob diabetic mice. In the sciatic nerve, insu- thresholds of diabetic ob/ob mice compared to nondiabetic lin produced 9.7 and 6.1 fold increase in Akt activation mice (Figure 2), consistent with sensory aberrations associ- in nondiabetic and ob/ob mice, respectively. ated with peripheral neuropathy as previously reported To confirm that these results were not dependent on in this genetic mouse strain [36]. the intrathecal route of delivery, a small number of mice were administered intraperitoneal insulin at a dose of Blunted insulin and IGF-1 Akt activation in ob/ob DRG 3.33 U/kg. PBS injections once again appeared to cause an and sciatic nerve increase in blood glucose levels from baseline, nondiabetic Insulin stimulation causes a robust activation of Akt in mice levels started at 7.0 ± 1.4 mmol/L and ended at 8.6 ± insulin-sensitive tissues like muscle and adipose, as well 1.4 mmol/L (p > 0.05), whereas ob/ob mice showed a as in neurons of both the peripheral and central nervous significant increase from 10.8 ± 0.8 mmol/L to 15.4 ± systems. Moreover, reduced insulin-induced activation of 1.0 mmol/L. IP insulin resulted in significantly lower blood glucose levels in nondiabetic mice after 30 minutes, 7.8 ± 0.6 versus 4.2 ± 0.5 mmol/L, respectively. Ob/ob mice blood glucose levels were not significantly altered by IP in- sulin injection 15.3 ± 3.0 versus 13.6 ± 3.9 mmol/L. Similar to the intrathecal delivery route, a significant increase in Akt activation was observed in the DRG and sciatic nerve of nondiabetic mice stimulated with insulin; however, no significant change was observed in either tissue from ob/ob mice. (Figure 4A,B). In the DRG, nondiabetic mice displayeda2.4foldchangeinAkt activation,comparedto a 1.5 fold change in ob/ob mice. IP insulin induced a 3.8 fold change in Akt in the sciatic nerve of nondiabetic mice, Figure 2 Ob/ob mice develop mechanical allodynia. Mechanical but only a 1.4 fold change in ob/ob in the sciatic nerve thresholds were tested using von Frey monofilaments at 8, 9, 10, from ob/ob mice. and 11 weeks of age. Ob/ob mice did not display significant differences from nondiabetic controls at 8, 9, or 10 weeks. However, IGF-1 and insulin activate many of the same intracellular at week 11, ob/ob mice had a significant decrease in their signaling pathways [21], and altered IGF-1 signaling has mechanical withdrawal thresholds. * = p < 0.05. n = 6 nondiabetic been demonstrated in states of insulin resistance [39]. Fur- mice, n = 6 ob/ob diabetic mice. thermore, IGF-1 resistance has recently been demonstrated Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 4 of 11 http://www.actaneurocomms.org/content/1/1/15 Figure 3 Intrathecal insulin-induced Akt activation is blunted in the PNS of ob/ob mice. DRG (A) and sciatic nerve (B) were harvested after an intrathecal injection of PBS (nondiabetic n = 10, ob/ob n = 7) or insulin (nondiabetic n = 10, ob/ob n = 9) was administered to nondiabetic control and ob/ob mice. Nondiabetic mice displayed a robust and significant increase in Akt activation with insulin stimulation; however insulin failed to significantly activate Akt in the DRG of ob/ob mice. Furthermore, the maximal increase in Akt activation with insulin stimulation was significantly lower in both the DRG and sciatic nerve of ob/ob mice. There were no differences in mice that received PBS in either the DRG or sciatic nerve. * = p < 0.05, *** = p < 0.001. to be associated with brain insulin resistance and cognitive versus 8.1 ± 0.3 mmol/L. Ob/ob mice that received IT IGF- decline in Alzheimer’s patients [40]. To investigate IGF-1 1 had similar blood glucose profiles to ob/ob mice that re- signal transduction in the PNS of ob/ob mice, a dose of ceived IT PBS, with a significant increase in blood glucose IGF-1 equimolar to 0.1U insulin was administered via an after 30 minutes, 16.3 ± 2.7 mmol/L as compared to 27.7 ± intrathecal injection. Blood glucose levels in both 1.2 mmol/L. Akt was significantly activated in the DRG nondiabetic control and ob/ob mice once again appeared from both nondiabetic (13.3 fold) and ob/ob diabetic mice to increase with intrathecal PBS injection, 7.7 ± 0.3 mmol/ (6.0 fold). However, Akt activation was significantly lower L at baseline as compared to 9.7 ± 0.8 mmol/L (p = 0.056) in the DRG from ob/ob mice compared to nondiabetic after 30 minutes for nondiabetic mice and 12.1 ± mice (Figure 5A). In the sciatic nerve of nondiabetic mice, 1.9 mmol/L at baseline to 23.7 ± 3.1 mmol/L after 30 mi- IGF stimulation produced a significant 2.8 fold increase in nutes for ob/ob mice. IT IGF-1 did not significantly alter Akt activation. In contrast, Akt was not significantly acti- blood glucose levels in nondiabetic mice, 9.3 ± 0.4 mmol/L vated in the sciatic nerve of ob/ob mice (Figure 5B). Figure 4 The PNS of ob/ob mice showed reduced insulin-induced Akt activation in response to intraperitoneally-delivered insulin. Nondiabetic and ob/ob diabetic mice were given intraperitoneal injections of PBS (nondiabetic n = 3, ob/ob n = 3) or insulin at a dose of 3.33 U/kg (nondiabetic n =3 and ob/ob n = 3). In both the DRG (A) and sciatic nerve (B) of nondiabetic mice, there was a significant increase in Akt activation in the insulin stimulated group as compared to mice that received PBS, yet no statistically significant changes were observed in the PNS from ob/ob mice. * = p < 0.05, ** = p < 0.01. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 5 of 11 http://www.actaneurocomms.org/content/1/1/15 Figure 5 The PNS of ob/ob mice displayed reduced Akt activation in response to intrathecal IGF-1. Similar to the results shown for intrathecal insulin, an intrathecal injection of IGF-1 produced a strong activation of Akt in both the DRG and sciatic nerve of nondiabetic mice, but the response was somewhat blunted in the PNS of ob/ob mice. In the DRG (A), there was a significant increase in Akt activation in both the nondiabetic and ob/ob mice; however, the activation level was significantly lower in the DRG from ob/ob mice. In the sciatic nerve (B), IGF-1 stimulation resulted in a significant Akt activation in nondiabetic mice, but not in the ob/ob mice. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n = 8 nondiabetic PBS, n = 9 nondiabetic IGF-1, n = 7 diabetic PBS, n = 10 diabetic IGF-1. Insulin signaling downstream of Akt in the DRG and The PNS of ob/ob mice display reduced insulin receptor sciatic nerve expression and increased JNK activation To assess whether diabetes-induced blunting of Akt To explore possible mechanisms responsible for reduced activation was maintained downstream, several other PNS insulin sensitivity, we investigated several pathways insulin-responsive proteins were investigated via known in other insulin-resistant tissues. One contributor Western blot analysis, including mTor (protein syn- to reduced insulin signaling is a downregulation of insu- thesis), p70S6K (protein synthesis), AS160 (glucose lin receptor expression induced by hyperinsulinemia uptake), and GSK3β (glycogen synthesis). At the in- [23]. As shown in Figure 6A, protein levels of the in- sulin dose (0.1U) and time point (30 minute stimula- sulin receptor subunit β were significantly lower in the tion) that were investigated, no statistical differences DRG of ob/ob mice compared to nondiabetic mice. How- (p > 0.05) were observed in the activation of these ever, there was no statistical difference in the expression proteins even in control mice (Table 1). Additionally, of insulin receptor between nondiabetic and ob/ob similar to Akt results, no differences were observed in mice in the sciatic nerve (Figure 6B). No significant baseline levels between all proteins investigated. However, differences between groups were observed in IGF-1 it is interesting to note that in both the DRG and sciatic receptor expression in either the DRG or sciatic nerve nerve from ob/ob mice, there is a consistent pattern of a (data not shown). reduced fold change in response to insulin as compared to Our previous studies in primary DRG cultures reported responses in nondiabetic mice. an upregulation of IRS2 serine phosphorylation [6], a Table 1 Downstream Akt pathway activation in the DRG and sciatic nerve after intrathecal insulin stimulation Protein of DRG Sciatic nerve interest Control nondiabetic ob/ob diabetic Control nondiabetic ob/ob diabetic Insulin-induced fold change Insulin-induced fold change Insulin-induced fold change Insulin-induced fold change mTor 1.52 1.00 1.13 0.98 AS160 1.47 1.28 2.13 1.22 p70S6K 1.00 1.04 1.09 0.93 GSK3β 1.26 1.17 1.56 0.88 Four proteins downstream of Akt that are known to be involved in the intracellular actions of insulin signaling were investigated in the PNS of nondiabetic and ob/ob mice. In both the DRG and sciatic nerve, there were no significant changes in the activation of mTor, p70S6K, or AS160 in either nondiabetic or ob/ob mice, nor was there a significant change in the inhibition of GSK3β. Data presented is the fold change of protein modification (measured with Western blot analysis) induced by insulin as compared to that observed in mice that received PBS. n = 7-10 for all groups. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 6 of 11 http://www.actaneurocomms.org/content/1/1/15 Figure 6 (See legend on next page.) Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 7 of 11 http://www.actaneurocomms.org/content/1/1/15 (See figure on previous page.) Figure 6 Possible mechanisms that may be contributing to insulin resistance in the PNS. A) In this study the expression of the beta subunit of the insulin receptor was significantly reduced in the DRG of ob/ob mice as compared to nondiabetic controls. B) No significant change in insulin receptor expression was observed in the sciatic nerve. C) The stress kinase JNK was not significantly activated in the DRG of ob/ob mice; however in the sciatic nerve (D) there was a significant upregulation of JNK in ob/ob mice. E, F) No differences in PTP1B expression profiles were observed in either the DRG or sciatic nerve between nondiabetic and diabetic groups. * = p < 0.05. n = 9 nondiabetic PBS, n = 9 nondiabetic insulin, n = 7 diabetic PBS, n = 9 diabetic insulin. recognized mechanism of insulin resistance in muscle and These experiments used an in vivo approach to sup- adipose. In the current study, we investigated both IRS1 port the mounting in vitro evidence pointing to PNS in- (muscle and adipose isoform) [41] and IRS2 (neural iso- sulin resistance in diabetes. Interestingly, Akt activation form) [6,42] serine phosphorylation. In contrast to neurons was very prominent in the DRG and sciatic nerve of in vitro, IRS serine phosphorylation does not appear to be nondiabetic mice, yet very few significant changes were significantly affected in the PNS in vivo within this model, seen in downstream signaling molecules. This may be (data not shown). Interestingly, there was significant activa- due to a temporal effect, as downstream mediators of tion of the stress kinase JNK (p(Thr183/Tyr185)JNK/total the Akt pathway may have not yet been activated during JNK) in the sciatic nerve of ob/ob mice compared to the 30-minute stimulation period used for this study. nondiabetic mice (Figure 6D) and a similar pattern of acti- However, it is also plausible that the downstream Akt vatedJNK wasobservedinthe DRGof ob/ob mice, how- signaling proteins explored in this study do not play a ever significance was not reached (nondiabetic vs. diabetic prominent role in insulin pathways within the DRG. In- p = 0.122) (Figure 6C). stead of driving protein synthesis through mTor and In addition to stress kinase activation and reduced in- p70S6K or regulation of GSK3β actions, insulin may be sulin receptor expression, insulin resistance can also be playing a more important role in lipid and glucose me- induced by over activation of tyrosine phosphatases [29]. tabolism, gene regulation, or mitochondrial maintenance However, PTP1B expression was not elevated in the in peripheral neurons. Further studies are underway to DRG or sciatic nerve of ob/ob mice, nor did insulin stimu- explore these other downstream components of insulin lation appear to alter its expression levels (Figure 6E,F, signaling and to define the temporal components of this respectively). signaling pathway. An additional caveat to this study is the use of leptin- Discussion deficient ob/ob mice. Leptin’s role in the nervous system Diabetic neuropathy is associated with profound loss of is receiving increasing attention, and it may have a distal limb sensation and/or pain, causing significant neuroprotective role [44]. It is not known how reduced decline in the quality of life and potential morbidity neuronal leptin may have contributed to our results. and mortality for patients. Currently, there are no clin- Thus, confirming these results in a high-fat diet model ical treatments that successfully improve neuropathic of obesity will be an important step to further investigat- damage to peripheral sensory nerve fibers, likely due to ing PNS insulin resistance. the multifactorial etiology of neuropathy development In experiments presented here, it appeared that in- and progression. Here, we have demonstrated in vivo sulin produced a stronger Akt activation in the sciatic PNS insulin resistance in ob/ob mice. These results are nerve compared to the DRG (Figure 3), whereas IGF- consistent with recent in vitro studies and support the 1 produced a stronger Akt activation in the DRG view that altered insulin signaling may contribute to compared to the sciatic nerve (Figure 5). These results DN [43]. A robust activation of insulin-sensitive path- point to an apparent separation in insulin/IGF-1 sig- naling support within the PNS. One plausible explan- ways was observed in the DRG and sciatic nerve of nondiabetic mice, with a blunted response in both tis- ation may be that insulin and IGF-1 have different sues from insulin-resistant ob/ob mice. While no one actions on the DRG soma and satellite cells compared to sensory axons, motor axons, and Schwann cells in mechanism of insulin resistance was clearly prevalent, significant changes were seen in two known pathways the peripheral nerve, leading to alternative signaling of insulin resistance, including increased JNK activity profiles. How this potential divergence in signaling may affect sensory neuron function is yet to be deter- and reduced insulin receptor expression. Although more research is needed to fully elucidate the path- mined and ongoing research is further delineating the ways leading to PNS insulin resistance, these results differential roles that insulin and IGF-1 may play in sensory nerve biology. suggest that cellular mechanisms of insulin resistance that have been defined in muscle may also play an im- In ob/ob mice, both the DRG and sciatic nerve displayed portant role in the PNS. reduced insulin-induced Akt activation, a classic indication Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 8 of 11 http://www.actaneurocomms.org/content/1/1/15 of insulin resistance. Several mechanisms of insulin resist- PTP1B knockout mice display increased insulin sensitiv- ance outlined in muscle also appear to be altered in the ity [48]. In the current study, we did not detect signifi- PNS, and may be contributing to the observed reduction cant upregulation of PTP1B in the DRG or sciatic nerve in insulin signal transduction. However, these results must of insulin resistant mice. While there was no change in be interpreted with caution as significant changes were not PTP1B expression, there still could be alterations in seen consistently across PNS tissues, and further research phosphatase activity and further studies are underway will need to be completed to fully establish a clear mechan- to explore this possibility. ism. Interestingly, no change in baseline Akt activation It will be important to put the current results in levels was observed between nondiabetic and ob/ob mice context with other contributory mechanisms of DN, as may be expected in states of insulin resistance. These re- including glucose and/or lipid mediated toxicity as sults are intriguing and suggest that future research focus- well as oxidative stress [49]. We postulate that the ing on pathways driving Akt signaling is warranted. metabolic dysfunction associated with hyperglycemia Hyperinsulinemia can promote insulin resistance and dyslipidemia in concert with reduced neuro- through downregulation of the insulin receptor [22]. This trophic support promotes deterioration and reduced effect was demonstrated in our data. The ob/ob mice in regeneration of the distal axon. Furthermore, the loss this cohort had serum insulin levels 34.3 fold higher than of appropriate insulin signaling could make neurons nondiabetic mice and the DRG of ob/ob mice displayed even more susceptible to these pathogenic cascades. It significantly lower insulin receptor expression. Thus, the should be noted that both intrathecal and intraperito- extreme hyperinsulinemia in the ob/ob mice may be neal insulin injections altered blood glucose levels. promoting insulin receptor downregulation and contrib- Thus, these results should be viewed in the context uting to PNS insulin resistance. This idea is supported that glucose levels were also transiently altered by in- by a recent study that reported a significant decrease in sulin administration. Further research into disrupted insulin receptor mRNA in cultured DRG neurons that PNS insulin signaling relative to other pathogenic displayed insulin resistance when treated with high levels mechanisms is needed, as this will be a key step in of insulin [7]. translating these basic science results into clinical An alternative mediator of insulin resistance is the applications. stress kinase JNK, which is activated in response to vari- ous cellular stressors, including low grade chronic in- flammation induced by obesity [33,45]. In fact, ob/ob Conclusions mice with a JNK null mutation have improved whole Insulin resistance is emerging as a potential mediator of body glucose tolerance and insulin sensitivity [31]. Add- several neurological syndromes (reviewed in [1]). This itionally, JNK activation has been implicated in altered study, along with recent data of in vitro DRG insulin re- neurofilament phosphorylation in the PNS of diabetic sistance, strongly supports altered insulin signaling as a rats [46]. JNK activation is proposed to promote insulin pathogenic mechanism in DN. While deficient insulin resistance through upregulation of IRS serine phosphor- signaling has been a proposed contributor to DN in type ylation, and IRS is a key common signaling component 1 models for some time [3,4,14,43,50], little has been of both the insulin and IGF-1 pathways. In the current known about insulin signaling effectiveness in type 2 study we observed increased JNK activation without a (hyperinsulinemic) models of DN. We observed re- significant elevation in either IRS1 or IRS2 serine phos- duced insulin signaling in vivo in the PNS of type 2 phorylation. Some controversy does exist as to which diabetic ob/ob mice and suggest possible mechanisms serine sites are most important in insulin resistance, thus that may be contributing to these changes. It is now the serine sites that we probed (p(ser731)IRS2 and becoming evident that decreased insulin neurotrophic (p(ser307)IRS1) may not be heavily involved in inhibiting supportinthe PNSis anintegralpartofDN and insulin signaling in the PNS. More powerful approaches, may be a congruent mechanism between type 1 and such as mass spectrometry, may be needed to establish a type 2 diabetic models of DN, as both have reduced global change in the IRS phosphorylation profile within insulin signaling either due to insulinopenia or neur- the PNS [47]. onal insulin resistance. Another possible component of the insulin receptor Future studies will focus on mechanisms through signaling pathway that could be affected in insulin which insulin supports proper PNS function, as reveal- resistance is PTP1B. PTP1B is the canonical member ing these pathways may provide insight into how de- of protein tyrosine phosphatases and serves an import- creased insulin support contributes to the pathogenesis ant role in insulin signaling regulation [29]. Over- of DN. Furthermore, delineating the details of PNS insu- expression of PTP1B has been linked to insulin lin signaling may open new avenues for therapeutic resistance in peripheral tissues of ob/ob mice [26] and intervention in patients with DN. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 9 of 11 http://www.actaneurocomms.org/content/1/1/15 Methods followed by an additional 4 more applications. The Animals withdrawal threshold was calculated using the formula All experiments were approved by the University of from the up-down method previously described [52]. Kansas Medical Center Institutional Animal Care and Use Committee. Male ob/ob leptin null mutant and age- Insulin and IGF-1 injections matched control mice (ob/+) were purchased from Jackson Sterile PBS (vehicle), 0.1U (~0.7 nmol) Humulin R insu- Laboratories (Bar Harbor, Maine) at 8 weeks of age. Mice lin, or recombinant IGF-1 equimolar to 0.1U insulin was were given access to food and water ad libitum and directly administered to both nondiabetic and ob/ob type housed on a 12-hour light/dark cycle. Weekly blood glu- 2 diabetic mice via a one-time intrathecal injection. Pre- cose (Glucose Diagnostic Assay Sigma-Aldrich, St. Louis, viously, intrathecal 0.1U insulin and equimolar IGF-1 MO), serum insulin (Insulin ELISA Alpco, Salem, NH) have been shown to have beneficial effects on the symp- and weights were monitored and mice were sacrificed at toms of DN [10]. All injections were 50 μL and adminis- 11 weeks of age. tered with a 1cc 28½ gauge insulin syringe between the L6 and S1 vertebrae. In an additional preliminary study, Glucose tolerance test sterile PBS or insulin was delivered through an intra- At 9 weeks of age, an intraperitoneal glucose tolerance peritoneal injection at a dose of 3.33 U/kg, such that test (IPGTT) was used to assess the response of mice to the total insulin administered was approximately 0.1 U a glucose challenge. After a 6-hour fast, mice were given for nondiabetic mice and 0.17U (~1.2 nmol) for ob/ob an intraperitoneal injection of glucose at 1g of glucose mice. The doses administered and stimulation time frames per kg body weight. Blood glucose levels were measured used were confirmed to be sufficient for Akt activation in via tail clip immediately prior to the glucose bolus and the PNS with dose curve and time course studies (Grote, then at 15, 30, 60, and 120 minutes after injection. unpublished observation). Insulin tolerance test Western blots At 10 weeks of age, mice underwent an insulin toler- After a 30 minute insulin stimulation period, the right ance test (ITT). Mice were fasted for 6 hours and then and left lumbar DRG and sciatic nerves were harvested administered IP insulin (Humulin R, Lilly, Indianapolis, for each sample from 11 week old mice and frozen Indiana) at a dosage of 1.5 U per kg body weight. Blood at −80°C. Tissues were sonicated in Cell Extraction Buffer glucose levels were monitored immediately prior to in- (Invitrogen, Carlsbad, CA) containing 55.55 μl/ml protease sulin injection and then at 15, 30, 60, and 120 minutes inhibitor cocktail, 200 mM Na VO , and 200 mM NaF. 3 4 thereafter. Following sonication, protein was extracted on ice for 60 minutes and vortexed every 10 minutes. After centrifuga- HOMA-IR tion, protein concentration of the supernatant was mea- Fasting insulin and fasting glucose levels were used to sured with a Bradford assay (Bio-Rad, Hercules, CA). calculate the homeostatic model assessment of insulin Samples were then boiled with Lane Marker Reducing resistance (HOMA-IR). Scores were calculated with the Sample Buffer (Thermo Scientific, Waltham, MA) for 3 following equation: (blood glucose (mg/dl) X (serum insulin minutes. Equal amounts of protein (30 μg) were loaded (uU/mL))/405) [51]. per lane and samples were separated on a 4-15% gradient tris-glycine gel (Bio-Rad), and then transferred to a nitro- Mechanical sensitivity cellulose membrane. Membranes were probed with the Mechanical behavioral responses to Semmes Weinstein-von following primary antibodies and all antibodies were Frey monofilaments (0.07 to 5.0 g) were assessed at 8, purchased from Cell Signaling (Danvers, MA) unless 9, 10, and 11 weeks of age. Mice underwent acclima- otherwise noted: total Akt (1:2000), p-(Ser473)Akt tion 2 days prior to the first day of behavioral testing. (1:500), total p70S6K (1:500), p-(Thr389)p70S6K (1:500), Mice were placed in individual clear plastic cages total GSK3β (1:1500), p-(Ser9)GSK3β (1:1000), total JNK (11×5×3.5 cm) on a wire mesh grid 55 cm above the (1:1000), p-(Thr183/Tyr185)JNK (1:500), total mTor table and were acclimated for 30 minutes prior to be- (1:500), p-(Ser2448)mTor (1:500), Insulin-like growth fac- havioral analysis. The filaments were applied perpen- tor 1 receptor β subunit (1:500), PTP1B (1:500) (Abcam, dicularly to the plantar surface of the hindpaw until Cambridge, MA), total AS160 (1:1000) (Millipore, Biller- the filament bent. Testing began with the 0.7 g fila- ica, MA), p-(Thr642)AS160 (1:500) (Millipore), Insulin ment, and in the presence of a response, the next Receptor β subunit (1:500) (Santa Cruz, Santa Cruz, CA), smaller filament was applied. If no response was ob- and α-tubulin (1:5000) (Abcam). Bands were visualized served, the next larger filament was used. Filaments with either anti-mouse or anti-rabbit HRP-conjugated sec- were applied until there was a change in response, ondary antibodies (Santa Cruz) and ECL with X-ray film. Grote et al. Acta Neuropathologica Communications 2013, 1:15 Page 10 of 11 http://www.actaneurocomms.org/content/1/1/15 Densitometry with ImageJ (NIH) was then used to analyze Received: 20 March 2013 Accepted: 19 April 2013 Published: 10 May 2013 each lane. All samples from each tissue were run simul- taneously across multiple gels and each group was equally represented on each gel (approximately 3 nondiabetic References 1. Kim B, Feldman EL: Insulin resistance in the nervous system. Trends Endocrinol PBS, 3 nondiabetic insulin, 3 ob/ob PBS, and 3 ob/ob insu- Metab 2012, 23(3):133–141. lin per gel). Data is presented as the ratio of integrated 2. 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Acta Neuropathologica CommunicationsSpringer Journals

Published: May 10, 2013

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