Peripheral nervous system insulin resistance in ob/ob mice

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.

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) At 10 weeks 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.

Figure 2

Ob/ob mice develop mechanical allodynia. Mechanical thresholds were tested using von Frey monofilaments at 8, 9, 10, and 11 weeks of age. Ob/ob mice did not display significant differences from nondiabetic controls at 8, 9, or 10 weeks. However, at week 11, ob/ob mice had a significant decrease in their mechanical withdrawal thresholds. * = p < 0.05. n = 6 nondiabetic mice, n = 6 ob/ob diabetic mice.

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.

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.

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.

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.