Murine model and mechanisms of treatment-induced painful diabetic neuropathy

Abstract

Diabetes mellitus represents a group of metabolic diseases that are characterized by hyperglycemia caused by either lack of insulin production or a reduced ability to respond to insulin. It is estimated that there were 347 million people worldwide who suffered from diabetes in 2008 and incidence is predicted to double by 2050. Neuropathy is the most common complication of long-term diabetes and approximately 30% of these subjects develop chronic neuropathic pain. A distinct acute, severe form of neuropathic pain, called insulin neuritis or treatment-induced painful neuropathy of diabetes (TIND), may also occur shortly after initiation of intensive glycemic control, with an incidence rate of up to 10.9%. The pathological mechanisms leading to TIND, which is mostly unresponsive to analgesics, are not yet understood, impeding the development of therapies. Studies to date have been clinical and with limited cohorts of patients. In the current study, we developed chronic and acute insulin-induced neuropathic pain in mice with type 2 insulin-resistant diabetes. Furthermore, we determined that insulin-induced acute allodynia is independent of glycemia levels, can also be induced with Insulin-like Growth Factor 1 (IGF1) and be prevented by inhibition of AKT, providing evidence of an insulin/IGF1 signaling pathway-based mechanism for TIND. This mouse model is useful for the elucidation of mechanisms contributing to TIND and for the testing of new therapeutic approaches to treat TIND.

Keywords: TIND; diabetes; insulin; insulin neuritis; neuropathy.

Figure 1. Glucose tolerance test demonstrating insulin resistance in HFD mice

Blood glucose levels in C57Bl6 mice fed a low fat diet (LFD) or a high fat diet (HFD) following an ip injection of glucose at 1.5g/kg after an overnight fast. Two-way repeated measures ANOVA followed by Sidak’s post-hoc test, **p<0.01, ***p<0.001, F(4,28)=9.008, n=6. Mean±s.e.m.

Figure 2. Daily insulin injection induces persistent allodynia

(A) Tactile responses to von Frey filaments 5–6 hrs after the last injection for mice fed a low fat diet (LFD), a high fat diet (HFD) or a HFD and receiving daily 1U insulin injection sc (HFD+Chronic Insulin). Two-way repeated measures ANOVA followed by Sidak’s post-hoc test versus HFD group, *p<0.05, **p<0.01***p<0.001, n=4–6. (B) Responses to thermal stimulation 6–7 hrs after the last injection for mice fed a low fat diet (LFD), a high fat diet (HFD) or a HFD and receiving daily 1U insulin injection sc (HFD+Chronic Insulin). Two-way repeated measures ANOVA followed by Sidak’s post-hoc test versus HFD group, *p<0.05, **p<0.01***p<0.001, n=4–6. B: Baseline, Day 5 to Day 32, measurements were performed 5–7hrs after insulin injection while Day 64, tactile and thermal responses were assessed 72 hrs after the last insulin injection. (C) Time course of tactile responses on day 60 after injection of 1U insulin to previously untreated HFD mice (HFD) and to HFD receiving daily insulin injection for the past 59 days (HFD+Chronic Insulin). Twoway repeated measures ANOVA followed by Sidak’s post-hoc test, ** p<0.01 chronic vs HFD, n=6 (D) Blood glucose levels on day 60 after injection of 1U insulin to previously untreated HFD mice (HFD) and to HFD receiving daily insulin injection for the past 59 days (HFD+Chronic Insulin). Two-way repeated measures ANOVA followed by Tukey’s post-hoc test, ***p<0.001 vs baseline, n=6. Mean±s.e.m.

Figure 3. Effect of Gabapentin

(A) Tactile responses to von Frey filaments were assessed in mice fed a low fat diet (LFD) ± gabapentin at 200mg/kg ip or a HFD and receiving daily 1U insulin injection sc (HFD+Chronic Insulin) + Gabapentin at 200mg/kg ip. (B) Area under the curve (AUC) of time courses in (A). One-way ANOVA followed by Dunnet’s post hoc test, *p<0.05 vs LFD, n=6. Mean+s.e.m.

Figure 4. Insulin acutely induces tactile allodynia in HFD mice

(A) Tactile responses to von Frey filaments for HFD mice receiving saline (HFD saline), insulin at 0.5U (HFD 0.5U insulin) or insulin at 2.0U (HFD 2.0U insulin). Two-way repeated measures ANOVA followed by Sidak’s post-hoc test, *p<0.05 vs baseline, n=8. (B) Blood glucose levels in HFD mice receiving saline (HFD saline), insulin at 0.5U (HFD 0.5U insulin) or insulin at 2.0U (HFD 2U insulin). Two-way ANOVA, **p<0.01, ***p<0.001 vs baseline followed by Tuley’s post hoc test, n=8. Mean+s.e.m.

Figure 5. Insulin acutely induces tactile allodynia in db/db mice

(A) Tactile responses to von Frey filaments for HFD mice receiving 2U insulin (HFD 2U insulin), db/db mice receiving 2U insulin (db/db 2U insulin) or saline (db/db Saline). Two-way repeated measures ANOVA followed by Tukey’s post hoc test, *p<0.05 vs baseline, n=11. (B) Blood glucose levels in HFD mice receiving 2U insulin (HFD 2U insulin), db/db mice receiving 2U insulin (db/db 2U insulin) or saline (db/db Saline). Two-way repeated measures ANOVA followed by Tukey’s post hoc test, *p<0.05, ***p<0.001 vs baseline, n=11. Mean+s.e.m.

Figure 6. Insulin acutely induces tactile allodynia in insulin receptor knockdown mice without affecting blood glucose levels

(A) Tactile responses to von Frey filaments for HFD mice receiving 2U insulin (HFD 2U insulin) or insulin receptor knockdown mice receiving 2U insulin (IR KD 2U insulin). Two-way repeated measures ANOVA followed by Tukey’s post hoc test, *p<0.05 vs baseline, n=8. (B) Blood glucose levels in HFD mice receiving 2U insulin (HFD 2U insulin) or insulin receptor knockdown mice receiving 2U insulin (IR KD 2U insulin). Two-way repeated measures ANOVA followed by Tukey’s post hoc test, ***p<0.001 vs baseline, n=8. Mean+s.e.m.

Figure 7. IGF1 acutely induces tactile allodynia in HFD mice without affecting blood glucose levels

(A) Tactile responses to von Frey filaments for HFD mice receiving saline (HFD saline), insulin at 2U (HFD 2U insulin) or IGF1 at 2mg/kg (HFD 2mg/kg IGF1). Two-way repeated measures ANOVA followed by Tukey’s post hoc test, *p<0.05, ***p<0.001 vs baseline, n=8. (B) Blood glucose levels in HFD mice receiving saline (HFD saline), insulin at 2U (HFD 2U insulin) or IGF1 at 2mg/kg (HFD 2mg/kg IGF1). Two-way repeated measures ANOVA followed by Tukey’s post hoc test, **p<0.01, ***p<0.001 vs baseline, n=8. Mean+s.e.m.

Figure 8. Effect of AKT inhibitor IV on acute insulin-induced tactile allodynia

(A) Tactile responses to von Frey filaments were assessed in HFD mice receiving 2U insulin and HFD receiving 2U insulin after pretreatment with AKT Inhibitor IV at 1 or 2.5 mg/kg ip. Two-way repeated measures ANOVA followed by Tukey’s post hoc test, *p<0.05 vs baseline, n=7–8. (B) Blood glucose levels in HFD mice receiving 2U insulin and HFD receiving 2U insulin after pretreatment with AKT Inhibitor IV at 1 or 2.5 mg/kg ip. Two-way repeated measures ANOVA followed by Tukey’s post hoc test, ***p<0.001 vs baseline, n=7–8. Mean+s.e.m.

Figure 9. Effect of gabapentin on acute insulin-induced tactile allodynia

(A) Tactile responses to von Frey filaments were assessed in HFD mice receiving 2U insulin and HFD receiving 2U insulin after pretreatment with gabapentin at 200mg/kg ip. Two-way repeated measures ANOVA followed by Tukey’s post hoc test, *p<0.05, **p<0.01, ***p<0.001 vs baseline, n=7–8. (B) Blood glucose levels in HFD mice receiving 2U insulin and HFD receiving 2U insulin after pretreatment with gabapentin at 200mg/kg ip. Two-way repeated measures ANOVA followed by Tukey’s post hoc test, ***p<0.001 vs baseline, n=7–8. Mean+s.e.m.