Peripheral nervous system insulin resistance in ob/ob mice

To quantify the extent of systemic insulin resistance in ob/ob mice, nondiabetic and diabetic ob/ob mice underwent an IPGTT at 9 weeks of age (Figure 1A). Blood glucose levels of the ob/ob mice were significantly higher than nondiabetic mice throughout the course of the experiment and the area under the curve (AUC) was also significantly elevated for ob/ob mice (Figure 1B). Results from the ITT indicated that nondiabetic, insulin-injected mice exhibited an expected physiological decrease in blood glucose in response to insulin; however, ob/ob mice displayed a transient elevation of glucose levels (Figure 1C). Statistical analysis of the data revealed that ob/ob mice maintained elevated glucose levels compared to nondiabetic controls throughout most of the study, and that the AUC was significantly higher for diabetic ob/ob mice (Figure 1D). The HOMA-IR, a measure of insulin resistance, was calculated using fasting blood glucose and serum insulin levels from 10 week old mice. Ob/ob mice had significantly higher blood glucose levels (14.3 ± 2.1 mmol/L) compared to nondiabetic mice (8.2 ±0.5 mmol/L) (Figure 1E). Fasting insulin levels were also significantly higher in diabetic ob/ob mice (6780 ± 1610 pmol/L) compared to nondiabetic mice (198 ± 25 pmol/L, Figure 1F). As such, ob/ob mice had a significantly elevated HOMA-IR as compared to nondiabetic mice (557 ± 130 compared to 10.1 ± 1.4, respectively, Figure 1G). These results demonstrate significant glucose intolerance and insulin resistance in ob/ob mice at this age.

To quantify a known behavioral abnormality associated with neuropathy in mice, mechanical sensitivity was assessed in nondiabetic and diabetic ob/ob mice at 8, 9, 10, and 11 weeks of age. There were no differences in mechanical thresholds between nondiabetic and ob/ob diabetic mice at 8, 9, or 10 weeks of age. However, at 11 weeks, there was a significant decrease in the mechanical thresholds of diabetic ob/ob mice compared to nondiabetic mice (Figure 2), consistent with sensory aberrations associated with peripheral neuropathy as previously reported in this genetic mouse strain [36].

Insulin stimulation causes a robust activation of Akt in insulin-sensitive tissues like muscle and adipose, as well as in neurons of both the peripheral and central nervous systems. Moreover, reduced insulin-induced activation of Akt is a hallmark of insulin resistance [5, 6, 37, 38]. Here, nondiabetic and diabetic ob/ob mice were administered either intrathecal PBS or insulin and the DRG and sciatic nerve were harvested 30 minutes later for Western blot analysis to assess Akt activation. Both nondiabetic control and ob/ob mice display significantly elevated blood glucose levels following intrathecal injection of PBS. Nondiabetic mice glucose levels increased from 6.6 ± 0.4 mmol/L to 8.6 ± 0.5 mmol/L, whereas ob/ob levels increased from 12.1 ± 2.4 mmol/L to 22.3 ± 2.4 mmol/L. Glucose levels in nondiabetic mice significantly decreased from 7.0 ± 0.3 mmol/L to 3.3 ± 0.3 mmol/L after intrathecal insulin injection. Ob/ob mice glucose levels 30 minutes after insulin injection were not significantly different from baseline, starting at 12.7 ± 0.9 mmol/L and ending at 12.3 ± 1.5 mmol/L after 30 minutes.

In nondiabetic mice, insulin produced a strong elevation in levels of activated Akt (p(ser473)Akt/total Akt) in both the DRG and sciatic nerve (Figure 3A,B). However in ob/ob mice, Akt activation was significantly lower in the DRG and sciatic nerve. In fact, insulin failed to significantly increase Akt activation over baseline in the DRG of ob/ob mice. For comparison, Akt activation in the DRG was increased 3.1 fold in nondiabetic mice and 1.6 fold in ob/ob diabetic mice. In the sciatic nerve, insulin produced 9.7 and 6.1 fold increase in Akt activation in nondiabetic and ob/ob mice, respectively.

To confirm that these results were not dependent on the intrathecal route of delivery, a small number of mice were administered intraperitoneal insulin at a dose of 3.33 U/kg. PBS injections once again appeared to cause an increase in blood glucose levels from baseline, nondiabetic mice levels started at 7.0 ± 1.4 mmol/L and ended at 8.6 ± 1.4 mmol/L (p > 0.05), whereas ob/ob mice showed a significant increase from 10.8 ± 0.8 mmol/L to 15.4 ± 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 insulin 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 displayed a 2.4 fold change in Akt activation, compared to 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, but only a 1.4 fold change in ob/ob in the sciatic nerve from ob/ob mice.

IGF-1 and insulin activate many of the same intracellular signaling pathways [21], and altered IGF-1 signaling has been demonstrated in states of insulin resistance [39]. Furthermore, IGF-1 resistance has recently been demonstrated to be associated with brain insulin resistance and cognitive decline in Alzheimer’s patients [40]. To investigate IGF-1 signal transduction in the PNS of ob/ob mice, a dose of IGF-1 equimolar to 0.1U insulin was administered via an intrathecal injection. Blood glucose levels in both nondiabetic control and ob/ob mice once again appeared to increase with intrathecal PBS injection, 7.7 ± 0.3 mmol/L at baseline as compared to 9.7 ± 0.8 mmol/L (p = 0.056) after 30 minutes for nondiabetic mice and 12.1 ± 1.9 mmol/L at baseline to 23.7 ± 3.1 mmol/L after 30 minutes for ob/ob mice. IT IGF-1 did not significantly alter blood glucose levels in nondiabetic mice, 9.3 ± 0.4 mmol/L versus 8.1 ± 0.3 mmol/L. Ob/ob mice that received IT IGF-1 had similar blood glucose profiles to ob/ob mice that received IT PBS, with a significant increase in blood glucose after 30 minutes, 16.3 ± 2.7 mmol/L as compared to 27.7 ± 1.2 mmol/L. Akt was significantly activated in the DRG from both nondiabetic (13.3 fold) and ob/ob diabetic mice (6.0 fold). However, Akt activation was significantly lower in the DRG from ob/ob mice compared to nondiabetic mice (Figure 5A). In the sciatic nerve of nondiabetic mice, IGF stimulation produced a significant 2.8 fold increase in Akt activation. In contrast, Akt was not significantly activated in the sciatic nerve of ob/ob mice (Figure 5B).

To assess whether diabetes-induced blunting of Akt activation was maintained downstream, several other insulin-responsive proteins were investigated via Western blot analysis, including mTor (protein synthesis), p70S6K (protein synthesis), AS160 (glucose uptake), and GSK3β (glycogen synthesis). At the insulin dose (0.1U) and time point (30 minute stimulation) that were investigated, no statistical differences (p > 0.05) were observed in the activation of these proteins even in control mice (Table 1). Additionally, similar to Akt results, no differences were observed in baseline levels between all proteins investigated. However, it is interesting to note that in both the DRG and sciatic nerve from ob/ob mice, there is a consistent pattern of a reduced fold change in response to insulin as compared to responses in nondiabetic mice.

To explore possible mechanisms responsible for reduced PNS insulin sensitivity, we investigated several pathways known in other insulin-resistant tissues. One contributor to reduced insulin signaling is a downregulation of insulin receptor expression induced by hyperinsulinemia [23]. As shown in Figure 6A, protein levels of the insulin receptor subunit β were significantly lower in the DRG of ob/ob mice compared to nondiabetic mice. However, there was no statistical difference in the expression of insulin receptor between nondiabetic and ob/ob mice in the sciatic nerve (Figure 6B). No significant differences between groups were observed in IGF-1 receptor expression in either the DRG or sciatic nerve (data not shown).

Our previous studies in primary DRG cultures reported an upregulation of IRS2 serine phosphorylation [6], a recognized mechanism of insulin resistance in muscle and adipose. In the current study, we investigated both IRS1 (muscle and adipose isoform) [41] and IRS2 (neural isoform) [6, 42] serine phosphorylation. In contrast to neurons in vitro, IRS serine phosphorylation does not appear to be significantly affected in the PNS in vivo within this model, (data not shown). Interestingly, there was significant activation of the stress kinase JNK (p(Thr183/Tyr185)JNK/total JNK) in the sciatic nerve of ob/ob mice compared to nondiabetic mice (Figure 6D) and a similar pattern of activated JNK was observed in the DRG of ob/ob mice, however significance was not reached (nondiabetic vs. diabetic p = 0.122) (Figure 6C).

In addition to stress kinase activation and reduced insulin receptor expression, insulin resistance can also be induced by over activation of tyrosine phosphatases [29]. However, PTP1B expression was not elevated in the DRG or sciatic nerve of ob/ob mice, nor did insulin stimulation appear to alter its expression levels (Figure 6E,F, respectively).

All experiments were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. Male ob/ob leptin null mutant and age-matched control mice (ob/+) were purchased from Jackson Laboratories (Bar Harbor, Maine) at 8 weeks of age. Mice were given access to food and water ad libitum and housed on a 12-hour light/dark cycle. Weekly blood glucose (Glucose Diagnostic Assay Sigma-Aldrich, St. Louis, MO), serum insulin (Insulin ELISA Alpco, Salem, NH) and weights were monitored and mice were sacrificed at 11 weeks of age.

At 9 weeks of age, an intraperitoneal glucose tolerance test (IPGTT) was used to assess the response of mice to a glucose challenge. After a 6-hour fast, mice were given an intraperitoneal injection of glucose at 1g of glucose per kg body weight. Blood glucose levels were measured via tail clip immediately prior to the glucose bolus and then at 15, 30, 60, and 120 minutes after injection.

At 10 weeks of age, mice underwent an insulin tolerance test (ITT). Mice were fasted for 6 hours and then administered IP insulin (Humulin R, Lilly, Indianapolis, Indiana) at a dosage of 1.5 U per kg body weight. Blood glucose levels were monitored immediately prior to insulin injection and then at 15, 30, 60, and 120 minutes thereafter.

Fasting insulin and fasting glucose levels were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). Scores were calculated with the following equation: (blood glucose (mg/dl) X (serum insulin (uU/mL))/405) [51].

Mechanical behavioral responses to Semmes Weinstein-von Frey monofilaments (0.07 to 5.0 g) were assessed at 8, 9, 10, and 11 weeks of age. Mice underwent acclimation 2 days prior to the first day of behavioral testing. Mice were placed in individual clear plastic cages (11×5×3.5 cm) on a wire mesh grid 55 cm above the table and were acclimated for 30 minutes prior to behavioral analysis. The filaments were applied perpendicularly to the plantar surface of the hindpaw until the filament bent. Testing began with the 0.7 g filament, and in the presence of a response, the next smaller filament was applied. If no response was observed, the next larger filament was used. Filaments were applied until there was a change in response, followed by an additional 4 more applications. The withdrawal threshold was calculated using the formula from the up-down method previously described [52].

Sterile PBS (vehicle), 0.1U (~0.7 nmol) Humulin R insulin, or recombinant IGF-1 equimolar to 0.1U insulin was directly administered to both nondiabetic and ob/ob type 2 diabetic mice via a one-time intrathecal injection. Previously, intrathecal 0.1U insulin and equimolar IGF-1 have been shown to have beneficial effects on the symptoms of DN [10]. All injections were 50 μL and administered with a 1cc 28½ gauge insulin syringe between the L6 and S1 vertebrae. In an additional preliminary study, sterile PBS or insulin was delivered through an intraperitoneal injection at a dose of 3.33 U/kg, such that the total insulin administered was approximately 0.1 U for nondiabetic mice and 0.17U (~1.2 nmol) for ob/ob mice. The doses administered and stimulation time frames used were confirmed to be sufficient for Akt activation in the PNS with dose curve and time course studies (Grote, unpublished observation).

After a 30 minute insulin stimulation period, the right and left lumbar DRG and sciatic nerves were harvested for each sample from 11 week old mice and frozen at −80°C. Tissues were sonicated in Cell Extraction Buffer (Invitrogen, Carlsbad, CA) containing 55.55 μl/ml protease inhibitor cocktail, 200 mM Na₃VO₄, and 200 mM NaF. Following sonication, protein was extracted on ice for 60 minutes and vortexed every 10 minutes. After centrifugation, protein concentration of the supernatant was measured with a Bradford assay (Bio-Rad, Hercules, CA). Samples were then boiled with Lane Marker Reducing Sample Buffer (Thermo Scientific, Waltham, MA) for 3 minutes. Equal amounts of protein (30 μg) were loaded per lane and samples were separated on a 4-15% gradient tris-glycine gel (Bio-Rad), and then transferred to a nitrocellulose membrane. Membranes were probed with the following primary antibodies and all antibodies were purchased from Cell Signaling (Danvers, MA) unless otherwise noted: total Akt (1:2000), p-(Ser473)Akt (1:500), total p70S6K (1:500), p-(Thr389)p70S6K (1:500), total GSK3β (1:1500), p-(Ser9)GSK3β (1:1000), total JNK (1:1000), p-(Thr183/Tyr185)JNK (1:500), total mTor (1:500), p-(Ser2448)mTor (1:500), Insulin-like growth factor 1 receptor β subunit (1:500), PTP1B (1:500) (Abcam, Cambridge, MA), total AS160 (1:1000) (Millipore, Billerica, MA), p-(Thr642)AS160 (1:500) (Millipore), Insulin Receptor β subunit (1:500) (Santa Cruz, Santa Cruz, CA), and α-tubulin (1:5000) (Abcam). Bands were visualized with either anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz) and ECL with X-ray film. Densitometry with ImageJ (NIH) was then used to analyze each lane. All samples from each tissue were run simultaneously across multiple gels and each group was equally represented on each gel (approximately 3 nondiabetic PBS, 3 nondiabetic insulin, 3 ob/ob PBS, and 3 ob/ob insulin per gel). Data is presented as the ratio of integrated density of the phosopho-protein normalized to the integrated density of the total protein. The normalized ratio was averaged for each group and the mean ± SEM is represented in the corresponding figures. Representative immunoblots are shown.

All data is expressed as means ± standard error of the mean. IPGTT, ITT, and behavior data were analyzed with a repeated measures analysis of variance (RM-ANOVA). In addition, the area under the curve (AUC) for IPGTT and ITT was analyzed using a Student’s t-test. Blood glucose changes at 30 minutes in response to insulin or IGF-1 were analyzed with a paired Student’s t-test. Western blot results were analyzed with 2-way ANOVA and Bonferroni’s post hoc analysis when appropriate. Outliers greater than or less than 2 standard deviations from the mean were not included in the analysis. All statistical tests were performed using SigmaPlot software and a P value <0.05 was considered significant.