Exendin-4 improves glycemic control, ameliorates brain and pancreatic pathologies, and extends survival in a mouse model of Huntington's disease

Abstract

Objective: The aim of this study was to find an effective treatment for the genetic form of diabetes that is present in some Huntington's disease patients and in Huntington's disease mouse models. Huntington's disease is a neurodegenerative disorder caused by a polyglutamine expansion within the huntingtin protein. Huntington's disease patients exhibit neuronal dysfunction/degeneration, chorea, and progressive weight loss. Additionally, they suffer from abnormalities in energy metabolism affecting both the brain and periphery. Similarly to Huntington's disease patients, mice expressing the mutated human huntingtin protein also exhibit neurodegenerative changes, motor dysfunction, perturbed energy metabolism, and elevated blood glucose levels.

Research design and methods: Huntington's disease mice were treated with an FDA-approved antidiabetic glucagon-like peptide 1 receptor agonist, exendin-4 (Ex-4), to test whether euglycemia could be achieved, whether pancreatic dysfunction could be alleviated, and whether the mice showed any neurological benefit. Blood glucose and insulin levels and various appetite hormone concentrations were measured during the study. Additionally, motor performance and life span were quantified and mutant huntingtin (mhtt) aggregates were measured in both the pancreas and brain.

Results: Ex-4 treatment ameliorated abnormalities in peripheral glucose regulation and suppressed cellular pathology in both brain and pancreas in a mouse model of Huntington's disease. The treatment also improved motor function and extended the survival time of the Huntington's disease mice. These clinical improvements were correlated with reduced accumulation of mhtt protein aggregates in both islet and brain cells.

Conclusions: Targeting both peripheral and neuronal deficits, Ex-4 is an attractive agent for therapeutic intervention in Huntington's disease patients suffering from diabetes.

FIG. 1.

Experimental design. A: Experimental timeline of the study. Male N171-82Q and age-matched wild-type mice were injected daily with either Ex-4 or saline (control). Body weight and glucose measurements were recorded weekly, and rotarod performance was assessed bi-weekly. On euthanization, cortex, pancreas, and plasma were collected for further analyses. B: Ex-4 is an agonist of the GLP-1 receptor. Several amino acids differ between GLP-1 and Ex-4 sequences, most importantly in the NH₂-terminal region where the substitution of alanine to glycine renders Ex-4 resistant to proteolysis by dipeptidyl peptidase-IV. Whereas GLP-1 has a half-life of <2 min in circulation, Ex-4 has biological activity for ∼6 h. This ensures that Ex-4 has potent and long-acting effects on both the periphery (pancreas) and the brain. C: Proposed mechanisms of action of Ex-4 in peripheral tissues and the brain. Ex-4 promotes pancreatic β-cell growth and insulin production and secretion and increases insulin sensitivity of muscle and liver cells. Ex-4 crosses the blood-brain barrier and acts on neurons in the brain to promote their survival and support their high energy demands.

FIG. 2.

Ex-4 normalizes plasma glucose levels in Huntington's disease mice (right panel). Weekly (A and B) and average (C and D) glucose levels of saline- and Ex-4–treated wild-type (left panel) and Huntington's disease mice are shown throughout the treatment period. Ex-4 treatment significantly reduced plasma glucose levels in Huntington's disease mice from the first week of treatment, and euglycemia was maintained throughout the course of the study period. Ex-4 also significantly reduced plasma glucose levels in wild-type mice. Weekly (E and F) and average (G and H) body weight measurements for saline- and Ex-4–treated wild-type and Huntington's disease mice throughout the study are shown. Ex-4 treatment caused a significant reduction in body weight in both wild-type and Huntington's disease mice. Values are means ± SE, n = 18–24 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 3.

Modification of plasma levels of energy-regulating hormones by Ex-4 treatment in wild-type (left panel) and Huntington's disease (right panel) mice. Plasma concentrations of insulin (A and B), leptin (C and D), ghrelin (E and F), and adiponectin (G and H) were measured in wild-type and Huntington's disease mice treated with either saline (control) or Ex-4. Ex-4 treatment significantly reduced plasma insulin levels in wild-type mice, compared with control wild-type animals (P < 0.05). In Huntington's disease mice, Ex-4 treatment did not significantly alter circulating levels of insulin compared with Huntington's disease control animals. Thus, despite having lower circulating levels of insulin, the Ex-4–treated wild-type and Huntington's disease mice exhibit improved insulin sensitivity, as demonstrated by the reduction in plasma glucose levels (A and B). B, inset: Using plasma insulin and glucose measurements, HOMA values were calculated. C and D: Ex-4 treatment significantly reduced leptin levels in both wild-type (P < 0.001) and Huntington's disease (P < 0.05) mice, consistent with the decrease in the body weight in the Ex-4–treated mice. E and F: There were no significant effects of Ex-4 on plasma ghrelin levels in wild-type and Huntington's disease mice, although there were trends toward decreased levels in the wild-type mice and increased levels in the Huntington's disease mice. G and H: There were no significant alterations in the plasma adiponectin levels with Ex-4 treatment for both the wild-type and Huntington's disease mice, and Huntington's disease mice had significantly lower plasma adiponectin levels compared with wild-type mice (P < 0.01). Values are means ± SE, n = 18–24 animals per group. *P < 0.05, ***P < 0.001.

FIG. 4.

Treatment with Ex-4 improves pancreatic islet physiology in Huntington's disease mice (right panel). Immunostaining of pancreatic tissue for the β-cell–derived hormone insulin and corresponding phase contrast images. A and B: Saline-treated Huntington's disease mice had small, significantly diminished islets compared with saline-treated wild-type mice (left panel). C and D: Treatment with Ex-4 restored islet size in Huntington's disease mice but did not significantly affect islet size in wild-type mice. Immunostaining for the α-cell–derived hormone glucagon confirmed these improvements in islet physiology with Ex-4 treatment. E and G: In nondiabetic mice, glucagon-positive cells are typically arranged in a “halo” around the edge of the islet. F: In the Huntington's disease mice, the islet structure was altered, and α-cells were displaced into the center of the islet. H: This α-cell abnormality was improved with Ex-4 treatment in Huntington's disease mice. Values are means ± SE, n = 6–8 animals per group. (Please see http://dx.doi.org/10.2337/db08-0799 for a high-quality digital representation of this figure.)

FIG. 5.

Ex-4 treatment improves motor coordination in Huntington's disease mice. Motor performance was measured bi-weekly using an accelerating rotarod apparatus. Mice were placed on the rotarod, which accelerated from 4 to 40 rpm over a 5-min test period, and latency to fall was recorded. A: Average time spent on the rotarod during the course of treatment for each study group is shown. Treatment with Ex-4 significantly increased the rotarod times of Huntington's disease mice (P < 0.05). Ex-4 treatment also increased the rotarod latencies of wild-type mice (P < 0.01). B: Analysis of percent change in rotarod performance from baseline confirmed that the performance of the Ex-4–treated Huntington's disease mice showed a much slower rate of motor control decline than the saline-treated Huntington's disease mice. Hence, at each time trial, Ex-4–treated animals showed a greater time on the rotarod compared with saline-treated animals. C: The relative week-to-week differences (modulus) in rotarod performance (percent change from baseline) between Ex-4–and control-treated Huntington's disease animals expressed as a ratio. Hence, an increase in the ratio denotes a relative increase in maintenance of rotarod performance in the Ex-4–treated animals compared with control. Values are means ± SE, n = 19–24 animals per group.

FIG. 6.

Ex-4 reduces mhtt aggregates in both the cortex and islets of Langerhans and significantly increases life span of Huntington's disease mice. A, left: Western immunoblot analysis with S830, an antibody against the Huntington's disease exon 1 transgene protein, showed that the amount of mhtt aggregation was significantly reduced in cortical tissue of Ex-4–treated Huntington's disease mice, compared with saline-treated controls (P < 0.01). Hsp-70 is a molecular chaperone whose expression is increased in times of cellular stress, and it acts to prevent protein misfolding and aggregation in response to environmental insults or disease. A, right: Western immunoblotting showed that cortical levels of Hsp-70 were slightly elevated with Ex-4 treatment in both Huntington's disease and wild-type mice. Immunostaining of pancreatic tissue with S830 antibody showed that Ex-4 treatment caused a significant decrease in the number of mhtt aggregates in the pancreatic islets of Langerhans. B: There was a significant decrease in the average number of mhtt aggregates per islet (P < 0.001) and an increase in the number of islets containing no mhtt aggregates in the Ex-4–treated Huntington's disease mice (bar charts). Representative images of both Huntington's disease control and Huntington's disease Ex-4–treated islets are shown. The Huntington's disease control mice generally had two types of islets, some very small and others large and irregularly shaped, and both types containing large numbers of mhtt aggregates. B: The small islets in Ex-4–treated Huntington's disease mice had a small number of mhtt aggregates, whereas large islets had a more regular structure and little or no mhtt aggregates. Ex-4 treatment caused a significant increase in the survival of Huntington's disease mice (P < 0.01). C: Ex-4–treated Huntington's disease mice lived an average of 25.3 days longer than saline-treated controls, an 18% increase in life span. Values are means ± SE, n = 21–24 animals per group. **P < 0.01, ***P < 0.001.