Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice

Abstract

In addition to neurological deficits, Huntington's disease (HD) patients and transgenic mice expressing mutant human huntingtin exhibit reduced levels of brain-derived neurotrophic factor, hyperglycemia, and tissue wasting. We show that the progression of neuropathological (formation of huntingtin inclusions and apoptotic protease activation), behavioral (motor dysfunction), and metabolic (glucose intolerance and tissue wasting) abnormalities in huntingtin mutant mice, an animal model of HD, are retarded when the mice are maintained on a dietary restriction (DR) feeding regimen resulting in an extension of their life span. DR increases levels of brain-derived neurotrophic factor and the protein chaperone heat-shock protein-70 in the striatum and cortex, which are depleted in HD mice fed a normal diet. The suppression of the pathogenic processes by DR in HD mice suggests that mutant huntingtin promotes neuronal degeneration by impairing cellular stress resistance, and that the body wasting in HD is driven by the neurodegenerative process. Our findings suggest a dietary intervention that may suppress the disease process and increase the life span of humans that carry the mutant huntingtin gene.

Figure 1

DR delays the onset of motor dysfunction, reduces weight loss, and increases the survival of HD N171-82 Q mice. (a) Eight-week-old male HD mice were maintained on an alternative day fasting regimen (DR) or an AL feeding regimen for 3 mo. Onset of disease was scored as the first date when limb tremors were detected. Values are the mean and SE (n = 12 mice per group); *, P < 0.001 (paired t test). (b) Survival plots for HD mice maintained on AL or DR diets. (c) Body weights for HD mice maintained on AL or DR diets. Values are the mean and SE (12 mice per group). (d and e) Motor behavioral performance was evaluated by using a rotarod apparatus in 16- and 20-wk-old nontransgenic (Non-Tg) and HD mice maintained on AL or DR diets. The number of times the mouse fell from the rod (d) and the total time spent on the rod (e) during a 5-min period were recorded. Values are the mean and SE (n = 6–15 mice per group). *, P < 0.01, compared with the non-Tg AL value; **, P < 0.01 compared with the HD AL value (ANOVA with Scheffé post hoc tests).

Figure 2

DR reduces brain atrophy, decreases huntingtin aggregate formation, and inhibits caspase-1 activation in HD mice. (a) Photomicrographs of brain sections of 20-wk-old HD mice and nontransgenic mice (Non-Tg) that had been maintained on AL or DR regimens for 3 mo. Brain sections were stained with cresyl violet. The enlargement of lateral ventricles in HD mice is reduced in HD mice maintained on DR. Scale bar: 4 mm. The examples presented are representative of all six mice examined in each group. (b) Brain sections from 20-wk-old mice were immunostained with EM-48 antibody, which recognizes mutant human huntingtin N-terminal fragment. Intranuclear inclusions of mutant huntingtin (arrows) were abundant in the cortex (bA and bB) and striatum (bE and bF) of HD mice fed AL. The number of cells with intranuclear inclusions was decreased in the cortex (bC and bD) and striatum (bG and bH) of HD mice that had been maintained on DR compared with those fed AL. Scale bars: 125 μm in bG (bA, bC, and bE), 50 μm in bH (bB, bD, and bF). The results presented are representative of all six mice examined in each group. (c and d) Immunoblots of proteins in samples of cortical and striatal tissue (50 μg per lane) from the indicated genotype and diet groups. (Upper) Representative blots with caspase-1 antibody, which recognize full length of caspase-1 (45 kDa) and cleavage product (33 kDa) in cortex (c) and striatum (d). (Lower) Densitometric analysis results of blots (n = 4 mice per group). *, P < 0.01 compared with the value for nontransgenic (NT)-AL 33-kDa cleavage product; **, P < 0.01 compared with the value for the HD-AL 33-kDa cleavage product. ANOVA with Scheffé post hoc tests.

Figure 3

DR ameliorates hyperglycemia in HD N171-82Q mice. Eight-week-old male HD mice and nontransgenic control mice (Non-Tg) were maintained on DR or AL feeding regimens for 3 mo. Blood glucose levels were measured either under feeding conditions (a and c) or after an overnight fast (b and d) in 16-wk-old mice (a and b) and 20-wk-old mice (c and d). The values are the mean and SE of determinations made in six mice. *, P < 0.01 compared with the value of nontransgenic (NT)-AL group. **, P < 0.01 compared with the value of HD-AL group. ANOVA with Scheffé post hoc tests. (e) Mice were fasted overnight and then administered d-glucose orally (2 g/kg body weight); blood samples were taken before and at 30, 60, and 120 min after glucose administration. The values are the mean and SE of determinations made in six mice per group.

Figure 4

DR normalizes BDNF levels and up-regulates HSP-70 protein levels in brains of HD N171-82Q mice. Eight-week-old male HD mice and nontransgenic mice (NT) were maintained on DR or AL feeding regimens for 3 mo. (a and b) BDNF protein levels were measured in striatal (a) and cortical (b) tissues by ELISA. The values are the mean and SE, n = 6. *, P < 0.01, **, P < 0.001 compared with value for NT-AL; #, P < 0.01 compared with the value for HD-AL. ANOVA with Scheffé post hoc tests. (c and d) HSP-70 protein levels were measured in striatal (c) and cortical (d) tissues by immunoblot analysis. (Upper) Representative immunoblots. (Lower) The densitometric analysis of samples from four different mice per group. The values are the mean and SE. *, P < 0.01 compared with the value for the corresponding AL group; #, P < 0.01 compared with the value for the NT-AL group. ANOVA with Scheffé post hoc tests.