Full-length huntingtin levels modulate body weight by influencing insulin-like growth factor 1 expression

Abstract

Levels of full-length huntingtin (FL htt) influence organ and body weight, independent of polyglutamine length. The growth hormone-insulin like growth factor-1 (GH-IGF-1) axis is well established as a regulator of organ growth and body weight. In this study, we investigate the involvement of the IGF-1 pathway in mediating the effect of htt on body weight. IGF-1 expression was examined in transgenic mouse lines expressing different levels of FL wild-type (WT) htt (YAC18 mice), FL mutant htt (YAC128 and BACHD mice) and truncated mutant htt (shortstop mice). We demonstrate that htt influences body weight by modulating the IGF-1 pathway. Plasma IGF-1 levels correlate with body weight and htt levels in the transgenic YAC mice expressing human htt. The effect of htt on IGF-1 expression is independent of CAG size. No effect on body weight is observed in transgenic YAC mice expressing a truncated N-terminal htt fragment (shortstop), indicating that FL htt is required for the modulation of IGF-1 expression. Treatment with 17beta-estradiol (17beta-ED) lowers the levels of circulating IGF-1 in mammals. Treatment of YAC128 with 17beta-ED, but not placebo, reduces plasma IGF-1 levels and decreases the body weight of YAC128 animals to WT levels. Furthermore, given the ubiquitous expression of IGF-1 within the central nervous system, we also examined the impact of FL htt levels on IGF-1 expression in different regions of the brain, including the striatum, cerebellum of YAC18, YAC128 and littermate WT mice. We demonstrate that the levels of FL htt influence IGF-1 expression in striatal tissues. Our data identify a novel function for FL htt in influencing IGF-1 expression.

Figure 1.

Levels of huntingtin modulate body weight and IGF-1 expression. Body weights (A) and whole-brain htt levels (B) of WT, YAC128 (line 53) and YAC18 (line 212) mice were assessed at 3 months of age (body weight: n = 7 for WT and YAC128, and 11 for YAC18; whole-brain htt: n = 3 per genotype). Both the body weights and htt levels of YAC18 mice are significantly higher compared with YAC128 mice, and the body weights and htt levels of YAC128 are significantly higher compared with WT mice. Plasma IGF-1 levels (C) in WT, YAC128 and YAC18 animals were assessed by ELISA at 2 months (n = 19 for WT; 27 for YAC128; 8 for YAC18). Levels of plasma IGF-1 follow the same pattern as levels of FL htt and body weight in these animals, with plasma IGF-1 levels being significantly higher in YAC18 compared with YAC128 mice, and levels of plasma IGF-1 being significantly higher in YAC128 compared with WT mice. To validate these observations in an independent transgenic FL htt mouse model of HD, plasma IGF-1 levels (D) and the body weight (E) were assessed in BACHD mice expressing ∼2 times the levels of FL htt observed in WT (15) compared with littermate control mice at 2 months (n = 13 for WT and 18 for BACHD). Plasma IGF-1 levels and body weights are significantly higher in BACHD mice compared with WT littermate mice. Data are represented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Reducing plasma IGF-1 levels by treatment with 17β-estradiol reduces the body weight of transgenic YAC128 mice. To examine the effect of reducing plasma IGF-1 levels on body weight, YAC128 and WT mice were treated with 17β-ED starting at 7.5 months of age, and their body weight was monitored on a biweekly-basis until 12 months of age. Control-treatment groups received either 17α-ED or placebo. (A) Treatment of YAC128 and WT animals with 17β-ED resulted in significantly lower plasma IGF-1 levels compared with placebo- and 17α-ED-treated controls. (B) At 12 months of age, the body weight of 17β-ED-treated YAC128 animals was significantly lower than that of placebo- and 17α-ED-treated YAC128s animals and did not differ from that of WT animals. (C) As plasma leptin levels per gram of fat in YAC128 and WT animals are not different, plasma levels of leptin were assessed as a measure of fat mass. Plasma leptin levels were significantly higher in placebo-treated YAC128 mice compared with placebo-treated WT mice, consistent with significantly increased fat mass in YAC128 mice. Plasma leptin levels were significantly lower in YAC128 and WT mice treated with 17β-ED compared with their respective placebo- and 17α-ED-treated controls. (D) Plasma htt levels in 17α-ED- and 17β-ED-treated YAC128 animals were measured by FRET assay and normalized to placebo-treated YAC128 animals. Htt levels in 17β-ED-treated YAC128 animals were not different compared with 17α-ED-treated YAC128 animals. Data are represented as means ± SEM; N for (A–C): WT n = 13 for Placebo, 13 for 17α-ED, 7 for 17β-ED; YAC128 n = 19 for Placebo, 19 for 17α-ED, 12 for 17β-ED; N for (D) YAC128 n = 11 for 17α-ED, 8 for 17β-ED; *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference.

Figure 3.

Relationship of IGF-1 expression in the striatum, cortex, cerebellum and hypothalamus to full-length levels of htt. To evaluate the influence of FL htt levels on IGF-1 expression in CNS tissues, IGF-1 mRNA levels were assessed in striatal, cortical, cerebellar and hypothalamic tissue of YAC18 and YAC128 mice. (A) IGF-1 mRNA levels were significantly higher in striatal, but not cortical, cerebellar or hypothalamic tissue of YAC18 mice compared with WT (n = 5–14 for WT; 9–10 for YAC18). (B) Similarly, levels of IGF-1 mRNA were significantly higher in striatal, but not cortical, cerebellar or hypothalamic tissue of YAC128 mice compared with WT (n = 5–12 for WT; 7–11 for YAC128). (C) To validate these measurements of IGF-1 mRNA levels on the protein level, primary striatal, cortical and cerebellar neuronal cultures were prepared from YAC128 and WT pups. Consistent with the increased levels of IGF-1 mRNA in striatal tissue of YAC128 animals, primary striatal neurons derived from YAC128 pups secreted significantly more IGF-1 compared with primary striatal neurons from WT pups. In contrast, the amount of IGF-1 secreted by primary cortical and cerebellar neurons derived from YAC128 pups was similar to that of WT (n = 3–6/genotype). (D) IGF-1 secreted by primary striatal neurons steadily accumulated over time, and was significantly higher in YAC128 cultures at day 6 and day 9 compared with WT cultures (n = 4/genotype). Data are represented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference; Str, striatum; Ctx, cortex; Cbl, cerebellum; Hyp, hypothalamus.

Figure 4.

Reduced IGF-1 expression in cellular and animal models exhibiting reduced htt protein levels. To examine the effect of reduced FL htt levels on IGF-1 expression clonal striatal knock-in STHdhQ7 and STHdhQ111 cells we used. (A) Significantly lower levels of FL htt are detected in the clonal striatal knock-in STHdhQ111 cells (Q111) compared with STHdhQ7 cells (Q7) as assessed by immunoblotting with two different antibodies to htt (n = 3/genotype). (B) Assessment of Q111 cells revealed significantly lower IGF-1 mRNA levels compared with Q7 cells (n = 7/genotype). (C) Similarly, significantly lower levels of IGF-1 protein were detected in culture media of Q111 cells compared with Q7 cells (n = 3/genotype). (D) To examine the impact of reduced htt levels in vivo, R6/2 HD mice which show progressive depletion of endogenous FL htt levels, at 7, 9 and 11 weeks of age were assessed. Significantly reduced plasma IGF-1 levels in R6/2 mice at 11 weeks of age (n = 3/genotype). (E) Consistent with published observations, R6/2 mice began to lose body weight starting at 9 weeks of age, reaching statistical significance at 11 weeks of age (n = 3/genotype). Data are represented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, n.s. = no significant difference.