Insulin and IGF-1 regularize energy metabolites in neural cells expressing full-length mutant huntingtin
Introduction
Huntington's disease (HD) in an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG triplets in the HTT gene, encoding for mutant huntingtin (mHTT in humans, or mHtt in mice). HD clinical symptoms include involuntary movements, dementia, dramatic weight loss, and eventually death. Neuropathologically, HD is characterized by selective dysfunction and death of GABAergic projection medium spiny neurons in the striatum. Moreover, the degree of striatal atrophy correlates with the degeneration of cerebral cortex during the latest stages (Gil and Rego, 2008). Although striatal death underlies many symptoms in advanced stages of the disease (Vonsattel and DiFiglia, 1998), early deficits, which seem to occur years before the evident movement disorder, are more likely associated with neuronal and synaptic dysfunction in the cortex (e.g. Rosas et al., 2005). An expansion of more than 39 CAG repeats in the HTT gene underlies several mechanisms of neurodegeneration, such as oxidative stress, mitochondrial dysfunction, and deficits in energy metabolism (Naia et al., 2011). In particular, mHTT impairs mitochondrial respiration and ATP production (Milakovic and Johnson, 2005, Seong et al., 2005, Silva et al., 2013). Elevated levels of lactate were also detected in the striatum and cerebral cortex of HD patients (Koroshetz et al., 1997, Jenkins et al., 1998), as well as decreased ATP and phosphocreatine (PCr) levels following glycolysis inhibition in HD cybrids, exhibiting bioenergetically dysfunctional mitochondria (Ferreira et al., 2011). Additionally, a diversity of metabolic enzymes are altered in HD, namely, pyruvate dehydrogenase (PDH) (Perluigi et al., 2005, Ferreira et al., 2011), pyruvate carboxylase (PC) (Butterworth, 1986, Lee et al., 2013), glucose-6-phosphate dehydrogenase (Ferreira et al., 2011), aconitase (Tabrizi et al., 2000), and aspartate aminotransferase (Perluigi et al., 2005).
During the last few years, our group analyzed the effects of insulin and insulin-like growth factor 1 (IGF-1) in HD, from peripheral to neural cells and also in in vivo models. The insulin/IGF-1 signaling has been studied for many years in several neurodegenerative disorders linked to toxic protein aggregation, but it has generated conflicting results. While some studies demonstrated that IGF-1 modulates the clearance of brain aggregation-prone proteins, such as amyloid-β peptide (Carro et al., 2002) or mHTT (Humbert et al., 2002), other authors claimed that reduced insulin/IGF-1 signaling regulate disaggregation of amyloid-β peptide, ataxin-3, and other polyglutamine proteins to promote cell survival (Morley et al., 2002, Cohen et al., 2006, Kapperler et al., 2008, Teixeira-Castro et al., 2011). Indeed, IGF-1 signaling was described to be deleterious in the regulation of lifespan in nematodes (Kenyon et al., 1993) and mouse models (Holzenberger et al., 2003); however, the ablation of IGF-1 or its receptor promoted brain growth retardation (D'Ercole et al., 2002), indicating that IGF-1 may stimulate neuronal development. A more recent study found no measurable effect between the inhibition of insulin/IGF-1 pathway and the decreased mHTT aggregation (Jakubik et al., 2014). Even so, IGF-1 plasma levels were shown to be increased in YAC128 mice (expressing human full-length mHTT), correlating with increased body weight (Pouladi et al., 2010), and high IGF-1 levels were associated with cognitive decline in HD patients (Saleh et al., 2010).
Counterweighting the previous data, recent findings in our group showed that peripheral administration of IGF-1 prevented metabolic abnormalities in a hemizygous R6/2 mouse model of HD, such as impaired glucose tolerance and age-related decrease in body weight by enriching blood insulin and IGF-1 levels (Duarte et al., 2011). Indeed, both insulin and IGF-1 may activate insulin/IGF-1 receptor (IR/IGF-1R), stimulating PI-3K/Akt signaling pathways, through direct phosphorylation of HTT by Akt at serine 421 (Humbert et al., 2002), which appears to promote mitochondrial function and modulate the expression of proteins involved in glucose metabolism and anti-apoptotic mechanisms (Duarte et al., 2008, Naia et al., 2015). The activation of the same pathway also counteracted the increase in lactate/pyruvate ratio in both YAC128 mice (Lopes et al., 2014) and HD human lymphoblasts (Naia et al., 2015). In addition, we showed that the activation of IGF-1/insulin signaling pathways in striatal cells expressing 111 glutamines precludes mitochondrial generation of reactive oxygen species and mitochondrial dysfunction, largely reducing apoptotic and senescent cells induced by mHtt expression (Ribeiro et al., 2014). Nevertheless, it remained unknown whether the improvement in mitochondrial function would also be linked to an improvement in cell bioenergetics. Therefore, we hypothesized that insulin or IGF-1 treatment could ameliorate metabolic function in the context of HD. Here, we show that both insulin and IGF-1 can rescue energy deficits in YAC128 primary cortical cells by ameliorating ATP, PCr, and lactate levels, the later involving modified lactate dehydrogenase (LDH) activity. Furthermore, IGF-1 alleviates anomalous pyruvate levels in homozygous STHdhQ111/Q111 striatal cells derived from HD knock-in mice.
Section snippets
Materials
Fetal bovine serum (FBS), B27 supplement and penicillin/streptomycin were from Gibco (Paisley, Scotland, UK). Insulin from porcine pancreas, DMEM medium, trypsin-type IX-S from porcine pancreas, IGF-1, glucose-6-phosphate, deoxy-d-glucose (DG), l-glutamine, poly-l-lysine, fatty acid free bovine serum albumin (BSA), trypan blue (0.4%), resazurin, adenine dinucleotide phosphate hydrate (NADP), nicotinamide adenine dinucleotide hydrate (NAD), ADP, glucose 6 phosphate dehydrogenase (G6P-DH),
Insulin and IGF-1 restore energy metabolic parameters in primary cortical cultures derived from YAC128 mice
We started by characterizing the energy levels in YAC128 versus wild-type mice primary cortical cells exhibiting similar levels of HTT protein (Supplementary Fig. S1). In these cells, we found a significant decrease in ATP/ADP ratio (Fig. 1A) that was accompanied by an increase in AMP levels (data not shown). Decreased ATP/ADP ratio was restored after insulin or IGF-1 (1 nM) exposure (Fig. 1A). Importantly, due to structural homology, insulin and IGF-1 can bind and activate both IR and IGF-1R,
Discussion
Under a great controversy (Carro et al., 2002, Morley et al., 2002, Cohen et al., 2006, Kapperler et al., 2008), insulin and IGF-1 have demonstrated important effects in the central nervous system, including neuronal survival, learning and memory, and animal life span (D'Ercole et al., 2002, Zemva and Schubert, 2014). Moreover, these peptides have been previously described to stimulate glucose uptake and intracellular metabolism in peripheral tissues (Andreassen et al., 2002, Duarte et al., 2011
Conflict of interest
The authors declare that there are no conflict of interests regarding this study.
Acknowledgements
This work was supported by “Fundação para a Ciência e Tecnologia” (FCT), Portugal, grant referencePTDC/SAU-FCF/66421/2006 and PTDC/SAU-FCF/108056/2008, and co-financed by COMPETE-“Programa Operacional Factores de Competitividade”, QREN, and the European Union (FEDER-“Fundo Europeu de Desenvolvimento Regional”). CNC was supported by project PEst-C/SAU/LA0001/2013-2014. L. Naia, M. Ribeiro, and C. Lopes are/were supported by Ph.D. fellowships from FCT (SFRH/BD/86655/2012, SFRH/BD/41285/899/2007,
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The authors contributed equally for this work.