Conceived and designed the experiments: AD FM PC. Performed the experiments: GC AD FM PC JB CC LP CG AV DR. Analyzed the data: AD FM FS JB CC YL JF DR. Contributed reagents/materials/analysis tools: GC FS CC LP YL CG AV DR. Wrote the paper: AD FM YL JF.
The authors have declared that no competing interests exist.
Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD have not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance (1H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. 1H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide.
Huntington disease (HD) is a neurodegenerative disease with autosomal dominant inheritance. The causal mutation is an abnormally expanded CAG trinucleotide repeat in the first exon of the HD1 gene on chromosome 4p, encoding a polyglutamine stretch in the huntingtin protein. Although transcriptional modifications, excitotoxicity, protein aggregation and loss of normal function of huntingtin, have been hypothesized to be responsible for the symptoms in patients
Despite the ubiquitous expression of mutated huntingtin, only the pathophysiology of brain-related symptoms, which are relatively inaccessible to investigation, have received attention. However, unlike other neurodegenerative disorders, affected HD patients are known to lose weight
Therefore, we conducted a thorough multiparametric investigation exploring the underlying causes of weight loss in a group of patients with no or little signs of the disease, compared to controls. This included the use of a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance (1H NMR) spectroscopy. We observed a systemic metabolic defect involving branched chain amino acids (BCAA) in HD, associated with early weight loss. The role of BCAA in mitochondrial intermediary metabolism may indicate a systemic attempt at compensating for the early energy deficit in HD. These results further support recent data relating altered mitochondrial energy metabolism in HD with defect in the regulation of transcription caused by mutated huntingtin
We included 32 individuals with abnormal CAG repeats expansions (>36) in the HD1 gene (19 women and 13 men, mean age of 42±11 years, ranging from 28 to 80) and 21 controls (13 women and 8 men, mean age of 37±9.5 years, ranging from 27 to 62). In the HD group, 3 subgroups were constituted on the basis of their symptoms determined by the Unified Huntington disease rating scale (UHDRS) score
Height and weight were recorded the day of the clinical examination. The body mass index (BMI) was obtained by dividing weight (in kilograms) by height (in meters) squared. Bioelectrical impedance (Tanita®) was measured to evaluate the lean mass and fat mass of all participants. Since this was a cross-sectional study, changes in body weight were not evaluated prospectively. Instead, the kinetics of body weight was determined by subtracting current weight from the weight recorded in medical records of each participant 5 years before inclusion in the study.
To determine caloric intake accurately, both opened and closed-ended questionnaires were used. The opened-ended questionnaire consisted of a semi-quantitative recording of the patients' regular food and beverage consumption during the three days preceding the consultation. The accuracy of this questionnaire was verified one month later with a closed-ended questionnaire that was sent to the homes of all participants. A series of diagrams was used, reflecting the amount of each class of food and beverage consumed over a 24-hour period. The same methodology was strictly applied to assess caloric intake in HD patients, presymptomatic carriers and controls. In order to interpret changes in body weight, major changes in eating habits during the past 5 years were also recorded, especially diets intended to gain or lose weight. Mean total calories, proteins, lipids and sugar intake for both the HD and control groups were calculated using an automated system (Diaeta software®).
A standardized protocol was designed to thoroughly evaluate all possible causes of weight loss and to avoid biases related to food intake and circadian fluctuations. It included sequentially: (i) a minimal 12-hour fast the night preceding the examination, (ii) collection of blood and urine at the same time (9 am), (iii) ingestion of a standardized meal (450 calories) over a period of 10 minutes, (iv) collection of a second blood sample to determine ghrelin values 1 hour after eating. Samples were stored on ice for immediate analyses or frozen at −80°C for further analyses.
Routine analyses of blood chemistry, including liver and kidney function, were performed. Serum insulin growth factor type 1 (IGF1) concentrations were measured using a specific immunoradiometric assay (IGF1 RIACT, Cis-Bio International, Gif- sur- Yvette, France). The three main axes involved in the regulation of weight balance were explored: (i) inflammation, by measuring of CRP, ESR, IL1β and IL6 by ELISA (Diaclone, Besançon, France), (ii) endocrine function, by measuring (at 9 am), fasting serum cortisol, insulin, tetraiodothyronine (T4L) and thyroid stimulating hormone (TSH) levels, fasting and fed ghrelin (8089-K, Linco Research, St Louis, USA) levels, and fasting leptin levels (HL-81HK, Linco Research, Paris, France); (iii) intermediary metabolism, by measuring plasma amino acids and acylcarnitines, as well as urinary organic acids
For comparison of means, ANOVA or non-parametric tests were used as appropriate (SPSS® software).
Plasma samples were prepared for 1H NMR spectroscopy with minimal handling as described
The 3 groups defined according to UHDRS scores showed similar ages at examination (
Presymptomatic | Early HD | Mild HD | ||
42±2.0 (39–46) | 45.8±4.7 (41–58) | 44.4±2.0 (42–48) | ||
41.4±12.9 (28–80) | 38.2±9.3 (28–53) | 49.3±6.0 (43–60) | ||
4.4±4.5 (0–16) | 10.4±8.5 (0–25) | 9.3±5.5 (3–18) | ||
0.2±0.6 (0–2) | 4.8±3.7 (0–12) | 16.9±5.8 (11–26) | ||
0 | 0 | 2.6±2.6 (0–7) | ||
13±0 (13) | 12.1±0.9 (11–13) | 10.3±2.2 (8–13) |
Chorea subscore 28 maximal, worst, dystonia subscore 20 maximal, worst
The values represent the mean±standard deviations. Ranges are in parentheses. Comparisons of means (ANOVA) were made among the 3 HD groups. UHDRS = Unified Huntington disease rating scale, 120 is the maximal score of severity, up to 4 is considered as normal. The HD group included 15 strictly presymptomatic carriers, 10 patients at an early stage of the disease and 7 mildly affected patients. Ages at examination did not differ significantly among groups; presymptomatic HD carriers were not younger than affected patients. Total functional score was close to maximal in all groups, even in mildly affected patients, in whom the mean TFC was greater than 10. However, the depression score was pathological, even in presymptomatic carriers, and worsened with disease progression.
Retrospective analysis of body weight during the 5 years preceding the study showed a significant weight loss in the HD group compared to controls (p<0.001). The difference remained significant when men (p = 0.002) and women (p = 0.003) were analyzed separately (
Total | Men | Women | |||||||
HD | Controls | HD | Controls | HD | Controls | ||||
−3.3±4.5 (−13 to 6) | 2.8±3.9 (−3 to 10) | −2.8±4.6 (−13 to 2) | 5.2±4.1 (0–10) | −3.6±4.6 (−12 to 6) | 1.5±3.3 (−3 to 10) | ||||
64.4±10.0 (48–89) | 70.1±17.7 (49–112) | 71.05±7.6 (61–88) | 87.9±15.4 (66–112) | 59.85±9.0 (48–78) | 59.2±6.8 (49–68) | ||||
168.7±8.1 (151–185) | 170.3±6.6 (160–183) | 175.7±5.7 (165–185) | 176.2±4.7 (171–183) | 163.8±5.6 (151–177) | 166.7±4.7 (160–175) | ||||
22.6±3.0 (18–29) | 24.0±5.0 (18–38) | 23.05±2.8 (18–29) | 28.35±5.3 (21–38) | 22.3±3.1 (18–29) | 21.3±2.1 (18–26) | ||||
50.7±8.3 (39–68) | 52.7±11.5 (39–72) | 58.9±4.6 (53–68) | 66.2±4.9 (57–72) | 44.4±3.4 (39–51) | 44.4±3.1 (39–48) | ||||
20.9±.8 (11–37) | 24.2±6.4 (13–38) | 12.2±4.8 (7–25) | 21.65±11.6 (9–43) | 14.9±7.2 (6–29) | 14.8±4.5 (9–22) | ||||
2020±530 (985–3040) | 1665±305 (1280–2235) | 2250±376 (1720–2960) | 1760±355 (1280–2235) | 1890±565 (983–3041) | 1610±265 (1330–2130) | ||||
81.3±23.7 (42–121) | 70.0±14.1 (50–102) | 86.3±23.3 (53–121) | 78.6±15.9 (62–102) | 78.4±24.1 (42–117) | 64.6±10.0 (50–86) | ||||
86.5±18.3 (53–120) | 65.7±18.0 (47–126) | 90.4±15.1 (61–114) | 68.9±11.9 (50–90) | 84.2±19.9 (53–120) | 62.4±21.2 (47–126) | ||||
216.0±77.1 (76–375) | 191.3±48.6 (123–310) | 257.5±60.2 (186–375) | 196.0±68.4 (123–310) | 192.0±76.8 (76–372) | 188.4±34.4 (130–256) |
The values represent the mean±standard deviations. Ranges are in parentheses. The HD group was compared to controls (ANOVA).
Determined retrospectively for a 5-year period.
Determined by bioelectric impedance.
Determined from a 3-day and a 24-hour questionnaire performed at a one-month interval. Weight loss was significant in the HD group, in both men and women. Despite significantly higher calories intake, carriers of huntingin mutations did not have a higher BMI (body mass index). In men, BMI and lean body mass were lower in the HD group despite higher food intake. These nutritional analyses demonstrate the existence of a hypermetabolic state in the early stages of HD.
ERS, CRP, serum interleukins IL 1ß (0.96±3.82 pg/ml versus 0.28±0.77 pg/ml) and IL 6 (0.54±2.50 pg/ml versus 0.39±0.89 pg/ml), on the one hand, and serum fasting cortisol (13.4±4.4 µg/100 ml versus 16.7±6.8 µg/100 ml), insulin (5.5±2.2 µU/ml versus 6.0±3.6 µU/ml), T4L (14.7±2.2 pmol/l versus 16.4±1.9 pmol/l) and TSH (1.92±0.90 mUI/l versus 1.80±0.65 mUI/l), on the other hand, were similar in the HD group and controls. Therefore, common causes of hypercatabolic state, i.e. inflammation and classical endocrine dysfunctions, were excluded. There was no glycosuria, and fasting blood glucose levels were in the normal range in the HD group (4.7±0.4 mmol/l).
PLS analyses distinguished HD individuals at different stages of the disease as shown in
Three groups of presymptomatic, early and mildly affected HD patients were constituted on the basis of their UHDRS scores, as described in the
Metabolic profiles of plasma from early affected HD patients were then compared to those of presymptomatic carriers, those of mildly affected HD patients to early HD patients. The spectral region that contributed to differences among the HD groups determined from PLS contribution plots is shown in
PLS-contribution plots allow comparison of plasma metabolic profiles in early affected HD patients and presymptomatic carriers. NMR variables that have the greatest weight (w*1; scaled in units of standard deviation), therefore contributing most to the separation between HD groups, are decreased concentrations (>2SD) of metabolites located between 0.85 and 1.0 ppm: valine, leucine and isoleucine. A similar contribution plot was obtained when plasma metabolic profiles from mildly affected HD patients were compared to early HD patients (data not shown).
To confirm that BCAA are affected in HD, we also measured their concentrations in plasma by ion exchange chromatography. Valine, leucine and isoleucine levels were significantly lower in the HD group compared to controls (209±47 µmol/l versus 245±44 µmol/l, p = 0.009; 118±24 µmol/l versus 144±23 µmol/l, p<0.001 and 56±12 µmol/l versus 68±15 µmol/l, p = 0.002, respectively). In addition, the levels of each BCAA were correlated with the observed weight loss in the patients (p = 0.007, 0.003 and 0.017), and, more importantly, negatively correlated with UHDRS values (p = 0.017, <0.0001 and 0.003) in both men (p = 0.035, 0.019 and 0.036) and women (p = 0.007 for leucine and 0.01 for isoleucine). The number of CAG repeats was also negatively correlated with valine (p = 0.015), leucine (p = 0.018) and isoleucine (p = 0.020). Although the BMI values of women with HD were similar to those of controls, they had significantly lower leucine (109±19 µmol/l versus 134±22 µmol/l, p = 0.002) and isoleucine (53±10 µmol/l versus 65±16 µmol/l, p = 0.014) levels (
The concentrations of valine, leucine and isoleucine in plasma were determined by ion exchange chromatography. Comparisons of means (ANOVA) were made between men or women with HD and their respective controls. In men, there was a significant decrease of valine, leucine and isoleucine in the HD group. In women, similar results are observed for leucine and isoleucine.
All other metabolic markers were similar in the HD group and in controls. However, IGF1 levels were significantly lower in the HD group than in controls (175±48 ng/ml versus 210±47 ng/ml, p = 0.011) and negatively correlated with UHDRS scores (p = 0.004). IGF1 levels were also correlated with leucine (p = 0.04) and isoleucine (p = 0.02) levels. There was no correlation between IGF1 levels and parameters associated with weight (BMI, lean and fat body mass) or caloric intake.
In men, fasting and postprandial ghrelin levels were higher in the HD group than in controls (1797±719 pg/ml versus 1077±453 pg/ml, p = 0.024 and 1365±538 pg/ml versus 860±291 pg/ml, p = 0.021 respectively). However, women with HD and controls had similar BMI values. Accordingly, ghrelin levels were negatively correlated with BMI (p = 0.008) but not with the UHDRS score. Fasting ghrelin also correlated with caloric intake in men (p = 0.029), consonant with its orexigenic activity. Importantly, the ratio between pre- and post-prandial ghrelin levels were strictly identical in the HD group compared to controls (1.35 versus 1.36) indicating that the negative feedback on hypothalamus induced by food intake is functional in HD. Leptin levels, which vary independently but inversely to ghrelin, were lower in men with HD compared to controls (4.4±3.3 ng/ml versus 12.4±11.5 ng/ml, p = 0.028), but not in women, and were correlated with weight (p<0.001) and fat body mass (p<0.001), but not with the UHDRS score.
This study is the first comprehensive investigation of the physiopathology of weight loss in HD. We retrospectively confirmed that weight loss begins early in the disease
Our 1H NMR spectroscopic analyses showed that a systemic metabolic defect is associated with the status of HD carriers, as well as disease progression. A selective decrease in branched chain amino acids plasma concentrations distinguished presymptomatic carriers from patients at an early stage of the disease and patients at an early stage from mildly affected patients. Decreased plasma BCAA levels were previously documented in more severely affected HD patients, but the authors did not evaluate the patients' diet or weight
The implication of BCAA in mitochondrial intermediary metabolism, both in brain and peripheral tissues, further supports an important role for energy deficit in HD. A reduction in ATP production was shown in brain of HD mice, including presymptomatic mice
BCAT (branched-chain aminotransferase) is the first enzyme involved in BCAA catabolism and is preferentially expressed in muscle. Since altered energy metabolism has been shown in muscle of HD patients
Finally, our study showed that low plasma BCAA levels are associated with reduced IGF1 levels in the HD group despite a higher protein and caloric intake than controls. The tight connection between IGF1 and essential amino acids has been repeatedly demonstrated
This study provides the first demonstration of a systemic metabolic defect in HD, associated with early weight loss. Plasma BCAA represent a promising and accessible biomarker that may be useful for the detection of disease onset and for monitoring therapeutic trials in patients at an early stage of the disease. Decreased plasma BCAA levels in HD strongly suggest a critical need in the brain for citric acid cycle substrates provided by peripheral organ metabolism.
The authors gratefully acknowledge Alexis Brice, Merle Ruberg and Charles Roe for their helpful suggestions. They also acknowledge Anne Faudet for her expert technical assistance and Anne Desfarge for her help with the dietary assessment of both the controls and HD patients.
This study was approved by the Committee de Protection des Personnes (CPP) from the Pitié-Salpêtrière Hospital Paris (France) on October 18, 2006 (project n# 17-06).