Journal of Cystic Fibrosis 12 (2013) 271 – 276 www.elsevier.com/locate/jcf

Original Article

Hypertriglyceridemia is associated with insulin levels in adult cystic fibrosis patients☆ Marie-Claire Ishimo a , Linda Belson a, b , Sophie Ziai a, b , Emile Levy b, e , Yves Berthiaume Lise Coderre a, c , Rémi Rabasa-Lhoret a, b, c, d, f,⁎

c, d

,

a

Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada b Department of Nutrition, Université de Montréal, Montréal, Québec, Canada c Department of Medicine, Université de Montréal, Québec, Canada d Cystic Fibrosis Clinic and Research Center, Centre hospitalier de l'Université de Montréal, Montréal, Québec, Canada e Research Centre, Centre hospitalier universitaire Sainte-Justine, Université de Montréal, Montréal, Québec, Canada f Montreal Diabetes Research Centre, Montréal, Québec, Canada Received 14 March 2012; received in revised form 14 May 2012; accepted 21 August 2012 Available online 25 September 2012

Abstract Background: Recent studies have identified hypertriglyceridemic cystic fibrosis patients (CF-TG). However, whether hypertriglyceridemia is associated with an altered metabolic profile remains unknown. Objective: To characterize CF-TG and determine whether triglycerides (TG) levels are associated with metabolic alterations. Methods: 210 adult CF subjects from the Montreal Cystic Fibrosis Cohort without known diabetes were included in the analysis. All subjects underwent an OGTT to assess glucose tolerance, insulin secretion (insulin AUC) and insulin sensitivity (Stumvoll index). Fasting lipid profiles, pulmonary function (%FEV1) and BMI were determined. Hypertriglyceridemia (TG N 1.7 mmol/L) was observed in 20 CF patients. These subjects were matched for age, sex and glucose tolerance category with 20 CF patients (CF-normal-TG) and 20 healthy controls that had TG levels below 1.7 mmol/L. Pearson correlations were performed in the complete study sample (n = 210) to examine the associations between TG levels and other parameters. Results: The prevalence of hypertriglyceridemia was 9.5%. Compared to CF-normal-TG, CF-TG subjects displayed significantly higher %FEV1, insulin AUC (AUC0–120, AUC0–30, AUC30–120), cholesterol levels and a higher ratio of total cholesterol to HDL-cholesterol. Pearson analysis demonstrated that TG levels were associated with BMI, %FEV1, fasting insulin, insulin AUC0–120 and AUC30–120, Stumvoll index, cholesterol levels and the ratio of total cholesterol to HDL-cholesterol. All these correlations remained significant after correction for BMI except %FEV1. Conclusion: TG levels are associated with a mild alteration of the metabolic profile. Whether these changes will increase the long-term risk of CF patients in developing cardiometabolic complications remains to be investigated. © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Dyslipidemia; Triglycerides; Insulin secretion; Insulin sensitivity; Body mass index; Lung function

1. Introduction ☆ Authors' roles: conceived and design of the study: YB, LC, RRL, performed the experiments: M-CI, LB, SZ; analyzed the data: M-CI, LB, LC; wrote the manuscript: MCI, LC, critically review the manuscript: EL, YB, RRL. ⁎ Corresponding author at: Institut de recherches cliniques de Montréal, 110 avenue des Pins Ouest, Montréal, QC, Canada, H2W 1R7. Tel.: + 1 514 987 5666; fax: + 1 514 987 5670. E-mail address: [email protected] (R. Rabasa-Lhoret).

Cystic fibrosis (CF) is the most common lethal autosomal disease affecting the Caucasian population with approximately one case per 3608 live births in Canada in 2000 [1]. With advances in clinical and nutritional therapy, the life expectancy of CF patients has gradually increased to reach a median survival of 37 years [2]. Increased longevity has led to an increased

1569-1993/$ -see front matter © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2012.08.012

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prevalence of CF-related co morbidities, including CF-related diabetes (CFRD). As the life expectancy of CF patients increases, we may start to observe additional cardiometabolic complications [3]. In most CF subjects, first-phase insulin secretion is absent leading to increased glucose excursions after a glucose load even in individuals with normal glucose tolerance (NGT) [4]. In addition, previous studies have reported that some CF patients displayed dyslipidemia. Figueroa et al. [5] reported that 16% of CF patients were hypertriglyceridemic whereas Rhodes et al. [6] found a prevalence of 5%. In addition, a small percentage of CF subjects displayed hypercholesterolemia (4%) [5]. Dyslipidemia is an important predictor of cardiovascular disease and is associated with an altered metabolic profile including glucose and insulin levels and insulin resistance [7–10]. Alterations of both glucose and lipid metabolism may favor the development of cardiometabolic complications in CF subjects [3]. The aim of this study was to characterize CF adults with hypertriglyceridemia and determine whether TG levels were associated with alterations of the cardiometabolic profile.

(FEV1) and predicted %FEV1 (Medgraphic 1870, St. Paul, MN, USA). Genotype status and pancreatic insufficiency were extracted from the medical files. To calculate BMI (kg/m 2), body weight and standing height were measured using an electronic scale (Tanita Corporation, Arlington Heights, IL, USA) and a wall stadiometer, respectively. 2.4. Oral glucose tolerance test (OGTT) All subjects underwent a 2-h OGTT. After an overnight fast, they ingested in less than 5 min, a glucose solution of 1.75 g/kg of body weight to a maximum of 75 g and glucose tolerance categories were determined according to the Canadian Diabetes Association Guidelines [11]. Blood samples were taken at 0, 30, 60, 90, and 120 min to measure plasma glucose and insulin levels. Plasma glucose concentrations were measured immediately in duplicate by the glucose oxidase method (YSI Glucose Analyzer 2, YSI Incorporated Life Sciences, Yellow Springs, OH, USA). Insulin levels were quantified in duplicate using a human insulin radioimmunoassay (Linco Research, Inc., St-Charles, MO, USA).

2. Materials and methods 2.5. Insulin secretion and insulin sensitivity assessment 2.1. Subjects The Montreal Cystic Fibrosis Cohort was established in 2004 as part of an ongoing systematic screening program to detect CFRD using the oral glucose tolerance test (OGTT) [4]. The main objective of this prospective ongoing observational cohort is to study mechanisms leading to glucose intolerance in adult CF patients as well as the association of pre-diabetic states with CF outcomes. The Research Ethics Committee of the Centre hospitalier de l'Université de Montréal (CHUM) and the Institut de recherches cliniques de Montréal approved the protocol and all subjects signed a written consent form. At the time of the analysis for this study (May, 2011), 210 CF subjects had been enrolled. We recruited 20 NGT control subjects of comparable age and body mass index (BMI). All subjects were over 18 years of age. CF patients were excluded if they had an exacerbation in the previous month, lung transplant, pregnancy, or had received oral or intravenous steroids therapy. Patients already diagnosed with CFRD were excluded from the study. 2.2. Subject classification Hypertriglyceridemia was defined as TG concentrations N 1.7 mmol/L (150 mg/dL) according to the Canadian Cardiovascular Society Guidelines [8]. Hypertriglyceridemic CF (CF-TG) patients were matched with the nearest-aged CF patients with normal TG (CF-normal-TG) subjects of the same sex and glucose tolerance category and were compared to healthy control subjects without CF or hypertriglyceridemia. 2.3. Clinical protocol Pulmonary function was measured by spirometry on the day of the OGTT using forced expiratory volume in 1 sec (L/s)

The area under the curve (AUC) for both glucose and insulin was calculated with Graph Pad Prism 4.0 for Windows. We used insulin AUC0–120, AUC0–30 and AUC30–120 as indices for total, early and late insulin secretion respectively [12]. Insulin sensitivity was calculated with the Stumvoll index 2001 (0.156 − 0.0000459 ∗ Ins120 (pmol/L) − 0.000321 ∗ Ins0 (pmol/L) − 0.00541 ∗ Glyc120 (mmol/L)) [13]. 2.6. Biochemical dosages TG, total cholesterol, and HDL-cholesterol were measured from fasting blood samples by enzymatic reaction (ADVIA1650, Bayer Health Care Diagnostics). LDL-cholesterol was calculated from the Friedewald equation [14]. LDL-cholesterol was not calculated for one individual because their TG was N 4.5 mmol/L. In the absence of other cardiovascular risk factors, the Canadian Cardiovascular Society recommends the following values to define a healthy lipid profile: TG b 1.7 mmol/L, total cholesterol b 5.17 mmol/L, LDL-cholesterol b 5 mmol/L, HDL-cholesterol N 1.03 mmol/L for men and N 1.30 mmol/L for women. 2.7. Statistical analysis Data are presented as mean ± standard deviation (SD). TG, fasting insulin, glucose AUC and insulin AUC (0–30; 30–120; 0–120) failed the Levene's homogeneity test of variance and were log transformed for the analysis. Group comparisons were performed by independent t-test and one-way ANOVA followed by LSD post-hoc test as appropriate. The association between TG levels and other variables were analyzed by Pearson correlation. We used SPSS for Windows (Version 16.0 SPSS, Chicago, IL) for statistical analysis. Significance was accepted at p b 0.05.

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Table 2 Characteristics of control and cystic fibrosis (CF) patients with normal triglyceride levels (CF normal-TG) and CF patients with hypertriglyceridemia (CF-TG).

3. Results 3.1. Subjects characteristics Baseline characteristics of the 210 CF subjects included in the study are presented in Table 1. TG levels above 1.7 mmol/L were found in 20 patients (9.5% of the cohort), 12 patients (5.7% of the cohort) had elevated total cholesterol concentrations (N 5.17 mmol/L) while three patients had both elevated TG and total cholesterol levels (1.4% of the cohort). The 20 CF-TG patients were then matched for age, sex, and glucose tolerance category with CF patients with normal TG levels (CF-normal-TG) and with healthy NGT controls for sex and age. As shown in Table 2, CF-TG and CF-normal-TG subjects displayed similar genotype, enzyme supplementation, and BMI. On the other hand, CF-TG showed an 18% higher %FEV1 compared to CF-normal-TG subjects. 3.2. Lipid profile As shown in Table 3, levels of HDL and LDL cholesterol were similar among the three groups. On the other hand, CF normal-TG subjects displayed lower total cholesterol levels than either CF-TG or control subjects while CF-TG subjects showed a higher ratio of total cholesterol to HDL cholesterol compared to CF normal-TG and control subjects. 3.3. Plasma glucose and insulin profiles during OGTT CF-TG subjects presented significantly higher fasting glucose levels compared to control subjects (Table 3). As illustrated in Table 1 Baseline descriptive characteristics of cystic fibrosis subjects (n = 210). Characteristics

Mean ± SD

Age (years) FEV1% (L/s) Sex (% women) Genotype Δ508 homozygote (%) Δ508 heterozygote (%) Other mutations (%) Glucose tolerance category Normal (%) Intolerant (%) Cystic fibrosis related diabetes (%) Pancreatic insufficiency (%) Body mass index (kg/m2) Fasting glucose (mmol/L) Glucose area under the curve Fasting insulin (μU/ml) Insulin area under the curve Stumvoll index Triglycerides (mmol/L) Cholesterol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Cholesterol/HDL cholesterol

26 ± 8 73 ± 22 46.6

Range 18–52 19–119

47.3 39.8 10.9 57.1 28.3 14.6 79.9 22.3 ± 3.0 5.0 ± 0.9 470 ± 229 10.0 ± 4.2 3622 ± 2141 0.08 ± 0.02 1.08 ± 0.58 3.54 ± 0.96 1.20 ± 0.31 1.85 ± 0.77 3.02 ± 0.73

%FEV1: predicted forced expiratory volume in 1 s.

Characteristics

Control N = 20

CF normal-TG N = 20

CF-TG N = 20

Age (years) BMI %FEV1 (L/s) Sex (men/women) Pancreatic insufficiency (%) Genotype Δ508 homozygote Δ508 heterozygote Other mutations Glucose tolerance category Normal Intolerance Cystic fibrosis-related diabetes

26 ± 4 22.2 ± 1.8 – 8/12 –

26 ± 8 21.0 ± 2.3 67.2 ± 22.6 6/14 75%

26 ± 9 22.7 ± 3.9 82.6±23.6* 6/14 75%

15 3 2

9 7 4

6 10 4

6 10 4

Values are mean ± SD. BMI: body mass index, %FEV1: predicted forced expiratory volume in 1 s. p b 0.05.

Fig. 1A, both CF-TG and CF normal-TG subjects had similar glucose excursions during the OGTT. They also exhibited significantly higher glucose excursions compared to controls as highlighted by their higher glucose AUC (Table 3). Next, we examined insulin levels and insulin AUC. As shown in Table 3, there was no significant difference in fasting insulin levels between controls, CF normal-TG and CF-TG. Compared to controls, CF-TG subjects display a 48% decrease in insulin AUC0–30 but similar insulin AUC30–120 and insulin AUC0–120 (Fig. 2A). Comparison of insulin AUC among CF groups showed that CF-TG showed a 71%, 74%, and 73% increase in insulin AUC0–30, AUC30–120, and AUC0–120 respectively compared to CF normal-TG. We next examined insulin sensitivity using the Stumvoll index. As shown in Fig. 2B, control subjects displayed a higher insulin sensitivity index then either CF-TG or CF normal-TG patients. While we observed slightly lower insulin sensitivity in CF-TG compared to CF normal-TG, this difference did not reach significance (p = 0.1).

Table 3 Metabolic characteristics of control and cystic fibrosis (CF) patients with normal triglyceride levels (CF normal-TG) and CF patients with hypertriglyceridemia (CF-TG).

13–31 3.0–9.0 89–1199 2.0–36.5 416–11906 0.02–0.13 0.35–5.05 1.72–7.06 0.59–2.28 0.63–4.87 1.6–5.6

Characteristics

Control N = 20

CF normal-TG N = 20

CF-TG N = 20

Triglycerides (mmol/l) Fasting glucose (mmol/l) Glucose area under curve Fasting insulin (μU/ml) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Ratio total cholesterol /HDL

0.87 ± 0.32 4.8 ± 0.6 158 ± 71 12.4 ± 6.0 4.05 ± 0.55 1.47 ± 0.39 2.19 ± 0.58 2.9 ± 0.9

0.78 ± 0.29 5.3 ± 0.5 533 ± 229 § 9.7 ± 2.7 3.38 ± 0.98 §, ¥ 1.26 ± 0.32 1.76 ± 0.80 2.7 ± 0.6

2.39 ± 0.75 ⁎, § 5.34 ± 0.5 § 500 ± 177 § 12.3 ± 5.1 4.16 ± 0.96 1.25 ± 0.36 1.89 ± 0.82 3.5 ± 0.7 ⁎

HDL: high-density lipoprotein, LDL: low-density lipoprotein. § p b 0.05 compared to control. ⁎ p b 0.05 compared to CF normal-TG. ¥ p b 0.05 compared to CF-TG.

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A

A 10000

Control CF-TG

8 6

*

CF-TG

6000 4000 2000

* *

*

0 0-30

2

30-120

0-120

Insulin AUC 0

30

60

90

120

Control

80

CF

70

CF-TG

60 50

0.08

0.04

0.00 Control

40 30 20 10 0

*

0.12

Insulin sensitivity (Stumvoll)

90

*

B

Time (min)

Plasma insulin (µU/ml)

8000

4

0

B

*

Control CF

CF

10

Arbitrary Units

Plasma glucose (mmol/L)

12

0

30

60

90

120

CF

CF-TG

Fig. 2. (A) Insulin area under the curve (AUC) in control, cystic fibrosis patients with normal TG (CF) and cystic fibrosis patients with TG above 1.7 mM (CF-TG). *p b 0.05. (B) Stumvoll index of insulin sensitivity in control, cystic fibrosis subjects with normal TG (CF) and cystic fibrosis subjects with TG above 1.7 mM (CF-TG). *p b 0.05.

Time (min) Fig. 1. Plasma glucose (A) and insulin (B) profile in control, cystic fibrosis patients with normal TG levels (CF) and cystic fibrosis patients with TG above 1.7 mM (CF-TG).

To further examine the relationship between TG levels and insulin secretion and insulin sensitivity, we used Pearson correlations in the complete study sample (n = 210). As shown in Table 4, TG levels were not associated with age. On the other hand, they were positively associated with BMI, %FEV1, total cholesterol levels, total cholesterol to HDL cholesterol ratio, fasting insulin, insulin AUC0–120 and AUC30–120 but not with insulin AUC0–30. In addition, we observed a negative correlation between the Stumvoll index of insulin sensitivity and TG levels. Because TG levels correlated with BMI, we examined whether these associations were maintained after correction for this variable. After correction, all variables remains significantly associated with TG levels except for %FEV1. 4. Discussion The aim of our study was to determine whether hypertriglyceridemia in CF subjects was associated with an alteration of their cardiometabolic profile. Our study demonstrated that CF-TG subjects have higher total cholesterol and total cholesterol to HDL cholesterol ratio as well as higher insulin secretion as assessed by insulin AUC (AUC0–120, AUC0–30, AUC30–120) compared to CF normal-TG. Further analysis of the complete

CF study sample (n = 210) highlighted an association with the same variables as well as with fasting insulin levels and the Stumvoll index. Together, these results suggest that in adult CF patients, hypertriglyceridemia is associated with mild but significant alteration of the metabolic profile compared to CF normal-TG. Whether these changes will increase the long-term risk of CF patients to develop cardiometabolic diseases and their complications remains to be investigated.

Table 4 Pearson correlation of triglycerides with clinical and metabolic factors in cystic fibrosis subjects (n = 210). Variables

r

Model corrected for BMI

Age BMI %FEV1 Total cholesterol HDL-cholesterol LDL-cholesterol Cholesterol/HDL cholesterol Fasting glucose Glucose AUC Fasting insulin Insulin AUC0–120 Insulin AUC0–30 Insulin AUC30–120 Stumvoll

− 0.03 0.17 ⁎ 0.16 ⁎ 0.35 ⁎ 0.00 0.15 ⁎ 0.37 ⁎ 0.12 0.02 0.26 ⁎ 0.20 ⁎ 0.14 0.20 ⁎ − 0.29 ⁎

− 0.03 – 0.13 0.33 ⁎ 0.02 0.10 0.35 ⁎ 0.01 0.03 0.18 ⁎ 0.20 ⁎ 0.14 0.20 ⁎ − 0.22 ⁎

BMI: body mass index, %FEV1: predicted forced expiratory volume in 1 s. ⁎ p b 0.05.

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Of the 210 subjects included in the analysis, 20 patients (9.5% of the cohort) had elevated TG levels. This is similar to other studies who reported a prevalence between 5 and 15% depending on the cut off used to define hypertriglyceridemia [5,6]. Furthermore, and similar to previous studies [5,6], hypertriglyceridemia was unrelated to genotype, glucose tolerance category, fasting glucose and glucose AUC. Interestingly, CF-TG patients displayed higher total cholesterol levels as well as a higher ratio of total cholesterol to HDL-cholesterol compared to CF normal-TG. We also found a positive relationship between TG levels and both parameters in our study sample (n = 210). Similar associations have in also been reported in the Physicians Health Study [15] and in CF subjects [5]. Despite this correlation, the prevalence of combined hypertriglyceridemia and hypercholesterolemia was low in our cohort (1.4% in the study sample). Both hypertriglyceridemia and the ratio of total cholesterol/ HDL-cholesterol are independent risk factor for the development of cardiovascular disease [7–10,16]. However, whether this also applies to CF subjects remains to be demonstrated since no deaths from cardiovascular disease have been reported in this population. However, subclinical changes in left ventricular function have been observed in CF patients [17] and it has been suggested that CF subjects may have a higher risk of ischemic heart disease [18,19]. Since the life expectancy of CF patients is increasing, it is possible that additional complications will emerge from this population. CF-TG shown an 18% higher %FEV1 compared to those with normal TG levels. We also observed that TG levels were associated with %FEV1 in the complete study sample (n = 210). However, this association did not remain significant after correction for BMI suggesting that TG levels do not directly affect lung function but there is no definite proof at this point. It should be noted, however, that in CF patients, the absence of a correlation between lung function and TG levels has also been reported by others [5,20]. We observed an increase in insulin AUC in CF-TG subjects compared to CF normal-TG subjects. We also observed a positive correlation between TG levels and insulin AUC30–120 and AUC0–120 but not between TG levels and insulin AUC0–30 in the complete study sample. It is well known that most CF subjects present delayed first phase insulin secretion [4,21,22]. The absence of a correlation between TG levels and insulin AUC0–30 may reflect the inability of the beta cells to adapt first phase insulin secretion to the metabolic state. In contrast to our results, Figueroa et al. [5] had reported similar insulin AUC0–120 between CF-TG and CF subjects. However, the age of the study participants differ significantly between the two cohorts as our study included only adult subjects whereas in the study of Figueroa et al. [5], half of the hypertriglyceridemic subjects were under 19 years old. Potentially, the difference observed between the two studies could be due to the age of the subjects included in the analysis. Numerous studies have highlighted an association between insulin sensitivity and TG levels [23–25]. Our results demonstrated that CF-TG subjects displayed higher insulin AUC (AUC0–120; AUC0–30; AUC30–120) but similar glucose AUC compared to CF-normal-TG patients suggesting insulin resistance. This result

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prompted us to further examine this relationship in CF patients. Using the Stumvoll index, we did not observe any significant difference in insulin sensitivity between CF-TG and CF normalTG groups. However, because the analysis involved a limited number of subjects (n = 20) we may not have had the power to detect differences in insulin sensitivity among groups. On the other hand, using Pearson correlation, we found a negative association between TG levels and insulin sensitivity in the complete CF study sample and this association was independent of BMI. This latter result suggests that, similar to what has been observed in other populations such as type 2 diabetic patients, hypertriglyceridemia is associated with increased insulin resistance in CF subjects. Further studies are needed to examine this relationship in the CF population. Insulin plays an important role in lipid metabolism by activating lipoprotein lipase, the key enzyme in adipose tissue TG storage [26,27]. Reduction of enzyme activity following insulin deficiency or insulin resistance impairs this process leading to increased TG levels. Moran et al. [28] have previously shown that insulin-mediated inhibition of lipolysis in CF patients tends to be diminished compared to controls suggesting insulin resistance. Our results demonstrated that CF-TG patients display higher insulin AUC than CF normal-TG subjects. Furthermore, we also observed a positive association between TG levels and insulin AUC in all CF patients. Thus, CF-TG subjects represent a subgroup of patients that are able to increase insulin levels, potentially to palliate insulin resistance. This is a cross-sectional study. Therefore, causal associations between TG and other parameters cannot be made. We included only CF subjects with de novo diabetes to avoid confounding factors such as insulin treatment as well as CF subjects with mild to moderate lung disease. Therefore, whether our findings can be extended to more severely affected CF subjects remain to be investigated. Despite these limitations, our results are strengthened by the use of standard techniques for the evaluation of various metabolic risk factors in a relatively large, well-characterized cohort of CF patients. In conclusion, the present study provides new evidence of an association of TG levels with insulin AUC and insulin sensitivity in a population of adult CF subjects with mild to moderate lung disease. In CF, hypertriglyceridemia is also associated with mild but significant cardiometabolic abnormalities. Further studies should examine the long-term significance of theses associations. Acknowledgment Dr. Rabasa-Lhoret is supported by a scholarship from the “Fonds de la recherche en santé du Québec” (FRSQ) and holds the J-A. De Sève clinical research chair. S.Z. holds a studentship from the FRSQ. This study was supported by Cystic Fibrosis Canada (CFRD team grant). References [1] Dupuis A, Hamilton D, Cole DE, Corey M. Cystic fibrosis birth rates in Canada: a decreasing trend since the onset of genetic testing. J Pediatr 2005 Sep;147(3):312-5.

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