The association between body composition, leptin levels and glucose dysregulation in youth with cystic fibrosis

Abstract

Background:

Optimization of nutritional status is recommended in patients with cystic fibrosis (CF) given the association between lower body mass index (BMI) and poor clinical outcomes. However, higher BMI and body fat correlate with glucose impairment and higher leptin levels in the general population. Differences in body composition and leptin levels between the categories of glucose tolerance were assessed in youth with CF and healthy controls.

Methods:

In a cross-sectional study, 59 adolescents and young adults with CF and 15 healthy controls matched by age and gender, underwent body composition analysis using dual energy X-ray absorptiometry (DXA) and a 2-hour oral glucose tolerance test (OGTT). Measures of insulin sensitivity, β-cell insulin secretion and fasting leptin levels were obtained.

Results:

Of the participants with CF, 62% were classified as abnormal glucose tolerant and 22% with cystic fibrosis related diabetes (CFRD). Patients with CFRD had a lower fat mass index (FMI) z-score, wt z-score and leptin levels compared to the control group (−1.86 vs. - 0.59, p=0.01; −1.86 vs 0.44, p=<0.001 and 7.9 vs vs. 27.7 μg/L, p=0.01). Leptin correlated positively with FMI z-score, BMI, weight z-score and indices of insulin secretion. FMI z-score correlated positively with higher insulin resistance (HOMA-IR), and lower insulin sensitivity (Matsuda index) (r=0.31; p =0.01 and r=−0.29; p=0.02, respectively) in the CF group.

Conclusions:

This study shows that despite new therapeutic strategies, youth with CF have lower body fat, weight z-score and leptin levels, particularly in subjects with early onset CFRD.

1. Introduction

With the increase in life expectancy of patients with Cystic Fibrosis (CF), Cystic Fibrosis Related Diabetes (CFRD) has emerged as a major non-pulmonary complication. CFRD profoundly affects nutritional status and lung function [1,2,3]. Studies have shown that during the years before overt diabetes, patients with CF present with an insidious decline in weight and Body Mass Index (BMI). Nutritional support with maintenance of an age- and sex specific BMI goal has been a vital component of care for patients with CF [4], given the association between malnutrition and lung disease progression [5]. However, this approach ignores the effects of body composition and fails to differentiate between the role of fat mass (FM) and muscle mass (Lean Body Mass (LBM)), which are key determinants of glucose dysregulation in children and adults with and without CF [6,7].

Prior studies have shown that patients with CF have reduced LBM compared to controls [8,9]. Similar to patients with various lung diseases [10], a progressive loss of LBM (sarcopenia) is associated with a risk of adverse outcomes in CF, such as poor lung function, disability, poor quality of life, and death [8,9]. In contrast, high adiposity in the presence of a normal BMI (also known as normal weight obesity) has been associated with severity of lung disease in adult CF patients [11].

In the CF population, decline in the nutritional status has been directly associated with insulin insufficiency in diabetes and abnormal glucose tolerance and diabetes [3]. Insulin is one of the main factors regulating body composition, muscle and adipose tissue function. In muscle, insulin stimulates protein synthesis to favor an anabolic process [12], while in fat, it exerts an inhibitory effect on lipolysis while stimulating lipogenesis [13]. Insulin is also an important regulator of leptin secretion, a hormone involved in various biological processes including the regulation of appetite, thermogenesis, immune responses, and energy expenditure [14]. Leptin is known to be secreted by fat cells and closely correlates with body fat mass [15,14]. Growing evidence suggests that leptin also plays a key role in glucose metabolism [16], and its impact on glucose homeostasis is generally anti-hyperglycemic, increasing insulin sensitivity of the liver and muscle cells [17,18,19]. Limited reports addressing the relationship between body composition and leptin levels with glucose dysregulation in youth with CF have had inconsistent results [20,21,22]. Leptin levels have been found to be reduced [20] and elevated [21] in children and young adults with CF. Therefore, this study aimed to evaluate the relationship between body composition, particularly body fat content; leptin levels; and the degree of glucose intolerance, including diabetes, in clinically stable youth with CF compared to healthy controls.

2. Research design and methods

2.1. Participants

59 patients, 10 to 25 years of age, with CF and mild lung disease and pancreatic exocrine insufficiency participated in the study. Patients attended the Washington University CF Center Clinics and the study was conducted at the Washington University Clinical Research Unit. All subjects and their parents or legal guardians signed informed consent or assent approved by the Washington University Human Research Protection Office.

Patients were required to be clinically stable, without evidence of deterioration from previous Pulmonary Function Tests (PFTs), CF exacerbation or hospitalizations for at least one month prior to the study visit. All participants were receiving ADEK vitamins and pancreatic enzyme replacement. Additionally, patients were required to have maintained stable weight within 5% variance for three months prior to participation. Exclusion criteria included FEV1%-predicted <50%, IV antibiotic or systemic steroid use within one month of the study visit, oral corticosteroid usage during the month prior to the study visit, history of lung or liver transplant, elevated transaminases, the inability to perform spirometry, or known diagnosis of CFRD. Patients receiving cystic fibrosis trans-membrane conductance (CFTR) modulators or potentiators were included in the study if the duration of therapy was longer than six months, given previously reported data of a potential role of these therapies on glucose homeostasis ^23,24. Age and gender matched healthy subjects were recruited to participate in this study.

2.2. Procedures

Lung Function

To assess clinical status, pulmonary function tests (FEV1 % predicted and FVC % predicted) were obtained on all subjects the day of the visit.

Anthropometric measurements

Weight was measured on a digital scale (SECA model 6841321107) with subjects wearing light clothing. Standing height without shoes was measured using a wall-mounted stadiometer (SECA model 240) and recorded to the nearest 0.1 cm. Z-scores for height (Ht z-score), weight (Wt z-score), and BMI were calculated for subjects 10.0 to 19.9 years old using the Centers for Disease Control and Prevention 2000 Growth Charts [25]. Ages of individuals aged >19.9 years were converted to 19.9 to permit calculation of Ht z-score, Wt z-score, and BMI z-score.

Pubertal assessment

Sexual maturation in all participants was determined by physical examination by a pediatric endocrinologist. Pubertal stage for breast in females and genitalia in males was assessed according to the Tanner stage (TS) criteria [26].

Dual-energy X-ray Absorptiometry (DXA)

Whole body DXA was performed using a Lunar Prodigy Advance (General Electric Healthcare), Encore Software Version 16. LBM and FM were measured based on their X-ray attenuation properties. The following whole-body derivative values were calculated: Fat Mass Index (FMI): FM/height² and Lean Body Mass Index (LBMI): lean mass/height²). LBMI and FMI were converted to sex- and age-adjusted z-scores for the participants in the study using contemporary reference data [27]. To assess fat mass distribution we calculated three ratios, including trunk fat mass / total fat mass, limbs fat mass/ total fat mass, limbs fat mass to trunk fat mass and visceral fat mass.

Oral glucose tolerance test

Following an 8 hour fast, measures of insulin sensitivity and β-cell insulin secretion in response to glucose were derived from a 2 h oral glucose tolerance test (OGTT). Venous samples for glucose, insulin, and c-peptide were drawn at 0, 30, 60, 90, and 120 minutes after starting ingestion of the glucose load (1.75 g/kg). Patients were classified into three glucose tolerance categories based on the fasting, 1 h and 2 h glucose concentration²⁸:

1. Normal glucose tolerance (NGT) was defined as fasting glucose <100 mg/dL 1 h glucose <200 mg/dL, and 2 h glucose 140 mg/dL.

2. Abnormal Glucose Tolerance (AGT): consisted of patients with either indeterminate glucose tolerance (INDET), impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). INDET is defined as having a fasting glucose <100 mg/dl and a 2 hour glucose <140 mg/dL, but a 1 h glucose ≥200 mg/dL. IGT is defined as having a fasting plasma glucose <100 mg/dL and a 2 h glucose between 140–199 mg/dL. IFG was defined as having a fasting glucose ≥ 100 mg/dL but <126 mg/dL and a 2 h glucose <140 mg/dL.

3. CFRD: was defined as a fasting glucose ≥ 126 mg/dL or a 2 h glucose ≥ 200 mg/dL

Calculations of Insulin Sensitivity and Secretion using OGTT- Derived Measurements

Glucose and C-peptide responses were calculated as the incremental area under the curve (iAUC) above fasting concentrations over the 2 h sampling period using the trapezoidal method. Two indices of β-cell function were calculated: the stimulated C-peptide response normalized to the glucose response (CP-iAUC/G-iAUC) [29], and the insulinogenic index [(Insulin _t30 – Insulin _t0) (mU/L) / (Glucose _t30 – Glucose _t0) (mg/dL)]^30,31 .

To estimate the degree of insulin resistance, the homeostasis model assessment index (HOMA-IR) ([Insulin _{t0’ (mU/L)} × Glucose _{t0’ (mmol/L)})] /22.5) [32] was obtained. However, given that in this population most patients with AGT and CFRD have normal fasting blood glucose levels but are unable to handle a glucose load, we also calculated the insulin sensitivity index - Matsuda (ISI_Matsuda) from the 2-hour OGTT, (10,000)/√[(Glucose_{t0’ (mg/dL)} × Insulin_{t0’ (mU/L)}) × (Glucose_mean × Insulin_mean)] [33]. ISI_Matsuda reflects a composite estimate of hepatic and muscle insulin sensitivity, which declines in the pre-diabetic metabolic state resulting in lower insulin sensitivity and thus, a lower ISI_Matsuda value. Unlike HOMA-IR, which is based solely on fasting levels, ISI_Matsuda considers the mean insulin and glucose levels during the 2 h OGTT following a glucose load.

Analytical Methods

Plasma glucose concentrations were measured using enzymatic technique. Insulin and C-peptide were measured using Electro-chemiluminescence (ECL) technology. Leptin was measured using a radioimmunoassay kit from Millipore.

3. Statistical Analysis

Descriptive statistics were performed on all variables. Differences in outcome measures were determined between the NGT, AGT and CFRD groups. Categorical variables were analyzed with Pearson Chi-square test and Fisher’s exact test as needed. After controling for sex and age, continuous variables were analyzed using an analysis of variance to compare the NGT, AGT, CFRD and control groups. To examine the sex differences in the CF cohort we used a t-test for normally distributed variables and Mann-Whitney test for non-normally distributed variables. A two-way factorial ANOVA’s was used to evaluate differenes in TS among the glucose tolerance groups and controls .

Multiple comparisons were adjusted using Tukey-Kramer adjustment. Glucose and C-peptide measurements at the different time points were analyzed using a mixed random effect repeated measures model with group, time, and group by time interaction as fixed effects; subject as a random effect; and variance components for each group to account for differences in variance. The association between the variables of body composition, anthropometric measurements, leptin and measurements of insulin sensitivity and β-cell function were assessed using a Pearson partial correlation while controlling for biological sex and age. Analyses were performed in SAS 9.4 using two-sided tests with alpha significance value of 0.05.

4. Results

4.1. Characteristic of Cystic fibrosis groups and controls

Fifty-nine patients with CF and fifteen healthy controls participated in the study.

There were no significant differences in age, gender, pubertal status, BMI z-score and LBMI z-score between the CF and control groups. Patients with CF had a lower Ht z-score (−0.18 vs 0.78, p = 0.001), Wt z-score (−0.27 vs. 0.44, p = 0.019), FMI z-score (−1.42 vs.−0.59 p = 0.03) and leptin levels (11.76 vs 27.70 μg/L, p = 0.002) compared to the control group. Based on the OGTT results, nine (15.2%) of the CF patients were classified as normal glucose tolerant, thirty seven (62.7%) as AGT and thirteen patients (22.1%) as de novo CFRD (Table 1). In the CF group, there were no significant differences in gender, TS BMI z-score,Ht z-score or LBMI z-score among the different glucose tolerance categories (table 1). Wt z-score and FMI z-score were lower in patients with CFRD compared to the control group. Likewise, leptin levels were three times lower in the CFRD group compared to the healthy control group and the results remained statistically significant after adjusting for age and sex (7.9 vs 27.7 μg/L respectively p = 0.01). Interestingly, females with CF had higher FMI z-score compared to males (−1.03 vs −1.80 respectively p = 0.024), and their leptin levels were higher compared to males (18.2 vs. 5.5 μg/L respectively, p = 0.002).

Table 1.

Physical and metabolic parameters of youth with cystic fibrosis and healthy controls.

	Controls (N = 15)	CF patients (n = 59)			p
		NGT (n = 9)	AGT (n = 37)	CFRD (n = 13)	NGT vs. Control	AGT vs. Control	CFRD vs. Control	NGT vs. AGT	NGT vs. CFRD	AGT vs. CFRD
Male % (n)	46 (7)	55(5)	54(20)	38(5)	0.95	0.97	0.98	0.86	0.77	0.97
Age (y)	17 ± 4	16 ± 3	15 ± 3	17 ± 3	0.98	0.97	0.95	0.98	0.93	0.65
TS n(%)III-IIIIV-V	04 (26.6)11 (73.3)	03 (22.3)6 (66.7)	3 (8.1)10 (27.03) 2 (84.7)	02(15.3)13 (84.6)	***	***	***	***	***	***
BMIz-score	−0.049 ± 1.35	0.004 ± 1.5	−0.04 ± 1.01	−0.75 ± 0.97	0.91	0.98	0.11	1.0	0.43	0.22
Wtz-score	0.44 ± 0.98	0.14 ± 0.83	−0.12 ± 0.97	−0.97 ± 1.1	0.47	0.06	< 0.001*	0.88	0.43	0.05*
FMIz-score	−0.59 ± 1.2	−1.59 ± 1.5	−1.23 ± 1.2	−1.86 ± 1.44	0.07	0.11	0.01*	0.87	0.96	0.44
LBMIz-score	−1.07 ± −1.12	−0.7 ± 1.1	−0.98 ± 0.99	−1.43 ± 0.57	0.36	0.76	0.34	0.85	0.42	0.31
Leptin (ug/L) §	27.7 ± 21.01	14.62 ± 14.3	12.05 ± 16.3	7.9 ± 7.3	0.12	0.11	0.01**	0.99	0.26	0.24
FEV1 (%) pred.	-				-	-	-	0.21	0.78	0.083
		84.89 ± 11.14	93.43 ± 18.75	85.31 ± 16.26

Data are expressed as means ± SD; NGT: normal glucose tolerance; AGT: abnormal glucose tolerance; CFRD: cystic fibrosis-related diabetes; TS: Tanner stage

^*p < 0.05

^**p < 0.001

^***TS group analysis combinations were not statically significant among the CF groups and control p < 0.05

^§Variables adjusted for age and sex

4.2. Glucose, measurements of insulin sensitivity, insulin resistance and β-cell function in patients with CF and control group

Mean glucose levels at the different time points of the OGTT and glucose area under the curve (G-iAUC) were significantly higher in the CF groups than controls (Table 2). Patients with CF including NGT, AGT and CFRD had a delayed and blunted C-peptide response during the OGTT compared to controls, demonstrating a loss of early insulin secretion, as previously described [34] (Fig. 1). Measurements of β-cell function were significantly lower in the CF groups compared to the healthy control group (Table 2). Furthermore, we observed that TS did not have a statistical significant relationship with ISI_Matsuda, HOMA-IR, and ISR.

Table 2.

Glucose levels, indices of insulin sensitivity and secretion in healthy controls and categories of glucose tolerance in youth with cystic fibrosis

		CF patients (n= 59)			p
	Controls (N = 15)	NGT (n = 9)	AGT(n = 37)	CFRD(n = 13)	NGT vs. control	AGT vs. Control	CFRD vs. Control	NGT vs. AGT	NGT vs. CFRD	AGT vs. CFRD
Glucose 0 min (mg/dL)	91 ± 7	91 ± 7	99 ± 1.4	101 ± 12	1.0	1.0	1.0	0.99	1.0	1.0
Glucose 120 min (mg/dL)	106 ± 18	101 ± 24	147.22 ± 27	228.3 ± 50.69	0.61	< 0.001*	< 0.001*	< 0.001*	< 0.001*	< 0.001*
G-iAUCmg/dL* min	15223 ± 2754	16675 ± 1475	19598 ± 2705	25565 ± 4146	0.62	< 0.001*	< 0.001*	0.38	< 0.001*	< 0.001*
CP-iAUC ng/mL*min	947.2 ± 342	658.8 ± 193	670.45 ± 238	712.5 ± 267.4	0.011	< 0.001*	0.028	0.99	0.96	0.96
CP-iAUC/G- iAUC	0.062 ± 0.018	0.039 ± 0.012	0.034 ± 0.01	0.028 ± 0.012	< 0.001*	< 0.001*	< 0.001*	0.644	0.23	0.60
ISIMatsuda §	4.7 ± 4.4	6.09 ± 3.3	5.86 ± 3.1	4.19 ± 1.6	0.64	0.62	0.62	0.99	0.58	0.47
HOMA-IR §	2.51 ± 1.58	1.83 ± 1.19	1.74 ± 1.19	1.96 ± 1.23	0.21	0.07	0.37	0.99	0.96	0.96
ISR §	2.07 ± 1.47	0.41 ± ± 0.32	0.36 ± 0.21	0.38 ± 0.29	< 0.001*	< 0.001*	< 0.001*	0.80	1.0	0.99

p < 0.05 statistically significant

^§Control for age and sex

Fig. 1.

Plot of Means and 95% confidence intervals of glucose (A) and C peptide concentrations (B) at the OGTT time points

4.3. Associations between body composition and insulin sensitivity, insulin resistance and β cell function in youth with CF

No statistically significant differences in body fat distribution were observed among the different groups (NGT, AGT, CFRD, and control). However, in the CF cohort, FMI z-score was negatively associated with insulin sensitivity (ISI_Matsuda )(r = −0.29, p = 0.02), and positively correlated with insulin resistance (HOMA-IR) (r = 0.31, p = 0.01). FMI z-score also correlated positively with the measurement of β-cell function CP iAUC/ G iAUC, and insulinogenic index (r = 0.44, p = 0.001; and r = 0.327 p = 0.013, respectively). No statistically significant associations were found between LBMI z-score and indices of insulin sensitivity.

4.4. Associations between Leptin levels and body composition, insulin secretion and glucose in youth with CF

As shown in Fig. 2, leptin levels positively correlate with FMI z-score, BMI z-score and Wt z-score. Moreover, leptin was positively correlated with CP iAUC/ G iAUC and insulinogenic index (r = 0.29, p = 0.03 and r = 0.28, p = 0.05), with a trend for a negative correlation with glucose iAUC (r = −0.27, p = 0.056).

Fig. 2.

Partial correlations between Leptin and FMI z-scores, BMI z-scores and Wt z-scores.

5. Discussion

This cross-sectional study demonstrates differences in body composition and leptin levels in clinically stable youth with CF across the spectrum of glucose tolerance (NGT, AGT, CFRD). Over-all, CF patients had lower fat mass, leptin levels and diminished indices of insulin secretion compared to healthy controls. Interestingly, the strong correlation between low leptin concentrations and diminished insulin secretion in the CF patients suggest that leptin may play a role in glucose dysregulation in this population. These findings resemble similar metabolic changes occurring in conditions with low leptin levels, and postulate a potential therapeutic role for leptin in these patients.

Lower BMI in CF patients has been shown to affect important clinical outcomes, including pulmonary function, frequency of hospital admission, and quality of life [35,36]. It is noteworthy that in our study, the CF groups and healthy controls were not different when assessed for BMI z-scores. However, the FMI z-score was different, especially in the CFRD group. Nutritional support and monitoring weight status is a vital component of care for patients with CF [37]. These recommendations have led to a remarkable improvement in the nutritional status of CF patients over the years, defined by a BMI above 50^th percentile in children and above 22 kg/m² for females or 23 kg/m² for males in adults with CF [4]. Focusing exclusively on BMI may miss important information on body composition. In this study, patients with CFRD had lower fat mass, which was associated positively with insulin secretion but inversely with insulin sensitivity. These observations suggest a compensatory mechanism of insulin secretion in the setting of insulin resistance in patients with CF. When this compensatory mechanisms is lost, hyperglycemia appears.

Given the cross-sectional nature of the study, it can not be determined if the lower fat mass is cause or effect. Nevertheless, increasing fat mass solely by promoting an increase in BMI, may increase proinflammatory adipocytokines that further promote insulin resistance and glucose dysregulation as well as confering potentially direct catabolic effects on muscle [38,39]. Therefore unrestrictive diets without specific nutritional recommendations regarding diet quality may be disadvantageous for this population.

In the current study, no statistically significant differences were evident in muscle mass between the CF participants and the healthy group or between the different groups of glucose tolerance within the CF group. These findings may be explained by the relatively healthy cohort studied. As glucose impairment worsens, the differences in muscle mass may become evident. Muscle mass depletion has been described as a complication of diabetic patients and has been attributed to disease duration, chronic inflammation and aging [40,41]. Given that skeletal muscle is the primary tissue responsible for insulin-mediated glucose disposal, muscle mass depletion could affect insulin sensitivity negatively in CF patients. Although insulin replacement is essential in CFRD and has been shown to improve BMI in several studies [42,43], further studies are needed to evaluate the effects of insulin replacement on body composition and leptin levels.

A challenge in evaluating body composition is that DXA is an imperfect method to measure body composition. Computed tomography (CT) and magnetic resonance imaging (MRI) are more specific for imaging muscle tissue, and they allow fat to be distinguished between muscle fibers [44,45]. However, it is more practical to determine body composition from DXA scans since they are routinely done in CF clinical practice to determine of bone mineral density, and the amount of radiation exposure is far less than the one received from CT.

Studies analyzing the impact of diabetes on body composition and leptin levels are lacking in the CF population. A study of adults with CF showed comparable levels of fat mass and leptin levels between CFRD and controls [22]. However, despite an association between insulin and leptin levels, the authors failed to explain why patients with CFRD, who have abnormal insulin secretion, had similar leptin levels compared to controls. Authors suggested that in the setting of impaired insulin secretion and repeated infections or subclinical inflammation, leptin levels may be higher than expected in CF due to the stimulatory effect of elevated cytokines on leptin.

The current study demonstrates an association between BMI, body fat mass and leptin concentrations as previously described [22,21]. Surprisingly, there was a gender-dependent dissociation in the range of leptin concentrations and body fat content, with higher leptin levels in girls who had lower fat mass content. Though gender differences in fat content have been reported [4,37], this discrepancy in leptin levels with fat mass is possibly due to an increase of leptin secretion or leptin resistance in females [46]. Moreover, our data clearly demonstrate a decrease in leptin levels in those patients with CFRD. Though this relationship is contrary to what is seen in patients with type 2 diabetes, in which increased leptin and insulin are due to insulin resistance [47], this data suggest that CF patients behave more like patients with conditions associated with leptin deficiency or low leptin levels, like lipodystrophy [48] or type 1 diabetes [49] respectively, in which the low leptin levels could be contributing to hyperglycemia by lack of direct inhibition of the pancreatic glucagon secretion [50,51,52]. Further studies in CF patients are needed to determine the association between leptin levels and glucagon. If further studies support this hypothesis, leptin replacement may represent a potential therapeutical target for these patients

This study highlights the impact of body composition and leptin concentrations in early glucose abnormalities in subjects with CF. Fat mass depletion and low leptin levels in those patients with lower insulin secretion, as observed in our study, may represent an early manifestation of a cascade for metabolic derangements that may ultimately result in negative clinical outcomes in patients with CF. Low leptin concentration may ultimately result in increased glucagon secretion or reduced immune function, decreased insulin signaling and therefore lead to alterations of glucose homeostasis [53,54]. The abnormalities in body composition in patients with CF suggest a distorted nutritional approach in long term CF care. Thus, longitudinal studies are warranted to further elucidate the effect of insulin impairment on body composition and leptin concentrations in patients with CF. Moreover, studies are needed to determine if insulin therapy and/or dietary interventions in CF patients with early glucose abnormalities are associated with changes in leptin and body composition and subsequent improvements in metabolic control.