Abstract
Objective
GH replacement lowers total cholesterol and low-density lipoprotein cholesterol (LDL-C) in GH-deficient adults, but effects on high-density lipoprotein (HDL) cholesterol (HDL-C) are variable. Both GH and glucocorticoids decrease cholesteryl ester transfer protein (CETP) activity, which is important in HDL metabolism. We determined the extent to which the changes in HDL-C in response to GH replacement are predicted by the −629C>A CETP promoter polymorphism, and questioned whether this association is modified by concomitant glucocorticoid treatment.
Design and methods
A total of 91 GH-deficient adults (63 receiving glucocorticoids) were genotyped for the −629 CETP C>A polymorphism. Fasting serum lipids were measured before and after 1.2±0.4 years of GH treatment (Genotropin, Pfizer Inc., Stockholm, Sweden).
Results
In the whole group, total cholesterol and LDL-C decreased (P<0.05) after GH treatment, but the changes in HDL-C were not significant. In CC carriers receiving glucocorticoids (n=19), HDL-C rose by 0.15±0.25 mmol/l (P=0.02; P<0.03 from unchanged HDL-C in −629 AA+CA carriers on glucocorticoids and from CC homozygotes not receiving glucocorticoids). Multivariate regression analysis showed that individual changes in HDL-C were predicted by the CETP polymorphism (CC versus AA+CC, P=0.006) in glucocorticoid users, independently of baseline HDL-C and other variables including apolipoprotein E4 carrier status; an opposite association with the CETP polymorphism was found in patients not receiving glucocorticoids (P=0.053).
Conclusions
We suggest a common CETP variant-glucocorticoid treatment interaction concerning the effect of GH replacement on HDL-C. This may explain some of the reported variation in the HDL-C response to GH.
Introduction
Cardiovascular morbidity and mortality are likely to be increased in patients with hypopituitarism (1). The increased cardiovascular risk in hypopituitary patients is in part attributable to GH deficiency, which is associated with an adverse cardiovascular risk profile, including abnormalities in body composition, high serum concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol (LDL-C), and triglycerides, as well as low high-density lipoprotein (HDL) cholesterol (HDL-C) (2). GH replacement therapy improves body composition, and favorably lowers serum total cholesterol and LDL-C (2, 3, 4, 5), but inconsistent effects of GH replacement on HDL-C have been reported. Smaller studies have demonstrated an increase, no change or even a decrease in HDL-C in adults with GH deficiency (2, 6, 7, 8). A surveillance report from the Hypopituitary Control and Complications Study (HypoCSS; Eli Lilly) showed an increase in HDL-C (3), whereas an analysis from KIMS (Pfizer International Metabolic Database) demonstrated a small decrease in HDL-C after 1 and 2 years of GH replacement in hypopituitary adults (4). A meta-analysis comprising placebo-controlled trials did not reveal a significant effect on HDL-C (9).
The serum total cholesterol and LDL-C response to GH therapy may be stronger in younger subjects and in patients with adult onset GH deficiency (9, 10), but the extent to which genetic factors may affect GH replacement effects on HDL-C is largely unknown. HDL metabolism is regulated by several processes, including cholesteryl ester transfer protein (CETP)-mediated neutral lipid transfer process (11, 12). CETP transfers cholesteryl esters from HDL toward triglyceride-rich lipoproteins. Consequently, increased CETP activity results in a lower HDL-C (11, 12, 13). Common genetic variations have been identified in CETP (14, 15), among which the −629 CETP C>A promoter polymorphism (rs1800775) has been shown to regulate CETP transcriptional activity in vitro (16). The more frequent C allele is associated with higher circulating CETP mass and activity and a lower HDL-C (17, 18). Of interest, GH replacement decreases CETP mass and activity in hypopituitary adults (6, 7). Moreover, circulating CETP mass and activity are probably lowered by glucocorticoids as well. CETP activity is considerably reduced in GH-deficient adults receiving conventional glucocorticoid substitution therapy (19, 20), which is considered to be unphysiological (21). This may explain why HDL-C is not decreased, despite higher serum triglycerides in glucocorticoid receiving hypopituitary patients who are treated with GH (22). Steroid treatment also attenuates CETP activity in other patient groups (23, 24), probably contributing to high HDL-C (25, 26).
The present study was initiated to test the hypothesis that the −629 CETP C>A polymorphism predicts the HDL-C response to GH replacement in hypopituitary adults. Since both GH and glucocorticoids are likely to affect the CETP pathway, we also determined whether such an association may be modified by concomitant glucocorticoid treatment for ACTH deficiency.
Subjects and methods
The study was approved by the medical ethics committees of the University Medical Centres Groningen and Utrecht, The Netherlands. All participants provided written informed consent, and GH deficiency was established according to the international consensus criteria, i.e. a peak GH in response to insulin-induced hypoglycemia <3 μg/l and/or an insulin-like growth factor 1 (IGF1) concentration >2 s.d.s below the age-adjusted reference value (4). The participants (aged ≥18 years) were recruited from the outpatient clinics of the Departments of Endocrinology from each University Medical Centre. Patients with adult-onset or childhood-onset GH deficiency were eligible. In case of childhood-onset GH deficiency, GH provocation tests were again performed before the (re)start of GH treatment in order to confirm persistent GH deficiency. Exclusion criteria were the use of lipid lowering drugs, severe illness necessitating clinical admission in the preceding year, pregnancy, and severe hypertriglyceridemia (fasting serum triglyceride level >5.0 mmol/l). All patients participated in the KIMS (Pfizer International Metabolic Database) protocol, and they were studied before and during GH (Genotropin, Pfizer Inc., Stockholm, Sweden) treatment, which was scheduled at 1 year, i.e. at the first time point at which protocolized data concerning serum lipids and body composition were obtained. If 1-year follow-up lipid data were not available, measurements obtained after more prolonged GH treatment were used. Dose titration that was aimed at normalizing the IGF1 level was applied in both centers.
ACTH deficiency was diagnosed using an 0800 h serum cortisol or a peak cortisol threshold in response to insulin-induced hypoglycemia as described (27). If an insulin tolerance test was contraindicated, a metyrapone test was used. Deficiencies of other pituitary hormones were diagnosed as described (28). Hormonal substitution therapy with glucocorticoids, thyroid hormone, sex steroids, and vasopressin (ADH) was administered when indicated. Data concerning the underlying disorder responsible for GH deficiency, age at onset of GH deficiency and number of additional pituitary hormone deficiencies were also retrieved from the medical records. Body fat mass was assessed using bioelectrical impedance, according to the algorithm provided by the manufacturer (29). Waist circumference was measured as the smallest girth between rib cage and iliac crest. Body mass index (BMI, kg/m2) was calculated as weight divided by height squared.
Laboratory methods
Serum samples were stored at −20 °C until assay. Serum lipids and IGF1 were measured in a central laboratory, according to the KIMS protocol. Cholesterol and triglycerides were measured enzymatically using routine biochemical methods. HDL-C was measured in the supernatant fraction after precipitation of apolipoprotein B-containing lipoproteins with sodium phosphotungstate and Mg2 (30). LDL-C was calculated by the Friedewald formula. Plasma glucose was measured in the laboratory of each center using standard clinical chemical methods.
Genotyping was performed at the University Medical Centre Groningen and at the Laboratory of Experimental Vascular Medicine, Academic Medical Centre Amsterdam, The Netherlands. DNA was extracted from whole blood using the Qiamp mini kit (Qiagen). The −629C>A CETP promoter polymorphism (rs1800775) was genotyped as described (17). Apolipoprotein E (ApoE) genotypes (rs429358 and rs7412) were determined using allelic discrimination on a TaqMan 7500 Real Time PCR system, using the SNP genotyping mixes C-3084793-20 and C-904973-10 and Taqman Universal PCR mastermix No AmpErase (Applied Biosystems, Nieuwerkerk a/d Ijssel, The Netherlands). The method has been validated against a previously described restriction isotyping procedure (31, 32).
Statistical analysis
Statistical analyses were carried out using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Data are given in mean±s.d., median (interquartile ranges), or in percentages. Between-group differences in variables were evaluated using unpaired t-tests, Mann–Whitney U tests, or ANOVA with subsequent Bonferroni correction for multiple comparisons. Between-group differences in (genotype) prevalences were determined using χ2 analysis. Changes in variables were tested with paired t-tests or Wilcoxon tests where appropriate. Multiple linear regression analysis was performed to disclose the independent relationship between variables. Assuming a mean change in HDL-C of 0.12±0.06 mmol/l after GH administration (derived from Leese et al. (6)), it was estimated that a between-group difference in HDL-C change of 0.06 mmol/l could be demonstrated in 11 patients per group with a power of 0.80 and a two-sided P value of 0.05. A two-sided P value <0.05 was considered to be significant.
Results
Ninety-one patients participated in the study (mean age 43±14 years; 47 men, 44 women). Twenty-eight patients were classified as ACTH sufficient (mean age 36±14 years; 11 men, 17 women), and 63 patients received glucocorticoids for ACTH deficiency (mean age 46±13 years, P<0.001; 36 men, 27 women, P=0.36), of whom 58 were treated with cortisone acetate and five with hydrocortisone. In most cases, glucocorticoids were administered in two daily doses. The mean glucocorticoid dose (in hydrocortisone equivalents) was 22±6 (range 6.0–30.0) mg/day, corresponding to 11.0±2.9 (range 3.0–17.2) mg/m2 of body surface area. In all participants, glucocorticoid dose remained unchanged during follow-up. Twenty-three patients (25.3%) had childhood-onset GH deficiency, which was present in 32.1% of ACTH-sufficient patients and in 22.2% of patients receiving glucocorticoids (P=0.46). In the whole group, 59 patients (64.8%) had TSH deficiency, 69 (75.8%) had gonadotropin deficiency, and 18 (19.8%) had diabetes insipidus (32.5%). TSH deficiency (84.1 vs 21.4%, P<0.001), gonadotropin deficiency (90.5 vs 42.5%, P<0.001), and ADH deficiency (28.6 vs 0%, P=0.004) were more prevalent in glucocorticoid receiving patients than in ACTH-sufficient patients. In all thyroid hormone-treated patients, serum free thyroxine levels were within the reference range before and during GH replacement. All hypogonadal men used testosterone supplementation. All hypogonadal women aged <50 years used oral estrogen preparations, but sex steroids were only used by one of the eight (13%) women aged >50 years. Transdermal estrogens were not used. The percentage of hypogonadal women which used estrogen supplementation was not different between ACTH-sufficient and glucocorticoid-treated patients (P=0.18). Baseline blood pressure was 127±14 mmHg systolic and 83±10 mmHg diastolic in the whole group with higher systolic (P=0.07) and diastolic (P=0.03) values in glucocorticoid-treated patients. Five patients had diabetes mellitus of whom four were treated with glucocorticoids (P=0.96). Fasting plasma glucose (4.8±1.2 mmol/l in the whole group) was not different between ACTH-sufficient and glucocorticoid-treated patients (P=0.84).
The participants were studied before and after 1.2±04 (range 0.9–2.8) years of GH replacement. Duration of follow up was not different between ACTH-sufficient and glucocorticoid-treated patients (P=0.14). GH dose at follow-up was 0.40±0.23 (range 0.20–1.60) mg/day in the whole group, and was not different between ACTH-sufficient patients (0.40±0.20 mg/day) and patients receiving glucocorticoids ((0.40±0.24) mg/day, P=0.89).
In the whole group, BMI and waist circumference remained unchanged after GH (Table 1). Fat mass decreased similarly in ACTH-sufficient and glucocorticoid-treated patients. Waist circumference decreased slightly in glucocorticoid-treated patients. Before GH treatment, serum IGF1 levels were lower in glucocorticoid-treated patients, but there was no difference in IGF1 when expressed as s.d. Z-scores. The IGF1 Z-scores attained after GH were similar in both groups. Before GH treatment, serum total cholesterol, LDL-C, and HDL-C were not different between patients with and without ACTH deficiency; triglycerides were higher in glucocorticoid-treated patients (Table 1). In the whole group, serum total cholesterol and LDL-C decreased after GH, but HDL-C and triglycerides remained unchanged. Consequently, the LDL/HDL-C ratio decreased. In ACTH-sufficient patients, the decrease in total cholesterol and LDL-C was significant. In glucocorticoid-treated patients, serum total cholesterol and LDL-C did not decrease significantly (P=0.08), although the LDL/HDL-C ratio decreased as well. HDL-C did not change in either ACTH-sufficient patients or in glucocorticoid-treated patients.
Clinical characteristics, insulin-like growth factor 1, plasma glucose and lipid levels in before and after GH treatment in 91 GH-deficient adults. Data in mean±s.d. or in median (interquartile ranges).
| Whole group (n=91) | ACTH sufficient (n=28) | On glucocorticoids (n=63) | ||||
|---|---|---|---|---|---|---|
| Before GH treatment | After GH treatment | Before GH treatment | After GH treatment | Before GH treatment | After GH treatment | |
| BMI (kg/m2) | 28.3±5.3 | 28.3±5.3 | 28.6±5.0 | 28.3±4.5 | 28.2±5.4 | 28.3±5.6 |
| Waist (cm) | 97±15 | 96±14 | 93±13 | 94±13 | 98±15 | 97±15* |
| Fat mass (kg) | 27.3±11.3 | 25.5±11.0‡ | 29.4±10.6 | 27.1±8.5* | 26.5±11.7 | 24.9±11.9† |
| IGF1 (μg/l) | 73±45 | 164±72‡ | 98±53 | 180±71‡ | 62±36§ | 156±71‡ |
| IGF1 Z-score | −2.39±1.32 | −0.16±1.34‡ | −2.20±1.38 | −0.30±1.28‡ | −2.48±1.29 | −0.09±1.37‡ |
| Total cholesterol (mmol/l) | 5.46±1.01 | 5.33±0.93* | 5.41±0.96 | 5.17±0.94* | 5.48±1.04 | 5.40±0.12 |
| LDL cholesterol (mmol/l) | 3.50±0.92 | 3.30±0.82† | 3.55±0.85 | 3.21±0.78* | 3.48±0.96 | 3.33±0.85 |
| HDL cholesterol (mmol/l) | 1.16±0.36 | 1.18±0.34 | 1.22±0.40 | 1.21±0.35 | 1.14±0.34 | 1.17±0.33 |
| LDL/HDL cholesterol ratio | 3.29±1.23 | 3.03±1.17† | 3.19±1.17 | 2.92±1.21* | 3.34±1.26 | 3.08±1.16* |
| Triglycerides (mmol/l) | 1.70 (1.30–2.10) | 1.80 (1.20–2.30) | 1.35 (1.10–1.80) | 1.40 (0.95–1.98) | 1.70 (1.30–2.10)∥ | 1.90 (1.50–2.40)§ |
BMI, body mass index; IGF1, insulin like growth factor 1; HDL, high-density lipoproteins. *P<0.05; †P<0.01: ‡P<0.001 from baseline; §P<0.05; ∥P<0.01 from patients not using glucocorticoids.
Table 2 shows baseline serum lipids according to the −629C>A CETP polymorphism, which was in Hardy–Weinberg equilibrium in the whole group (P>0.90). The CETP genotype distribution was not different in glucocorticoid-using patients compared to ACTH-sufficient patients (P=0.36). In the whole group and in patients not receiving glucocorticoids, HDL-C was lowest in CC carriers, but the CETP genotype effects were not significant (P>0.20). In glucocorticoid-treated patients, HDL-C was essentially similar across the CETP genotype groups. Serum total cholesterol, LDL-C, and triglycerides did not vary according to the −629C>A CETP polymorphism (Table 2). Fifty-nine patients had the ApoE3/E3 genotype, and eight were ApoE2/E3 carriers (non-ApoE4 carriers, n=67). Three patients were ApoE2/E4 carriers, 19 were ApoE3/E4 carriers, and two were ApoE4/E4 homozygotes (ApoE4 carriers, n=24). The presence of the ApoE4 allele was not different between −629 CETP AA+CA subjects using glucocorticoid or not, nor between −629 CETP CC carriers using glucocorticoid or not (Table 2, P>0.50 for both comparisons). In the whole group and in glucocorticoid-treated patients separately, baseline serum cholesterol, LDL-C, HDL-C, and triglyceride levels were not significantly different between the non-ApoE4 carriers and the ApoE4 carriers (P>0.30 for all comparisons; data not shown). Among patients not receiving glucocorticoids, baseline serum total cholesterol and LDL-C were higher in ApoE4 carriers (n=9; 6.14±0.58 and 4.18±0.49 mmol/l respectively) than in non-ApoE4 carriers (n=19; 5.07±0.91 mmol/l, P=0.003 and 4.18±0.49 mmol/l, P=0.005 respectively); there were no further differences in baseline serum lipids according to ApoE4 carrier status in this group (data not shown).
Baseline plasma lipids and apolipoprotein (apo) E4 carrier status according to −629C>A cholesteryl ester transfer protein (CETP) promoter polymorphism in 91 GH-deficient adults. Data in mean±s.d. or in median (interquartile ranges).
| AA+CA | AA | CA | CC | |
|---|---|---|---|---|
| Whole group (n=91) | n=61 | n=18 | n=43 | n=30 |
| Apo E4 carriers (no/yes) | 44/17 | 8/10 | 36/7 | 23/7 |
| Total cholesterol (mmol/l) | 5.46±1.04 | 5.58±0.78 | 5.40±1.09 | 5.46±1.04 |
| LDL cholesterol (mmol/l) | 3.47±0.90 | 3.57±0.71 | 3.42±0.98 | 3.58±0.96 |
| HDL cholesterol (mmol/l) | 1.19±0.39 | 1.21±0.35 | 1.18±0.41 | 1.10±0.28 |
| Triglycerides (mmol/l) | 1.60 (1.30–2.10) | 1.50 (1.20–1.85) | 1.80 (1.30–2.20) | 1.70 (1.10–1.93) |
| ACTH sufficient (n=28) | n=17 | n=9 | n=8 | n=11 |
| Apo E4 carriers (no/yes) | 11/6 | 4/5 | 7/1 | 8/3 |
| Total cholesterol (mmol/l) | 5.39±0.92 | 5.76±0.86 | 4.98±0.84 | 5.45±1.06 |
| LDL cholesterol (mmol/l) | 3.49±0.80 | 3.77±0.67 | 3.17±0.86 | 3.65±0.94 |
| HDL cholesterol (mmol/l) | 1.29±0.46 | 1.31±0.35 | 1.27±0.59 | 1.10±0.23 |
| Triglycerides (mmol/l) | 1.30 (0.95–1.65) | 1.30 (1.10–1.75) | 1.30 (0.80–1.48) | 1.70 (1.10–2.00) |
| On glucocorticoids (n=63) | n=44 | n=9 | n=35 | n=19 |
| Apo E4 carriers (no/yes) | 33/11 | 4/5 | 29/6 | 15/4 |
| Total cholesterol (mmol/l) | 5.48±1.05 | 5.41±0.69 | 5.50±1.13 | 5.46±1.06 |
| LDL cholesterol (mmol/l) | 3.46±0.95 | 3.39±0.74 | 3.48±1.00 | 3.54±1.00 |
| HDL cholesterol (mmol/l) | 1.15±0.35 | 1.11±0.34 | 1.17±0.37 | 1.10±0.31 |
| Triglycerides (mmol/l) | 1.85 (1.50–2.20) | 1.50 (1.45–2.55) | 1.90 (1.50–2.20) | 1.70 (1.30–1.90) |
HDL, high-density lipoproteins. All comparisons between the three genotype groups and between −629 CETP AA+CA carriers and CC homozygotes were not significant (P>0.10).
The changes in serum total cholesterol, LDL-C, and triglycerides in response to GH replacement did not differ significantly according to the −629C>A CETP polymorphism (Table 3). Of note, HDL-C increased in response to GH replacement in glucocorticoid receiving patients with the −629CC CETP genotype (P=0.02); this increase was different from the changes in HDL-C in −629 CETP AA+CA glucocorticoid-treated patients (P<0.03) and from −629 CETP CC patients with ACTH sufficiency (P<0.03; Table 3, individual changes in HDL-C are shown in Fig. 1). The changes in serum lipids after GH did not differ according to ApoE4 carrier status (data not shown).
Changes in plasma lipids in response to GH treatment according to −629C>A cholesteryl ester transfer protein (CETP) promoter polymorphism in 91 GH-deficient adults. Changes in mean±s.d. or in median (interquartile range).
| Whole group | ACTH sufficient | On glucocorticoids | ||||
|---|---|---|---|---|---|---|
| AA+CA (n=61) | CC (n=30) | AA+CA (n=17) | CC (n=11) | AA+CA (n=44) | CC (n=19) | |
| Total cholesterol (mmol/l) | −0.14±0.63 | −0.10±0.73 | −0.17±0.85 | −0.36±0.32 | −0.13±0.54 | 0.05±0.86 |
| LDL cholesterol (mmol/l) | −0.18±0.63 | −0.27±0.63 | −0.28±0.66 | −0.44±0.31 | −0.14±0.63 | −0.17±0.75 |
| HDL cholesterol (mmol/l) | 0.00±0.07 | −0.07±0.25 | 0.02±0.17 | −0.06±0.19 | −0.01±0.18 | 0.15±0.25*,† |
| Triglycerides (mmol/l) | 0.10 (−0.40 to 0.40) | 0.20 (−0.23 to 0.63) | 0.00 (−0.40 to 0.25) | 0.20 (−0.20 to 0.60) | 0.10 (−0.48 to 0.48) | 0.20 (−0.30 to 0.70) |
HDL, high-density lipoproteins. *P=0.02 from baseline; †P<0.03 from −629 CETP AA+CA carriers on glucocorticoids and from CETP CC homozygotes not using glucocorticoids.
Individual changes in high-density lipoprotein (HDL) cholesterol in response to GH replacement according to −629 CETP C>A promoter polymorphism and concomitant glucocorticoid treatment. *P=0.02 from baseline; **P<0.03 from −629 CETP CA+AA carriers receiving glucocorticoids, and from ACTH sufficient −629 CETP CC.
Citation: European Journal of Endocrinology 162, 2; 10.1530/EJE-09-0742
Individual changes in high-density lipoprotein (HDL) cholesterol in response to GH replacement according to −629 CETP C>A promoter polymorphism and concomitant glucocorticoid treatment. *P=0.02 from baseline; **P<0.03 from −629 CETP CA+AA carriers receiving glucocorticoids, and from ACTH sufficient −629 CETP CC.
Citation: European Journal of Endocrinology 162, 2; 10.1530/EJE-09-0742
Individual changes in high-density lipoprotein (HDL) cholesterol in response to GH replacement according to −629 CETP C>A promoter polymorphism and concomitant glucocorticoid treatment. *P=0.02 from baseline; **P<0.03 from −629 CETP CA+AA carriers receiving glucocorticoids, and from ACTH sufficient −629 CETP CC.
Citation: European Journal of Endocrinology 162, 2; 10.1530/EJE-09-0742
Multiple regression analysis was performed to determine whether the association of the GH-induced changes in HDL-C with the −629 CETP C>A polymorphism was independent of clinical factors, baseline HDL-C, changes in triglycerides and in LDL-C as well as ApoE4 carrier status (Table 4). In glucocorticoid-receiving patients, the changes in HDL-C were positively predicted by the −629 CETP genotype (CC versus AA+CA; P=0.006), independently of baseline HDL-C and age. This CETP gene effect was also independent of glucocorticoid dose (expressed per m2 of body surface area, P=0.92). In patients not receiving glucocorticoids, in contrast, the individual changes in HDL-C were negatively predicted by the −629 CETP genotype (CC versus AA+CA; P=0.053). In both groups, these age-adjusted associations remained essentially similar (glucocorticoid receiving patients: P<0.014 in all analyses; no glucocorticoid receiving patients: P<0.075 in all analyses) after adjustment for either gender, childhood-onset versus adult-onset GH deficiency, treatment for other pituitary hormone deficiencies, ApoE4 carrier status, changes in IGF1, in waist circumference, in triglycerides, and in LDL-C (data not shown). When the −629 CETP polymorphism was divided into three allelic groups (CC, CA and AA), again opposing trends were found between the association of baseline HDL-C-adjusted changes in HDL-C with the −629 CETP polymorphism in glucocorticoid-treated patients (β=0.225, P=0.059) compared to patients not receiving glucocorticoids (β=−0.368, P=0.036). When glucocorticoid treatment (either as a categorical variable or as glucocorticoid dose per m2 of body surface area), the −629 CETP genotype (CC versus AA+AC), and the interaction of these variables were forced in a multivariate regression model, a positive interaction between the −629 CETP C>A genotype and glucocorticoid treatment on the HDL-C response to GH was found in all patients combined (P=0.002 and P<0.001 respectively), again independently of age (P>0.40) and baseline HDL-C (P=0.001).
Association of changes in high-density lipoprotein (HDL) cholesterol in response to GH therapy according to −629C>A cholesteryl ester transfer protein (CETP) promoter polymorphism by multiple linear regression analysis.
| ACTH sufficient (n=28) | On glucocorticoids (n=63) | |||
|---|---|---|---|---|
| β | P value | β | P value | |
| Age (years) | −0.127 | 0.46 | −0.054 | 0.66 |
| HDL cholesterol | −0.521 | 0.006 | −0.360 | 0.005 |
| −629C>A CETP polymorphism CC versus CA+AA | −0.345 | 0.053 | 0.323 | 0.006 |
β, Standardized regression coefficient; −629CC, CA, AA: CETP −629C>A promoter polymorphism.
Discussion
The present study has shown that HDL-C does not change in response to GH replacement in hypopituitary patients with and without an intact pituitary–adrenal axis. In the whole group, the individual changes in HDL-C were also not significantly associated with the −629 CETP C>A polymorphism. Remarkably, HDL-C increased in glucocorticoid-treated patients with the −629 CETP CC genotype, contrasting unchanged HDL-C levels in glucocorticoid patients with the −629 CETP AA+CA genotype and −629 CETP CC carriers with intact ACTH secretion. Furthermore, multivariate regression analysis showed that the presence of the −629 CETP C allele predicted an increase in HDL-C even independently of its baseline concentration in glucocorticoid-treated patients, whereas an opposite association was observed in ACTH-sufficient patients. Thus, our findings agree with the contention that there is an association of the HDL-C response to GH administration with this common CETP gene variation, which is modified by concomitant glucocorticoid administration.
Current conventional glucocorticoid replacement regimens often exceed the endogenous cortisol production rate in adults with intact adrenal reserve, which approximates 6–10 mg/m2 per day (33, 34). In the present cohort, the mean glucocorticoid substitution regimen (in hydrocortisone equivalents) was 11.0 mg/m2 per day; 60% of patients used a dose higher than 10 mg/m2 per day. Thus, despite some expected acceleration of peripheral cortisol metabolism during GH replacement (35, 36), glucocorticoid substitution doses should still be regarded to be high in many patients. Importantly, an increased risk of stroke and coronary heart disease may be related to concomitant glucocorticoid substitution treatment in GH-replaced hypopituitary patients (37). Taken together, these considerations provide a clinical rationale to elucidate whether effects of GH replacement on serum lipids is modified by concomitant glucocorticoid administration. It is obvious that the possible cardiovascular benefit of the increase in HDL-C in response to GH, averaging 0.15 mmol/l in glucocorticoid receiving hypopituitary patients who were homozygous for the −629 CETP C allele remains uncertain. A meta-analysis comprising prospective observational studies has demonstrated that mortality from ischemic heart disease is 33% lower for each 0.33 mmol/l higher HDL-C (38). Extrapolating these data (38) to the present findings would translate into a 15% risk reduction in this subgroup of patients, besides expected benefit from GH-induced LDL-C lowering.
Circulating CETP levels are highest in −629 CETP CC allele carriers (14, 16, 17, 18), whereas both GH and glucocorticoids are able to reduce serum CETP (7, 8, 20). The increase in HDL-C after GH replacement in glucocorticoid-treated hypopituitary patients homozygous for the −629 CETP CC allele may thus be explained in part by assuming that GH elicits a more pronounced decrease in circulating CETP levels in these patients. Although we did not measure serum CETP mass or activity in the present study, it is plausible that the GH-induced increase in HDL-C is attributable to CETP lowering, resulting in decreased transfer of cholesteryl esters out of HDL particles (11, 12, 13). Notably, it is likely that other mechanisms also contribute to the effects of glucocorticoids on HDL metabolism. Glucocorticoids increase hepatic synthesis of apolipoprotein A–I, the major HDL protein constituent (39, 40), and reduce hepatic lipase activity (41), thereby maintaining or even increasing HDL-C. Several interdependent metabolic processes may, therefore, be involved in an effect modification of concomitant glucocorticoid treatment on the HDL-C response to GH replacement.
The association of HDL-C with common CETP variations can be modified by environmental factors, such as diet composition and alcohol (reviewed in Ref. (15)). Our present findings support the possibility of a CETP gene-glucocorticoid treatment interaction with respect to the HDL-C response to GH replacement. Gene–environment interaction has also been suggested for ApoE (42). For example, ApoE gene variation predicts the LDL-C response to cholesterol feeding (32), whereas ApoE may interact with circulating CETP mass on the HDL-C response to a cholesterol-rich diet (43). Of potential interest, one study in hypopituitary patients suggested that the HDL-C changes after GH replacement are dependent on the ApoE genotype (6), although no such effects of the ApoE phenotype were observed in another report (44). The ApoE4 genotype did not predict changes in HDL-C response to GH replacement, and ApoE4 carrier status did not modify the association of the changes in HDL-C after GH with the −629 CETP C>A polymorphism in the present study. It seems unlikely that ApoE gene variation has a major impact on GH replacement-induced changes in HDL metabolism. Our report did show expected (45) differences in baseline serum total cholesterol and LDL-C between ApoE4 carriers and non-ApoE4 carriers, which reached statistical significance in patients who were not treated with glucocorticoids. This supports the presence of ApoE–serum lipid relationships in hypopituitary patients.
Some other aspects of our study need to be considered. The −629 CETP C allele frequently amounted to 57% in this cohort of hypopituitary adults as compared to 52% in a population-based survey in The Netherlands (46). This suggests that this CETP polymorphism did not result in an important cardiovascular morbidity-related selection of hypopituitary patients participating in the current study. The present study was sufficiently powered to be able to demonstrate statistically effects on the HDL-C in response to GH, which was our primary outcome variable. Of note, the number of hypopituitary patients included in our study was not large enough to demonstrate significant differences in HDL-C at baseline according to CETP genotype groups. Yet among patients not receiving glucocorticoids, the point estimates of the mean differences in HDL-C across CETP genotype groups were comparable to those reported recently (18).
In conclusion, this report suggests a common CETP variant-glucocorticoid treatment interaction concerning the effect of GH replacement on HDL-C. This may explain some of the reported variation in the HDL-C response to GH treatment, and could influence potential cardiovascular benefit.
Declaration of interest
The study is investigator driven, and the authors do not have any financial or other conflict of interest to declare.
Funding
This study is financially supported by Pfizer Inc., Stockholm, Sweden.
Acknowledgements
The help of Mrs Inge E Pop in data collection is greatly appreciated.
References
- 1↑
Tomlinson JW, Holden N, Hills RK, Wheatley K, Clayton RN, Bates AS, Sheppard MC, Stewart PM. Association between premature mortality and hypopituitarism. West Midlands Prospective Hypopituitary Study Group. Lancet 2001 357 425–431.
- 2↑
Carroll PV, Christ ER, Bengtsson BA, Carlsson L, Christiansen JS, Clemmons D, Hintz R, Ho K, Laron Z, Sizonenko P, Sonksen PH, Tanaka T, Thorne M. Growth hormone deficiency in adulthood and the effects of growth hormone replacement: a review. Growth Hormone Research Society Scientific Committee. Journal of Clinical Endocrinology and Metabolism 1998 83 382–395.
- 3↑
Attanasio AF, Bates PC, Ho KK, Webb SM, Ross RJ, Strasburger CJ, Bouillon R, Crowe B, Selander K, Valle D, Lamberts SWHypopituitary Control and Complications Study International Advisory Board. Human growth hormone replacement in adult hypopituitary patients: long-term effects on body composition and lipid status – 3-year results from the HypoCCS Database. Journal of Clinical Endocrinology and Metabolism 2002 87 1600–1606.
- 4↑
Abs R, Feldt-Rasmussen U, Mattsson AF, Monson JP, Bengtsson BA, Goth MI, Wilton P, Koltowska-Haggstrom M. Determinants of cardiovascular risk in 2589 hypopituitary GH-deficient adults – a KIMS database analysis. European Journal of Endocrinology 2006 155 79–90.
- 5↑
Molitch ME, Clemmons DR, Malozowski S, Merriam GR, Shalet SM, Vance ML, Endocrine Society's Clinical Guidelines Subcommittee, Stephens PA. Evaluation and treatment of adult growth hormone deficiency: an Endocrine Society Clinical Practice Guideline. Journal of Clinical Endocrinology and Metabolism 2006 91 1621–1634.
- 6↑
Leese GP, Wallymahmed M, Wieringa G, VanHeyningen C, MacFarlane IA. Apo E phenotype and changes in serum lipids in adult patients during growth hormone replacement. European Journal of Endocrinology 1999 140 174–179.
- 7↑
Beentjes JA, van Tol A, Sluiter WJ, Dullaart RPF. Effect of growth hormone replacement therapy on plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities in growth hormone-deficient adults. Journal of Lipid Research 2000 41 925–932.
- 8↑
Carrilho AJ, Cunha-Neto MB, Nunes VS, Lottenberg AM, Medina WL, Nakandakare ER, Musolino NR, Bronstein MD, Quintao EC. Plasma cholesteryl ester transfer protein and lipoprotein levels during treatment of growth hormone-deficient adult humans. Lipids 2001 36 549–554.
- 9↑
Maison P, Griffin S, Nicoue-Beglah M, Haddad N, Balkau B, Chanson P. Impact of growth hormone (GH) treatment on cardiovascular risk factors in GH-deficient adults: a metaanalysis of blinded, randomized, placebo-controlled trials. Journal of Clinical Endocrinology and Metabolism 2004 89 2192–2199.
- 10↑
Attanasio AF, Lamberts SW, Matranga AM, Birkett MA, Bates PC, Valk NK, Hilsted J, Bengtsson BA, Strasburger CJ. Adult growth hormone (GH)-deficient patients demonstrate heterogeneity between childhood onset and adult onset before and during human GH treatment. Adult Growth Hormone Deficiency Study Group. Journal of Clinical Endocrinology and Metabolism 1997 82 82–88.
- 12↑
Borggreve SE, de Vries R, Dullaart RPF. Alterations in high-density lipoprotein metabolism and reverse cholesterol transport in insulin resistance and type 2 diabetes mellitus: role of lipolytic enzymes, lecithin:cholesterol acyltransferase and lipid transfer proteins. European Journal of Clinical Investigation 2003 33 1051–1069.
- 13↑
Dullaart RPF, Dallinga-Thie GM, Wolffenbuttel BHR, van Tol A. CETP inhibition in cardiovascular risk management: a critical appraisal. European Journal of Clinical Investigation 2007 37 90–98.
- 14↑
Boekholdt SM, Thompson JF. Natural genetic variation as a tool in understanding the role of CETP in lipid levels and disease. Journal of Lipid Research 2003 44 1080–1093.
- 15↑
Dullaart RPF, Sluiter WJ. Common variation in the CETP gene and the implications for cardiovascular disease and its treatment: an updated analysis. Pharmacogenomics 2008 9 747–763.
- 16↑
Dachet C, Poirier O, Cambien F, Chapman J, Rouis M. New functional promoter polymorphism, CETP/−629, in cholesteryl ester transfer protein (CETP) gene related to CETP mass and high density lipoprotein cholesterol levels: role of Sp1/Sp3 in transcriptional regulation. Arteriosclerosis, Thrombosis, and Vascular Biology 2000 20 507–515.
- 17↑
Borggreve SE, Hillege HL, Wolffenbuttel BHR, de Jong PE, Bakker SJ, van der Steege G, van Tol A, Dullaart RPF. The effect of cholesteryl ester transfer protein −629C→A promoter polymorphism on high-density lipoprotein cholesterol is dependent on serum triglycerides. Journal of Clinical Endocrinology and Metabolism 2005 90 4198–4204.
- 18↑
Thompson A, Di Angelantonio E, Sarwar N, Erqou S, Saleheen D, Dullaart RPF, Keavney B, Ye Z, Danesh J. Association of cholesteryl ester transfer protein genotypes with CETP mass and activity, lipid levels, and coronary risk. Journal of the American Medical Association 2008 299 2777–2788.
- 19↑
Beentjes JAM, van Tol A, Sluiter WJ, Dullaart RPF. Low plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities in growth hormone deficient and acromegalic men: role in altered high density lipoproteins. Atherosclerosis 2000 153 491–498.
- 20↑
Beentjes JAM, van Tol A, Sluiter WJ, Dullaart RPF. Decreased plasma cholesterol esterification and cholesteryl ester transfer in hypopituitary patients on glucocorticoid replacement therapy. Scandinavian Journal of Clinical and Laboratory Investigation 2000 60 189–198.
- 21↑
Crown A, Lightman S. Why is the management of glucocorticoid deficiency still controversial: a review of the literature. Clinical Endocrinology 2005 63 483–492.
- 22↑
Dullaart RPF, Schols JL, van der Steege G, Zelissen PM, Sluiter WJ, van Beek AP. Glucocorticoid replacement is associated with hypertriglyceridaemia, elevated glucose and higher non-HDL cholesterol and may diminish the association of HDL cholesterol with the −629C>A CETP promoter polymorphism in GH-receiving hypopituitary patients. Clinical Endocrinology 2008 69 359–366.
- 23↑
Atger V, Leclerc T, Cambillau M, Guillemain R, Marti C, Moatti N, Girard A. Elevated high density lipoprotein concentrations in heart transplant recipients are related to impaired plasma cholesteryl ester transfer and hepatic lipase activity. Atherosclerosis 1993 103 29–41.
- 24↑
Georgiadis AN, Papavasiliou EC, Lourida ES, Alamanos Y, Kostara C, Tselepis AD, Drosos AA. Atherogenic lipid profile is a feature characteristic of patients with early rheumatoid arthritis: effect of early treatment – a prospective, controlled study. Arthritis Research & Therapy 2006 8 R82.
- 25↑
García-Gómez C, Nolla JM, Valverde J, Narváez J, Corbella E, Pintó X. High HDL-cholesterol in women with rheumatoid arthritis on low-dose glucocorticoid therapy. European Journal of Clinical Investigation 2008 38 686–692.
- 26↑
Dullaart RPF. Comment on high HDL-cholesterol in women with rheumatoid arthritis on low-dose glucocorticoid therapy. European Journal of Clinical Investigation 2009 39 533.
- 27↑
Dullaart RPF, Pasterkamp SH, Beentjes JA, Sluiter WJ. Evaluation of adrenal function in patients with hypothalamic and pituitary disorders: comparison of serum cortisol, urinary free cortisol and the human-corticotrophin releasing hormone test with the insulin tolerance test. Clinical Endocrinology 1999 50 465–471.
- 28↑
Tjeerdsma G, Sluiter WJ, Hew JM, Molenaar WM, de Lange WE, Dullaart RPF. Hyperprolactinaemia is associated with a higher prevalence of pituitary–adrenal dysfunction in non-functioning pituitary macroadenoma. European Journal of Endocrinology 1996 135 299–308.
- 29↑
Lukaski HC, Johnson PE. A simple, inexpensive method of determining total body water using a tracer dose of D2O and infrared absorption of biological fluids. American Journal of Clinical Nutrition 1985 41 391–398.
- 30↑
Lopez-Virella M, Stone P, Ellis S, Colwell J. Cholesterol determination in high-density lipoproteins separated by three different methods. Clinical Chemistry 1977 23 882–884.
- 31↑
Reymer PW, Groenemeyer BE, van de Burg R, Kastelein JJ. Apolipoprotein E genotyping on agarose gels. Clinical Chemistry 1995 41 1046–1047.
- 32↑
Blaauwwiekel EE, Beusekamp BJ, Sluiter WJ, Hoogenberg K, Dullaart RPF. Apolipoprotein E genotype is a determinant of low-density lipoprotein cholesterol and of its response to a low-cholesterol diet in type 1 diabetic patients with elevated urinary albumin excretion. Diabetic Medicine 1998 15 1031–1035.
- 33↑
Esteban NV, Loughlin T, Yergey AL, Zawadzki JK, Booth JD, Winterer JC, Loriaux DL. Daily cortisol production rate in man determined by stable isotope dilution/mass spectrometry. Journal of Clinical Endocrinology and Metabolism 1991 72 39–45.
- 34↑
Kraan GP, Dullaart RPF, Pratt JJ, Wolthers BG, Drayer NM, De Bruin R. The daily cortisol production reinvestigated in healthy men. The serum and urinary cortisol production rates are not significantly different. Journal of Clinical Endocrinology and Metabolism 1998 83 1247–1252.
- 35↑
Agha A, Monson JP. Modulation of glucocorticoid metabolism by the growth hormone–IGF1 axis. Clinical Endocrinology 2007 66 459–465.
- 36↑
Beentjes JA, Kerstens MN, Dullaart RPF. Effects of growth hormone replacement on cortisol metabolism in hypopituitary patients treated with cortisone acetate. Scandinavian Journal of Clinical and Laboratory Investigation 2001 61 277–286.
- 37↑
Filipsson H, Monson JP, Koltowska-Häggström M, Mattsson A, Johannsson G. The impact of glucocorticoid replacement regimens on metabolic outcome and comorbidity in hypopituitary patients. Journal of Clinical Endocrinology and Metabolism 2006 91 3954–3961.
- 38↑
Prospective Studies Collaboration Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55 000 vascular deaths. Lancet 2007 370 1829–1839.
- 39↑
Hargrove GM, Junco A, Wong NC. Hormonal regulation of apolipoprotein AI. Journal of Molecular Endocrinology 1999 22 103–111.
- 40↑
Berg AL, Nilsson-Ehle P. Direct effects of corticotropin on plasma lipoprotein metabolism in man – studies in vivo and in vitro. Metabolism 1994 43 90–97.
- 41↑
Jansen H, van Tol A, Auwerx J, Skretting G, Staels B. Opposite regulation of hepatic lipase and lecithin: cholesterol acyltransferase by glucocorticoids in rats. Biochimica et Biophysica Acta 1992 1128 181–185.
- 42↑
Talmud PJ, Humphries SE. Gene:environment interaction in lipid metabolism and effect on coronary heart disease risk. Current Opinion in Lipidology 2002 13 149–154.
- 43↑
Martin LJ, Connelly PW, Nancoo D, Wood N, Zhang ZJ, Maguire G, Quinet E, Tall AR, Marcel YL, McPherson R. Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype. Journal of Lipid Research 1993 34 437–446.
- 44↑
Johannsson G, Oscarsson J, Rosén T, Wiklund O, Olsson G, Wilhelmsen L, Bengtsson B-Å. Effects of 1 year of growth hormone therapy on serum lipoprotein levels in growth hormone-deficient adults. Influence of gender and Apo(a) and ApoE phenotypes. Arteriosclerosis, Thrombosis, and Vascular Biology 1995 15 2142–2150.
- 45↑
Bennet AM, Di Angelantonio E, Ye Z, Wensley F, Dahlin A, Ahlbom A, Keavney B, Collins R, Wiman B, de Faire U, Danesh J. Association of apolipoprotein E genotypes with lipid levels and coronary risk. Journal of the American Medical Association 2007 29 1300–1311.
- 46↑
Borggreve SE, Hillege HL, Wolffenbuttel BHR, de Jong PE, Zuurman MW, van der Steege G, van Tol A, Dullaart RPF. An increased coronary risk is paradoxically associated with common cholesteryl ester transfer protein gene variations that relate to higher high-density lipoprotein cholesterol: a population-based study. Journal of Clinical Endocrinology and Metabolism 2006 91 3382–3388.
