Growth Hormone & IGF Research 24 (2014) 174–179
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Long-term effects of growth hormone replacement therapy on liver function in adult patients with growth hormone deficiency Ryusaku Matsumoto a, Hidenori Fukuoka b, Genzo Iguchi b, Hitoshi Nishizawa a, Hironori Bando a, Kentaro Suda a, Michiko Takahashi a, Yutaka Takahashi a,⁎ a b
Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Division of Diabetes and Endocrinology, Kobe University Hospital, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 15 July 2014 Accepted 23 July 2014 Available online 31 July 2014 Keywords: Adult growth hormone deficiency (AGHD) Growth hormone replacement therapy Nonalcoholic steatohepatitis (NASH) Nonalcoholic fatty liver disease (NAFLD)
a b s t r a c t Objective: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are frequently observed in patients with adult growth hormone deficiency (AGHD) and short-term GH replacement therapy (GHRT) has reportedly been efficacious in NAFLD and NASH. The aim of this study was to investigate whether long-term GHRT is an effective treatment for the hepatic comorbidities in AGHD. Design: This is a retrospective observational study. We recruited 54 consecutive hypopituitary patients with AGHD. Among them, 31 patients who had received GHRT for more than 24 months were compared with 19 ageand sex-matched patients without GHRT. We also analyzed the long term effect of GHRT on 14 patients diagnosed with NASH by liver biopsy. In addition, we subdivided the GHRT group into GH-responder and GH-non-responder groups and analyzed the factors associated with the efficacy of the treatment. Results: For a period of 24 months, the significant reduction of serum liver enzyme levels and a fibrotic marker was observed in patients receiving GHRT compared with the control group. Furthermore, GHRT also improved liver enzyme levels in AGHD patients with NASH. The GH-non-responder group showed a higher proportion of patients who gained weight during the study period. Conclusions: These results indicate that GHRT is efficacious for improving serum liver enzyme levels for at least 24 months in patients with AGHD. To optimize this effect, it is important to avoid body weight gain during the treatment. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction A deficiency in growth hormone (GH) secretion in adults results in visceral obesity, abnormal lipid profile, and insulin resistance, leading to an increased risk of cardiovascular disease [1–3]. Furthermore, patients with adult GH deficiency (AGHD) exhibit decreased bone mineral density and impaired quality of life (QOL) [4,5]. Recently, an essential role of GH in liver has emerged [6]. In a murine model, liver-specific deletion of the GH receptor resulted in insulin resistance, glucose intolerance, and severe hepatic steatosis, indicating the physiological importance of GH signaling in the liver [7]. Regarding the downstream signaling of the GH receptor, liver-specific janus kinase (JAK)-2 or signal transducer and activator of transcription (STAT)-5 deficient mice also develop hepatic steatosis [8,9]. In addition, a spontaneous dwarf rat, in which GH is deficient, exhibits steatohepatitis and administration of insulin-like growth factor-I (IGF-I) as well as GH reversed these changes [10], suggesting that IGF-I may also play an important role in the liver.
⁎ Corresponding author. Tel.: +81 78 382 5861; fax: +81 78 382 2080. E-mail address: [email protected] (Y. Takahashi).
http://dx.doi.org/10.1016/j.ghir.2014.07.002 1096-6374/© 2014 Elsevier Ltd. All rights reserved.
A case report published in 1997 noted improvements in fatty liver associated with panhypopituitarism after GH administration, suggesting that fatty liver is at least partly attributable to GH deficiency [11]. Ichikawa et al. compared 5 hypopituitary patients without AGHD and 13 patients with AGHD, and found that nonalcoholic fatty liver disease (NAFLD) was more prevalent in the AGHD group [12]. Furthermore, Fukuda et al. reported that the incidence of metabolic comorbidities including NAFLD increased after the cessation of GH administration in adults with childhood-onset GHD depending on its duration [13]. Intriguingly, GH replacement therapy (GHRT) drastically reversed nonalcoholic steatohepatitis (NASH) in a case of AGHD, suggesting a beneficial effect of GH [14]. While Gardner et al. [15] reported that NAFLD is equally common in obese patients with GHD and in age- and body mass index (BMI)-matched control subjects, Nishizawa et al. [16] recently reported that the prevalence of NAFLD was significantly higher in patients with AGHD compared to age-, sex-, and BMI-matched controls, and at least 21% of patients with AGHD were diagnosed with NASH. Moreover, 6 months of GHRT significantly reduced serum liver enzyme levels in patients with AGHD and improved histological changes in the liver, concomitant with a reduction in fibrotic marker concentrations in patients with NASH. Collectively, these data indicate that NAFLD and
R. Matsumoto et al. / Growth Hormone & IGF Research 24 (2014) 174–179
NASH are one of the important comorbidities in patients with AGHD, and short-term GHRT may be beneficial. It has been shown that long-term GHRT improves body composition, lipid profile, carotid intima thickness, bone mineral density, QOL, and mortality due to cardiovascular disease in AGHD patients [17–20]. However, whether long-term GHRT is beneficial for the NAFLD and NASH in AGHD patients remains unknown. Therefore, we performed a retrospective study comparing liver function between AGHD patients with or without GHRT for 24 months and analyzed the factors associated with the efficacy of treatment. 2. Materials and methods 2.1. Study subjects This is a retrospective and observational study, which was approved by the Kobe University Graduate School of Medicine Ethics Committee. We screened 139 consecutive outpatients with hypopituitarism at Kobe University Hospital between January 2002 and March 2013. The study design is described in Fig. 1. For the diagnosis of AGHD, each patient was subjected to an insulin tolerance test (peak GH b3 ng/mL) or the GH releasing peptide-2 test (peak GH b9 ng/mL) [21]. The hypothalamo–pituitary–adrenal axis, hypothalamo–pituitary–thyroid axis, and hypothalamo–pituitary–gonadal axis were evaluated as previously described [22]. Exclusion criteria included alcohol consumption of more than 20 g/day for women and more than 30 g/day for men. Subjects were excluded if they had hepatitis B or C, autoimmune hepatitis, other liver diseases, or if they were receiving drugs known to cause steatohepatitis. Patients with acromegaly and Cushing's disease were also excluded. Accordingly, we compared 31 patients with GHRT and 19 age- and sex-matched patients without GHRT (Fig. 1). The diagnosis of NAFLD was made by ultrasonography assessing hepatic fatty change, including hepato-renal contrast, impaired visualization of the hepatic vein borders or the diaphragm [23]. The diagnosis of NASH was made by a liver biopsy with written informed consent according to the standard clinical indication for liver disease [24]. Liver biopsy specimens
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were evaluated independently by 2 pathologists blinded to the patients' clinical histories. NASH was diagnosed when the pathological findings exhibited type 3 or 4 of Matteoni's classification [25]. 2.2. GHRT The GHRT group received recombinant human GH administration according to clinical practice guidelines [26,27]. In brief, the initial dose of GHRT was 0.1–0.2 mg/day. The dose was titrated in 2–3 month intervals by an attending physician according to serum IGF-I levels, subjective symptoms, QOL, and side effects. The target for the IGF-I standard deviation score (SDS) level was within −2.0 to +2.0. The mean dose of GHRT at 24 months was 0.23 ± 0.15 mg/day (male, 0.17 ± 0.15 mg/day, female, 0.26 ± 0.14 mg/day). 2.3. Glucocorticoid and thyroxine replacement therapy Most of the patients received glucocorticoid and thyroxine replacement therapy. Both replacement therapies were performed according to clinical consensus or clinical practice guideline [28,29]. In brief, they received 10–20 mg/day of oral hydrocortisone, which were titrated according to their well-being clinical data including metabolic state. Thyroxine administration was started with 12.5 to 25 μg/day and gradually increased until free-T4 levels reached within the high normal range. During GHRT, there were no patients in whom glucocorticoid and thyroxine doses were changed. 2.4. Measures Data were retrospectively collected from patients' medical records. In the control group, we defined January 2010 as the baseline. Primary outcome of this study was longitudinal change of serum liver enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). AST, ALT, g-GTP, ALP, and LDH levels were measured using a standard method (AST and ALT, Kanto Chemical Co., Inc., Tokyo, Japan; γ-GTP, ALP, and LDH, Shino-Test Co., Tokyo, Japan). Secondary outcome of this study was longitudinal change of serum hyaluronic acid level as a fibrotic marker, which was measured using latex agglutination-turbidimetric immunoassay (Mitsubishi Chemical Medicine Co., Tokyo, Japan). The laboratory measurements were performed at each occasion when the patients visited the hospital in the same laboratory during the study period. Measurements of liver enzymes were performed in Kobe University Hospital Clinical Laboratory, and measurements of hyaluronic acid were entrusted to SNL Inc. The coefficients of variation for the assays of liver enzymes and hyaluronic acid are the following; AST 0.79%, ALT 0.6%, γ-GTP 0.65%, LDH 0.64%, ALP 0.8%, hyaluronic acid 2.42%, respectively, for the sample within each normal range. 2.5. The definition of GH-responder and GH-non-responder in patients with GHRT In order to clarify the factors associated with GHRT efficacy, we subdivided the GHRT group into GH-responder group and GH-nonresponder group, and compared various indices between these groups. The GH-responder group was defined as patients in whom more than half of the abnormal liver enzymes (AST, ALT, γ-GTP, ALP, LDH) at baseline improved to within normal limit through the study period. We also analyzed the rate of patients who gained weight at 24 months compared with at the baseline in these groups. 2.6. Statistical analysis
Fig. 1. Study design.
Data are appropriately expressed as mean ± standard deviation, mean ± standard error of mean, or median [1st quartile, 3rd quartile].
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R. Matsumoto et al. / Growth Hormone & IGF Research 24 (2014) 174–179
Categorical data were compared by χ2 test or Fisher's exact test and continuous data were compared by Student's t-test or a Mann–Whitney test, as appropriate. Homogeneity of variance was tested by Levene's test. Longitudinal data of both groups were compared by 1-way repeated measures analysis of variance (ANOVA) followed by a Tukey–Kramer honestly significant difference test or 2-way repeated measures ANOVA, followed by a pairwise multiple-comparison procedure. The two sided P values of 0.05 or less were considered significant. Statistical analyses were performed using JMP Statistical Database Software version 8. 0. 1 (SAS Institute, Inc., Cary, NC, USA). 3. Results 3.1. Baseline characteristics
Gender (male/female) Age (years) BMI IGF-I SDS Hypertension (%) Dyslipidemia (%) HbA1c (%) FPG (mg/dL) HOMA-IR Total-C (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) TG (mg/dL) NAFLD (%) Glucocorticoid replacement (%) Thyroxine replacement (%) Gonadal replacement (%) n
3.2. Effects of GHRT on liver function At 24 months, the GHRT group showed a significant increase in the IGF-I SDS, from − 4.3 ± 3.7 at baseline to − 1.0 ± 2.1, than the control group (from − 2.9 ± 2.1 at baseline to − 3.3 ± 1.8) (Fig. 2A; P b 0.0001). BMI remained unchanged in each group (GHRT group, from 27.9 ± 4.8 kg/m2 at baseline to 28.6 ± 4.7 kg/m2 at 24 months, P = 0.15; control group, from 25.0 ± 2.8 kg/m2 to 26.6 ± 3.4 kg/m2, P = 0.25) and there was no significant difference between them
Control group
P value
11/20 44.9 ± 18.6 27.9 ± 4.8 −4.3 ± 3.7 32.3 71.0 5.9 ± 0.8 97.9 ± 24.9 3.0 [1.9, 6.2] 228.2 ± 49.6 137.9 ± 40.6 64.0 ± 18.1 196 [123, 240] 65.5 93.6 93.6 M 27.3 F 30.0 31
8/11 52.3 ± 13.3 25.0 ± 2.8 −2.6 ± 2.2 31.6 73.7 5.6 ± 0.4 93.1 ± 13.9 1.2 [0.8, 5.2] 201.6 ± 46.0 128.9 ± 35.8 51.4 ± 18.0 147 [107, 213] 61.1 89.5 94.7 M 30.0 F 15.4 19
0.64 0.14 0.07 0.07 0.96 0.84 0.31 0.45 0.19 0.16 0.49 0.05* 0.24 0.76 0.61 0.86 0.89 0.34
(Fig. 2B; P = 0.53). At baseline, the levels of AST, ALT, γ-GTP, and LDH were significantly higher in the GHRT group than in the control group (Fig. 3A, B, C, D, and E). Subsequently, we analyzed the longitudinal changes in liver enzyme levels and fibrotic marker for 24 months. Intriguingly, the levels of AST, ALT, γ-GTP, and LDH gradually increased during the observation period in the control group. In a sharp contrast, these enzyme levels were rapidly decreased after 3 months of GHRT, and
A
P < 0.0001 1 0 -1 -2 -3 -4 -5
0
3
6
12
24
(month)
P = 0.53
B 34 32
Table 1 Etiology of hypopituitarism.
Craniopharyngioma Pituitary adenoma Sheehan's syndrome Rathke's cleft cyst Germinoma Invisible stalk Empty sella Hypophysitis Langerhans cell histiocytosis Pituitary apoplexy Malignant lymphoma Meningioma Unknown cause n
GHRT group
Data were compared by the χ2 test, Fisher's exact test, Student's t-test, or Mann–Whitney test, as appropriate. * indicates P value b0.05. FPG: fasting plasma glucose; HOMA-IR: homeostasis model assessment-insulin resistance; C: cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglyceride; M: male; F: female.
IGF -I SDS
After screening 139 consecutive outpatients with hypopituitarism, it was shown that 61 patients exhibited adult GH deficiency, in whom 38 patients received GHRT. Among these patients with GHRT, 6 patients had received GHRT for less than 24 months. One patient discontinued GHRT because of a malignancy. No patients discontinued because of a lack of effectiveness. Twenty-three patients did not receive GHRT, despite there was an indication for GHRT. Although the reason why these patients did not receive GHRT varies according to the individuals, most patients declined because of a hesitation in injection and some patients did not feel necessity irrespective of their understanding for the medical importance. As a result, 31 AGHD patients with GHRT and 19 age- and sex-matched AGHD patients without GHRT were enrolled in this study (Fig. 1). Table 1 shows the etiology of hypopituitarism. Craniopharyngioma was more prevalent in the GHRT group; however, there was no statistically significant difference between these groups (P = 0.25). The baseline characteristics of these groups are shown in Table 2. Although body mass index (BMI) tended to be higher in the GHRT group, it was not significant (P = 0.07). HbA1c and HOMA-IR levels were comparable in both groups. In lipid profiles, high density lipoprotein (HDL) was significantly higher in the GHRT group (P = 0.05). IGF-I SDS was lower in the GHRT group, which may indicate that the GHRT group had more severe GHD, but there was no statistical difference (P = 0.07).
Table 2 Baseline characteristics of the GHRT group and control group.
Control group
8 3 3 4 4 2 1 1 2 1 1 1 0 31
1 5 3 1 1 1 2 2 0 1 0 0 2 19
BMI
30 GHRT group
28 26 24 22
0
3 6
12
24
(month) Fig. 2. Longitudinal changes in serum IGF-I and BMI in the GHRT group and control group. The solid line indicates the GHRT group and the dotted line indicates the control group. These groups were compared by repeated measures 2-way analysis of variance (ANOVA) for GHRT and time, followed by a pairwise multiple-comparison procedure. * indicates P value b 0.05, ** indicates P value b 0.01. Error bars show standard error. A. IGF-I standard deviation score (SDS) B. BMI.
R. Matsumoto et al. / Growth Hormone & IGF Research 24 (2014) 174–179
B
P < 0.001
50
60
20 10
24
0 3 6
(month)
D
P = 0.012 120 100 80 60 40 20 0 3 6
12
24
320 300 280 260 240 220 200 180
240 220 200 180 0 3 6
12
24
Hyaluronic acid (ng/mL)
LDH (IU)
260
160
30
C
P = 0.003
0 3 6
12
24
P = 0.046
100
24
0 3 6 12 (month)
24
D
P = 0.008 260
400
220
360
180 140 100 20
40
0 3 6
(month)
12
24
P < 0.001
E
20
Fig. 3. Longitudinal changes in serum hepatic enzyme levels and fibrotic marker in the GHRT group and control group. The solid line indicates the GHRT group and the dotted line indicates the control group. These groups were compared by repeated measures 2way analysis of variance (ANOVA) for GHRT and time, followed by a pairwise multiplecomparison procedure. * indicates P value b 0.05, ** indicates P value b 0.01. Error bars show standard error. A. Aspartate aminotransferase (AST) B. Alanine aminotransferase (ALT) C. γ-Glutamyl transpeptidase (γ-GTP) D. Alkaline phosphatase (ALP) E. Lactate dehydrogenase (LDH) F. Hyaluronic acid.
sustained at a lower level for 24 months in the GHRT group (Fig. 3A, B, C, D, and E. AST, P b 0.001; ALT, P b 0.001; γ-GTP, P = 0.012; ALP, P = 0.053; and LDH, P = 0.003, respectively). At the end of the study, the levels of the liver enzymes AST, ALT, and LDH, were significantly higher in the control group. In addition, the levels of the fibrotic marker hyaluronic acid, gradually increase in the control group but remained unchanged in the GHRT group (Fig. 3F; P = 0.048).
0 3 6 12 (month)
340 300 260 220 180 140
0 3 6 12 (month)
24
F
24
(month)
280
200
0 3 6 12 (month)
60
320
240
60
80
0
0 3 6 12 (month)
100 90 80 70 60 50 40 30 20
P = 0.22
F
P = 0.003
40 20
(month)
280
50
P = 0.053
(month)
E
24
(month)
ALP (IU)
γ-GTP (IU)
C
12
γ-GTP (IU)
12
LDH (IU)
0 3 6
ALT (IU)
70
30
ALP (IU)
30
80
40
AST (IU)
40
20
B
P = 0.002
60
ALT (IU)
AST (IU)
50
A
P < 0.001
Hyarulonic acid (ng/mL)
A
177
24
P = 0.46 50 40 30 20 10 0
0 3 6 12 (month)
24
Fig. 4. Longitudinal changes in serum hepatic enzyme levels and fibrotic marker in patients with NASH during GHRT. Data were analyzed by repeated measures 1-way analysis of variance (ANOVA) followed by a Tukey-Kramer honestly significant difference test. * indicates P value b 0.05, ** indicates P value b 0.01. Error bars show standard error. A. Aspartate aminotransferase (AST) B. Alanine aminotransferase (ALT) C. γ-Glutamyl transpeptidase (γ-GTP) D. Alkaline phosphatase (ALP) E. Lactate dehydrogenase (LDH) F. Hyaluronic acid.
3.3. Effects of GHRT on AGHD patients with NASH
3.4. Factors associated with GHRT efficacy
Liver biopsy was performed in 16 patients. Of these patients, 14 were diagnosed with NASH, and all of them were treated with GH [14]. We analyzed the long-term effect of GHRT in 13 patients with NASH, who were treated for more than 24 months (Fig. 4). The levels of the liver enzymes AST, ALT, γ-GTP, and LDH were significantly decreased after 3 months of GHRT, and these effects were sustained until 24 months (Fig. 4A, B, C, D and E. AST, P = 0.002; ALT, P = 0.003; γ-GTP, P = 0.008; ALP, not significant; and LDH, P b 0.001, respectively). The levels of hyaluronic acid tended to decrease during 12 months; however, including the levels at 24 months, there was no significant improvement in the patients with NASH (Fig. 4F; P = 0.46).
To clarify the factors associated with the efficacy of GHRT on the liver, we subdivided the GHRT group into GH-responder group and GH-non-responder group by the effect on liver dysfunction, and compared various indices between these groups at baseline. There were no differences in the characteristics between the GH-responder group and GH-non-responder group at the baseline; however, the proportion of the patients who gained body weight during GHRT was significantly lower in the GH-responder group (Table 3; 44% vs. 82%, P = 0.04). Regarding the other hormone replacement therapy, the female patients in the GH-non-responder group showed a higher proportion of gonadal replacement therapy (Table 3; P = 0.02).
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Table 3 Comparison of the GH-responder group and GH-non-responder group.
Sex (male/female) Age (years) BMI Body weight gain (%) IGF-I SDS Hypertension (%) Dyslipidemia (%) HbA1c (%) HOMA-IR Glucocorticoid replacement (%) Thyroxine replacement (%) Gonadal replacement (%) Dose of GHRT (mg/day) Peak GH with ITT (ng/mL) Peak GH with GHRP-2 (ng/mL) AST (IU/L) ALT (IU/L) γ-GTP (IU/L) ALP (IU/L) LDH (IU/L) Hyaluronic acid (ng/mL) n
GH-responder group
Non-responder group
P value
14/6 45.0 ± 16.3 27.4 ± 5.5 44 −5.0 ± 0.8 35 65 5.9 ± 0.9 3.2 [1.6, 5.4] 100 95 M 16.7 F 0.0 M 0.11 ± 0.09 F 0.26 ± 0.10 0.1 [0, 0.2] 0.73 [0.12, 1.07] 40.9 ± 7.2 44.1 ± 39.2 61.2 ± 33.2 257.6 ± 24.1 236.1 ± 91.3 38.3 ± 17.4 20
6/5 44.6 ± 22.9 28.5 ± 4.0 82 −3.1 ± 1.1 27 82 5.9 ± 0.7 2.8 [2.2, 7.3] 82 91 M 40.0 F 42.9 M 0.25 ± 0.10 F 0.28 ± 0.09 0.0 [0.0, 0.35] 0.14 [0.03, 0.41] 40.6 ± 9.6 48.1 ± 39.2 113.5 ± 43.7 267.4 ± 31.7 241.0 ± 46.0 40.1 ± 20.0 11
0.39 0.96 0.60 0.04* 0.19 0.66 0.31 0.88 0.76 0.12 1.0 0.39 0.02* 0.18 0.78 0.96 0.19 0.99 0.79 0.35 0.81 0.87 0.95
Data were compared by the χ2 test, Fisher's exact test, Student's t-test, or Mann–Whitney test, as appropriate. * indicates P value b0.05. Body weight (BW) gain is defined as BW at 24 months N BW at baseline. ITT: insulin tolerance test; GHRP-2: growth hormone releasing peptide-2; M: male; F: female.
4. Discussion In the present study, we demonstrated that GHRT was efficacious for improving serum liver enzyme levels and fibrotic marker for at least 24 months in patients with AGHD. In addition, long-term GHRT was also effective for treating AGHD patients with NASH in terms of liver dysfunction. Although it was difficult to predict the effect of GHRT before treatment, we showed that body weight gain during treatment was associated with the deteriorated effect of GHRT, suggesting that the control of body weight is important. Notably, in contrast to the control group, in which liver enzyme levels gradually increased, the GHRT group exhibited an improvement in these markers, indicating the progression of liver dysfunction in the patients without GHRT and the significance of GHRT to protect against it. In contrast to the established efficacy of GHRT on visceral obesity and QOL in patients with AGHD [30,31,17], its effect on the liver has not been fully elucidated. Several case reports and clinical studies have reported the efficacy of GHRT on NAFLD and NASH in patients with AGHD [32,14,16]. On the other hand, Gardner et al. reported that GHRT did not reduce liver fat measured by magnetic resonance imaging. However, when analysis was restricted to patients with NASH, there was a trend towards a reduction in liver fat content (P = 0.07). In addition, although it was demonstrated that serum liver enzyme levels were unchanged before and after 6 months of GHRT, body weight tended to increase during treatment (+2.3 kg) in their study [15]. Taken together with the present data, in which weight gain during GHRT deteriorates the effect on the liver, this may explain the discrepancy between Gardner's and our data. In the present study, we first demonstrated the long-term effects of GHRT on the liver. Our results suggest that the liver is an important target tissue for GH and when considering the indication of GHRT in patients with AGHD, the presence of NAFLD or NASH as a comorbidity should be taken into account. The role of the GH-IGF-I axis in the liver has recently emerged [6]. In an animal model, liver-specific deletion of the GH receptor and the downstream signaling molecules JAK-2 and STAT-5 results in steatosis [9,8]. GH receptor loss of function mutations in humans (Laron syndrome) also manifests as NAFLD in adults, and chronic replacement
of IGF-I does not influence the NAFLD status [33], suggesting that GH has a direct effect on the liver, particularly in the prevention of steatosis in hepatocytes. On the other hand, a spontaneous dwarf rat, in which GH is deficient, exhibits hepatic steatosis and fibrosis. Interestingly, IGF-I as well as GH administration was able to reverse these changes in the liver [10], suggesting the presence of a GH-independent action of IGF-I. Many reports indicate the significance of serum IGF-I levels in liver disease. NAFLD is associated with lower circulating levels of IGF-I [34]. Interleukin-6 and IGF-I are independent prognostic factors of NAFLD in morbidly obese patients [35]. In addition, the levels of hyaluronic acid, a marker of fibrosis, showed a negative correlation with IGF-I and the IGF-I/IGF binding protein-3 ratio in patients with NAFLD [36]. These data suggest that not only GH but also IGF-I plays a pivotal role in the liver. In a clinical setting, it is important to predict and improve the efficacy of GHRT. Younger age is reported to be a predictor of the effectiveness of GHRT, in terms of improvements in abnormal lipid profile and body composition [17]. In this study, we examined the factors which are predictive of GHRT efficacy on the liver. Although we could not detect any pre-treatment indices that predict GHRT effectiveness, body weight gain during the treatment deteriorated the efficacy. Given that obesity itself is an essential risk factor for the development of NAFLD, one can safely reason that the body weight gain during treatment has a negative impact on the effect of GHRT. Our results also suggest the importance of body weight control during GHRT to obtain an optimal effect. We cannot exclude the possibility that the other pituitary hormone deficiencies especially sex steroid, may interfere with the effects of GHRT on the liver. Female patients in the GH-non-responder group had higher proportion of gonadal replacement therapy. It is known that estrogen blocks the ability of GH to generate IGF-I in the liver [37]. Although it was not a significant difference, lower serum IGF-I levels between patients with or without gonadal replacement therapy (−2.2 ± 1.8 vs. −1.5 ± 2.6 SDS, respectively, P = 0.22) maybe associated with the effect of GHRT. These results suggest that female patients who have gonadal replacement therapy may need more dose of GH in terms of the improvement of liver dysfunction. There are several limitations in this study. First, because of the retrospective and non-randomized study design, there was a selection bias between the control and GHRT groups. Although we recruited ageand sex-matched control group, BMI tended to be higher in the GHRT group. In addition, there was a tendency towards a decreased IGF-I SDS and higher prevalence of craniopharyngioma in the GHRT group. These differences might be reflected by a preference towards GHRT in younger patients and those with more severe disease. As a result, there were significant differences in serum liver enzyme levels at baseline. Nevertheless, we observed a reversal change of serum liver enzyme levels during treatment between the control and GHRT group, further supporting the efficacy of GHRT. Secondly, although we have shown that fibrotic marker hyaluronic acid levels were significantly decreased in the GHRT group compared with the control group (Fig. 3F), the effect of GHRT on fibrosis is inconclusive. There have been several markers for evaluating liver fibrosis and serum hyaluronic acid level is reportedly a useful marker, especially for severe liver fibrosis [38]. For example, it has been reported that serum hyaluronic acid level showed a significant positive correlation with the degrees of liver fibrosis and the cut-off value of 46.1 ng/mL exhibited a sensitivity of 85% and a specificity of 80% for identifying severe fibrosis. However, because of the sensitivity and specificity, liver biopsy is gold standard for the evaluation of liver fibrosis at present and it remains unclear whether longitudinal changes in serum hyaluronic acid levels reflect precise status of liver fibrosis. Thus, accurate evaluation of liver fibrosis requires liver biopsy; however, because of the invasive procedure, enough number of patients in this study had not undergone liver biopsy after GHRT for the histological evaluation. Further investigation is necessary to clarify the long-term effect of GHRT on fibrosis. Thirdly, patients in the GHRT group received a relatively low dose of GH (0.23 ± 0.15 mg/day) compared to the previous studies [30,18,17]. Serum IGF-I SDS during the
R. Matsumoto et al. / Growth Hormone & IGF Research 24 (2014) 174–179
treatment ranged from − 1.0 to 0, suggesting that the effect was not optimized in the GHRT group. Nonetheless, hepatic markers dramatically improved, even at 3 months after the initiation of GHRT. These data suggest that the threshold of IGF-I concentration in the liver may be different from other tissues, such as adipose tissue. Another possible explanation is a higher local concentration of IGF-I in the liver than in other organs [39], and a paracrine effect of IGF-I may be important to improve liver dysfunction. In conclusion, we demonstrated that GHRT has a beneficial effect on improving serum liver enzyme levels for at least 24 months in hypopituitary patients with AGHD. Body weight gain has a negative impact on the effect of GHRT. Although further study is necessary to clarify the long-term prognosis and significance of GHRT in AGHD patients with NAFLD or NASH, these data emphasize the importance of NAFLD and NASH as a comorbidity in patients with AGHD. Conflict of interest disclosures Dr. Takahashi reports receiving research funding and payments for lectures from Ili Lily. No other potential conflict of interest relevant to this manuscript was reported. Author contributions Ryusaku Matsumoto: collection, analysis, and interpretation of data and drafting of the manuscript. Hidenori Fukuoka, Genzo Iguchi, Hitoshi Nishizawa, Hironori Bando, Kentaro Suda, and Michiko Takahashi: collection and assembly of data. Yutaka Takahashi: conception, study design, and critical revision of the manuscript for important intellectual content. Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology 23659477, 23591354, and 22591012, Grantsin-Aid for Scientific Research (research on hypothalamic–hypophyseal disorders) from the Ministry of Health, Labor, and Welfare, Japan, Daiichi-Sankyo Foundation of Life Science, and Naito Foundation. The authors are grateful to C. Ogata and K. Imura for their excellent technical assistance. References [1] J.O. Johansson, J. Fowelin, K. Landin, I. Lager, B.A. Bengtsson, Growth hormonedeficient adults are insulin-resistant, Metabolism 44 (1995) 1126–1129. [2] T. Rosén, S. Edén, G. Larson, L. Wilhelmsen, B.-Å. Bengtsson, Cardiovascular risk factors in adult patients with growth hormone deficiency, Acta Endocrinol. (Copenh) 129 (1993) 195–200. [3] V. Markussis, S.A. Beshyah, D.G. Johnston, C. Fisher, A.N. Nicolaides, P. Sharp, Detection of premature atherosclerosis by high-resolution ultrasonography in symptomfree hypopituitary adults, Lancet 340 (1992) 1188–1192. [4] T. Rosén, T. Hansson, H. Granhed, J. Szucs, B.-Å. Bengtsson, Reduced bone mineral content in adult patients with growth hormone deficiency, Acta Endocrinol. (Copenh) 129 (1993) 201–206. [5] T. Rosen, L. Wiren, L. Wilhelmsen, I. Wiklund, B.A. Bengtsson, Decreased psychological well-being in adult patients with growth hormone deficiency, Clin. Endocrinol. (Oxf.) 40 (1994) 111–116. [6] Y. Takahashi, Essential roles of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in the liver [review], Endocr. J. 59 (2012) 955–962. [7] Y. Fan, R.K. Menon, P. Cohen, et al., Liver-specific deletion of the growth hormone receptor reveals essential role of growth hormone signaling in hepatic lipid metabolism, J. Biol. Chem. 284 (2009) 19937–19944. [8] B.C. Sos, C. Harris, S.M. Nordstrom, et al., Abrogation of growth hormone secretion rescues fatty liver in mice with hepatocyte-specific deletion of JAK2, J. Clin. Invest. 121 (2011) 1412–1423. [9] J.L. Barclay, C.N. Nelson, M. Ishikawa, et al., GH-dependent STAT5 signaling plays an important role in hepatic lipid metabolism, Endocrinology 152 (2011) 181–192. [10] H. Nishizawa, M. Takahashi, H. Fukuoka, G. Iguchi, R. Kitazawa, Y. Takahashi, GHindependent IGF-I action is essential to prevent the development of nonalcoholic steatohepatitis in a GH-deficient rat model, Biochem. Biophys. Res. Commun. 423 (2012) 295–300.
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Yakar, Mechanisms of disease: metabolic effects of growth hormone and insulin-like growth factor 1, Nat. Clin. Pract. End. Met. 3 (2007) 302–310. [21] K. Chihara, A. Shimatsu, N. Hizuka, et al., A simple diagnostic test using GH-releasing peptide-2 in adult GH deficiency, Eur. J. Endocrinol. 157 (2007) 19–27. [22] J.W. Tomlinson, N. Holden, R.K. Hills, et al., Association between premature mortality and hypopituitarism. West Midlands Prospective Hypopituitary Study Group, Lancet 357 (2001) 425–431. [23] S. Saadeh, Z.M. Younossi, E.M. Remer, et al., The utility of radiological imaging in nonalcoholic fatty liver disease, Gastroenterology 123 (2002) 745–750. [24] L.A. Adams, J.A. Talwalkar, Diagnostic evaluation of nonalcoholic fatty liver disease, J. Clin. Gastroenterol. 40 (2006) S34–S38. [25] C.A. Matteoni, Z.M. Younossi, T. Gramlich, N. Boparai, Y.C. Liu, A.J. McCullough, Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity, Gastroenterology 116 (1999) 1413–1419. [26] M.E. Molitch, D.R. Clemmons, S. Malozowski, et al., Evaluation and treatment of adult growth hormone deficiency: an Endocrine Society Clinical Practice Guideline, J. Clin. Endocrinol. Metab. 91 (2006) 1621–1634. [27] K.K. Ho, GH Deficiency Consensus Workshop Participants, Consensus guidelines for the diagnosis and treatment of adults with GH deficiency II: a statement of the GH research society in association with the European Society for Pediatric Endocrinology, Lawson Wilkins Society, European Society of Endocrinology, Japan Endocrine Society, and Endocrine Society of Australia, Eur. J. Endocrinol. 157 (2007) 695–700. [28] A.B. Grossman, Clinical review#: the diagnosis and management of central hypoadrenalism, J. Clin. Endocrinol. Metab. 95 (2010) 4855–4863. [29] J.R. Garber, R.H. Cobin, H. 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Long-term effects of growth hormone replacement therapy on liver function in adult patients with growth hormone deficiency Ryusaku Matsumoto a, Hidenori Fukuoka b, Genzo Iguchi b, Hitoshi Nishizawa a, Hironori Bando a, Kentaro Suda a, Michiko Takahashi a, Yutaka Takahashi a,⁎ a b
Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Division of Diabetes and Endocrinology, Kobe University Hospital, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 15 July 2014 Accepted 23 July 2014 Available online 31 July 2014 Keywords: Adult growth hormone deficiency (AGHD) Growth hormone replacement therapy Nonalcoholic steatohepatitis (NASH) Nonalcoholic fatty liver disease (NAFLD)
a b s t r a c t Objective: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are frequently observed in patients with adult growth hormone deficiency (AGHD) and short-term GH replacement therapy (GHRT) has reportedly been efficacious in NAFLD and NASH. The aim of this study was to investigate whether long-term GHRT is an effective treatment for the hepatic comorbidities in AGHD. Design: This is a retrospective observational study. We recruited 54 consecutive hypopituitary patients with AGHD. Among them, 31 patients who had received GHRT for more than 24 months were compared with 19 ageand sex-matched patients without GHRT. We also analyzed the long term effect of GHRT on 14 patients diagnosed with NASH by liver biopsy. In addition, we subdivided the GHRT group into GH-responder and GH-non-responder groups and analyzed the factors associated with the efficacy of the treatment. Results: For a period of 24 months, the significant reduction of serum liver enzyme levels and a fibrotic marker was observed in patients receiving GHRT compared with the control group. Furthermore, GHRT also improved liver enzyme levels in AGHD patients with NASH. The GH-non-responder group showed a higher proportion of patients who gained weight during the study period. Conclusions: These results indicate that GHRT is efficacious for improving serum liver enzyme levels for at least 24 months in patients with AGHD. To optimize this effect, it is important to avoid body weight gain during the treatment. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction A deficiency in growth hormone (GH) secretion in adults results in visceral obesity, abnormal lipid profile, and insulin resistance, leading to an increased risk of cardiovascular disease [1–3]. Furthermore, patients with adult GH deficiency (AGHD) exhibit decreased bone mineral density and impaired quality of life (QOL) [4,5]. Recently, an essential role of GH in liver has emerged [6]. In a murine model, liver-specific deletion of the GH receptor resulted in insulin resistance, glucose intolerance, and severe hepatic steatosis, indicating the physiological importance of GH signaling in the liver [7]. Regarding the downstream signaling of the GH receptor, liver-specific janus kinase (JAK)-2 or signal transducer and activator of transcription (STAT)-5 deficient mice also develop hepatic steatosis [8,9]. In addition, a spontaneous dwarf rat, in which GH is deficient, exhibits steatohepatitis and administration of insulin-like growth factor-I (IGF-I) as well as GH reversed these changes [10], suggesting that IGF-I may also play an important role in the liver.
⁎ Corresponding author. Tel.: +81 78 382 5861; fax: +81 78 382 2080. E-mail address: [email protected] (Y. Takahashi).
http://dx.doi.org/10.1016/j.ghir.2014.07.002 1096-6374/© 2014 Elsevier Ltd. All rights reserved.
A case report published in 1997 noted improvements in fatty liver associated with panhypopituitarism after GH administration, suggesting that fatty liver is at least partly attributable to GH deficiency [11]. Ichikawa et al. compared 5 hypopituitary patients without AGHD and 13 patients with AGHD, and found that nonalcoholic fatty liver disease (NAFLD) was more prevalent in the AGHD group [12]. Furthermore, Fukuda et al. reported that the incidence of metabolic comorbidities including NAFLD increased after the cessation of GH administration in adults with childhood-onset GHD depending on its duration [13]. Intriguingly, GH replacement therapy (GHRT) drastically reversed nonalcoholic steatohepatitis (NASH) in a case of AGHD, suggesting a beneficial effect of GH [14]. While Gardner et al. [15] reported that NAFLD is equally common in obese patients with GHD and in age- and body mass index (BMI)-matched control subjects, Nishizawa et al. [16] recently reported that the prevalence of NAFLD was significantly higher in patients with AGHD compared to age-, sex-, and BMI-matched controls, and at least 21% of patients with AGHD were diagnosed with NASH. Moreover, 6 months of GHRT significantly reduced serum liver enzyme levels in patients with AGHD and improved histological changes in the liver, concomitant with a reduction in fibrotic marker concentrations in patients with NASH. Collectively, these data indicate that NAFLD and
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NASH are one of the important comorbidities in patients with AGHD, and short-term GHRT may be beneficial. It has been shown that long-term GHRT improves body composition, lipid profile, carotid intima thickness, bone mineral density, QOL, and mortality due to cardiovascular disease in AGHD patients [17–20]. However, whether long-term GHRT is beneficial for the NAFLD and NASH in AGHD patients remains unknown. Therefore, we performed a retrospective study comparing liver function between AGHD patients with or without GHRT for 24 months and analyzed the factors associated with the efficacy of treatment. 2. Materials and methods 2.1. Study subjects This is a retrospective and observational study, which was approved by the Kobe University Graduate School of Medicine Ethics Committee. We screened 139 consecutive outpatients with hypopituitarism at Kobe University Hospital between January 2002 and March 2013. The study design is described in Fig. 1. For the diagnosis of AGHD, each patient was subjected to an insulin tolerance test (peak GH b3 ng/mL) or the GH releasing peptide-2 test (peak GH b9 ng/mL) [21]. The hypothalamo–pituitary–adrenal axis, hypothalamo–pituitary–thyroid axis, and hypothalamo–pituitary–gonadal axis were evaluated as previously described [22]. Exclusion criteria included alcohol consumption of more than 20 g/day for women and more than 30 g/day for men. Subjects were excluded if they had hepatitis B or C, autoimmune hepatitis, other liver diseases, or if they were receiving drugs known to cause steatohepatitis. Patients with acromegaly and Cushing's disease were also excluded. Accordingly, we compared 31 patients with GHRT and 19 age- and sex-matched patients without GHRT (Fig. 1). The diagnosis of NAFLD was made by ultrasonography assessing hepatic fatty change, including hepato-renal contrast, impaired visualization of the hepatic vein borders or the diaphragm [23]. The diagnosis of NASH was made by a liver biopsy with written informed consent according to the standard clinical indication for liver disease [24]. Liver biopsy specimens
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were evaluated independently by 2 pathologists blinded to the patients' clinical histories. NASH was diagnosed when the pathological findings exhibited type 3 or 4 of Matteoni's classification [25]. 2.2. GHRT The GHRT group received recombinant human GH administration according to clinical practice guidelines [26,27]. In brief, the initial dose of GHRT was 0.1–0.2 mg/day. The dose was titrated in 2–3 month intervals by an attending physician according to serum IGF-I levels, subjective symptoms, QOL, and side effects. The target for the IGF-I standard deviation score (SDS) level was within −2.0 to +2.0. The mean dose of GHRT at 24 months was 0.23 ± 0.15 mg/day (male, 0.17 ± 0.15 mg/day, female, 0.26 ± 0.14 mg/day). 2.3. Glucocorticoid and thyroxine replacement therapy Most of the patients received glucocorticoid and thyroxine replacement therapy. Both replacement therapies were performed according to clinical consensus or clinical practice guideline [28,29]. In brief, they received 10–20 mg/day of oral hydrocortisone, which were titrated according to their well-being clinical data including metabolic state. Thyroxine administration was started with 12.5 to 25 μg/day and gradually increased until free-T4 levels reached within the high normal range. During GHRT, there were no patients in whom glucocorticoid and thyroxine doses were changed. 2.4. Measures Data were retrospectively collected from patients' medical records. In the control group, we defined January 2010 as the baseline. Primary outcome of this study was longitudinal change of serum liver enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). AST, ALT, g-GTP, ALP, and LDH levels were measured using a standard method (AST and ALT, Kanto Chemical Co., Inc., Tokyo, Japan; γ-GTP, ALP, and LDH, Shino-Test Co., Tokyo, Japan). Secondary outcome of this study was longitudinal change of serum hyaluronic acid level as a fibrotic marker, which was measured using latex agglutination-turbidimetric immunoassay (Mitsubishi Chemical Medicine Co., Tokyo, Japan). The laboratory measurements were performed at each occasion when the patients visited the hospital in the same laboratory during the study period. Measurements of liver enzymes were performed in Kobe University Hospital Clinical Laboratory, and measurements of hyaluronic acid were entrusted to SNL Inc. The coefficients of variation for the assays of liver enzymes and hyaluronic acid are the following; AST 0.79%, ALT 0.6%, γ-GTP 0.65%, LDH 0.64%, ALP 0.8%, hyaluronic acid 2.42%, respectively, for the sample within each normal range. 2.5. The definition of GH-responder and GH-non-responder in patients with GHRT In order to clarify the factors associated with GHRT efficacy, we subdivided the GHRT group into GH-responder group and GH-nonresponder group, and compared various indices between these groups. The GH-responder group was defined as patients in whom more than half of the abnormal liver enzymes (AST, ALT, γ-GTP, ALP, LDH) at baseline improved to within normal limit through the study period. We also analyzed the rate of patients who gained weight at 24 months compared with at the baseline in these groups. 2.6. Statistical analysis
Fig. 1. Study design.
Data are appropriately expressed as mean ± standard deviation, mean ± standard error of mean, or median [1st quartile, 3rd quartile].
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Categorical data were compared by χ2 test or Fisher's exact test and continuous data were compared by Student's t-test or a Mann–Whitney test, as appropriate. Homogeneity of variance was tested by Levene's test. Longitudinal data of both groups were compared by 1-way repeated measures analysis of variance (ANOVA) followed by a Tukey–Kramer honestly significant difference test or 2-way repeated measures ANOVA, followed by a pairwise multiple-comparison procedure. The two sided P values of 0.05 or less were considered significant. Statistical analyses were performed using JMP Statistical Database Software version 8. 0. 1 (SAS Institute, Inc., Cary, NC, USA). 3. Results 3.1. Baseline characteristics
Gender (male/female) Age (years) BMI IGF-I SDS Hypertension (%) Dyslipidemia (%) HbA1c (%) FPG (mg/dL) HOMA-IR Total-C (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) TG (mg/dL) NAFLD (%) Glucocorticoid replacement (%) Thyroxine replacement (%) Gonadal replacement (%) n
3.2. Effects of GHRT on liver function At 24 months, the GHRT group showed a significant increase in the IGF-I SDS, from − 4.3 ± 3.7 at baseline to − 1.0 ± 2.1, than the control group (from − 2.9 ± 2.1 at baseline to − 3.3 ± 1.8) (Fig. 2A; P b 0.0001). BMI remained unchanged in each group (GHRT group, from 27.9 ± 4.8 kg/m2 at baseline to 28.6 ± 4.7 kg/m2 at 24 months, P = 0.15; control group, from 25.0 ± 2.8 kg/m2 to 26.6 ± 3.4 kg/m2, P = 0.25) and there was no significant difference between them
Control group
P value
11/20 44.9 ± 18.6 27.9 ± 4.8 −4.3 ± 3.7 32.3 71.0 5.9 ± 0.8 97.9 ± 24.9 3.0 [1.9, 6.2] 228.2 ± 49.6 137.9 ± 40.6 64.0 ± 18.1 196 [123, 240] 65.5 93.6 93.6 M 27.3 F 30.0 31
8/11 52.3 ± 13.3 25.0 ± 2.8 −2.6 ± 2.2 31.6 73.7 5.6 ± 0.4 93.1 ± 13.9 1.2 [0.8, 5.2] 201.6 ± 46.0 128.9 ± 35.8 51.4 ± 18.0 147 [107, 213] 61.1 89.5 94.7 M 30.0 F 15.4 19
0.64 0.14 0.07 0.07 0.96 0.84 0.31 0.45 0.19 0.16 0.49 0.05* 0.24 0.76 0.61 0.86 0.89 0.34
(Fig. 2B; P = 0.53). At baseline, the levels of AST, ALT, γ-GTP, and LDH were significantly higher in the GHRT group than in the control group (Fig. 3A, B, C, D, and E). Subsequently, we analyzed the longitudinal changes in liver enzyme levels and fibrotic marker for 24 months. Intriguingly, the levels of AST, ALT, γ-GTP, and LDH gradually increased during the observation period in the control group. In a sharp contrast, these enzyme levels were rapidly decreased after 3 months of GHRT, and
A
P < 0.0001 1 0 -1 -2 -3 -4 -5
0
3
6
12
24
(month)
P = 0.53
B 34 32
Table 1 Etiology of hypopituitarism.
Craniopharyngioma Pituitary adenoma Sheehan's syndrome Rathke's cleft cyst Germinoma Invisible stalk Empty sella Hypophysitis Langerhans cell histiocytosis Pituitary apoplexy Malignant lymphoma Meningioma Unknown cause n
GHRT group
Data were compared by the χ2 test, Fisher's exact test, Student's t-test, or Mann–Whitney test, as appropriate. * indicates P value b0.05. FPG: fasting plasma glucose; HOMA-IR: homeostasis model assessment-insulin resistance; C: cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglyceride; M: male; F: female.
IGF -I SDS
After screening 139 consecutive outpatients with hypopituitarism, it was shown that 61 patients exhibited adult GH deficiency, in whom 38 patients received GHRT. Among these patients with GHRT, 6 patients had received GHRT for less than 24 months. One patient discontinued GHRT because of a malignancy. No patients discontinued because of a lack of effectiveness. Twenty-three patients did not receive GHRT, despite there was an indication for GHRT. Although the reason why these patients did not receive GHRT varies according to the individuals, most patients declined because of a hesitation in injection and some patients did not feel necessity irrespective of their understanding for the medical importance. As a result, 31 AGHD patients with GHRT and 19 age- and sex-matched AGHD patients without GHRT were enrolled in this study (Fig. 1). Table 1 shows the etiology of hypopituitarism. Craniopharyngioma was more prevalent in the GHRT group; however, there was no statistically significant difference between these groups (P = 0.25). The baseline characteristics of these groups are shown in Table 2. Although body mass index (BMI) tended to be higher in the GHRT group, it was not significant (P = 0.07). HbA1c and HOMA-IR levels were comparable in both groups. In lipid profiles, high density lipoprotein (HDL) was significantly higher in the GHRT group (P = 0.05). IGF-I SDS was lower in the GHRT group, which may indicate that the GHRT group had more severe GHD, but there was no statistical difference (P = 0.07).
Table 2 Baseline characteristics of the GHRT group and control group.
Control group
8 3 3 4 4 2 1 1 2 1 1 1 0 31
1 5 3 1 1 1 2 2 0 1 0 0 2 19
BMI
30 GHRT group
28 26 24 22
0
3 6
12
24
(month) Fig. 2. Longitudinal changes in serum IGF-I and BMI in the GHRT group and control group. The solid line indicates the GHRT group and the dotted line indicates the control group. These groups were compared by repeated measures 2-way analysis of variance (ANOVA) for GHRT and time, followed by a pairwise multiple-comparison procedure. * indicates P value b 0.05, ** indicates P value b 0.01. Error bars show standard error. A. IGF-I standard deviation score (SDS) B. BMI.
R. Matsumoto et al. / Growth Hormone & IGF Research 24 (2014) 174–179
B
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Fig. 3. Longitudinal changes in serum hepatic enzyme levels and fibrotic marker in the GHRT group and control group. The solid line indicates the GHRT group and the dotted line indicates the control group. These groups were compared by repeated measures 2way analysis of variance (ANOVA) for GHRT and time, followed by a pairwise multiplecomparison procedure. * indicates P value b 0.05, ** indicates P value b 0.01. Error bars show standard error. A. Aspartate aminotransferase (AST) B. Alanine aminotransferase (ALT) C. γ-Glutamyl transpeptidase (γ-GTP) D. Alkaline phosphatase (ALP) E. Lactate dehydrogenase (LDH) F. Hyaluronic acid.
sustained at a lower level for 24 months in the GHRT group (Fig. 3A, B, C, D, and E. AST, P b 0.001; ALT, P b 0.001; γ-GTP, P = 0.012; ALP, P = 0.053; and LDH, P = 0.003, respectively). At the end of the study, the levels of the liver enzymes AST, ALT, and LDH, were significantly higher in the control group. In addition, the levels of the fibrotic marker hyaluronic acid, gradually increase in the control group but remained unchanged in the GHRT group (Fig. 3F; P = 0.048).
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Fig. 4. Longitudinal changes in serum hepatic enzyme levels and fibrotic marker in patients with NASH during GHRT. Data were analyzed by repeated measures 1-way analysis of variance (ANOVA) followed by a Tukey-Kramer honestly significant difference test. * indicates P value b 0.05, ** indicates P value b 0.01. Error bars show standard error. A. Aspartate aminotransferase (AST) B. Alanine aminotransferase (ALT) C. γ-Glutamyl transpeptidase (γ-GTP) D. Alkaline phosphatase (ALP) E. Lactate dehydrogenase (LDH) F. Hyaluronic acid.
3.3. Effects of GHRT on AGHD patients with NASH
3.4. Factors associated with GHRT efficacy
Liver biopsy was performed in 16 patients. Of these patients, 14 were diagnosed with NASH, and all of them were treated with GH [14]. We analyzed the long-term effect of GHRT in 13 patients with NASH, who were treated for more than 24 months (Fig. 4). The levels of the liver enzymes AST, ALT, γ-GTP, and LDH were significantly decreased after 3 months of GHRT, and these effects were sustained until 24 months (Fig. 4A, B, C, D and E. AST, P = 0.002; ALT, P = 0.003; γ-GTP, P = 0.008; ALP, not significant; and LDH, P b 0.001, respectively). The levels of hyaluronic acid tended to decrease during 12 months; however, including the levels at 24 months, there was no significant improvement in the patients with NASH (Fig. 4F; P = 0.46).
To clarify the factors associated with the efficacy of GHRT on the liver, we subdivided the GHRT group into GH-responder group and GH-non-responder group by the effect on liver dysfunction, and compared various indices between these groups at baseline. There were no differences in the characteristics between the GH-responder group and GH-non-responder group at the baseline; however, the proportion of the patients who gained body weight during GHRT was significantly lower in the GH-responder group (Table 3; 44% vs. 82%, P = 0.04). Regarding the other hormone replacement therapy, the female patients in the GH-non-responder group showed a higher proportion of gonadal replacement therapy (Table 3; P = 0.02).
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Table 3 Comparison of the GH-responder group and GH-non-responder group.
Sex (male/female) Age (years) BMI Body weight gain (%) IGF-I SDS Hypertension (%) Dyslipidemia (%) HbA1c (%) HOMA-IR Glucocorticoid replacement (%) Thyroxine replacement (%) Gonadal replacement (%) Dose of GHRT (mg/day) Peak GH with ITT (ng/mL) Peak GH with GHRP-2 (ng/mL) AST (IU/L) ALT (IU/L) γ-GTP (IU/L) ALP (IU/L) LDH (IU/L) Hyaluronic acid (ng/mL) n
GH-responder group
Non-responder group
P value
14/6 45.0 ± 16.3 27.4 ± 5.5 44 −5.0 ± 0.8 35 65 5.9 ± 0.9 3.2 [1.6, 5.4] 100 95 M 16.7 F 0.0 M 0.11 ± 0.09 F 0.26 ± 0.10 0.1 [0, 0.2] 0.73 [0.12, 1.07] 40.9 ± 7.2 44.1 ± 39.2 61.2 ± 33.2 257.6 ± 24.1 236.1 ± 91.3 38.3 ± 17.4 20
6/5 44.6 ± 22.9 28.5 ± 4.0 82 −3.1 ± 1.1 27 82 5.9 ± 0.7 2.8 [2.2, 7.3] 82 91 M 40.0 F 42.9 M 0.25 ± 0.10 F 0.28 ± 0.09 0.0 [0.0, 0.35] 0.14 [0.03, 0.41] 40.6 ± 9.6 48.1 ± 39.2 113.5 ± 43.7 267.4 ± 31.7 241.0 ± 46.0 40.1 ± 20.0 11
0.39 0.96 0.60 0.04* 0.19 0.66 0.31 0.88 0.76 0.12 1.0 0.39 0.02* 0.18 0.78 0.96 0.19 0.99 0.79 0.35 0.81 0.87 0.95
Data were compared by the χ2 test, Fisher's exact test, Student's t-test, or Mann–Whitney test, as appropriate. * indicates P value b0.05. Body weight (BW) gain is defined as BW at 24 months N BW at baseline. ITT: insulin tolerance test; GHRP-2: growth hormone releasing peptide-2; M: male; F: female.
4. Discussion In the present study, we demonstrated that GHRT was efficacious for improving serum liver enzyme levels and fibrotic marker for at least 24 months in patients with AGHD. In addition, long-term GHRT was also effective for treating AGHD patients with NASH in terms of liver dysfunction. Although it was difficult to predict the effect of GHRT before treatment, we showed that body weight gain during treatment was associated with the deteriorated effect of GHRT, suggesting that the control of body weight is important. Notably, in contrast to the control group, in which liver enzyme levels gradually increased, the GHRT group exhibited an improvement in these markers, indicating the progression of liver dysfunction in the patients without GHRT and the significance of GHRT to protect against it. In contrast to the established efficacy of GHRT on visceral obesity and QOL in patients with AGHD [30,31,17], its effect on the liver has not been fully elucidated. Several case reports and clinical studies have reported the efficacy of GHRT on NAFLD and NASH in patients with AGHD [32,14,16]. On the other hand, Gardner et al. reported that GHRT did not reduce liver fat measured by magnetic resonance imaging. However, when analysis was restricted to patients with NASH, there was a trend towards a reduction in liver fat content (P = 0.07). In addition, although it was demonstrated that serum liver enzyme levels were unchanged before and after 6 months of GHRT, body weight tended to increase during treatment (+2.3 kg) in their study [15]. Taken together with the present data, in which weight gain during GHRT deteriorates the effect on the liver, this may explain the discrepancy between Gardner's and our data. In the present study, we first demonstrated the long-term effects of GHRT on the liver. Our results suggest that the liver is an important target tissue for GH and when considering the indication of GHRT in patients with AGHD, the presence of NAFLD or NASH as a comorbidity should be taken into account. The role of the GH-IGF-I axis in the liver has recently emerged [6]. In an animal model, liver-specific deletion of the GH receptor and the downstream signaling molecules JAK-2 and STAT-5 results in steatosis [9,8]. GH receptor loss of function mutations in humans (Laron syndrome) also manifests as NAFLD in adults, and chronic replacement
of IGF-I does not influence the NAFLD status [33], suggesting that GH has a direct effect on the liver, particularly in the prevention of steatosis in hepatocytes. On the other hand, a spontaneous dwarf rat, in which GH is deficient, exhibits hepatic steatosis and fibrosis. Interestingly, IGF-I as well as GH administration was able to reverse these changes in the liver [10], suggesting the presence of a GH-independent action of IGF-I. Many reports indicate the significance of serum IGF-I levels in liver disease. NAFLD is associated with lower circulating levels of IGF-I [34]. Interleukin-6 and IGF-I are independent prognostic factors of NAFLD in morbidly obese patients [35]. In addition, the levels of hyaluronic acid, a marker of fibrosis, showed a negative correlation with IGF-I and the IGF-I/IGF binding protein-3 ratio in patients with NAFLD [36]. These data suggest that not only GH but also IGF-I plays a pivotal role in the liver. In a clinical setting, it is important to predict and improve the efficacy of GHRT. Younger age is reported to be a predictor of the effectiveness of GHRT, in terms of improvements in abnormal lipid profile and body composition [17]. In this study, we examined the factors which are predictive of GHRT efficacy on the liver. Although we could not detect any pre-treatment indices that predict GHRT effectiveness, body weight gain during the treatment deteriorated the efficacy. Given that obesity itself is an essential risk factor for the development of NAFLD, one can safely reason that the body weight gain during treatment has a negative impact on the effect of GHRT. Our results also suggest the importance of body weight control during GHRT to obtain an optimal effect. We cannot exclude the possibility that the other pituitary hormone deficiencies especially sex steroid, may interfere with the effects of GHRT on the liver. Female patients in the GH-non-responder group had higher proportion of gonadal replacement therapy. It is known that estrogen blocks the ability of GH to generate IGF-I in the liver [37]. Although it was not a significant difference, lower serum IGF-I levels between patients with or without gonadal replacement therapy (−2.2 ± 1.8 vs. −1.5 ± 2.6 SDS, respectively, P = 0.22) maybe associated with the effect of GHRT. These results suggest that female patients who have gonadal replacement therapy may need more dose of GH in terms of the improvement of liver dysfunction. There are several limitations in this study. First, because of the retrospective and non-randomized study design, there was a selection bias between the control and GHRT groups. Although we recruited ageand sex-matched control group, BMI tended to be higher in the GHRT group. In addition, there was a tendency towards a decreased IGF-I SDS and higher prevalence of craniopharyngioma in the GHRT group. These differences might be reflected by a preference towards GHRT in younger patients and those with more severe disease. As a result, there were significant differences in serum liver enzyme levels at baseline. Nevertheless, we observed a reversal change of serum liver enzyme levels during treatment between the control and GHRT group, further supporting the efficacy of GHRT. Secondly, although we have shown that fibrotic marker hyaluronic acid levels were significantly decreased in the GHRT group compared with the control group (Fig. 3F), the effect of GHRT on fibrosis is inconclusive. There have been several markers for evaluating liver fibrosis and serum hyaluronic acid level is reportedly a useful marker, especially for severe liver fibrosis [38]. For example, it has been reported that serum hyaluronic acid level showed a significant positive correlation with the degrees of liver fibrosis and the cut-off value of 46.1 ng/mL exhibited a sensitivity of 85% and a specificity of 80% for identifying severe fibrosis. However, because of the sensitivity and specificity, liver biopsy is gold standard for the evaluation of liver fibrosis at present and it remains unclear whether longitudinal changes in serum hyaluronic acid levels reflect precise status of liver fibrosis. Thus, accurate evaluation of liver fibrosis requires liver biopsy; however, because of the invasive procedure, enough number of patients in this study had not undergone liver biopsy after GHRT for the histological evaluation. Further investigation is necessary to clarify the long-term effect of GHRT on fibrosis. Thirdly, patients in the GHRT group received a relatively low dose of GH (0.23 ± 0.15 mg/day) compared to the previous studies [30,18,17]. Serum IGF-I SDS during the
R. Matsumoto et al. / Growth Hormone & IGF Research 24 (2014) 174–179
treatment ranged from − 1.0 to 0, suggesting that the effect was not optimized in the GHRT group. Nonetheless, hepatic markers dramatically improved, even at 3 months after the initiation of GHRT. These data suggest that the threshold of IGF-I concentration in the liver may be different from other tissues, such as adipose tissue. Another possible explanation is a higher local concentration of IGF-I in the liver than in other organs [39], and a paracrine effect of IGF-I may be important to improve liver dysfunction. In conclusion, we demonstrated that GHRT has a beneficial effect on improving serum liver enzyme levels for at least 24 months in hypopituitary patients with AGHD. Body weight gain has a negative impact on the effect of GHRT. Although further study is necessary to clarify the long-term prognosis and significance of GHRT in AGHD patients with NAFLD or NASH, these data emphasize the importance of NAFLD and NASH as a comorbidity in patients with AGHD. Conflict of interest disclosures Dr. Takahashi reports receiving research funding and payments for lectures from Ili Lily. No other potential conflict of interest relevant to this manuscript was reported. Author contributions Ryusaku Matsumoto: collection, analysis, and interpretation of data and drafting of the manuscript. Hidenori Fukuoka, Genzo Iguchi, Hitoshi Nishizawa, Hironori Bando, Kentaro Suda, and Michiko Takahashi: collection and assembly of data. Yutaka Takahashi: conception, study design, and critical revision of the manuscript for important intellectual content. Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology 23659477, 23591354, and 22591012, Grantsin-Aid for Scientific Research (research on hypothalamic–hypophyseal disorders) from the Ministry of Health, Labor, and Welfare, Japan, Daiichi-Sankyo Foundation of Life Science, and Naito Foundation. The authors are grateful to C. Ogata and K. Imura for their excellent technical assistance. References [1] J.O. Johansson, J. Fowelin, K. Landin, I. Lager, B.A. Bengtsson, Growth hormonedeficient adults are insulin-resistant, Metabolism 44 (1995) 1126–1129. [2] T. Rosén, S. Edén, G. Larson, L. Wilhelmsen, B.-Å. Bengtsson, Cardiovascular risk factors in adult patients with growth hormone deficiency, Acta Endocrinol. (Copenh) 129 (1993) 195–200. [3] V. Markussis, S.A. Beshyah, D.G. Johnston, C. Fisher, A.N. Nicolaides, P. Sharp, Detection of premature atherosclerosis by high-resolution ultrasonography in symptomfree hypopituitary adults, Lancet 340 (1992) 1188–1192. [4] T. Rosén, T. Hansson, H. Granhed, J. Szucs, B.-Å. Bengtsson, Reduced bone mineral content in adult patients with growth hormone deficiency, Acta Endocrinol. (Copenh) 129 (1993) 201–206. [5] T. Rosen, L. Wiren, L. Wilhelmsen, I. Wiklund, B.A. Bengtsson, Decreased psychological well-being in adult patients with growth hormone deficiency, Clin. Endocrinol. (Oxf.) 40 (1994) 111–116. [6] Y. Takahashi, Essential roles of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in the liver [review], Endocr. J. 59 (2012) 955–962. [7] Y. Fan, R.K. Menon, P. Cohen, et al., Liver-specific deletion of the growth hormone receptor reveals essential role of growth hormone signaling in hepatic lipid metabolism, J. Biol. Chem. 284 (2009) 19937–19944. [8] B.C. Sos, C. Harris, S.M. Nordstrom, et al., Abrogation of growth hormone secretion rescues fatty liver in mice with hepatocyte-specific deletion of JAK2, J. Clin. Invest. 121 (2011) 1412–1423. [9] J.L. Barclay, C.N. Nelson, M. Ishikawa, et al., GH-dependent STAT5 signaling plays an important role in hepatic lipid metabolism, Endocrinology 152 (2011) 181–192. [10] H. Nishizawa, M. Takahashi, H. Fukuoka, G. Iguchi, R. Kitazawa, Y. Takahashi, GHindependent IGF-I action is essential to prevent the development of nonalcoholic steatohepatitis in a GH-deficient rat model, Biochem. Biophys. Res. Commun. 423 (2012) 295–300.
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