Conditions: NAFLD
Keywords: obesity adolescence fatty liver growth hormone
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 24
Healthy volunteers: no

Brief summary:
Fatty liver disease is an increasing problem in overweight and obese young adults. The purpose of this study is to test the effect of growth hormone on liver fat in obese young adults ages 18-29y with increased liver fat.

Detailed description:
Non-alcoholic fatty liver disease (NAFLD) is a significant health problem in obese adolescents. Obese children and adolescents have significant reductions in growth hormone secretion, and we hypothesize that augmenting growth hormone in this population will decrease liver fat. Growth hormone inhibits hepatic de novo lipogenesis, which is an important source of hepatic lipid. Patients with pituitary GH deficiency have a higher prevalence of NAFLD and non-alcoholic steatohepatitis (NASH) than the general population, and replacement of GH in these individuals reduces signs of liver damage. The purpose of this study is to test the hypothesis that growth hormone treatment will decrease liver fat quantity in young adults who begin the trial with more than 5% liver fat measured by magnetic resonance spectroscopy.

Arms:
arm 1: Somatropin given by daily subcutaneous injection. Dose will begin at 1mg and be titrated based on insulin-like growth factor 1 (IGF-1) levels. arm 2: (no study treatment - observation only)

Interventions: intervention 1: somatropin
Intervention descriptions: intervention 1: Norditropin (growth hormone) given by injection using a pen-device

Protocol document:
Prot_SAP_000.pdf: 
Study protocol with embedded statistical analysis plan 
 
Augmenting Growth Hormone to Ameliorate Nonalcoholic Fatty Liver Disease in Adolescents 
 
NCT02726542 


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 Augmenting Growth Hormone to Ameliorate Nonalcoholic Fatty Liver Disease in 
Young Adults 
PI:  Takara Stanley, MD 
Version Date 07/31/2019 
 
I.  Background and Significance 
 
Summary 
 
Non-alcoholic fatty liver disease (NAFLD) is a significant health problem in obese adolescents, and 
there are currently no recommended pharmacological treatments for this condition.  We and others have 
reported significant reductions in growth hormone (GH) in obese adolescents (1-3), and, as described below, 
reduced GH is highly likely to contribute to the pathogenesis of hepatic lipid accumulation.  In an adult 
population with HIV-associated visceral obesity, we have demonstrated that augmentation of GH significantly 
reduces liver fat (4).  The effect of augmenting GH to reduce liver fat in young adults has never been tested.  
We propose a randomized clinical trial to test the effects of physiologic replacement with recombinant human 
GH (rhGH), Norditropin, to reduce liver fat in obese young adults with NAFLD (defined as ≥5% hepatic lipid on 
1H-magnetic resonance spectroscopy) who have relative reductions in GH secretion.   
 
Epidemiology and Health Consequences of Pediatric NAFLD 
 
NAFLD, which encompasses both steatosis and steatohepatitis, is the leading cause of liver disease in 
adolescents, with an estimated prevalence of approximately 10% (5, 6).  Although it may occur in individuals of 
normal weight, NAFLD is largely a co-morbidity of obesity, found most commonly in the setting of increased 
visceral adiposity and insulin resistance. Over the past 3 decades, in conjunction with rising obesity, the 
prevalence of NAFLD in adolescents has more than doubled (5) such that the current prevalence among 
obese adolescents is estimated at approximately 40% (6).  Given the potential adverse consequences of 
NAFLD with respect to future liver health, this figure is alarming. More than 20% of adolescents with fatty liver 
show signs of non-alcoholic steatohepatitis (NASH), which is characterized by inflammation, hepatocellular 
injury and fibrosis (6-10). In many, NASH will progress to advanced fibrosis and cirrhosis.  An early report 
described fibrosis-cirrhosis in 75% of children presenting with NASH (11).  In a large autopsy study, 9% of 
children with NASH already had advanced fibrosis or cirrhosis (6).  Further, a longitudinal study showed a 
significantly increased standardized mortality ratio of 13.6 (95% CI 3.8-34.8) in children with NAFLD, with two 
of the 66 members of the cohort dying and two requiring liver transplantation for decompensated cirrhosis over 
20 year follow-up (12).  Histological findings of NAFLD in children differ from those in adults (9, 13, 14), 
suggesting unique pathophysiological mechanisms in the pediatric age group. Further, a recent study suggests 
that severely obese adolescents with NAFLD may have more severe liver damage compared to adults 
matched for BMI, gender, race, and features of metabolic syndrome (15).  Due to FDA requirement, the 
current study is limited to those ≥18 years of age, with the intention of pursuing further studies in the 
pediatric age group if data from this pilot study demonstrate benefit. 
In addition to conveying risk of progressive liver disease, NAFLD is strongly associated with 
dyslipidemia (16, 17), insulin resistance (IR) (18, 19), and cardiovascular disease (20-24).  Although causal 
pathways have not been definitively established, evidence suggests a bidirectional relationship between 
NAFLD and both dyslipidemia and IR, whereby NAFLD contributes to dyslipidemia and IR, both of which in 
turn may drive further accumulation of hepatic lipid (16-19, 25).  Additionally, large studies in adults show 
associations between NAFLD and CVD events, independent of traditional risk factors and metabolic syndrome 
components, suggesting that NAFLD may also play an etiologic role in CVD (23, 24, 26).  Thus treatment 
strategies for NAFLD in young adults are critical both to prevent liver disease and to ameliorate 
cardiometabolic risk in adulthood.   
 
Lack of Effective Therapies for NAFLD  
 
Effective treatment options for NAFLD in young adults are limited.  Although lifestyle modification 
appears to be effective (27-30), exercise and weight loss are difficult to sustain.  To date, no highly effective 


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pharmacologic treatment options exist to reduce liver fat in obese adolescents. Vitamin E has modest benefit in 
adults with NAFLD (31) and showed initial benefit in the pediatric population (32). In the TONIC trial – a large, 
randomized, controlled study of Vitamin E or metformin for NAFLD in the pediatric age group – Vitamin E 
resulted in histological improvement of liver disease but showed no benefit over placebo to consistently reduce 
ALT (33). Similarly, metformin has shown benefit in some studies (34, 35), but also proved no better than 
placebo in the TONIC trial with respect to ALT reduction (33).  Other treatment strategies, including 
ursodeoxycholic acid (36), cysteamine (37), probiotics (38), and polyunsaturated fatty acids (39), have been 
investigated, but to date there is no definitive evidence of efficacy. Thus no pharmacologic agents are currently 
standard of care for pediatric NAFLD. The 2012 Practice Guideline for diagnosis and management of NAFLD 
endorses lifestyle modification as the only recommended treatment in pediatrics, mentioning possible benefit of 
Vitamin E but concluding that confirmatory studies are necessary before its use can be recommended in 
clinical practice (40).  
 
Treatment strategies for NAFLD have thus far focused on ameliorating oxidative stress (e.g., with 
Vitamin E) or reducing insulin resistance (e.g., with metformin).  Whereas both oxidative stress and insulin 
resistance play a significant role in NAFLD and steatohepatitis, we argue that a treatment strategy targeting 
hepatic lipid accumulation itself should also be considered.  In this respect, as described below, Norditropin 
(rhGH), will be explored as a potential treatment to reduce liver fat in obese young adults with NAFLD.   
 
Reduced GH contributes directly to NAFLD 
 
NAFLD is defined by macrovesicular accumulation of triglyceride (TG) in the cytoplasm of at least 5% 
of hepatocytes.  Investigation with stable isotope tracers has quantified three major sources of hepatic TG:  (i) 
uptake of circulating free fatty acid (FFA), responsible for about 59% of hepatic TG; (ii) hepatic de novo 
lipogenesis (DNL) whereby palmitate is synthesized from glucose, particularly during conditions of 
carbohydrate excess, responsible for about 25% of hepatic TG; and (iii) dietary fat, responsible for about 15% 
of hepatic TG (41, 42).  We hypothesize that relative growth hormone (GH) deficiency of obesity contributes to 
the pathogenesis of NAFLD through two mechanisms.   
 
(i) Relative reductions in GH are associated with increased visceral adipose tissue (VAT) (3, 43), which 
causes increased free fatty acid flux through the liver.  VAT quantity is strongly associated with NAFLD 
and NASH in adults (44, 45), and lipolysis of VAT but not subcutaneous adipose tissue (SAT) is 
significantly associated with increased liver fat (46, 47).  Further, FFA derived from VAT are a primary 
contributor to increased hepatic very low density lipoprotein (VLDL) production in individuals with 
NAFLD (48).   
 
(ii) GH plays a critical role in the regulation of hepatic de novo lipogenesis (DNL), and increased 
hepatic DNL is strongly associated with NAFLD.  The rate of DNL is more than twice as high in 
individuals with high liver fat compared to controls (49).  GH administration suppresses hepatic DNL in 
animal models (50-54), and the same effect has been demonstrated in human models (55).  
 
Studies of GH deficient individuals demonstrate the importance of GH in preventing net accumulation of 
liver fat.  Patients with pituitary GH deficiency have a higher prevalence of NAFLD and NASH than the general 
population, and replacement of GH reduces transaminases and improves histological findings (56-60).  
Further, a study of individuals with childhood onset GH deficiency in whom treatment was stopped at 
attainment of final height demonstrated a high incidence of NAFLD, which developed as soon as 1 year after 
discontinuation of therapy and at a mean BMI of 26kg/m2, arguing against obesity per se as an etiologic 
mediator and suggesting instead a causal role of GH deficiency (61).  Just as individuals with GH deficiency 
have a higher prevalence of NAFLD, individuals with NAFLD appear to have reductions in GH, as 
demonstrated by lower concentrations of insulin-like growth factor 1 (IGF-1) (62).  Further, among individuals 
with NAFLD, IGF-1 levels are inversely associated with degree of steatosis and fibrosis (63-65), and hepatic 
liver IGF-1 mRNA expression is a negative predictor of histological severity of NAFLD (63).  In vitro and animal 
models support these findings and provide mechanistic insight. Transgenic mice that overexpress GH have 
decreased liver fat, whereas liver-specific GH-receptor (GHR) knock-out mice have severe hepatic steatosis 


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(66).  In the liver-specific GHR knock-out, hepatic lipogenesis and hepatic triglyceride output are significantly 
increased, and adenovirus-mediated rescue of GHR expression reverses these changes (66). Another model 
of GH deficiency, the spontaneous dwarf rat (SDR), also demonstrates hepatic steatosis and fibrosis that can 
be reversed by treatment with GH (67). Taken together, both human and animal models of GH deficiency 
demonstrate the critical role of GH in hepatic lipid metabolism and illustrate the consequences of GH 
deficiency with respect to increased hepatic lipid accumulation. 
 
II.  Specific Aims 
1.  In a randomized study of obese young adults with ≥5% liver fat by MRS (NAFLD) and reduced 
GH, administration of physiologic doses of Norditropin (rhGH) over 24 weeks will reduce liver 
fat as compared to no treatment.   
a. Compared to no treatment, Norditropin will reduce hepatic lipid as measured by 1H-MRS  
b. Compared to no treatment, Norditropin will significantly reduce markers of hepatocellular injury and 
steatohepatitis, including transaminases, gamma glutamyl transferase (GGT), and cytokeratin -18 
(CK18).   
 
2. Physiologic replacement of GH with Norditropin will be neutral to glucose homeostasis and will 
improve other measures of cardiometabolic risk. 
a. Fasting and 2-hour glucose, HOMA-IR, and Matsuda index will not be significantly different from 
baseline after 24 weeks of Norditropin treatment 
b. Norditropin will result in a reduction in VAT (measured by MRI) and trunk fat (measured by DXA) 
compared to no treatment 
c. Norditropin will result in an increase in adiponectin and a reduction in CRP compared to no 
treatment  
 
III.  Subject Selection 
24 obese young adults (M and F) with liver fat ≥5% on 1H-MRS and relative reduction in GH but without a 
known clinical diagnosis of growth hormone deficiency will be recruited for the RCT. We anticipate screening 
up to 150 subjects to identify 24 who meet enrollment criteria. Relative reduction in GH will be assessed by 
IGF-1 z-score, with subjects whose IGF-1 z-scores are ≤ 0 eligible for the study.  Many previous studies have 
defined relative GH deficiency as an IGF-1 z-score ≤0 (68, 69). 
Inclusion criteria:   
1. Males and Females ages 18-29yo 
2. BMI ≥95th percentile and/or ≥30kg/m2 
3. Hepatic fat ≥5% by 1H-MRS 
4. IGF-1 standard deviation score (SDS) ≤0  
 
Exclusion criteria:   
1. Alcohol consumption of >14 drinks per week (F) or >21 drinks per week (M) (40)   
2. Use of insulin or oral anti-diabetic medications, or HbA1c >7% or fasting glucose ≥126mg/dL 
3. Use of corticosteroid, gonadal steroids, or methotrexate ≤ 3 months prior to baseline visit 
4. Known diagnosis of alpha-1 antitrypsin deficiency, Wilson’s disease, hemochromatosis, or autoimmune 
hepatitis  
5. HgB < 11.0 g/dL or wt < 50kg 
6. AST or ALT >2.5x upper limit of normal (ULN), total bilirubin > ULN, positive Hep B sAg, or Hep C Ab 
7. Routine MRI exclusion criteria (including weight >450 lbs)  
8. Use of weight-loss medications or previous weight loss surgery 
9. Pregnant or breastfeeding, or, for sexually-active females, unwillingness to use an appropriate form of 
contraception during the study  
10. Use of oral combined estrogen/progesterone contraceptives or depot progesterone formulations unless 
stable use for 1 year prior to baseline visit.  Hormone-containing IUDs are permitted. 


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11. Known cirrhosis or clinical evidence of cirrhosis or portal hypertension on imaging or exam  
12. Use of GH or GHRH within the past 1 year 
13. Change in lipid lowering or anti-hypertensive medications within 3 months of screening  
14. Change in vitamin E or ursodiol <6 months before screen; subjects on stable doses of Vitamin E and/or 
Ursodiol for ≥6 months will be eligible.  
15. History of malignancy or active malignancy  
16. History of hypopituitarism, head irradiation or any other condition or chronic illness known to affect the 
GH axis 
 
IV.  Subject Enrollment 
 
Methods of Recruitment 
Subjects will be recruited from the greater Boston area through advertisements for obese young adults and 
through MGH ambulatory care practices including pediatric and adult gastroenterology and obesity clinics.  
Websites such as Facebook, craigslist, and Partners sponsored clinical-trial posting sites will also be utilized, 
as will other tools made available by the MGH and Partners. Obese subjects with known diagnoses of NAFLD 
and/or known chronic elevations in AST or ALT will be sought to optimize the likelihood of that subjects who 
undergo the screening visit will qualify for the study.  In addition, as approximately 40% of obese young adults 
in the general population will have NAFLD, obese individuals without previous evaluation for NAFLD may be 
screened.   
 
Methods of Enrollment 
Subjects who learn of the study through their physician or through advertisement and are interested will be 
asked to initiate contact with the study staff. Patients who respond to website advertising may elect to fill out a 
survey regarding eligibility that will ask them to provide contact information if they are willing to be contacted.  
Alternatively, if a patient’s physician obtains the patient’s permission for study staff contact, we may contact 
potential participants who have been identified as suitable by local providers.  For patients seen clinically by 
the PI or other physician investigators involved in the study, the following procedures will be followed to ensure 
that subjects do not feel obligated to participate because of the involvement of their regular physician:  The 
provider may mention the study to the patient and provide a flyer or consent form for the subject to take home.  
If this occurs, the provider will not talk further about the study unless the patient initiates the conversation by 
expressing interest.  Alternatively, the patient’s provider may mention the study and either ask the patient’s 
permission for a different study provider to contact him/her, or provide contact information of a different study 
provider whom the patient can contact if interested. 
 
Preliminary eligibility will be 
determined based on study staff 
interviews of interested subjects 
over the phone. Eligible subjects will 
then be scheduled for a screening 
visit.  
 
Informed Consent 
Written informed consent will be 
obtained by a licensed physician 
investigator prior to screening 
evaluation and testing. Subjects will 
be informed that they may withdraw 
from participation in the study at any 
point. 
 
Norditropin (dose-adjusted to target IGF-1)
No Treatment
1:1 Randomization
0
2wk
18wk
24wk
Screen 
(N=48)
N=24
12wk
4wk 6wk
Comprehensive assessment
H&P and interval laboratory assessment
Safety visits for Norditropin subjects only


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V.   Study Procedures 
The study will be conducted over 2 years, with subject recruitment complete after 18 months.  Each eligible 
subject will participate in the study for 24 weeks.  Following the screening visit, eligible subjects will return to 
the Massachusetts General Hospital Clinical Research Center (CRC) for as shown in the schema.  Subjects in 
the Norditropin group will have 7 additional visits, and subjects in the No Treatment group will have 3 additional 
visits.  Subjects who discontinue treatment for any reason will be asked to continue to return for study visits per 
their original randomization assignment.   
 
For all female subjects, urine pregnancy tests will be performed at all visits, and a positive pregnancy test will 
result in immediate discontinuation. At baseline, 12 weeks, and 24 weeks, subjects will be counseled regarding 
lifestyle modification (see Methods).  Subjects will continue to receive care from their regular medical 
providers, including hepatologists, throughout the study. Any subject not under the care of a hepatologist who 
is found at screen to have ≥5% liver fat and significantly elevated AST or ALT will be offered referral to the 
hepatology clinic.   
 
Screen: (1) Informed consent; (2) Detailed H&P, including medications and alcohol habits; (3) blood sampling 
for IGF-1, CBC, HbA1c, fasting glucose, AST, ALT, total bilirubin, Hepatitis B sAg, Hepatitis C antibody; (4) 1H-
MRS/MRI of abdomen in fasting state for quantification of liver fat. (This scan will also be used to assess VAT 
and SAT for those eligible subjects participating in the baseline visit.)   
 
Randomization and Study Drug: After the screening visit, eligible patients who wish to participate will be 
randomized in a 1:1 ratio to Norditropin vs. no treatment.  The study will not be blinded and placebo will not be 
used to avoid daily injections without benefit for subjects randomized to no treatment.  Randomization will be 
stratified by gender and Vitamin E use. The starting dose of Norditropin 
will be 0.5 mg daily, which is a recommended dose for restarting growth 
hormone in young adults with GHD during the transition from 
adolescence to adulthood (70).  IGF-1 will be checked at 2, 4, 6, 12, 
and 18 weeks, and will be reviewed by the PI. Active dose adjustments 
will be performed per algorithm as follows.  A 20% dose increase will be 
performed for any Norditropin subject with IGF-1 SDS ≤ 0.  Conversely, 
a 20% decrease in dose will be performed for any Norditropin subject 
with IGF-1 SDS >2.   
 
Baseline Visit:  
Patients will arrive after an 8-hour fast.  For females, urine pregnancy test will be performed prior to the start of 
any procedures.  When possible, women with regular menses will be studied within 10 days of the onset of the 
menstrual cycle.   
1. Within one week prior to the baseline visit, subjects will come in the fasting state for a clonidine/arginine 
stimulation test to assess GH secretion (see methods). 
2. Interval history and physical exam  
3. Indirect calorimetry in the fasting state  for the first 10 randomized subjects only 
4. Fasting blood sample for glucose, insulin, IGF-1, AST, ALT, GGT, lipid panel, CRP, adiponectin, CK18 
5. Standard 75g oral glucose tolerance test 
6. DXA for quantification of total and regional fat and lean mass, VAT and SAT 
7. Anthropometrics including height, weight, waist and hip circumferences, neck, mid-arm, and mid-thigh 
circumferences 
8. Analysis of 24-hour food recall for macro- and micronutrient composition 
9. Assessment of physical activity and lifestyle habits (including alcohol consumption) using questions 
from the 2013 Standard High School Youth Risk Behavior Survey (YRBS) 
10. Instruction on reconstituting and administering Norditropin for subjects randomized to rhGH, with first 
injection prior to discharge 
IGF-1 SDS ≤ 0
0 < IGF-1 SDS  ≤ 2
IGF-1 SDS > 2
20% dose 
increase
maintain dose
20% dose 
reduction*
Norditropin Dosing Algorithm
(starting dose 0.5 mg)
*Subjects with persistent IGF-1 SDS > 2 after 2 dose 
reductions will be discontinued from the study.


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Safety Visits (2, 4, 6, and 18 weeks, Norditropin subjects only):  (1) Interval H&P and safety assessment; (2) 
IGF-1, fasting glucose (3) Collection of used study drug and dispensation of new supply. 
 
12 Week Visit:  (1) Interval H&P and safety assessment; (2) IGF-1, fasting glucose (3) Collection of used study 
drug and dispensation of new supply for Norditropin subjects; (4) AST and ALT.  
 
24 Week Visit: Subjects receiving treatment will be instructed to take their final Norditropin injection the night 
before the visit.  Girls with regular menses will be studied within 10 days of the onset of the menstrual cycle.  
This visit will be identical to baseline with the addition of 1H-MRS/MRI in the fasting state prior to OGTT.  
(clonidine/arginine stimulation test will not be repeated.)  Indirect calorimetry will be performed only for the first 
10 randomized subjects. 
 
VI.  Methods 
 
1H Magnetic Resonance Spectroscopy(MRS) & MRI: Liver 1H-MRS will be performed using a 1.5 Tesla 
(Siemens Trio; Siemens Medical Systems) MRI system. After an overnight fast, each subject will undergo 1H-
MRS supervised by a radiologist who will review voxel placement and quality of spectra before patient is 
discharged. Subjects will be supine and feet first in the magnet bore. A body matrix phased array coil will be 
positioned over the abdomen and a tri-plane gradient echo localizer pulse sequence [repetition time (TR), 15 
msec; echo time (TE), 5 msec; slice thickness, 3 mm] will be obtained to localize the liver. A breath-hold True 
FISP sequence (TR, 3.8 msec; TE, 1.9 ms; slice thickness, 10 mm; imaging time, 12 seconds) will be obtained 
for detailed assessment of hepatic anatomy. A 20 × 20 × 20 mm voxel (8 mL) will be placed in the right hepatic 
lobe, avoiding vessels or artifacts, followed by automated shimming. The voxel placement will be registered by 
screen captures for consistency in follow-up studies. Single breath-hold single-voxel 1H-MRS data will be 
acquired in mid-expiration using point-resolved single voxel spectroscopy (PRESS) pulse sequence without 
water suppression: TR, 1,500 msec; TE, 30 msec; 8 averages; 1024 data points; and receiver bandwidth, 2000 
Hz; acquisition time: 18 seconds. This will be repeated with a voxel placement in the left hepatic lobe. 
Subsequently, liver 1H-MRS sequence with 2D PACE motion correction will be performed, triggered on the 
quiet and expiration phase of respiratory cycle with a pre-defined acceptance window during free breathing. A 
navigator image will be centered at the liver/diaphragm interface and a voxel measuring 20 × 20 × 20 mm (8 
mL) will be placed in the right (followed by left) hepatic lobe in a similar location as breath-hold study. A 
PRESS pulse sequence using TR, 1,500 msec; TE, 30 msec; 64 averages; and receiver bandwidth, 2000 Hz 
will be obtained. In our experience, reproducibility of both breath-hold and respiratory-gated 1H-MRS is high. 
Correlation analysis of data before and after repositioning is r= 0.99, p= 0.0003 for breath-hold and r= 0.93, p= 
0.007 for respiratory-gated 1H-MRS. Bland Altman analysis showed mean difference between same-day scans 
of 0.29% (95% CI: −1.46 and 2.05%) for breath-hold and −0.54% (95% CI: −2.53 and 1.45%) for respiratory 
gated 1H-MRS. The concordance correlation coefficient for breath-hold 1H-MRS is 0.97 and for respiratory-
gated 1H-MRS, 0.91 (71).  Fitting of all 1H-MRS data will be performed using LCModel (72). Data will be 
transferred from the MR scanner to a Linux workstation and metabolite quantification using eddy current 
correction and water scaling. A customized fitting algorithm for liver analysis provides estimates for all lipid 
signals (0.9, 1.3, and 2.3 ppm) scaled to unsuppressed water peak (4.7 ppm). Proton density fat fraction will be 
calculated from integral lipid and water peak areas as previously described (73, 74). Volume of abdominal fat 
depots (VAT and SAT) will be obtained in the same imaging session using an 8-channel phased-array torso 
coil covering the xyphoid process to the symphisis pubis. A modified 3-dimensional Dixon chemical-shift 6-
multiecho pulse sequence will be utilized automatically generating inline water-only and fat-only images, 
allowing differentiation of VAT/SAT from abdominal wall muscles, bowel and solid organs. Parameters include 
coronal slice/lab orientation, TR 9-13 ms, TE/echo-spacing 1.2-1.4/1.0-1.2, flip angle, 3o, slice thickness 3mm, 
150 slices. In addition, a T1 water-suppressed axial single slice at the level of L4 will be obtained for 
measurement of VAT/SAT areas. VAT/SAT areas and volume will be measured by semi-automated tracing 
with manual adjustments (Aquarius 3D Workstation, TeraRecon Inc, San Mateo, CA).   


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75 g Oral Glucose Tolerance Test will be performed after a 12-hour fast with sampling for insulin and glucose 
at 0 and 120 min.  
 
Whole Body DXA will be done to determine whole body and regional fat including DXA assessed VAT and SAT 
(75, 76).   
 
Clonidine/Arginine stimulation test will be performed after 12h fast according to standardized protocol.  At time 
0, subjects will take clonidine 150mcg/m2 (max 250mcg) PO x 1 and will receive intravenous administration of 
arginine hydrochloride (0.5g/kg with max dose of 30g; 30g/300 cc infusion) over 30 minutes.  GH sampling will 
be done at baseline and 30, 60, 90 and 120 minutes.   
 
Nutrition and Activity: 24-hour food recall will be performed and analyzed by registered dietitians at the Clinical 
Research Center to assess micronutrient and macronutrient intake.  Questions from the CDC 2013 Standard 
High School Youth Risk Behavior Survey (ftp://ftp.cdc.gov/pub/data/yrbs/2013/2013_hs_questionnaire.pdf) will 
be used to assess physical activity (Q80-84), eating habits (Q71-79), drinking habits (Q41-45), and recent 
dieting (Q66-70).   
 
Labs will be run using standard validated assays. 
 
Study Drug: For those randomized to treatment, Norditropin (rhGH) will be started at a dose of 0.5mg daily.  
The GH dosing strategy (see above) is designed to achieve IGF-1 levels between 0-2 SDS.  This strategy 
represents a modification of the dosing strategy in a previous pilot study, which began with 0.4mg daily at 
baseline, increased to 0.6mg at week 1 and 0.8mg GH daily by week 2, and then included subsequent dose 
adjustments at 1 and 3 months using the same algorithm proposed above.  This strategy resulted in a mean 
IGF-1 SDS of 0 in the treatment group, and thus proved somewhat too conservative.  The recommended dose 
for restarting GH during the transition period in young adults with GH deficiency is 0.2-0.5mg/day (70).  Based 
on this recommendation and the experience from our previous study, we will start with a baseline dose of 
0.5mg and include the opportunity for further adjustments at 2, 4, 6, 12, and 18 weeks. At the Principal 
Investigator’s discretion, subjects receiving Norditropin whose IGF-1 z-score is rising near 2 may be asked to 
come for an additional blood draw for IGF-1 between 12-24 weeks, to ensure that levels remain below a z-
score of 2.  Subjects will be instructed to administer Norditropin at night before bedtime. GH was safe and well-
tolerated in the previous pilot study. Safety will be closely monitored. Although no dosing reductions were 
required in the pilot study and we do not anticipate this circumstance, subjects with IGF-1 SDS >2 will undergo 
a 20% dose reduction.  If a subject undergoes 2 dose reductions and continues to have IGF-1 SDS >2, s/he 
will be discontinued from the study.  Subjects with fasting glucose ≥126mg/dL will have the value confirmed on 
a repeat draw and, if hyperglycemia is confirmed, will be discontinued from the study. Subjects reporting 
significant adverse effects including significant arthralgia, myalgia, or edema will also be discontinued. These 
did not occur in the pilot study and are not anticipated to occur with the physiologic dosing strategy that we will 
utilize.  
 
Compliance will be assessed using a daily study drug diary as well as vial count of returned used and unused 
study drug.  
 
Lifestyle Counseling:  Subjects will receive lifestyle counseling from study MDs or nutritionists at the baseline, 
12 week, and 24 week visits, with recommendations and resources provided based on the 2010 Dietary 
Guidelines for Americans 
(http://www.cnpp.usda.gov/sites/default/files/dietary_guidelines_for_americans/PolicyDoc.pdf) and the 2008 
Physical Activity Guidelines for Americans (http://health.gov/paguidelines/guidelines/summary.aspx). Emphasis 
will be placed on physical activity (150+ minutes weekly); reduction of saturated fats, total fats, and sugary 
beverages; and increased consumption of fruits and whole grains.   


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VII.  Biostatistical Analysis 
 
Statistical Consideration – Data Interpretation:  The primary endpoint will be change in hepatic fat as measured 
by 1H-MRS after 24 weeks of randomized treatment.  Secondary endpoints will include changes in ALT, AST, 
GGT, VAT, CRP, adiponectin, IGF-1, lipids, fasting and 2-hour glucose and insulin, HbA1c, and respiratory 
quotient measured by indirect calorimetry.  Permuted block randomization will be performed, stratified by 
gender and use of Vitamin E.  
Analysis will be intention-to-treat, using all available data from all subjects.  Wilk-Shapiro test will be 
used to assess for normality of distribution of all variables, and variables that are not normally distributed will 
be appropriately transformed. Baseline variables will be compared between treatment groups, and any 
baseline variable that is statistically different between treatment groups will be adjusted for in subsequent 
analysis. Changes in variables measured only at baseline and 6 months, including the primary endpoint of liver 
fat, will be initially assessed using a two-sample t-test comparing mean changes between treatment groups. A 
critical alpha level of 0.05 will be used as the threshold for significance, and all statistical tests will be two-
tailed.  Subsequently, adjusted analyses will be performed including as covariates any baseline characteristics 
that differ between treatment groups and any differential changes in nutritional intake or physical activity 
between treatment groups over the study duration. For variables measured at ≥3 timepoints throughout the 
study (e.g., IGF-1, AST and ALT), we will analyze the longitudinal data using general linear mixed effects 
modeling, testing for time x treatment group interaction.  The primary analysis will be an intention to treat 
analysis using all available data. We will consider secondary multiple imputation based analyses if necessary. 
 
A supplementary analysis will be performed assessing stimulated growth hormone levels in response to 
Arginine testing.  Using peak GH as the independent variable, we will assess the hypothesis that peak GH will 
be negatively associated with liver fat quantity. 
 
Sample Size and Power Calculations:  The study is designed to yield pilot data, with the primary endpoint 
being change in hepatic fat fraction. As we do not have preliminary data on this measure in an adolescent 
population, we used a published standard deviation (SD) of 3.1%.  A sample size of 24 subjects, with up to 
20% discontinuation rate, yielding 19 evaluable patients, has 80% power to detect a 4.2 percentage point 
treatment effect on liver fat (e.g., a reduction from 7% liver fat to 2.8% liver fat), which is the minimum 
difference that would be clinically relevant, as absolute reductions of ≥ 4-5% liver fat have been considered 
clinically relevant in previous publications (77, 78).  For secondary variables of interest, the study is has 80% 
power to detect a treatment difference of approximately 1.4 SD.  
 
VIII.  Risks and Discomforts 
 
Radiation:  Subjects will have 2 DXA scans over 6 months, for a total radiation risk from both scans of 2 
microSieverts.  This radiation exposure is minimal and does not pose excessive risk to subjects. Female 
subjects must have a negative pregnancy test before DXA scanning. 
 
Blood Drawing:  Blood sampling volumes will be as follows: screen 27cc, baseline and 24 week visit 99cc, 
safety visits 5-8cc each (5 cc at 2 and 4 weeks, 8cc at 6, 12, 18 weeks).  The total blood drawing will be 286cc 
over 6 months.  The maximum blood drawn during any 8 week period (screen+baseline+2wk+4wk) will be no 
more than 140cc.  Body weight ≥50kg and Hgb ≥ 11g/dL will be required to participate, which will ensure that 
the blood volume sampled is within guidelines and does not pose significant risk to subjects.   
 
Blood drawing may cause discomfort, pain, and bruising. 
 
MRI/MR spectroscopy:  MRI and MR spectroscopy will be performed using FDA approved devices and pulse 
sequences, and there are no known foreseeable risks associated with exposure to MRI.  Subjects will be 
carefully screened for the presence of metallic implants (e.g., vascular clamps) prior to MR scanning.  Some 


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subjects report claustrophobia during MRI scans, and if any patient experiences discomfort during the scan, 
the procedure will be aborted and not repeated without the subject’s consent.  
 
Oral Glucose Tolerance Test:  Oral glucose tolerance test has no significant risks.  Some subjects may 
experience nausea or lightheadedness during the procedure, and supportive nursing care will be provided. 
 
Clonidine/Arginine Stimulation:   
Administration of arginine for diagnostic purposes can, on rare occasions, cause nausea, lightheadedness, 
numbness, and tingling.  Clonidine for diagnostic purposes may cause sleepiness and a mild decrease in blood 
pressure.  The stimulation test will be performed on the Clinical Research Center by nursing staff who are 
highly experienced in conducting this test.  Supportive care will be provided if needed. 
 
Other procedures:  There are no appreciable risks of the following procedures:  indirect calorimetry and 
questionnaire administration.  If subjects express or demonstrate discomfort during any research procedure, 
they will be reassured that the research is optional and they may choose to quit any procedure at any time. 
 
Growth Hormone (rhGH, Norditropin):  
 
Norditropin will be administered to subjects via subcutaneous injection.  The dosing strategy has been 
formulated based on extensive clinical experience of Drs. Stanley (PI) and Misra (Co-I) as well as prior 
research experience administering rhGH in this population.  To be eligible for the research, subjects must have 
IGF-1 SDS ≤0, demonstrating relative reductions in GH secretion and ensuring that the dosing strategy will 
result in physiologic increases in IGF-1.  The starting dose of rhGH will be 0.5mg daily.  IGF-1 will be checked 
at 2, 4, 6, 12, and 18 weeks, and dose adjustments will be made as described above.  The goal IGF-1 is 
between 0-2 SDS, and frequent IGF-1 assessment and dose adjustment will ensure physiologic dosing, such 
that any potential side effects are minimized.     
 
Possible side effects of rhGH include headache, edema, myalgia, arthralgia, and hyperglycemia.  
Subjects will be asked to contact study physicians right away with any potential side effects, and subjects who 
experience concerning side effects will be advised to discontinue the medication.  A physician will be available 
on-call 24 hours/day, 7 days/week, to all study participants for any questions or concerns. Fasting blood sugar 
will be checked at 2, 4, 6, and 12 weeks, and any subject with a fasting blood sugar ≥126mg/dL that is 
confirmed on recheck will be discontinued from the study, with appropriate follow-up to ensure return to 
euglycemia.  RhGH, particularly at physiologic dosing, is incredibly well-tolerated in the pediatric population, 
and there were no significant GH-related side effects in our pilot study of obese adolescents.  When side 
effects, including hyperglycemia, do occur with rhGH, these resolve with discontinuation of treatment unless 
there is an underlying, non-GH-related, etiology.   
 
As stated elsewhere, a pregnancy test will be performed at each visit for females able to become 
pregnant, and subjects will be informed of the study-related risks of pregnancy and the need not to become 
pregnant during the study.  If a subject is found to be pregnant, she will be asked to discontinue the study drug 
immediately, and the event will be reported to Novo Nordisk as below.  She will be referred for continued 
primary care and OBGYN care. 
 
Subjects will be carefully instructed regarding reconstitution, safe injection technique, and appropriate 
storage of the study drug and disposal of sharps.  A study investigator will be on call 24/7 for any questions or 
concerns. 
 
Persistent elevations in blood sugar after discontinuing growth hormone are not expected.  However, if 
any subject is discontinued for fasting glucose ≥126mg/dL, confirmed on repeat draw (see below), we will 
arrange for a repeat fasting glucose approximately 2-4 weeks after stopping growth hormone.  We will arrange 
this to be performed either at MGH through the study or locally with the patient’s primary care doctor.  If this is 
still elevated, the patient will be referred to his/her primary care doctor for continued follow-up and, if desired, 
we will also arrange for follow-up with endocrinology at MGH.   
 
Individual and Study Stopping Rules 
Individuals will be discontinued from the study if 


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• 
s/he undergoes 2 dose reductions and continues to have IGF-1 SDS >2 
• 
s/he has fasting glucose ≥126mg/dL confirmed on a repeat draw 
• 
s/he reports significant adverse effects including significant arthralgia, myalgia, or edema  
 
The study will be stopped if  
• 
More than 2 individuals in the GH group have persistent fasting glucose ≥126mg/dL 
 
The study will be halted and the dosing algorithm reconsidered (and lowered) if 
• 
More than 2 individuals in the GH group have IGF-1 SDS >2 after 2 dose reductions 
 
Alternative Treatments 
 
There are no FDA-approved highly effective treatments for NAFLD in young adults, and research into 
novel treatments is urgently needed.  Vitamin E, ursodiol, and metformin are sometimes used for NAFLD, and 
vitamin E has been demonstrated to improve liver histology in individuals with NASH.  Subjects who wish to 
participate and have been receiving Vitamin E or ursodiol at a stable dose for ≥6 months will be eligible for the 
study with continued use of these medications (under the ongoing care of their hepatologist) during the study.  
Metformin use is not allowed as it will confound assessment of glucose homeostasis.  We will not ask any 
subject to discontinue any medication in order to participate in the study.   
 
As we are not performing liver biopsy during the current study, the study procedures will not result in 
new diagnosis of NASH (which requires histology).  However, subjects without known NAFLD who 
demonstrate ≥5% liver fat on MRS and who have elevated ALT or AST are likely to have NASH, and, if not 
already under the care of a hepatologist, these subjects will be offered a clinical referral to pediatric 
hepatology.  Subjects who are found to have labs suggesting significant liver disease (AST or ALT > 2.5x 
upper limit of normal (ULN) or total bilirubin > ULN) will be encouraged to seek clinical care from a hepatologist 
and will not be eligible for the study.  AST and ALT will be carefully monitored (0, 6, 12, 18, and 24 weeks). 
 
VIII.  Potential Benefits 
 
Participants will receive information about their nutritional and metabolic health.  In addition, subjects 
randomized to Norditropin may experience benefits from this treatment including decrease in fat mass and 
possibly decreases in liver fat and systemic inflammation.  All subjects will receive lifestyle counseling and 
information about healthy nutrition and physical activity.  Further, the information obtained from this study will 
inform the potential use of rhGH and possibly other strategies to treat NAFLD in adolescents.   
 
The risks of the study are minimized by close communication between study investigators and subjects, careful 
monitoring of IGF-1 levels, and frequent monitoring for adverse events.  The study may provide benefit to 
individual patients, as above, and is also expected to provide information that may lead to the development of 
new treatment strategies for NAFLD, which is a significant public health problem.  Therefore, benefits are felt to 
outweigh the risks.  
 
IX.  Adverse Event, Unanticipated Problem, and Deviation Definitions  
Adverse Event (AE) 
An adverse event is any untoward or unfavorable medical occurrence in a human subject, including any 
abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or disease, temporally 
associated with the subject’s participation in the research, whether or not considered related to the research. 
 
Adverse Reaction (AR) 
An adverse event that is caused by an investigational agent (drug or biologic). 
 


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Suspected Adverse Reaction (SAR) 
An adverse event for which there is a reasonable possibility that the investigational agent caused the adverse 
event. ‘Reasonable possibility’ means that there is evidence to suggest a causal relationship between the drug 
and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than 
adverse reaction which implies a high degree of certainty. 
 
Serious Adverse Event (SAE) 
A Serious Adverse Event is an AE that results in one or more of the following outcomes: 
• 
death 
• 
a life threatening (i.e., an immediate threat to life) event 
• 
an inpatient hospitalization or prolongation of an existing hospitalization 
• 
a persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions 
• 
a congenital anomaly/birth defect 
• 
a medically important event* 
 
* Medical and scientific judgment will be exercised in deciding whether expedited reporting is appropriate in 
other situations, such as important medical events that may not be immediately life threatening or result in 
death or hospitalization but that may jeopardize the subject or may require intervention to prevent one of the 
other outcomes listed above.  
 
Serious Adverse Drug Reaction (SADR) 
An SADR is defined as a serious adverse event that has a reasonable suspected causal relationship to the 
study drug.  For the purposes of reporting during this protocol, any serious adverse event occuring in a subject 
randomized to study drug that is judged by the investigator to be possibly related, probably related, or definitely 
related (see below) to study drug will be considered in this category. 
 
Unexpected Adverse Event 
An AE is unexpected if it is not listed in the Package Insert for norditropin or is not listed at the specificity or 
severity that has been observed.   
 
Unanticipated Problem (UP) 
An Unanticipated Problem is any event, incident, experience, or outcome that is 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and informed consent 
document; Investigator’s Brochure or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. possibly, probably, or definitely related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, economic, or social 
harm) than was previously known or recognized.  
Protocol Deviation: Any change, divergence, or departure from the IRB approved study procedures in a 
research protocol. Protocol deviations are designated as serious or non-serious and further characterized as 
1. Those that occur because a member of the research team deviates from the protocol. 
2. Those that are identified before they occur, but cannot be prevented. 
3. Those that are discovered after they occur 
 
Pregnancy will be reported as described below if it occurs in any subject receiving norditropin or if it occurs in 
a subject’s partner whose fetus or baby develops an adverse event considered to be related to norditropin. 
 
Relatedness will be determined as follows: 
Definitely Related 
   
• 
reasonable temporal relationship 


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• 
follows a known response pattern 
• 
clear evidence to suggest a causal relationship 
• 
there is no alternative etiology 
 
Probably Related 
 
• 
reasonable temporal relationship 
• 
follows a suspected response pattern (based on similar agents) 
• 
no evidence of a more likely alternative etiology 
 
Possibly Related 
 
• 
reasonable temporal relationship 
• 
little evidence for a more likely alternative etiology 
 
Unlikely Related 
 
• 
does not have a reasonable temporal relationship  
OR 
• 
good evidence for a more likely alternative etiology 
 
Not Related 
 
• 
does not have a temporal relationship 
         
OR 
• 
definitely due to an alternative etiology 
 
 
X.  Reporting Requirements 
Dr. Stanley (PI) will be responsible for the reporting of adverse events, unanticipated problems, protocol 
deviations, and other necessary events as follows: 
 
Reporting to Novo Nordisk  
Serious Adverse Drug Reactions and pregnancies will be reported to Novo Nordisk as soon as possible and no 
later than 7 days following investigator awareness of the event.  Dr. Stanly will assist Novo Nordisk with follow-
up of any reports as necessary. 
 
Reporting to IRB (Partners Human Research Committee) 
The following will be reported within 7 calendar days of investigator awareness: 
• 
Internal adverse events that are unexpected, and related or possibly related to the research and that 
indicate there are new or increased risks to subjects 
• 
Deviation from the approved research protocol or plan without IRB approval in order to eliminate 
apparent immediate hazard to subjects or harm to others 
• 
Deviation from the approved research protocol or plan that placed subjects or others at an increased 
risk of harm regardless of whether there was actual harm to subjects or others 
• 
Breach of confidentiality or violation of HIPAA (e.g., lost or stolen laptop) 
• 
Medication, procedural or laboratory error (e.g., errors in drug administration or dosing, surgical or other 
procedure, or testing of samples or test results) regardless of whether subjects experienced any harm 
• 
Interim analysis, safety monitoring report, publication in a peer-reviewed journal, or other finding that 
indicates that there are new or increased risks to subjects or others or that subjects are less likely to 
receive any direct benefits from the research 
• 
Change in FDA labeling (e.g., black box warning), withdrawal from market, manufacturer alert from 
Novo Nordisk, or recall of an FDA-approved drug, device, or biologic used in the research 


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• 
Complaint by/on behalf of a research subject that indicates that the rights, welfare, or safety of the 
subject have been adversely affected or that cannot be resolved by the investigator 
• 
Incarceration of a research subject during participation in research that is not approved for involvement 
of prisoners as subjects 
• 
Noncompliance with applicable regulations or requirements or determinations of the IRB identified by 
the research team or others (e.g., FDA Form 483 or Warning Letter) that indicates that the rights, 
welfare, or safety of subjects have been adversely affected 
• 
Suspension or termination of the research, in whole or in part, based on information that indicates that 
the research places subjects at an increased risk of harm than previously known or recognized (e.g., 
FDA clinical hold) 
• 
Suspension or disqualification of an investigator by FDA, Novo Nordisk, or others 
• 
Scientific misconduct  
• 
Any other problem that indicates that the research places subjects or others at an increased risk of 
harm or otherwise adversely affect the rights, welfare or safety of subjects or others.  
 
The following items will be reported to the MGH IRB in summary at the time of Continuing Review: 
- 
Serious and non-serious unanticipated problems  
- 
Expected serious adverse events that are possibly, probably, or definitely related to the research 
- 
Serious adverse events that are not related to the research 
- 
All adverse events 
- 
Serious and Non-Serious Protocol deviations  
- 
Serious, continuing, and minor non-compliance 
- 
Any trends or events which in the opinion of the investigator should be reported 
 
XI.  Monitoring and Quality Assurance 
 
Full IRB approval will be obtained by the Partners Human Research Committee before any interactions with 
subjects take place for the purposes of this protocol.  The protocol will be conducted adhering to the standards 
of Good Clinical Practice. 
 
Written, informed consent will be obtained prior to any procedures being performed on a subject for the 
purposes of this protocol.  This consent will be obtained by a licensed medical provider (MD or NP). 
 
The principal investigator (Stanley) will monitor all data collected for the studies. Data will be stored securely, 
with access restricted to co-investigators and study staff. Binders with subject information will be labeled with a 
coded enrollment number to protect confidentiality. Electronic databases will be locked and password-
protected, with access available only to study staff.  Data will not be saved on the hard drive of any laptop or 
desktop computers or on any removable data storage devices such as flash drives or CDs.  
 
The risks to subjects will be minimized by using procedures which are consistent with sound research design. 
Subjects will be carefully screened to minimize potential risk of study procedures and treatments. A pregnancy 
test will be performed at each visit for female subjects of childbearing potential, and patients who become 
pregnant will be asked to discontinue participation in the study. Most importantly, the safety of subjects will be 
monitored closely by the principal investigator, co-investigators and DSMB, and subjects will be discontinued 
from the study if the risk to the subject is felt to be excessive.  
 
Data and Safety Monitoring Plan  
An independent data and safety monitoring board will be established to review safety data and adverse events. 
The DSMB will include a statistician, a gastroenterologist, and a pediatric endocrinologist, and will meet every 
6 months. The studies will also be monitored continuously by the principal investigator (Stanley).  
 


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Subjects will be instructed to report any adverse events immediately. A physician or nurse practitioner will be 
available on-call 24 hours/day, 7 days/week, to all study participants for any questions or concerns.  
 
Clinical Trials Registration 
The trial will be registered on clinicaltrials.gov, with Dr. Stanley serving as the responsible party for registration 
and reporting of results. 
 
XII.  Dissemination of Results 
Upon completion of the protocol, an abstract summarizing the primary results will be submitted to an 
appropriate national meeting (e.g., Pediatric Academic Societies or ENDO), and a manuscript comprehensively 
reporting the results will be submitted to an appropriate journal (e.g., Journal of Clinical Endocrinology and 
Metabolism).  Preparation and submission of the primary manuscript reporting results is anticipated to be 
complete within 4 months of the last study visit for the last patient. 
 
 
XIII.  References Cited 
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response to provocative testing in children with short stature. J Clin Endocrinol Metab. 2009 Dec;94(12):4875-
81. 
2. 
Argente J, Caballo N, Barrios V, Munoz MT, Pozo J, Chowen JA, et al. Multiple endocrine 
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3. 
Misra M, Bredella MA, Tsai P, Mendes N, Miller KK, Klibanski A. Lower growth hormone and higher 
cortisol are associated with greater visceral adiposity, intramyocellular lipids, and insulin resistance in 
overweight girls. Am J Physiol Endocrinol Metab. 2008 Aug;295(2):E385-92. 
4. 
Stanley TL, Feldpausch MN, Oh J, Branch KL, Lee H, Torriani M, et al. Effect of tesamorelin on 
visceral fat and liver fat in HIV-infected patients with abdominal fat accumulation: a randomized clinical trial. 
JAMA. 2014 Jul 23-30;312(4):380-9. 
5. 
Welsh JA, Karpen S, Vos MB. Increasing prevalence of nonalcoholic fatty liver disease among United 
States adolescents, 1988-1994 to 2007-2010. J Pediatr. 2013 Mar;162(3):496-500 e1. 
6. 
Schwimmer JB, Deutsch R, Kahen T, Lavine JE, Stanley C, Behling C. Prevalence of fatty liver in 
children and adolescents. Pediatrics. 2006 Oct;118(4):1388-93. 
7. 
Manco M, Bedogni G, Marcellini M, Devito R, Ciampalini P, Sartorelli MR, et al. Waist circumference 
correlates with liver fibrosis in children with non-alcoholic steatohepatitis. Gut. 2008 Sep;57(9):1283-7. 
8. 
Xanthakos S, Miles L, Bucuvalas J, Daniels S, Garcia V, Inge T. Histologic spectrum of nonalcoholic 
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9. 
Schwimmer JB, Behling C, Newbury R, Deutsch R, Nievergelt C, Schork NJ, et al. Histopathology of 
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Molleston JP, Schwimmer JB, Yates KP, Murray KF, Cummings OW, Lavine JE, et al. Histological 
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Rashid M, Roberts EA. Nonalcoholic steatohepatitis in children. J Pediatr Gastroenterol Nutr. 2000 
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Kleiner DE, Brunt EM. Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in 
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14. 
Carter-Kent C, Brunt EM, Yerian LM, Alkhouri N, Angulo P, Kohli R, et al. Relations of steatosis type, 
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15. 
Holterman AX, Guzman G, Fantuzzi G, Wang H, Aigner K, Browne A, et al. Nonalcoholic fatty liver 
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Apr;49(4):755-65. 
17. 
Adiels M, Westerbacka J, Soro-Paavonen A, Hakkinen AM, Vehkavaara S, Caslake MJ, et al. Acute 
suppression of VLDL1 secretion rate by insulin is associated with hepatic fat content and insulin resistance. 
Diabetologia. 2007 Nov;50(11):2356-65. 
18. 
Seppala-Lindroos A, Vehkavaara S, Hakkinen AM, Goto T, Westerbacka J, Sovijarvi A, et al. Fat 
accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free 
fatty acids independent of obesity in normal men. J Clin Endocrinol Metab. 2002;87(7):3023-8. 
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Bugianesi E, Gastaldelli A, Vanni E, Gambino R, Cassader M, Baldi S, et al. Insulin resistance in non-
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20. 
Kim D, Choi SY, Park EH, Lee W, Kang JH, Kim W, et al. Nonalcoholic fatty liver disease is associated 
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21. 
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22. 
Brea A, Mosquera D, Martin E, Arizti A, Cordero JL, Ros E. Nonalcoholic fatty liver disease is 
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23. 
Hamaguchi M, Kojima T, Takeda N, Nagata C, Takeda J, Sarui H, et al. Nonalcoholic fatty liver disease 
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26. 
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27. 
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disease. Pediatr Gastroenterol Hepatol Nutr. 2012 Dec;15(4):256-65. 
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29. 
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30. 
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exercise reduces intrahepatic lipid content and visceral fat and improves insulin sensitivity in obese adolescent 
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Sanyal AJ. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 
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32. 
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33. 
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vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC 
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34. 
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the treatment of liver abnormalities in obese children. J Pediatr. 2000 Jun;136(6):739-43. 
37. 
Dohil R, Schmeltzer S, Cabrera BL, Wang T, Durelle J, Duke KB, et al. Enteric-coated cysteamine for 
the treatment of paediatric non-alcoholic fatty liver disease. Aliment Pharmacol Ther. 2011 May;33(9):1036-44. 
38. 
Vajro P, Mandato C, Licenziati MR, Franzese A, Vitale DF, Lenta S, et al. Effects of Lactobacillus 
rhamnosus strain GG in pediatric obesity-related liver disease. J Pediatr Gastroenterol Nutr. 2011 
Jun;52(6):740-3. 
39. 
Nobili V, Bedogni G, Alisi A, Pietrobattista A, Rise P, Galli C, et al. Docosahexaenoic acid 
supplementation decreases liver fat content in children with non-alcoholic fatty liver disease: double-blind 
randomised controlled clinical trial. Arch Dis Child. 2011 Apr;96(4):350-3. 
40. 
Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and 
management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological 
Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. 
Gastroenterology. 2012 Jun;142(7):1592-609. 
41. 
Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. Sources of fatty acids 
stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005 
May;115(5):1343-51. 
42. 
Kawano Y, Cohen DE. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver 
disease. J Gastroenterol. 2013 Apr;48(4):434-41. 
43. 
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