Clinical trial NCT01775813
Conditions: Obesity Insulin Resistance Gonadal Dysfunction Type 2 Diabetes
Keywords: Insulin resistance Puberty Obesity Hypogonadism Gonadal dysfunction Metformin
Sponsor: University of Colorado, Denver
Start date: 2011-06-01; Completion date: 2018-05-31
Enrollment: 104 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The Health Influences of Puberty (HIP) Study is designed to explore the relationships between puberty and the onset of type 2 diabetes in adolescents. The results of this study will help us better understand how to prevent type 2 diabetes in these youth. Children go through many changes during puberty, including important hormonal and behavioral alterations. Among these changes, it has long been known that, during puberty, insulin does not work as well as it does before and after puberty. This is called physiologic insulin resistance. In healthy children, this does not cause diabetes or affect blood sugar in any way because the body is able to compensate by making more insulin. Indeed, this is thought to be an important part of the adolescent growth spurt. However, in some children with increased risk for developing type 2 diabetes due to obesity and genetics, the worsening insulin resistance of puberty cannot be compensated for and these youth get diabetes early. The investigators believe this is because type 2 diabetes is rarely, if ever, seen before puberty begins, and the peak of diabetes onset in adolescents occurs at the time of the worst insulin resistance. This specific research project has two goals: 1. To examine effects of obesity on how well the body's insulin works during puberty, and 2. To see if treatment of obese children during this critical period of puberty with a medication that improves insulin resistance (metformin) will help prevent early onset type 2 diabetes.

Detailed description:
Specific Aims:

Pediatric insulin resistance and related disorders, such as type 2 diabetes mellitus (T2DM), are increasing in prevalence, and portend significant end-organ and cardiovascular morbidity and mortality. Thus, measures aimed at understanding its causes and preventing its onset are critical. The physiologic decrease in insulin sensitivity in all adolescents during puberty is well-established. It is also known that obese adolescents start out less insulin sensitive at the onset of puberty than lean adolescents, and that their insulin sensitivity worsens as puberty progresses. While there are both longitudinal and cross-sectional data confirming the natural recovery of pre-pubertal insulin sensitivity in normal weight adolescents after puberty is completed, it is unknown whether obese adolescents recover their pre-pubertal insulin sensitivity. Failure to regain pre-pubertal insulin sensitivity at the end of puberty, and failure of compensatory insulin secretion, may accelerate progression from obesity to insulin resistance to T2DM in at-risk youth and contribute to long-term cardiovascular risk.

In addition, obesity and insulin resistance are associated with earlier onset of puberty and premature adrenarche in females. Insulin resistance also contributes to the gonadal dysfunction of polycystic ovarian disease in fully pubertal females and is associated with hypogonadism in older adult males. Little is known about effects of obesity and insulin resistance on gonadal function in young males. However, persistent metabolic changes at the end of puberty may contribute to gonadal dysfunction in obese youth. Currently, there are few longitudinal studies in either sex that evaluate the interactions among obesity, insulin resistance and gonadal function during puberty.

The investigators' long-term goal is to better understand the metabolic changes that occur during puberty, their underlying mechanisms, and their potential contribution to adult disease. The overall aim is to evaluate the effects of obesity on the evolution of insulin sensitivity and gonadal function during puberty. In addition, because improvement in insulin action during puberty may slow β-cell deterioration, the investigators will evaluate whether compensatory insulin secretion is also affected in obese adolescents and whether treatment with metformin improves β-cell response.

HYPOTHESES:

1. Obese adolescents will show decreased improvement in insulin sensitivity from Tanner stage 2/3 to Tanner 5 when compared with lean counterparts.
2. Obese adolescents treated with metformin will have greater improvement in insulin sensitivity from Tanner stage 2/3 to Tanner 5 vs. those treated with placebo. (See hypothesis schematics below)

To test these hypotheses, we propose to address the following Specific Aims:

SPECIFIC AIM 1 (Observational Arm):

1. To compare longitudinal changes in insulin sensitivity and secretion and their correlates in obese and normal weight adolescents during puberty.

   1. Primary outcome: Change in insulin sensitivity (Si), as measured by frequently sampled intravenous glucose tolerance test (IVGTT), from early puberty to puberty completion in obese and normal weight adolescents.
   2. Secondary outcomes: Change in insulin secretion (AIR) and disposition index (DI) as measured by IVGTT, body composition, fat distribution, markers of gonadal function, and inflammatory markers over time in these groups.

   SPECIFIC AIM 2 (Treatment Arm):
2. To compare longitudinal changes in insulin sensitivity and secretion and their correlates in obese adolescents treated with metformin or placebo during puberty.

   1. Primary outcome: Change in Si from early puberty to puberty completion in obese controls and obese adolescents treated with metformin.
   2. Secondary outcome: Change in AIR and DI, body composition, fat distribution, markers of gonadal function, and inflammatory markers over time in these groups.

Arms: arm 1: Double-blinded placebo-controlled trial of metformin during puberty, treatment arm Dosage form: Metformin 1000 mg tablets Dosage: 1000 mg by mouth twice daily Duration: From early puberty (Tanner 2-3) until puberty completion (Tanner 5), approximately 3 years arm 2: Double-blinded placebo-controlled trial of metformin during puberty, placebo arm Dosage form: Placebo stamped to match 1000 mg metformin tablets Placebo comparator: Stamped placebo pill matching metformin dose Dosage: 1000 mg by mouth twice daily Duration: From early puberty (Tanner 2-3) until puberty completion (Tanner 5), approximately 3 years arm 3: Comparator group for the observational comparison of metabolic changes in youth with normal weight and obesity as they progress through puberty.

Duration: From early puberty (Tanner 2-3) until puberty completion (Tanner 5), approximately 3 years arm 4: Comparator group for the observational comparison of metabolic changes in youth with normal weight and obesity as they progress through puberty.

Duration: From early puberty (Tanner 2-3) until puberty completion (Tanner 5), approximately 3 years
Interventions: intervention 1: Metformin intervention 2: Placebo
Intervention descriptions: intervention 1: After randomization, the study drug (metformin or placebo) is gradually titrated to full dose of 1000 mg BID (or to maximum tolerated, at least 500 mg BID) over a period of 4 weeks to minimize adverse gastrointestinal effects. Participants are seen every three months to measure compliance and dispense new study drug. Every 6 months, they also have a physical examination in order to determine puberty staging. Study measurements (IVGTT, bloodwork, DXA) are performed at Tanner 4 puberty and Tanner 5 (puberty completion), at which time the study drug is stopped. Study measurements will be performed again 6 months after study drug is completed to assess if effects are persistent after study drug is stopped. During the treatment period, all participants receive standard lifestyle counseling. intervention 2: Stamped placebo pill to look like the 1000 mg metformin pill Dosage: 1 pill taken orally twice daily Duration: From early puberty (Tanner 3-4) until puberty completion (Tanner 5), approximately 3 years

Protocol documents:
Prot_SAP_000.pdf: 
PHS 398/2590 (Rev. 09/04, Reissued 4/2006) 
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Official Title: The Health Influences of Puberty (HIP) Study 
NCT Number: NCT01775813 
Document Date: 03/25/2016 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Title: COMBINED INFLUENCE OF PUBERTY AND OBESITY ON INSULIN RESISTANCE IN 
ADOLESCENTS  
 
A. STATEMENT OF HYPOTHESES AND SPECIFIC AIMS: 
   Pediatric type 2 diabetes mellitus (T2DM) is increasing in prevalence, and leads to significant morbidity 
and mortality.  Although the exact incidence in youth is largely unknown, it has been increasing over time.  
Studies suggest that the prevalence of T2DM in adolescents is somewhere between 3 and 7/1000, and that it 
represents approximately one-third of new onset cases of diabetes in adolescents. Thus, measures aimed at 
understanding its causes and preventing its onset are in critical need. The physiologic decrease in insulin 
sensitivity (IS) in all adolescents during puberty is well-established.  It is also known that obese adolescents 
start out less insulin sensitive at the onset of puberty than lean adolescents, and that their insulin sensitivity 
decreases further as puberty progresses.  While there are both longitudinal and cross-sectional data 
confirming the natural recovery of pre-pubertal insulin sensitivity in normal weight adolescents after puberty 
is completed, it is unknown whether obese adolescents follow the same pattern.  Furthermore, the 
characteristics that differentiate the natural decrease in insulin sensitivity during puberty from the pathologic 
decrease in insulin sensitivity seen in obese children are poorly understood.   If obese youth fail to regain 
their pre-pubertal insulin sensitivity at the end of puberty, the resulting stress on the pancreatic β-cell 
required to compensate with increased insulin secretion may contribute to the progression from obesity to 
insulin resistance to T2DM in at-risk youth.  Our long-term goal is to better understand the metabolic changes 
that occur during puberty, their underlying mechanisms, and their potential contribution to adult disease. In 
particular, we hypothesize that insulin sensitivity and its correlates will change adversely in obese 
adolescents during puberty, but will not recover after puberty is complete.  Furthermore, we hypothesize that 
treatment with metformin during puberty will help obese adolescents improve pubertal insulin sensitivity, will 
help preserve insulin secretion, and that such changes will be sustained after cessation of metformin.   
 
Our unique combination of pediatric and adult endocrinology skills, laboratory expertise in insulin signaling, 
and clinical research experience in insulin sensitivity testing in obese youth make us perfectly poised to 
address these hypotheses.  In addition, the use of a longitudinal study design will allow us to examine 
differences in percent body fat, body fat distribution and changes in BMI over time in the observational arm of 
the study.  It also allows to examine whether treatment of obese children during this time can have beneficial 
effects on insulin sensitivity and secretion in the treatment arm.  Enhancing our understanding of longitudinal 
changes of insulin sensitivity in obese adolescents and the response to metformin will help us identify 
potential interventions aimed at the prevention of T2DM. 
 
The studies in this proposal will test the following hypotheses: 
 
HYPOTHESES:  
1.  Obese adolescents, unlike normal weight controls, fail to improve insulin sensitivity at the end of 
puberty. 
2. Treatment of obese adolescents with metformin during puberty will help these individuals result in 
sustained improvement in insulin sensitivity at the end of puberty, decrease compensatory 
hyperinsulinemia, and preserve insulin secretion, thereby improving their disposition index (DI). 
 
To test these hypotheses, we propose to address the following Specific Aims: 
 
SPECIFIC AIM 1 (Observational Arm): 
1. To compare longitudinal changes in insulin sensitivity and secretion and their correlates in obese 
and normal weight adolescents during puberty.   
a. To compare changes in insulin sensitivity and secretion as measured by frequently 
sampled intravenous glucose tolerance test (IVGTT) from early puberty to puberty 
completion between obese and normal weight adolescents. 
COMIRB # 07-0988 
VERSION DATE: 3/25/2016 
 


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b. To compare changes in body composition, fat distribution, and inflammatory markers over 
time between these groups. 
 
Under this Specific Aim, insulin sensitivity and secretion will be measured by frequently sampled IVGTT in 
early puberty and at puberty completion in obese and normal weight adolescents.  Changes in insulin 
sensitivity will be correlated with changes in inflammatory markers in all participants and body composition in 
a subset of these participants. 
 
SPECIFIC AIM 2 (Treatment Arm):  
2. To compare changes in insulin sensitivity and secretion and their correlates, in obese 
adolescents treated with metformin or placebo during puberty. 
a. To compare changes in insulin sensitivity and secretion from early puberty to puberty 
completion between obese controls and obese adolescents treated with metformin. 
b. To compare changes in body composition, fat distribution, and inflammatory markers 
over time between these groups. 
 
Under this Specific Aim, obese participants from Specific Aim 1 will be randomized to receive placebo or 
metformin treatment during puberty.   Frequently sample sampled IVGTT will be performed to measure 
insulin sensitivity and secretion in early puberty, at the end of puberty when treatment is stopped, as well 
as six months after treatment completion. Changes in body composition will also be measured in a 
subset of participants and changes in inflammatory markers will be measured in all participants at these 
time points.  
 
The nested study design (Fig 1) will efficiently address four important knowledge deficits with a single 
recruited cohort: 1) Whether obese adolescents recover their pre-pubertal insulin sensitivity at the end of 
puberty; 2) The relationships between longitudinal changes in insulin sensitivity and changes in body 
composition in obese and normal weight youth; 3) Whether treatment with metformin during puberty can 
improve  insulin sensitivity in obese adolescents to the level seen in normal weight pubertal adolescents; and 
4) Whether treatment of obese adolescents during puberty will result in prolonged improvement of insulin 
sensitivity and secretion.  


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B. BACKGROUND AND SIGNIFICANCE 
Insulin Resistance and T2DM in Youth 
 
The proportion of adolescents who are obese has been increasing markedly in developed countries in 
the past 20 years (1), with a parallel increase in the incidence of type 2 diabetes mellitus (T2DM), so much 
so that it has been dubbed an “epidemic” by the American Diabetes Association (ADA)(2). Obesity is thought 
to promote the development of T2DM and related disorders at least partly  through the development of 
decreased insulin sensitivity, a condition in which maintenance of normal blood glucose requires higher than 
normal insulin concentrations (3;4). This results in hyperinsulinemia as the pancreas compensates by 
increasing insulin production. Overt T2DM develops when the pancreas is unable to compensate for IR by 
increasing insulin production to the levels necessary to maintain normal carbohydrate metabolism. This 
results in relative insulin deficiency, while circulating insulin levels may still be normal or elevated. Thus, 
progressive β-cell failure in insulin-resistant, primarily obese youth is proposed to be a critical factor in the 
pathophysiology of childhood T2DM.  IR correlates with increasing body weight and decreasing physical 
activity, as well as increased deposition of fat in the visceral cavity and skeletal muscle (5;6).   
 
Insulin Resistance in puberty 
 
The vast majority of youth who develop T2DM are in mid-puberty at the time of clinical presentation 
(7;8)  Puberty, similarly to pregnancy, is known to be a period of physiologic decrease in insulin sensitivity 
(9). Amiel et al (9),  reported a 36% reduction in insulin sensitivity in pubertal non-diabetics when compared 
with pre-pubertal non-diabetics, using the hyperinsulinemic euglycemic clamp technique.  Interestingly, 
insulin sensitivity of adult subjects does not differ from pre-pubertal children, suggesting that insulin 
sensitivity improves again after puberty. Multiple cross-sectional and limited longitudinal studies have verified 
this phenomenon (10-17). The largest study involved 357 hyperinsulinemic-euglycemic clamps performed on 
children in Tanner stages I (pre-pubertal) through V (fully pubertal) and was cross-sectional in nature (16).  
Normal Weight 
Control Group 
Obese Placebo 
Group 
Obese 
Metformin 
Group 
Comparison of longitudinal 
changes in insulin 
sensitivity and secretion 
during puberty between 
lean and obese children 
Comparison of longitudinal changes 
in insulin sensitivity and secretion 
during puberty between obese 
children treated with metformin and 
those treated with placebo 
SPECIFIC AIM 1 
Observation Arm 
SPECIFIC AIM 2 
Treatment Arm 
Fig. 1 


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This study found that insulin sensitivity decreases during puberty independently of changes in body mass 
index (BMI) and body fat (as determined by skinfold thickness), but that obese participants were more insulin 
resistant at all points before and during puberty.  This study did not report comparison data between obese 
children in Tanner V and in Tanner I, did not measure insulin secretion, and did not follow the same subjects 
over time.   
   Longitudinal studies allow investigators to control for intra-individual changes in body fat distribution, BMI 
and hormonal changes throughout puberty.  Several longitudinal trials have confirmed a decrease in insulin 
sensitivity during puberty, which seems to be most impaired at Tanner stage III and returns to baseline after 
puberty is completed (11;13-15).  
Multiple studies investigating insulin sensitivity in puberty have used the frequently sampled IVGTT 
(11;13;15;18-20), including one sentinel study performed at our institution (17). This method allows for 
calculation of both insulin sensitivity and insulin secretion using the minimal model method (21).  The IVGTT 
has been used extensively in both obese and normal weight children both by our group and others and has 
been shown to correlate well with the gold-standard measures of insulin sensitivity and insulin secretion: the 
hyperinsulinemic-euglycemic clamp and the hyperglycemic clamp techniques, respectively (22;23).   
   Although it is clear that insulin sensitivity decreases during puberty, the majority of adolescents maintain 
normal glucose homeostasis.  Studies show almost universally that insulin secretion increases during 
puberty and DI, a measure of glucose homeostasis, calculated by the ratio of insulin sensitivity to insulin 
secretion (21), is maintained, therefore pubertal adolescents preserve normal glucose tolerance. That is, in 
normal subjects, if there is a physiologic decrease in insulin sensitivity, such as occurs during puberty or 
pregnancy, insulin secretion increases to compensate and DI is maintained, leading to maintenance of  
normoglycemia.  Conversely, DI decreases as patients progress from normal insulin sensitivity to insulin 
resistance with normoglycemia, to T2DM. 
In contrast, insulin secretion and/or DI decreases as puberty progresses in adolescents who have risk 
factors for developing diabetes, such as in African-Americans or Hispanics (7).  Ball et al analyzed glucose 
metabolism longitudinally in 46 Caucasians and 46 African-Americans using the frequently sampled IVGTT 
and reported a steeper decline in insulin secretion in blacks than in whites as puberty progresses (11).  Both 
ethnic groups showed a decline in DI between Tanner stages I and V, however the only significant predictor 
of impaired DI in Caucasians, as determined by linear regression, was fat mass, whereas Tanner stage 
impacted DI independently in African-Americans.  In another cross-sectional study performed by the same 
group, 214 overweight Hispanic children with a family history of T2DM were examined by IVGTT across all 
stages of puberty (18).    These patients had multiple risk factors for diabetes including obesity, ethnicity and 
family history.  insulin sensitivity did not differ significantly according to Tanner stage, but subjects did tend to 
have a reduced DI by Tanner stage, suggesting declining β-cell function in these at-risk children.  These 
results suggest that there is some element of β-cell failure occurring as puberty progresses in at-risk youth.  
However, obesity as a risk factor has not been specifically investigated in any of these studies. 
    
Treatment of Insulin Resistance 
 
 
If T2DM results from a progressive decrease in insulin sensitivity, leading to compensatory insulin 
hypersecretion and eventual β-cell failure, improvements in insulin sensitivity through pharmacologic 
intervention should reduce the need for insulin secretion, prevent β-cell fatigue and prevent or delay diabetes 
onset.  Evidence to support this hypothesis was first demonstrated in adults by the Diabetes Prevention 
Program, where subjects with impaired glucose tolerance were randomized to placebo, lifestyle intervention 
or metformin (27). After an average 2.8 years of treatment, both lifestyle and metformin were able to prevent 
conversion to diabetes compared to placebo. In participants older than 45 years, lifestyle was more effective 
than metformin in preventing conversion to T2DM, whereas, in younger participants, the two treatments were 
equivalent.   Although lifestyle changes and weight loss are preferable treatments for obesity and decreased 
insulin sensitivity, this intensive intervention is expensive and success has been limited in a real world 
setting, especially among adolescents who already struggle with the developmental challenges inherent to 
the teenage years.   
 
Many pediatric providers have responded to this evidence by prescribing metformin to pediatric patients 
with obesity and/or hyperinsulinism.  There are, however, several problems with extrapolating adult data to 


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the pediatric population.  First, mechanisms underlying the insulin sensitivity changes during puberty may be 
different and, therefore, metformin may not have the same benefit in adolescents as in adults.  Second, 
beginning metformin in adolescence would be potentially committing pediatric patients to a much longer 
treatment period without any definable endpoint.  It is much more difficult to study diabetes prevention in 
adolescents because conversion rates to diabetes are much lower than in adults with impaired glucose 
tolerance (IGT).  Thus, conversion to diabetes is an unrealistic research endpoint.   
 
Metformin acts by decreasing endogenous hepatic glucose production, thereby lowering insulin 
requirements (28).  Several studies of metformin have been performed on insulin resistant children, with 
variable outcomes.  Three of these studies were controlled, randomized, double-blind and specifically 
targeted obese, insulin resistant adolescents with normal glucose homeostasis (29-31).  Srinivasan et al 
treated 28 adolescents aged 9-18 years in a one-year double-blind crossover trial (6 mos. metformin at a 
dose of 1g BID, 6 mos. placebo).  Fasting insulin, glucose and BMI standard deviation scores (SDS) were 
significantly decreased after the treatment period compared with the placebo period.  However, insulin 
sensitivity, insulin secretion and DI, as measured by Bergman’s minimal model method, did not change (31).  
Similarly, 32 obese adolescents aged 12 to 19 years with a family history of T2DM had a significant decrease 
in BMI SDS, fasting glucose and fasting insulin after 6 months of 500 mg metformin BID compared with 
placebo (29).  Again there was no change in insulin sensitivity as measured by the minimal model.  Data on 
insulin secretion and DI were not reported.  Finally, Kay et al assessed effects of 850 mg metformin BID on 
obesity and insulin sensitivity in 24 obese adolescents in a randomized, placebo controlled trial. Those in the 
treatment group had greater weight and body fat loss than the controls and had a decrease in area under the 
curve (AUC) for insulin (as measured by OGTT), but no change in glucose AUC.  They also had a significant 
decrease in triglycerides.  Insulin secretion was not measured.  In all of these studies the treatment period 
was relatively short and none of them targeted the most resistant time of puberty.  Furthermore, the 
Freemark study used a metformin dose that is approximately one half the standard dose used to treat 
diabetes or PCOS. 
 
Ibanez et al, in Spain, have used metformin in multiple clinical trials of female children and adolescents 
with premature pubarche (PP) associated with a history of low birth weight.  Being small for gestational age 
is thought to predispose certain infants to developing decreased insulin sensitivity later in life. The girls 
participating in these studies had evidence for reduced insulin sensitivity, as assessed by elevated insulin 
levels, but were not obese (32-37).  Treatment length and doses were variable, as well as timing of treatment 
in relation to puberty.  Furthermore, none of these trials was randomized or double-blinded, and measures of 
insulin sensitivity were not consistent.  Regardless, treatment with metformin in all these trials resulted in 
improvements in insulin levels and triglycerides and in several, a reduction in BMI.  Most interestingly, one 
trial showed a persistence of beneficial effects 12-18 months after the metformin was stopped (32).  In this 
trial, 22 girls with a history of PP and low birth weight were randomized to receive metformin treatment (850 
mg daily) or no treatment within one year of starting breast development and treated for 36 months (32;35).  
At the end of the treatment period, change in BMI, IR as measured by HOMA, triglycerides, abdominal fat 
mass, leptin and total fat mass were all improved in the metformin group compared with the untreated group.  
Furthermore, these differences between the groups persisted for 12 months after the treatment was stopped.  
Despite some design flaws, this study suggests that short-term treatment with metformin during puberty can 
result in sustained metabolic benefit in adolescents at increased risk for developing T2DM.   
 
Inflammatory markers and cytokines: relation to IR 
 
 
 
There is a well-established association in adults between obesity and a low-grade inflammatory state, as 
evidenced by elevated C-reactive protein (CRP) and tumor necrosis factor- α (TNF-α).  Furthermore, obesity 
is associated with dysfunctional adipocytes that secrete higher levels of pro-inflammatory cytokines, such as 
interleukin-6 (IL-6) and decreased levels of protective cytokines, such as adiponectin (38;39).  Adiponectin 
levels have been demonstrated to be inversely related to IR, obesity, CRP and TNF-α (40-42).  Adiponectin 
levels strongly inversely correlate with degree of insulin sensitivity in adults, even after adjustment for other 
risk factors for T2DM such as BMI and fat mass.  In fact, adiponectin may even have a stronger relationship 
with insulin sensitivity than adiposity (42).  


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An increasing number of studies have shown that these relationships also exist in children of all ages 
(43-47).  One recent study paired obese subjects with IR (as demonstrated by hyperinsulinemic euglycemic 
clamp technique), but normal glucose tolerance, with obese, insulin-sensitive adolescents of similar gender, 
pubertal status, weight and percent body fat (48).  The IR subjects had lower adiponectin levels than their 
insulin-sensitive counterparts, indicating that adipocyte dysregulation may precede development of IR and 
may be a marker of increased risk for IR and eventual development of T2DM.  Other studies have confirmed 
a positive correlation between adiponectin and insulin sensitivity (49;50). Furthermore, BMI was found to be 
the best predictor of CRP levels according to the National Health and Nutrition Examination Survey (51-53) 
and fasting insulin levels have been shown to correlate with CRP levels (54;55).  In summary, it is well-
documented that obesity-related IR is associated with an inflammatory response in both adults and children.  
However, it is not know if physiologic pubertal IR is also associated with inflammation. 
 
In summary, the temporary fall in insulin sensitivity that occurs during puberty is well-recognized.  This is 
typically overcome by an increase in insulin secretion.  However, certain risk factors for T2DM seem to affect 
the ability of the pancreas to compensate for this change in insulin sensitivity. The mechanism of pubertal IR 
remains poorly understood, with suggestions that GH/IGF-1 and sex steroids might play a causative role.  
Furthermore, little is known about the affect of obesity on decreased pubertal insulin sensitivity.  If obesity is 
superimposed on the already IR state of puberty, the resulting amplified IR may stress the beta cells and 
lead to the development of T2DM. Thus, puberty may be an ideal time to treat IR, with the hope of preventing 
or at least delaying onset beta cell failure.  In the current proposal we will examine the longitudinal changes 
in insulin sensitivity, body composition and inflammatory markers of normal weight and obese children during 
puberty.  Furthermore, we will compare these changes in obese children with and without metformin 
treatment during this time.  These studies will increase our understanding of the contribution of puberty to the 
progression of obesity to IR and to DM in adolescents. This knowledge will, in turn, allow to better target our 
preventive efforts.   
 
 
Liver fat, Body Composition and IR 
 
 
In a normally insulin sensitive subject, excess calories are stored in adipose tissue as a result of insulin 
release, or burned for fuel in skeletal muscle. Fat is stored preferentially in the adipose depot, not in the 
viscera, liver or skeletal muscle tissue. However, in humans with insulin resistance, an abnormal pattern of 
fat distribution is seen, with ectopic deposition of lipid in the liver, viscera and skeletal muscle, specifically as 
intramyocellular lipid (IMCL) (38), and appears to play an important role in the pathogenesis of reduced 
insulin sensitivity. IMCL by muscle biopsy (39) and by nuclear magnetic resonance (NMR) (40) are strongly 
correlated with insulin resistance, including in adolescents (41). Resistance is strongly associated with 
visceral adiposity as demonstrated by various methods including MRI and waist-to-hip ratio (42-44). 
Furthermore, data in adults have shown that accumulation of visceral fat poses a greater health risk than 
accumulation of subcutaneous fat (45). In T2DM, visceral fat content is associated with a decrease in 
peripheral insulin sensitivity and an increase in gluconeogensis (46;47), and is highly correlated with fatty 
liver disease in this population (48). Finally, fatty liver disease is thought to be the most common cause of 
liver pathology in obese children (49) and resistance is thought to play a role in the pathogenesis of fatty liver 
disease in both children and adults (50;51). Changes in total body fat and fat distribution occur during 
puberty, and these changes may correlate with changes in insulin sensitivity, however this has not been 
clearly defined. 
 
C. PRELIMINARY DATA 
1. Studies of metformin treatment in obese adolescents 
Our previous research using metformin in conjunction with personal goal setting in 85 IR adolescents 
was the largest trial to date using metformin in obese adolescents without T2DM(52). This study was 
conducted by our group, including my co-mentor Dr. Phil Zeitler and my consultant, Dr. Kristen Nadeau. 
Kathy Love-Osborne, M.D., one of the consultants on this application, was the primary investigator.  The 
mean age of subjects was 15.7 years. 76% of participants completed the study. Mean body mass index 
(BMI) was 39.7 kg/m2. 71% were female, 58% were Hispanic and 34% Black. Overall, there was no 


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significant difference in weight loss among subjects receiving metformin or placebo.  However, 11 (23%) 
subjects on metformin had a BMI decrease of 5% or more, while no subjects on placebo were able to attain 
this degree of weight loss. Of 11 subjects decreasing BMI by 5%, 82% were female. 45% were Hispanic, 
27% Black, 18% Caucasian and 9% Asian. 
All subjects had elevated fasting insulin levels at baseline, with an average level of 39.8 μIU/ml.  At 
the end of 6 months, the metformin group had a significant decrease in fasting insulin compared with the 
placebo group (-9.8 +3.4 μIU/ml vs. -3.1 +2.6 μIU/ml, p<0.05). Furthermore, the metformin group had 
significantly decreased fasting triglycerides at the 6 month follow-up compared with the placebo group (-26.2 
vs. -0.2, p<0.05) (unpublished data) and triglycerides were shown to be a significant predictor of IGT in this 
cohort (53).  Finally, the metformin group had a significantly improved fatty liver score, as measured by 
ultrasound (mean change -0.4 +0.1 vs. +0.25 +0.28, p<0.05) (54). 
Subjects on metformin were slightly less likely to have good pill compliance (68% vs. 78% on 
placebo).  29% of subjects on metformin reported side effects: nausea or vomiting (14%), diarrhea (14%), or 
abdominal pain (11%) vs. 22% of subjects on placebo: nausea (11%) or diarrhea (11%) but no subjects 
required dose reductions. There were no significant differences in attrition between the groups. 
Thus, metformin may be a candidate for primary prevention of T2DM in high-risk adolescents.  
Extended release metformin is available as a generic medication; it is relatively inexpensive and accessible.  
Furthermore, this trial shows that long-term treatment with metformin is safe and feasible in our adolescent 
population. 
 
2. Studies of Intramyocellular lipid deposition in relation to Insulin Sensitivity 
In a study designed to evaluate the association of exercise dysfunction with insulin sensitivity, my 
consultant, Dr. Nadeau, examined 37 adolescents (13 T2DM, 11 obese, and 11 lean) of similar age, sex, 
Tanner stage and activity level.  Muscle lipid content was assessed by muscle biopsy as well as NMR, a 
noninvasive imaging technique that allows separation of intramyocellular lipid, which has been negatively 
correlated with insulin sensitivity, and extramyocelluar lipid, and was well tolerated by adolescents. In 
addition, insulin sensitivity was measured by hyperinsulinemic euglycemic clamp. In this study, obese 
adolescents had significantly decreased insulin sensitivity when compared with normal weight controls, had 
significantly increased soleus extramyocellular lipid (EMCL) as measured by NMR (p=0.017) and had a trend 
toward increased soleus IMCL (2014 +947 in obese vs. 1166 +458 in lean controls).  Maximal exercise 
capacity (VO2max/kg) significantly positively correlated with insulin sensitivity (mg/kg/min) (r=0.82, 
p<0.0001) and negatively with soleus IMCL (r=-0.645, p<0.0001) (55).  These data demonstrate the 
experience of this institution at using NMR to detect soleus IMCL and EMCL. The NMR imaging in Dr. 
Nadeau’s protocol was performed by Dr. Mark Brown, who is consulted in this proposal, and the muscle 
biopsies and insulin clamps were performed by Dr. Nadeau at our pediatric Clinical Translational Research 
Center.  
These preliminary data show the feasibility and tolerability of these procedures in adolescents.  These 
studies also demonstrate our group’s ability to recruit and maintain study participation in a group of obese 
adolescents receiving treatment with metformin. 
Although I was not actively involved in these projects, I am a co-investigator on Dr. Love-Osborne’s 
current study, which is an expansion of the previous metformin project and I am involved in the data analysis 
for Dr. Nadeau’s study.   
 
D. RESEARCH DESIGN AND METHODS: 
 
HYPOTHESIS 1:  
1.  Obese adolescents, unlike normal weight controls, fail to improve insulin sensitivity at the end of 
puberty. 
 
SPECIFIC AIM 1 (Observational Arm): 
2. To compare longitudinal changes in insulin sensitivity and secretion and their correlates in obese 
and normal weight adolescents during puberty.   


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a. To compare changes in insulin sensitivity and secretion as measured by frequently 
sampled intravenous glucose tolerance test (IVGTT) from early puberty to puberty 
completion between obese and normal weight adolescents. 
b. To compare changes in body composition, fat distribution, and inflammatory markers over 
time between these groups. 
 
RATIONALE 
 
In normal weight adolescents there is a physiologic decrease in insulin sensitivity, beginning at Tanner 
stage I (T1), as well as changes in body composition. Insulin sensitivity progressively decreases to a nadir at 
T3 and then returns to pre-pubertal levels after puberty is completed (T5) (9;11;13;14).  Obese adolescents 
are more insulin resistant going into puberty, and also show a pubertal decrease in insulin sensitivity that is 
independent of BMI and body composition (16).  It is still unclear whether obese, insulin resistant 
adolescents recover insulin sensitivity when puberty is completed. 
 
DESIGN 
 
Participants: 
1. 104 Obese participants:  
    a.) BMI ≥ 95th percentile 
    b.) Tanner stage II – III at baseline 
    c.) Age ≥ 9 years 
    d.) Absence of IGT, impaired fasting glucose (IFG) or T2DM, as determined by oral glucose tolerance test 
(OGTT) 
2. 52 Normal weight participants: 
     a.) BMI ≥ 5th percentile and ≤ 85th percentile 
     b.) Tanner stage II – III at baseline 
     c.) Age ≥ 9 years 
     d.) Absence of IGT, IFG or T2DM, as determined by OGTT 
 
Other inclusion criteria: Every effort will be made to recruit study participants that are representative of 
the racial and ethnic make-up in Colorado. This means that a significant number of Spanish-speaking 
only participants that may be recruited. The methods of appropriately recruiting and consenting these 
subjects in a manner that will ensure that we are appropriately communicating the study protocol and 
risks and benefits of participating in this study.  
 
Exclusion criteria for both groups: 
a). Specific genetic syndrome or disorder known to affect glucose tolerance other than diabetes 
b). Inhaled steroids at dose above 1000 mcg daily, inhaled fluticasone or equivalent. 
c). Oral steroids within last 60 days or oral steroids more than 20 days during the past year 
d). Medication(s) which are known to cause weight gain or weight-loss within the last 30 days, including 
atypical antipsychotics. 
e). Refractory hypertension: average systolic blood pressure > 150 mmHg or average diastolic blood 
pressure > 95 mmHg despite appropriate medical therapy. 
f). Refractory hyperlipidemia: total cholesterol > 300 mg/dL or LDL > 190 mg/dL or triglycerides > 800 mg/dL, 
despite appropriate medical therapy. 
g). Evidence of proteinuria 
h). Patients on any form of insulin sensitizer within the last year. 
i). Admitted use of anabolic steroids within the past 60 days. 
j). Anemia: Hematocrit < 30.0%, a hemoglobin < 11 gm%/dL 
k). Weight >300 lbs., due to limits of the NMR and DEXA tables 
l). Other significant organ system illness or condition (including psychiatric or developmental disorder) that 
would prevent participation in the opinion of the investigator.   
 


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Approach: 
We will recruit obese and normal weight subjects who are in early puberty as determined by Tanner 
staging of breast development and pubic hair in girls and by Tanner staging of pubic hair and estimation of 
testicular size in the boys.  Adolescents whose pubertal status is at least Tanner II (T2), but no more than 
Tanner III (T3) will be asked to participate.  In two studies that assessed insulin sensitivity across all stages 
of puberty, it was already significantly decreased from prepubertal insulin sensitivity at T2 and, while it had 
not yet completely recovered in early T5, it was still significantly increased from T4 insulin sensitivity (11;16).  
We will recruit through our metabolic syndrome and Good Life clinics, through advertisements, and from local 
pediatric clinics, including Denver Health Medical Center.  Because of the ethnic make-up of our clinics, there 
will be Spanish-speaking only participants included in the study. The study coordinator is a native Spanish-
speaker and, therefore, has the ability to consent the participants in Spanish using our consent and HIPAA 
forms that have been translated into Spanish. Lifestyle intervention materials are also available in Spanish. 
When the study coordinator is not available, an official Spanish interpreter will be used for the consent 
process and all study visits. Because of the anticipated high screen-failure rates due to failure to meet 
physical examination criteria (most notable Tanner staging criteria and BMI criteria), we will need to consent 
at least 40% more participants than we ultimately intend to enroll. Therefore, we anticipate that 225 
participants will need to be consented for screening to be able to enroll 156 participants. The timeline of our 
experimental approach is shown below (Fig 2).  A fasting baseline screening visit, to be performed at the 
pediatric Clinical Translational Research Center (CTRC), will include assessment of obesity status as 
determined by height, weight, BMI, waist circumference and hip circumference.  An OGTT will be performed 
in all obese subjects to rule out IGT, IFG and diabetes in all subjects.  The following screening labs will also 
be performed to examine for any significant underlying illness: AST, ALT, electrolytes, CBC, BUN, creatinine, 
lipids, and urinalysis.   
 
After determination of eligibility, participants will have a one-day CTRC admission for baseline 
laboratory studies, IVGTT, and body composition assessment (Study Visit 1) following an overnight fast.  
Upon arrival they will have an IV placed for blood draws and injections.  Fasting blood glucose and insulin 
will be drawn at this time.  Sex steroids and inflammatory markers will also be drawn at this time. (After the 
blood draw, they will undergo a frequently sampled IVGTT (specific methods for above described after 
Specific Aim 2).  Up to 10 obese subjects will participate in the arm, as pilot subjects, who are not part of the 
treatment arm (i.e. they will not be taking a placebo). 
In the afternoon of the Study Visit 1, a dual-energy x-ray absorptiometry (DEXA) scan will be 
performed to determine body composition.  In a subset of participants (13 per group), soleus muscle nuclear 
magnetic resonance (NMR) and abdominal fast magnetic resonance imaging (MRI) sequence will also be 
performed to measure body fat distribution in terms of IMCL, liver fat content and visceral adipose content.   
The next study visit (Study Visit 2) will occur when the participant has reached Tanner stage IV, 
approximately one year after the baseline visit.  At this time the baseline laboratory draw, imaging studies 
and IVGTT will be repeated.  For those subjects randomized to the MRI, it will not be repeated at this visit.  
The next study visit (Study Visit 3) will occur when the participant has reached Tanner stage V, 
approximately one year after Study Visit 2.  At this time the baseline laboratory draw, imaging studies and 
IVGTT will be repeated. This is the last long visit for the lean subjects. The obese subjects will stop taking the 
study medication at this time point.   
The next study visit (Study Visit 4) will occur 6 months after the obese participants have reached 
Tanner stage V. At this time the baseline laboratory draw, imaging studies and IVGTT will be repeated. This 
is the last long visit for the obese subjects. For those subjects randomized to the MRI, it will not be repeated 
at this visit. 
 
Between the visits, participants will be asked to come in every 6 months for anthropometric 
measurements and Tanner staging. Obese participants in the treatment arm (randomized to either Metformin 
or placebo) will also be asked to come in quarterly for pill counts to measure medication compliance and to 
receive a refill on their medication.  
 


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Lean subjects will be asked to undergo a total of 3 DEXA scans throughout the course of the study, 
and for those randomized to the MRI, they will be asked to undergo a total of 2 MRI’s. Obese subjects will be 
asked to undergo a total of 4 DEXA scans through the study, and for those randomized to the MRI, they will 
be asked to undergo a total of 2 MRI’s.  
 
Please see the tables below for a more detailed explanation of the study procedures.  
 
Specific methodology that will be used in this study is described after Specific Aim 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
Treatment table for obese subjects: 
 
 
 
*Only those in the group who are asked to have an MRI/MRS 
 
 
No Treatment table for lean subjects/obese pilot no treatment subjects:  
 
 
 
 
 
 
 
 
 
 
 
*Only participants who are overweight 
Visit Number 
Puberty 
Exam 
Height & 
Weight 
Pill 
Count 
OGTT 
IVGTT DEXA MRI* Total Time 
Screening Visit 
x 
x 
 
x 
 
 
 
4 hr 
Study Visit 1 
 
x 
 
 
x 
x 
x 
5-8 hr 
Short Visits 
(Every 
 
3 
months) 
x 
x 
x 
 
 
 
 
1 hr 
Study Visit 2 
x 
x 
 
 
x 
x 
 
5-8 hr 
Study Visit 3 
 
x 
 
 
x 
x 
x 
5-8 hr 
Study Visit 4 
 
x 
 
 
x 
x 
 
5-8 hr 
Visit Number 
Puberty 
Exam 
Height & 
Weight 
OGTT* IVGTT 
DEXA 
MRI/ 
MRS** Total Time 
Screening Visit 
x 
x 
x 
 
 
 
2-4 hr 
Study Visit 1 
 
x 
 
x 
x 
x** 
5-8 hr 
Short Visits 
x 
x 
 
 
 
 
1 hr 
Study Visit 2 
x 
x 
 
x 
x 
 
5-8 hr 
Study Visit 3 
x 
x 
 
x 
x 
x** 
5-8 hr 


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**Only those in the group who are asked to have an MRI/MRS 
 
 
Fig. 2 Timeline of Study Participation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HYP
OTHE
SIS 2: 
2. 
Treat
ment 
of 
obes
e 
adole
scent
s 
with 
metformin during puberty will help these individuals recover insulin sensitivity at the end of puberty, 
decrease compensatory hyperinsulinemia, and preserve insulin secretion, thereby improving their 
disposition index (DI). 
 
 
SPECIFIC AIM 2: 
2. To compare changes in insulin sensitivity and secretion and their correlates in obese adolescents 
treated with metformin or placebo during puberty. 
a. To compare changes in insulin sensitivity, secretion and DI from early puberty to puberty 
completion between obese controls and obese adolescents treated with metformin. 
b. To compare changes in body composition, fat distribution, and inflammatory markers over 
time between these groups. 
 
RATIONALE: 
 
The goal of this aim is to see whether treatment with metformin can improve insulin sensitivity and reduce 
compensatory insulin secretion during puberty.  The thought is that, by improving insulin sensitivity and 
decreasing insulin secretion requirements during this temporary critical period of reduced insulin sensitivity, 
*Obese only 
Screening visit: 
All Groups 
• OGTT* 
• Screening labs 
(AST, ALT, BMP, 
CBC, UA) 
Study Visit 1: T2/3 
All Groups 
• IVGTT 
• Lab studies 
• Anthropometric 
measures 
• Body composition 
Study Visit 3: T5, 
Treatment Group 
Only 
• IVGTT 
• Anthropometric 
measures 
Study Visit 2: T5 
All Groups 
• IVGTT 
• Lab studies 
• Anthropometric 
measures 
• Body composition 
Randomization of 
obese participants: 
Metformin vs. Placebo 
2 years 
6 months 
Treatment 
completion 
Post-treatment 


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β-cell function will be preserved.  This would make puberty a potential treatment period to target prevention 
of T2DM.   
 
 
DESIGN 
 
Participants: 
1. 104 Obese participants:  
    a.) BMI ≥ 95th percentile 
    b.) Tanner stage II – III at baseline 
    c.) Age ≥ 9 years 
    d.) Absence of impaired glucose tolerance (IGT), impaired fasting glucose (IFG) or T2DM, as determined 
by oral glucose tolerance test (OGTT) 
 
Exclusion criteria: 
See Specific Aim 1 
Approach: 
   All obese subjects who are recruited will be randomized after Study Visit 1 to receive metformin or 
placebo. The 52 participants randomized to the placebo group will then also serve as the obese subjects for 
the longitudinal observational studies of Specific Aim 1 (Please see Fig 2 for further clarification). In other 
words, 52 obese participants will be included in both Specific Aims 1 and 2, in order to minimize the total 
number of participants needed. Randomization will be stratified by: 1) Family history in a first degree relative 
with T2DM or no family history and 2) BMI between the 95th and 99th percentile or BMI greater than the 99th 
percentile. The extended release metformin will be started at 500 mg daily for one week and then increased 
by 500 mg a week until the target dose of 2000 mg a day is attained. The extended release preparation 
allows patients to take the medication once a day to promote improved adherence and is better tolerated. All 
obese participants will receive standard lifestyle counseling, focusing primarily on dietary improvements and 
increased physical activity. The total treatment period will be two years to include, on average, the most 
resistant stages of puberty. In order to monitor metformin compliance closely, the participants will come in for 
monthly pill-counts. In addition, each participant will be provided with a pill dispenser and a calendar to 
record each dose that is taken to assist with and track compliance. Furthermore, parents will be encouraged 
to remind the participant to take his/her medication. Participants will be given a point each time they bring in 
their pill bottle, whether or not they have been compliant, and will be eligible for an extra gift card for every 6 
points they obtain. Our group has extensive experience in maintaining metformin compliance in this 
population (52). Tanner staging, anthropometric measurements and lifestyle counseling will also be 
performed at quarterly visits. 
   The CTRC study visit described previously for Study Visit 1 will be repeated twice more: at the end of this 
treatment period (Study Visit 2) and six months after the treatment is stopped (Study Visit 3).  IVGTT, DEXA, 
abdominal fast MRI and soleus muscle NMR will also be repeated.  We anticipate that the majority of 
patients will be Tanner stage V (T5) at the end of the 2-year treatment period, as the average time from the 
onset of breast development until menarche is 2-3 years.  If T5 has not been reached after two years, the 
treatment will continue until the individual is at T5.  Follow-up visits for Tanner staging will occur quarterly.   
 
The final visit (Study Visit 3) will be to determine whether insulin sensitizer treatment will result in 
persistent improvement in insulin senstivity and insulin secretion, even after treatment is stopped.  The 
subjects will again be given a 3-day standard macronutrient diet and will fast overnight prior to admission.  
Only the IVGTT and anthropometric measurements will be performed at this study visit. 
 
Specific Methods: 
 
Screening laboratory studies: ALT, AST, electrolytes, fasting lipids, CBC, BUN and urinalysis. 
 
Laboratory studies drawn at Visits 1, 2 and (for obese subjects only) 3 : sex steroids, c-reactive protein 
(CRP), interleukin-6 (IL-6), adiponectin and fasting lipids. Sex steroids (free testosterone, estradiol, 


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androstenedione and DHEAS) will be drawn in order to confirm or refute previously found associations 
between sex steroids and IR.  Inflammatory markers and other factors that have been associated with IR will 
also be drawn at this visit, including c-reactive protein, interleukin-6, adiponectin and fasting lipids (LDL, HDL 
and triglycerides).   
 
Anthropometric measurements: Height, weight, waist-to-hip ratio, and supine abdominal height are measured 
by standard techniques. 
 
Oral Glucose Tolerance Test: Glucose and insulin drawn at fasting (0), and at 30, 60, 120 and 180 minutes 
after ingestion of glucola (1.75g/kg, max 75g) 
 
Dual-energy X-ray absorptiometry (DEXA Scan): Body composition will be measured supine using the DEXA 
technique on the pediatric CRC and will be used to derive fat-free mass (92).  This technique relies on the 
absorption of dual electron wavelengths for the assessment of body fat, lean tissue, and bone mineral 
density.  
 
Fast Magnetic Resonance Imaging (MRI) sequence: Rapid MRI will be used to assess hepatic fat fraction by 
the modified Dixon technique, previously described (56).  In this technique, in- and out-of phase images of 
the liver will be obtained using a 1.5 Tesla magnet.  The subject will be placed in a supine position on the 
body coil.  The scan parameters include relaxation time = 9.3 ms (in-phase) and 7.3 ms (out-of-phase), echo 
time = 4.2 ms (in-phase) and 2.1 ms (out-of-phase), flip angle = 30 degrees, slice thickness = 10 mm, field of 
view = 32 x 16 cm, matrix size = 256 x 128, and number of excitations = 8.  Four image slice pairs (in- and 
out-of-phase) will be obtained through the liver.  Fat fraction will be calculated from the mean pixel signal 
intensity (SI) using the formula: Fat fraction = (SI inphase – SIoutofphase).  A single slice at the level of the 
umbilicus will be used for determination of abdominal adipose distribution.  Fast spin coil with a flip angle of 
70 degrees, repetition time = 120 ms, echo time = 1.6 mg, field of view = 36 x 27 cm, slice thickness = 5.0 
mm, matrix = 256 x 128, and number of excitations = 1. 
 
  
Nuclear Magnetic Resonance Imaging (NMR): Dr. Mark Brown, of UCHSC radiology, will perform soleus 
NMR on a 3.0 T whole–body MRI scanner (GE Medical Systems, Waukesha, WI). The subject is positioned 
in the MR magnet feet first, prone, with the soleus muscle centered in the extremity coil.  Scout images 
(usually T1-weighted) position the volume-of interest (usually 2cm x 2cm x 2cm), to avoid regions of gross 
adipose and vascular structures. Spectroscopy acquisition is performed using the PRESS pulse sequence 
without water suppression (TR/TE = 2000/24 ms, 64 averages, total acquisition time 2 minutes), and 
analyzed by SAGE spectroscopy analysis (GE). The water peak is fit as a Lorentzian line using Marquardt 
routine, and subtracted from the spectrum.  The remaining peaks (creatinine and choline) are then fit using 
the Marquardt routine. IMC and EMC triglyceride peaks (1.3 and 1.5 ppm, respectively) are obtained from the 
fit results, corrected for T1 and T2 relaxation, and expressed as a percentage of the water content (96). 
Subjects need to hold reasonably still during the scan and cannot weigh >300 lbs.  
 
All of the preceding imaging studies avoid radiation exposure and are regularly available at our institution. 
 
Standard macronutrient diet: 3 days of a diet composed of 55% carbohydrates, 30% fat, and 15% protein will 
be provided by the CTRC. This diet is the standard used by the UCHSC Center for Human Nutrition prior to 
insulin clamps or VO2max testing as variations in dietary intake affect insulin sensitivity.  
  
Frequently sampled IVGTT:  Prior to admission, they will be given a 3-day standard macronutrient diet by the 
CTRC and will also be restricted from intense exercise for seven days.  They will be asked to fast overnight 
before admission.  An IV catheter will be inserted into each arm.  At times -15 and zero, glucose and insulin 
will be drawn.  Participants will then be infused with 0.3g/kg of 25% dextrose over 60 seconds.  Glucose and 
insulin will be sampled from the contralateral arm at times 2, 3, 4, 5, 6, 8,10,12,14,16,19, 22, 25, 30, 35, 40, 


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50, 60, 70, 80, 90,100,120, 140,160, and 180 minutes.  Bergman’s minimal model program will be used to 
calculate insulin sensitivity (Si), insulin secretion (AIRglucose) and DI (DI). 
 
Sample size calculation:  A recruitment goal of 52 participants per group has been set (normal weight 
controls, obese controls, obese on metformin—a total of 156 participants).  Assuming a 30% drop-out rate, 
this will give a final sample size of 30 per group.  Power was run in PASS (NCSS Software; Kayesville, UT) 
and was calculated using a one-way ANOVA with a two-sided significance level of 0.05.  Variability of the 
estimates was based on data available in an article by Buchanan et al. (57) in which IVGTT insulin sensitivity 
was measured in high-risk Hispanic women at baseline and 3 months for a placebo (n=109) and a 
troglitazone group (n=108).  The 3-month change was -0.17 and 1.16 min -1 per μU/ml x 10-4, for the placebo 
and treatment groups, respectively.  Assuming a correlation of 0.5, the estimated overall standard deviation 
of the 3-month change for both the placebo and treatment groups was 1.85 min-1 per μU/ml x 10-4.   For this 
study, we assume similar variability and expect the 2-year change for the obese control and metformin 
groups to also be similar to those seen in the Buchanan et al. groups.  The 2-year insulin sensitivity change 
in normal controls is expected to be similar to the change seen in Ball et al. (11) for 46 white adolescents 
from Tanner II / Tanner III to Tanner V, which was 1.6 min -1 per μU/ml x 10-4.   Therefore, with a standard 
deviation of 1.85 and expected 2-year changes of 1.16, -0.17 and 1.6 for the metformin, obese control and 
normal control groups, respectively, a sample size of 30 per group achieves 93% power to detect an overall 
difference between the groups.  
 
Statistical analysis:  All analyses will assume a two-sided test of hypothesis with an overall significance level 
of 0.05 and will be performed in either SAS v9.1.3 or above (SAS Institute, Inc., Cary NC) or Splus v7.0 or 
above (Insightful Corp, Seattle WA).  
Simple descriptive statistics will be calculated for all variables (mean, median, standard deviation, 
minimum and maximum values for continuous variables; number and percentage of subjects for categorical 
variables).  Linear regression will be used to compare 2-year change in insulin senstivity, insulin secretion, 
and DI between groups for Specific Aims 1 and 2 (normal controls vs. obese controls and obese treated vs. 
obese controls).  Potential confounders such as sex, 2-year change in percent body fat and 2-year change in 
BMI will be included in each model.  In order to determine if the benefit of treatment persists beyond stopping 
treatment, the above analysis will be repeated on the change from baseline to 6 months post-treatment for 
the obese treated and obese control groups only.  The mean change in each outcome from treatment 
stoppage to 6 months post treatment will also be calculated with 95% confidence intervals (CI) in order to 
assess maintenance of the level of each outcome.  Although we are not powered for equivalence (to test the 
hypothesis that the 6-month change is equal to zero), the precision of the estimate and whether the 
difference is clinically significant will be evident from the 95% CI.  A CTRC biostatistician will be available to 
consult on all matters of statistics. 
 
Potential Difficulties and Limitations/Alternatives 
In adolescence, the number of routine visits to the pediatrician tends to drop.  Therefore, we foresee 
a potential difficulty in recruiting children who are just starting puberty.  For this reason, we are including 
children who are both Tanner 2 and Tanner 3 at study entry.  In both of these stages of puberty, insulin 
sensitivity has been shown to be significantly decreased when compared with Tanner 1 and Tanner 5.  The 
length of time required for progression through puberty varies in individuals.  It is important to measure the 
post-treatment insulin sensitivity in Tanner stage 5, because insulin sensitivity in Tanner 4 may not be 
significantly different from earlier in puberty and only at T5 insulin sensitivity returns to its pre-pubertal level.  
For this reason, if a subject has not reached Tanner 5 in two years, they will not have their Study Visit 3 until 
Tanner 5 has been attained.  This means that some subjects may need to be treated for a longer period of 
time.  However, in order to compare differences in insulin sensitivity after treatment is stopped between 
subjects, the interval from treatment stop (Study Visit 2) to post-treatment follow-up (Study Visit 3), should be 
consistent. 
The natural recovery of pubertal insulin sensitivity may confound measurement of metformin effects 
on insulin sensitivity as puberty progresses.  In order to control for this, we have included a placebo-
controlled group and a group of normal weight controls.  This will allow us to compare changes in insulin 


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sensitivity from baseline to the end of puberty between obese treated subjects and both normal-weight and 
obese subjects who have not been treated. 
The length of this study and potential difficulty with attrition are also limitations of this study.  To limit 
the number of participants lost to follow-up, we will maintain frequent contact with participants with quarterly 
visits for anthropometric measurements, monthly visits for pill-counts and phone contact at least once 
between these visits. In addition to these visits, the participants in the treatment group will be contacted by 
telephone at least monthly to inquire about medication side effects and to review changes in diet and 
exercise.  Due to our involvement in the TODAY study and other trials of metformin, our staff are 
experienced with working on long-term projects with obese and T2DM adolescents, with excellent study 
retention out as far as four years. 
Adverse effects of metformin include nausea, anorexia, diarrhea and vomiting.  These adverse effects 
could potentially interfere with patient compliance.  These effects have been shown to be reduced when the 
dose is titrated up slowly, as described in the Approach section of Specific Aim 2.  Furthermore, in our 
experience with metformin, adverse effects did not result in decreased dose or significant patient attrition.  
Participants in the metformin group were only slightly less compliant than the placebo group in our blinded 
study of metformin in obese adolescents. 
Finally, IVGTT is not the gold standard for measurement of insulin sensitivity. However, it has been 
shown to correlate well with the hyperinsulinemic-euglycemic clamp, which is the gold standard (21). It was 
chosen for this study as it is slightly less invasive and allows measurement of both insulin sensitivity and 
insulin secretion. In this population, which is as young as 9 years old and will be undergoing repeated 
measures, it is necessary to avoid time intensity and invasive procedures as much as possible. Therefore, it 
was felt that the benefits of IVGTT far outweigh the limitations in this protocol. 
 
Summary  
The temporary fall in insulin sensitivity that occurs during puberty is well-recognized. This is typically 
overcome by an increase in insulin secretion. However, certain risk factors for T2DM appear to affect the 
ability of the pancreas to compensate for this change in insulin sensitivity. The mechanism of pubertal insulin 
resistance remains poorly understood, with suggestions that GH/IGF-1 and sex steroids might play a 
causative role. Furthermore, little is known about the affect of obesity on decreased pubertal insulin 
sensitivity. If obesity is superimposed on the decreased insulin sensitivity of puberty, the resulting amplified 
insulin requirements may stress the β cells and lead to the development of T2DM. Thus, puberty may be a 
critical time to treat resistance, with the hope of preventing or at least delaying onset of β-cell failure. In the 
current proposal we will examine the longitudinal changes in insulin sensitivity, insulin secretion and body 
composition of normal weight and obese children during puberty. Furthermore, we will compare these 
changes in obese children with and without metformin treatment. These studies will increase our 
understanding of the contribution of puberty to the progression from obesity to IR and DM in adolescents. 
This knowledge will, in turn, allow to better target our preventive efforts.  
 
 


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Reference List 
 
 1.  Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of overweight and 
obesity in the United States, 1999-2004. JAMA 2006; 295(13):1549-1555. 
 2.  Type 2 diabetes in children and adolescents. American Diabetes Association. Diabetes Care 2000; 
23(3):381-389. 
 3.  Gungor N, Hannon T, Libman I, Bacha F, Arslanian S. Type 2 diabetes mellitus in youth: the complete 
picture to date. Pediatr Clin North Am 2005; 52(6):1579-1609. 
 4.  Pinhas-Hamiel O, Zeitler P. Clinical presentation and treatment of type 2 diabetes in children. Pediatr 
Diabetes 2007; 8 Suppl 9:16-27. 
 5.  Olefsky JM, Nolan JJ. Insulin resistance and non-insulin-dependent diabetes mellitus: cellular and 
molecular mechanisms. [Review] [52 refs]. Am J Clinical Nutrition 1995; 61(4:Suppl):Suppl-986S. 
 6.  Shulman GI. Cellular mechanisms of insulin resistance in humans. [Review] [28 refs]. American Journal 
of Cardiology 1999; 84(1A):3J-10J. 
 7.  Dabelea D, Pettitt DJ, Jones KL, Arslanian SA. Type 2 diabetes mellitus in minority children and 
adolescents. An emerging problem. Endocrinol Metab Clin North Am 1999; 28(4):709-29, viii. 
 8.  Kahn CR. Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 
1994; 43(8):1066-1084. 
 9.  Amiel SA, Sherwin RS, Simonson DC, Lauritano AA, Tamborlane WV. Impaired insulin action in 
puberty. A contributing factor to poor glycemic control in adolescents with diabetes. New England 
Journal of Medicine 315(4):215-9, 1986. 
 10.  Arslanian SA, Kalhan SC. Correlations between fatty acid and glucose metabolism. Potential 
explanation of insulin resistance of puberty. Diabetes 43(7):908-14, 1994. 
 11.  Ball GD, Huang TT, Gower BA et al. Longitudinal changes in insulin sensitivity, insulin secretion, and β-
cell function during puberty.[see comment]. Journal of Pediatrics 148(1):16-22, 2006. 
 12.  Caprio S, Plewe G, Diamond MP et al. Increased insulin secretion in puberty: a compensatory 
response to reductions in insulin sensitivity. Journal of Pediatrics 114(6):963-7, 1989. 
 13.  Goran MI, Gower BA. Longitudinal study on pubertal insulin resistance. Diabetes 50(11):2444-50, 2001. 
 14.  Hannon TS, Janosky J, Arslanian SA. Longitudinal study of physiologic insulin resistance and metabolic 
changes of puberty. Pediatric Research 60(6):759-63, 2006. 
 15.  Hoffman RP, Vicini P, Sivitz WI, Cobelli C. Pubertal adolescent male-female differences in insulin 
sensitivity and glucose effectiveness determined by the one compartment minimal model. Pediatric 
Research 48(3):384-8, 2000. 
 16.  Moran A, Jacobs DR, Jr., Steinberger J et al. Insulin resistance during puberty: results from clamp 
studies in 357 children. Diabetes 48(10):2039-44, 1999. 
 17.  Travers SH, Jeffers BW, Bloch CA, Hill JO, Eckel RH. Gender and Tanner stage differences in body 
composition and insulin sensitivity in early pubertal children. Journal of Clinical Endocrinology & 
Metabolism 80(1):172-8, 1995. 


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Continuation Format Page 
 18.  Ball GD, Weigensberg MJ, Cruz ML, Shaibi GQ, Kobaissi HA, Goran MI. Insulin sensitivity, insulin 
secretion and β-cell function during puberty in overweight Hispanic children with a family history of type 
2 diabetes. International Journal of Obesity 29(12):1471-7, 2005. 
 19.  Hoffman RP, Vicini P, Cobelli C. Comparison of insulin sensitivity and glucose effectiveness determined 
by the one- and two-compartment-labeled minimal model in late prepubertal children and early 
adolescents. Metabolism: Clinical & Experimental 51(12):1582-6, 2002. 
 20.  Travers SH, Labarta JI, Gargosky SE, Rosenfeld RG, Jeffers BW, Eckel RH. Insulin-like growth factor 
binding protein-I levels are strongly associated with insulin sensitivity and obesity in early pubertal 
children. Journal of Clinical Endocrinology & Metabolism 83(6):1935-9, 1998. 
 21.  Bergman RN. Minimal model: perspective from 2005. [Review] [24 refs]. Hormone Research 2005; 
64:Suppl-15. 
 22.  Bergman RN. Minimal model: perspective from 2005. [Review] [24 refs]. Hormone Research 2005; 
64:Suppl-15. 
 23.  Prigeon RL, Kahn SE, Porte D, Jr. Reliability of error estimates from the minimal model: implications for 
measurements in physiological studies. Am J Physiol 1994; 266(2 Pt 1):E279-E286. 
 24.  Moran A, Jacobs DR, Jr., Steinberger J et al. Association between the insulin resistance of puberty and 
the insulin-like growth factor-I/growth hormone axis. Journal of Clinical Endocrinology & Metabolism 
87(10):4817-20, 2002. 
 25.  Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K. Adiponectin and adiponectin receptors in 
insulin resistance, diabetes, and the metabolic syndrome. J Clin Invest 2006; 116(7):1784-1792. 
 26.  Goran MI, Ball GD, Cruz ML. Obesity and risk of type 2 diabetes and cardiovascular disease in children 
and adolescents. [Review] [131 refs]. Journal of Clinical Endocrinology & Metabolism 88(4):1417-27, 
2003. 
 27.  Knowler WC, Barrett-Connor E, Fowler SE et al. Reduction in the incidence of type 2 diabetes with 
lifestyle intervention or metformin. N Engl J Med 2002; 346(6):393-403. 
 28.  Gunton JE, Delhanty PJ, Takahashi S, Baxter RC. Metformin rapidly increases insulin receptor 
activation in human liver and signals preferentially through insulin-receptor substrate-2. J Clin 
Endocrinol Metab 2003; 88(3):1323-1332. 
 29.  Freemark M, Bursey D. The effects of metformin on body mass index and glucose tolerance in obese 
adolescents with fasting hyperinsulinemia and a family history of type 2 diabetes. Pediatrics 2001; 
107(4):E55. 
 30.  Kay JP, Alemzadeh R, Langley G, D'Angelo L, Smith P, Holshouser S. Beneficial effects of metformin 
in normoglycemic morbidly obese adolescents. Metabolism 2001; 50(12):1457-1461. 
 31.  Srinivasan S, Ambler GR, Baur LA et al. Randomized, controlled trial of metformin for obesity and 
insulin resistance in children and adolescents: improvement in body composition and fasting insulin. 
Journal of Clinical Endocrinology & Metabolism 91(6):2074-80, 2006. 
 32.  Ong K, de ZF, Valls C, Dunger DB, Ibanez L. Persisting benefits 12-18 months after discontinuation of 
pubertal metformin therapy in low birthweight girls. Clin Endocrinol (Oxf) 2007. 


PHS 398/2590 (Rev. 09/04, Reissued 4/2006) 
Page   19 
    
Continuation Format Page 
 33.  Ibanez L, Valls C, Potau N, Marcos MV, de Zegher F. Sensitization to insulin in adolescent girls to 
normalize hirsutism, hyperandrogenism, oligomenorrhea, dyslipidemia, and hyperinsulinism after 
precocious pubarche.[see comment]. Journal of Clinical Endocrinology & Metabolism 85(10):3526-30, 
2000. 
 34.  Ibanez L, Ferrer A, Ong K, Amin R, Dunger D, de Zegher F. Insulin sensitization early after menarche 
prevents progression from precocious pubarche to polycystic ovary syndrome.[see comment]. Journal 
of Pediatrics 144(1):23-9, 2004. 
 35.  Ibanez L, Valls C, Ong K, Dunger DB, de Zegher F. Metformin therapy during puberty delays 
menarche, prolongs pubertal growth, and augments adult height: a randomized study in low-birth-
weight girls with early-normal onset of puberty. Journal of Clinical Endocrinology & Metabolism 
91(6):2068-73, 2006. 
 36.  Ibanez L, Ong K, Valls C, Marcos MV, Dunger DB, de ZF. Metformin treatment to prevent early puberty 
in girls with precocious pubarche. J Clin Endocrinol Metab 2006; 91(8):2888-2891. 
 37.  Ibanez L, Valls C, Marcos MV, Ong K, Dunger DB, de ZF. Insulin sensitization for girls with precocious 
pubarche and with risk for polycystic ovary syndrome: effects of prepubertal initiation and postpubertal 
discontinuation of metformin treatment. J Clin Endocrinol Metab 2004; 89(9):4331-4337. 
 38.  Kuhlmann J, Neumann-Haefelin C, Belz U et al. Intramyocellular lipid and insulin resistance: a 
longitudinal in vivo 1H-spectroscopic study in Zucker diabetic fatty rats. Diabetes 2003; 52(1):138-144. 
 39.  Pan DA, Lillioja S, Kriketos AD et al. Skeletal muscle triglyceride levels are inversely related to insulin 
action. Diabetes 1997; 46(6):983-988. 
 40.  Krssak M, Falk PK, Dresner A et al. Intramyocellular lipid concentrations are correlated with insulin 
sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia 1999; 42(1):113-116. 
 41.  Sinha R, Dufour S, Petersen KF et al. Assessment of skeletal muscle triglyceride content by (1)H 
nuclear magnetic resonance spectroscopy in lean and obese adolescents: relationships to insulin 
sensitivity, total body fat, and central adiposity. Diabetes 2002; 51(4):1022-1027. 
 42.  Mourier A, Gautier JF, de KE et al. Mobilization of visceral adipose tissue related to the improvement in 
insulin sensitivity in response to physical training in NIDDM. Effects of branched-chain amino acid 
supplements. Diabetes Care 1997; 20(3):385-391. 
 43.  Richelsen B, Pedersen SB, Moller-Pedersen T, Schmitz O, Moller N, Borglum JD. Lipoprotein lipase 
activity in muscle tissue influenced by fatness, fat distribution and insulin in obese females. Eur J Clin 
Invest 1993; 23(4):226-233. 
 44.  Weiss R, Dufour S, Taksali SE et al. Prediabetes in obese youth: a syndrome of impaired glucose 
tolerance, severe insulin resistance, and altered myocellular and abdominal fat partitioning. Lancet 
2003; 362(9388):951-957. 
 45.  Busetto L. Visceral obesity and the metabolic syndrome: effects of weight loss. Nutr Metab Cardiovasc 
Dis 2001; 11(3):195-204. 
 46.  Gastaldelli A, Miyazaki Y, Pettiti M et al. Metabolic effects of visceral fat accumulation in type 2 
diabetes. J Clin Endocrinol Metab 2002; 87(11):5098-5103. 


PHS 398/2590 (Rev. 09/04, Reissued 4/2006) 
Page   20 
    
Continuation Format Page 
 47.  Miyazaki Y, Glass L, Triplitt C, Wajcberg E, Mandarino LJ, DeFronzo RA. Abdominal fat distribution and 
peripheral and hepatic insulin resistance in type 2 diabetes mellitus. Am J Physiol Endocrinol Metab 
2002; 283(6):E1135-E1143. 
 48.  Kelley DE, McKolanis TM, Hegazi RA, Kuller LH, Kalhan SC. Fatty liver in type 2 diabetes mellitus: 
relation to regional adiposity, fatty acids, and insulin resistance. Am J Physiol Endocrinol Metab 2003; 
285(4):E906-E916. 
 49.  Schwimmer JB, Deutsch R, Kahen T, Lavine JE, Stanley C, Behling C. Prevalence of fatty liver in 
children and adolescents. Pediatrics 2006; 118(4):1388-1393. 
 50.  Schwimmer JB, Deutsch R, Rauch JB, Behling C, Newbury R, Lavine JE. Obesity, insulin resistance, 
and other clinicopathological correlates of pediatric nonalcoholic fatty liver disease. J Pediatr 2003; 
143(4):500-505. 
 51.  Kawasaki T, Hashimoto N, Kikuchi T, Takahashi H, Uchiyama M. The relationship between fatty liver 
and hyperinsulinemia in obese Japanese children. J Pediatr Gastroenterol Nutr 1997; 24(3):317-321. 
 52.  Love-Osborne K, Sheeder J, Zeitler P. Addition of Metformin to a Lifestyle Modification Program in 
Adolescents with Insulin Resistance. The Journal of Pediatrics 2008; 152(6):817-822. 
 53.  Love-Osborne K, Butler N, Gao D, Zeitler P. Elevated fasting triglycerides predict impaired glucose 
tolerance in adolescents at risk for type 2 diabetes. Pediatric Diabetes 2006; 7(4):205-210. 
 54.  Nadeau KJ, Ehlers L, Zeitler P, Love-Osborne K. Nonalcoholic Liver Disease in Insulin Resistant 
Adolescents and Response to Metformin. Diabetes 2007; 56, Suppl. 1, A278. 
 55.  Nadeau KJ, Ehlers L, Draznin B, Regensteiner J, Reusch J. Exercise Capacity is Abnormal in Youth 
with Type 2 Diabetes. Diabetes 2007; 56, Suppl. 1, A278. 
 56.  Fishbein MH, Gardner KG, Potter CJ, Schmalbrock P, Smith MA. Introduction of fast MR imaging in the 
assessment of hepatic steatosis. Magnetic Resonance Imaging 1997; 15(3):287-293. 
 57.  Xiang AH, Peters RK, Kjos SL et al. Effect of pioglitazone on pancreatic β-cell function and diabetes 
risk in Hispanic women with prior gestational diabetes. Diabetes 2006; 55(2):517-522.