Impact of Fructose on Metabolism, Energy Homeostasis and MR Biomarkers in NAFLD

Like obesity, NAFLD and NASH are closely linked to nutrition and the "Western diet" which is
rich in saturated fats and refined sugars. Although fat consumption has remained relatively
stable, the marked increase in dietary fructose consumption (more than doubling in the past
30 years alone) supports the role of fructose in NAFLD and the metabolic syndrome. Although
the mechanism(s) for fructose-related liver injury is not yet well defined, fructose-related
hepatic adenosine triphosphate (ATP) depletion may contribute to liver injury. Observations
in animals suggest that fructose induces metabolic syndrome and NAFLD independent of energy
intake. One key difference in fructose metabolism (as opposed to glucose) relates to ATP
depletion and the necessity of adenosine monophosphate (AMP) kinase to replenish ATP stores.
As opposed to glucose, initial fructose metabolism involves phosphorylation of fructose to
fructose-1-phosphate by fructokinase (ketohexokinase, KHK) using the substrate ATP. Unlike
glucokinase, the phosphorylation of fructose by KHK is specific for fructose and not rate
limited. Replenishment of ATP stores requires phosphorylation of AMP back to ATP via AMP
kinase (which is inhibited in insulin resistance (common in patients with NAFLD) or
conversion to uric acid via xanthine dehydrogenase resulting in hyperuricemia. The high
activity of KHK in phosphorylating fructose to fructose-1-phosphate in the liver, could
result in hepatic ATP depletion with habitual fructose consumption.

Published animal and human studies support our hypothesis that fructose is a risk factor for
NAFLD and NAFLD-related liver disease progression. In animal models, diets high in fructose
induce features of the metabolic syndrome including weight gain, insulin resistance,
hypertriglyceridemia, and hypertension. Similar effects are not observed with the
administration of other simple sugars such as glucose. Fructose (or sucrose) administration
to humans also causes features of metabolic syndrome which are quite typical of patients with
NAFLD. Fructose is lipogenic, stimulates triglyceride synthesis and causes hepatic steatosis.
As previously reported in animals, our group reported that increased fructose consumption
(assessed as fructose-containing beverages only) is a risk factor of metabolic syndrome and
biopsy-proven NAFLD and that patients with NAFLD consume 3-4 times more fructose than age,
gender, and mass index (BMI) matched controls without liver disease.

In addition to increased fructose consumption being a risk factor for NAFLD, fructose has
been implicated in NAFLD disease progression. The administration of a diet with 25% of total
energy as sucrose (which contains 50% fructose) resulted in a rise in liver aminotransferase
levels within 18 days. This study, performed nearly 25 years ago, is all the more alarming as
current sugar intake of Americans is in this same range. In our study of 427 patients with
biopsy-proven NAFLD, increased consumption of fructose-containing beverages was univariately
associated with decreased age (P < 0.0001), male sex (P < 0.0001), hypertriglyceridemia (P <
0.04), low high density lipoprotein (HDL) cholesterol (<0.0001), decreased serum glucose (P <
0.001), increased calorie intake (P < 0.0001), and hyperuricemia (P < 0.0001). After
controlling for age, sex, BMI, and total calorie intake, daily fructose consumption was
associated with lower steatosis grade and higher fibrosis stage (P < 0.05 for each). Being
that triglyceride synthesis requires ATP, we hypothesize that lower hepatic steatosis may
reflect deceased ATP availability. Additionally, in older adults (age ≥ 48 years), daily
fructose consumption was associated with increased hepatic inflammation (P < 0.05), and
hepatocyte ballooning (P< 0.05). However, the mechanism(s) by which fructose causes liver
injury remains unknown.

In support of our hypothesis that ATP depletion underlies liver injury in patients with
NAFLD, our group has demonstrated that patients with biopsy-proven NAFLD have increased
hepatic mRNA expression of KHK compared to matched controls. Indeed, in human pilot studies,
intravenous (IV) fructose administration is associated with hepatic ATP depletion which can
be assessed by 31P magnetic resonance spectroscopy (MRS). Reduced hepatic ATP stores are more
prevalent in overweight and obese subjects than in lean subjects. Furthermore, recovery from
fructose-induced ATP depletion was found to be delayed in patients with NAFLD (n=8). However,
a limitation to this existing work is the small sample size and the inability to assess a
cause-effect relationship(s) between BMI, NAFLD, energy homeostasis, and histologic features
of liver injury. In liver cells, ATP depletion could perpetuate chronic liver injury by
making fatty hepatocytes less proliferative. Hepatic ATP depletion also encourages the
expansion of liver progenitor populations, causes arrest in protein synthesis, induces
inflammatory and prooxidative changes, increases endoplasmic reticulum stress, promotes
activation of stress-related kinases, induces mitochondrial dysfunction, and increases
apoptotic activity. This supporting data suggests that fructose may be associated with NAFLD,
NASH, and progressive fibrosis. Further, a study by Loguercio et al. demonstrated that
increased uric acid levels above the basal level after IV fructose infusion was significantly
higher (p < 0.01) in patients with cirrhosis (3 mg/dl) and NASH (1.9 mg/dl) than in healthy
controls (1.2 mg/dl). This effect was completely reversed by fructose 1,6-diphosphate which
could replenish the ability to resynthesize ATP from ADP. Therefore, an IV fructose challenge
could effectively differentiate healthy subjects, from chronic hepatitis, from cirrhosis.

NAFLD lacks accurate and robust non-invasive biomarkers to grade and stage histologic disease
activity. This is a critical barrier to understanding the influence of this important
environmental risk factor (increased/habitual fructose consumption) on the pathogenesis and
progression of NAFLD. Currently, reliable assessment NAFLD requires liver biopsy and
interpretation of histology. Serum aminotransferase levels and conventional imaging methods
can detect liver fat but cannot grade or stage NAFLD. Furthermore, current developments in
biomarker are cross-sectional in nature and do not characterize the dynamic changes which
underlie liver injury in patients with NAFLD. In vivo 31P MRS permits the evaluation of
dynamic changes of individual phosphorus-containing metabolites in the liver parenchyma, such
as phosphomonoester (PME), ATP, and inorganic phosphate (Pi). Intravenous fructose load
alters phosphorus metabolites and allows assessment of liver function by 31P MRS. Other
investigators have demonstrated that fructose loading could be used effectively as a tool to
investigate change in metabolic steps of hepatic metabolism in humans with alcohol-related
liver disease. Further, IV fructose loading causes significantly higher ATP degradation and
uric acid production in cirrhotic patients than in healthy controls. The associations between
fructose, increased uric acid, and hepatic ATP depletion has been previously described.
Increased uric acid is an independent risk factor for NAFLD and in keeping with our
hypothesis, hyperuricemia may be a surrogate marker of impaired hepatic energy homeostasis in
patients with NAFLD. The proposed mechanism for fructose-related hepatic ATP depletion,
NAFLD, NASH and the associated hyperuricemia is depicted in Figure 1 is novel, innovative,
scientifically rigorous and address an important public health concern—the impact of fructose
on the rising epidemic of NAFLD.

Inclusion Criteria:

Patients must satisfy all of the following criteria to be eligible for enrollment:

- Age greater than 18 years as of the initial screening interview and provision of
consent

- Healthy control as defined by:

- normal liver aminotransferases AND

- no evidence of NAFLD on radiologic imaging studies AND

- no history of chronic liver disease OR

- liver biopsy (if one had been historically performed for evaluation of suspected
liver disease).

OR • Patient with clinically suspected NAFLD as assessed by standard of care measures (risk
factors for NAFLD, abnormal liver enzymes and/or fatty liver on imaging studies) who are
scheduled to will undergo liver biopsy for the purpose of grading / staging the severity of
their underlying liver disease

Exclusion Criteria:

Patients who satisfy any of the following exclusion criteria will be ineligible for
enrollment:

- Current or history of significant alcohol consumption for a period of more than 3
consecutive months within 1 year prior to screening (significant alcohol consumption
is defined as more than 20 g/day in females and more than 30 g/day in males, on
average)

- Inability to reliably quantify alcohol consumption based upon local study physician
judgment

- Use of drugs historically associated with NAFLD (amiodarone, methotrexate, systemic
glucocorticoids, tetracyclines, tamoxifen, estrogens at doses greater than those used
for hormone replacement, anabolic steroids, valproic acid, and other known
hepatotoxins) for more than 2 weeks in the past year prior to randomization

- Prior or planned (during the study period) bariatric surgery

- Uncontrolled diabetes defined as HbA1c 9.5% or higher within 60 days prior to
enrollment

- A platelet count below 90,000/mm3

- Clinical evidence of hepatic decompensation as defined by the presence of any of the
following abnormalities:

- Serum albumin greater than 3.2 g/dL, INR greater than 1.3, bilirubin greater than
2.0 mg/dL

- History of esophageal varices, ascites or hepatic encephalopathy

- Evidence of other forms of chronic liver disease

- Serum alanine aminotransferase (ALT) greater than 300 U/L

- Serum creatinine of 2.0 mg/dL or greater

- Unstable therapy for components of the metabolic syndrome (ie. recent starting or
stopping of insulin sensitizing agent, lipid lowering agent, and/or antioxidant
therapy) . Recent starting or stopping (for more than 7 days) the use of a
thiazolidinedione (pioglitazone or rosiglitazone) 90 days before the entry biopsy or
anytime thereafter

- Use of any prescription or over-the-counter medication or herbal remedy that are
believed to improve or treat NASH or liver disease or obesity for the 90 days prior to
baseline liver biopsy or prior to randomization

- Patients must not take any other agent to treat NASH except the treatment assigned
after randomization.

- Inability to safely obtain a liver biopsy

- Active substance abuse including inhaled or injection drugs in the year prior to
screening

- Pregnancy, planned pregnancy, potential for pregnancy and unwillingness to use
effective birth control during the trial, breast feeding

- Any other condition which, in the opinion of the investigator, would impede compliance
or hinder completion of the study

- A contraindication to MRI examinations

- Extreme claustrophobia

- Weight or girth exceeds the scanner capabilities

- Any condition or circumstance that, in the opinion of the site investigator, would
interfere with completion of MR examinations

- Failure to give informed consent