Prevalence of Non-alcoholic Fatty Liver Disease (NAFLD) in Hispanics With Diabetes Mellitus Type 2 (T2DM) and Role of Treatment

Many NAFLD studies have found that the progression from "benign" steatosis to severe
necroinflammation and cirrhosis as observed in NASH varies widely depending upon the initial
stage at diagnosis, as well the presence or absence of specific risk factors associated with
disease progression. The factors that promote necroinflammation and fibrosis development are
complex, but are frequently associated with the presence of long-standing obesity, metabolic
syndrome, and in particular, of T2DM. Indeed, hyperglycemia has been identified as the single
most consistent factor for disease progression in many studies (Angulo et al, Hepatology
1999) Marceau et al, JCEM 1999; Luyckx et al, Obes Relat Metab Disord, 1998; Mofrad et al,
Hepatology 2003; many others; reviewed by Cusi, Current Diabetes Reports, 2009).

Given the worse prognosis of NASH in patients with T2DM, it is quite surprising that few
studies have focused on the prevalence of the disease and on early screening and treatment of
patients with diabetes for NASH. A prospective study conducted by Gupte et al
(Gastroenterology & Hepatology, 2004) reported biopsy-proven NASH in 87% of diabetics, 22%
having moderate to severe disease. In a retrospective analysis of 44 patients with T2DM
worked-up for NAFLD, Younussi et al also found that cirrhosis was more prevalent in diabetics
vs. nondiabetics (25% vs. 10%, p<0.001) (Hepatology 2004). In recent years, the diagnosis of
fatty liver has been made easier with the standardization of liver magnetic resonance and
spectroscopy (MRS) which has allowed a fast and highly reproducible test for NAFLD. With this
screening tool we have found that NAFLD is present in >80% of unselected patients with T2DM.
In non-diabetic patients a handful of small studies with paired biopsies indicate that
fibrosis progresses over time in 32-41% of patients with NAFLD (reviewed by Ali & Cusi,
Annals of Medicine, 2009). Obesity and T2DM were the 2 most prominent factors of poor
prognosis, while elevated liver enzymes (ALT or AST/ALT ratio) were of lesser value (Mofrad
et al, Hepatology 2003; Sorrentino et al, Hepatology 2004; Kunde et al, Hepatology 2005).

This study aims at establishing the role of pioglitazone and of vitamin E in VA patients.
Weight loss remains the standard of care because no therapy has conclusively proven to be
effective in the long-term. Pharmacological therapies with modest effects have included
pentoxifylline, orlistat, cytoprotective agents, ursodeoxycholic acid and lipid-lowering
agents, while insulin-sensitizers such as metformin and thiazolidinediones have yielded more
provocative results in small uncontrolled studies in NASH. Our research group recently
demonstrated in a randomized, double-blind, placebo-controlled trial, that pioglitazone
treatment for 6 months in patients with T2DM and NASH significantly improved glycemic
control, glucose tolerance, insulin sensitivity and systemic inflammation (Belfort et al,
NEJM 2006). This was associated with a ~50% decrease in steatohepatitis (p<0.001) and a 37%
reduction of fibrosis within the pioglitazone-treated group (-37%, p<0.002), although this
fell short of statistical significance when compared with placebo (p=0.08). Our results
provided "proof-of-principle" that pioglitazone may be the first agent capable of altering
the natural history of the disease. However, definitive proof requires establishing its
safety and efficacy in a large number of subjects treated for a longer period of time. The
CRN is conducting the PIVENS trial (www.ClinicalTrials.gov; NCT 00063622) examining the role
of pioglitazone, vitamin E or placebo in NASH, but the study design excluded diabetics, only
~5% of patients were Hispanic and studied a younger population than that typical from VA
Medical Centers. Also, this important multicenter trial did not perform the in-depth
metabolic measurements this trial will carry out (i.e., insulin clamps with glucose turnover
measurements, indirect calorimetry, etc.).

Understanding the long-term impact of thiazolidinediones and of vitamin E in patients with
NASH and T2DM, who are believed to be at the highest risk for liver disease progression, will
have important implications not only for the treatment of NASH but for drug selection and
treatment algorithms in T2DM, as an insulin-sensitizer approach of pioglitazone (in addition
to metformin) would be preferred over therapies such as sulfonylureas or insulin, if proven
to be effective to treat NASH in T2DM. However, currently the most common strategy to treat
T2DM is an insulin secretion-based approach (i.e., sulfonylureas and/or insulin) that has
little impact on liver fat and promotes weight gain without a major improvement in insulin
sensitivity, promoting chronic hyperinsulinemia and self-perpetuating the metabolic milieu
that promotes hepatic lipogenesis and fatty liver disease. Therefore, understanding the role
of pioglitazone and vitamin combined, of vitamin e alone (plus pioglitazone placebo tablets
as control) and compared to a third arm with placebo of both (pioglitazone and vitamin E) is
important to move the field forward.

Of note, the study started at the San Antonio VAMC, TX where ~60% of the population was
Hispanic. However, once Dr. Kenneth Cusi (principal investigator) moved to the Gainesville
VAMC, FL the study was transferred to Gainesville and recruitment continued in this new site
where the prevalence of Hispanics is only 5% (75% Caucasians, 20% African American).
Therefore, the final patient mix will reflect more the latter ethnic mix.

Inclusion Criteria:

- Be able to communicate meaningfully with the Investigator and be legally competent to
provide written informed consent.

- Subjects of both genders from within the Veterans Administration Healthcare System
with an age range between 18 to 70 years (inclusive).

- Have type 2 diabetes mellitus as defined by the American Diabetes Association
guidelines.

- Female volunteers must be non-lactating and must either be at least one year
post-menopausal, or be using adequate mechanical contraceptive precautions (i.e.
intrauterine device, diaphragm with spermicide, condom with spermicide), or be
surgically sterilized (i.e. bilateral tubal ligation, bilateral oophorectomy). Female
patients who have undergone a hysterectomy are eligible for participation in the
study. Female patients (except for those patients who have undergone a hysterectomy or
a bilateral oophorectomy) are eligible only if they have a negative pregnancy test
throughout the study period.

- The following laboratory values:

- Hemoglobin at least 12 gm/dl in males or 11 gm/dl in females, WBC count 3,000/mm3
(neutrophil count 1,500/mm3) and platelets 100,000/mm3

- Albumin equal or greater than 3.0 g/dl

- Serum creatinine less than 1.8 mg/dl

- AST and ALT up to 3.0 times upper limit of normal and alkaline phosphatase 2.5
times ULN

Exclusion Criteria:

- Any cause of chronic liver disease other than NASH (such as -but not restricted to-
alcohol or drug abuse, medication, chronic hepatitis B or C, autoimmune,
hemochromatosis, Wilson's disease, alpha1-antitrypsin deficiency).

- Any clinical evidence or history of ascitis, bleeding varices, or spontaneous
encephalopathy.

- History of alcohol abuse (alcohol consumption greater than 20 grams of ethanol per
day) or a positive AUDIT screening questionnaire.

- Prior surgical procedures to include gastroplasty, jejunoileal or jejunocolic bypass.

- Prior exposure to organic solvents such as carbon tetrachloride.

- Total parenteral nutrition (TPN) within the past 6 months.

- Subjects with type 1 diabetes mellitus.

- Patients on chronic medications with known adverse effects on glucose tolerance levels
unless the patient has been on a stable dose of such agents for 4 weeks before entry
into the study.

- Patients on drugs known to cause hepatic steatosis: estrogens or other hormonal
replacement therapy, tamoxifen, raloxifene, oral glucocorticoids, chloroquine and
others.

- Patients with a history of clinically significant heart disease (New York Heart
Classification greater than grade II), peripheral vascular disease (history of
claudication), or diagnosed pulmonary disease (dyspnea on exertion of one flight or
less; abnormal breath sounds on auscultation).

- Patients with severe osteoporosis (-3.0 at the level of spine and hip).

- Patients who have clinically significant acute or chronic medical conditions not
specifically written in the protocol, but that based in the investigator's clinical
judgment he/she considers unlikely that he will be able to complete study
participation or that such participation may be potentially detrimental to his
well-being.