Hyperpolarized C-13 magnetic resonance spectroscopy (MRS) to assess hepatic metabolism in nonalcoholic fatty liver disease (NAFLD) has not been reported. This study searched for cellular metabolism-based biomarkers for NAFLD induced by a high-fat diet (HFD) in rats. Also, correlations of the biomarkers with enzyme levels and histopathology were identified during a 6-week follow-up. Six rats were fed a control diet (CD) and seven rats were fed the HFD for 6 weeks. Hyperpolarized 13C dynamic MRS was performed on rat liver following an injection of hyperpolarized [1-C-13] pyruvate. Compared with CD-fed rats, HFD-fed rats showed significant increases in the levels of serum alanine aminotransferase and low-density lipoprotein cholesterol at weeks 4 and 6 of follow-up. After the 6 week HFD, the ratios of [1-C-13] alanine/pyruvate and [1-C-13] lactate/pyruvate were significantly increased, as were the levels of alanine aminotransferase and lactate dehydrogenase, which are potentially associated with hepatosteatosis. The results implicate [1-C-13] alanine and [1-C-13] lactate as potentially useful noninvasive biomarkers of hepatosteatosis occurring in NAFLD. (C) 2016 Published by Elsevier Inc.