The pathogenesis of type 2 diabetes is characterized by impaired insulin action and increased hepatic glucose production (HGP). Despite the importance of hepatic metabolic aberrations in diabetes development, there is currently no molecular probe that allows measurement of hepatic gluconeogenic pathways in vivo and in a noninvasive manner. In this study, we used hyperpolarized carbon 13 ( 13 C)-labeled pyruvate magnetic resonance spectroscopy (MRS) to determine changes in hepatic gluconeogenesis in a high-fat diet (HFD)-induced mouse model of type 2 diabetes. Compared with mice on chow diet, HFD-fed mice displayed higher levels of oxaloacetate, aspartate, and malate, along with increased 13 C label exchange rates between hyperpolarized [1-13 C]pyruvate and its downstream metabolites, [1-13 C]malate and [1-13 C]aspartate. Biochemical assays using liver extract revealed up-regulated malate dehydrogenase activity, but not aspartate transaminase activity, in HFD-fed mice. Moreover, the 13 C label exchange rate between [1-13 C]pyruvate and [1-13 C]aspartate (k pyr->asp ) exhibited apparent correlation with gluconeogenic pyruvate carboxylase (PC) activity in hepatocytes. Finally, up-regulated HGP by glucagon stimulation was detected by an increase in aspartate signal and k pyr->asp , whereas HFD mice treated with metformin for 2 weeks displayed lower production of aspartate and malate, as well as reduced k pyr->asp and 13 C-label exchange rate between pyruvate and malate, consistent with down-regulated gluconeogenesis. Conclusion: Taken together, we demonstrate that increased PC flux is an important pathway responsible for increased HGP in diabetes development, and that pharmacologically induced metabolic changes specific to the liver can be detected in vivo with a hyperpolarized 13 C-biomolecular probe. Hyperpolarized 13 C MRS and the determination of metabolite exchange rates may allow longitudinal monitoring of liver function in disease development. (HEPATOLOGY 2013;57:515-524) T he coordinated actions of insulin and glucagon ensure that glucose homeostasis is maintained across a wide range of physiological conditions. In obesity-associated type 2 diabetes, control of glucose metabolism by these two regulatory hormones is impaired, resulting in hepatic insulin resistance and excessive endogenous glucose production. 1 To date, it has not been possible to evaluate this metabolic dysfunction in the liver by a noninvasive in vivo method.Carbon-13 ( 13 C) magnetic resonance spectroscopy (MRS) has been used to study hepatic gluconeogenesis since the 1980s. However, its inherent low sensitivity has largely limited its application to the study of steady-state metabolism in perfused livers with long Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; 13 C, carbon-13; ChREBP, carbohydrate response element-binding protein; FAS, fatty acid synthase; G-6-Pase, glucose-6-phosphatase; HFD, high-fat diet; HGP, hepatic glucose production; IHTG, intrahepatic triglyceride; IPGTT, intraperitoneal glucose toleranc...