Type 2 diabetes (T2D) and metabolic dysfunction-associated steatotic liver disease (MASLD) are increasingly prevalent conditions. MASLD begins with hepatic steatosis and increases T2D risk. T2D, characterised by peripheral insulin resistance and chronic hyperglycaemia, accelerates MASLD progression. We have observed that MASLD impairs hepatic synthesis of endogenous creatine for delivery to the heart, impairing creatine-mediated ATP resuscitation. As the liver is the main endogenous source of creatine, dietary supplementation may improve hepatic health by reducing the hepatic burden for fuel resources to high-energy tissues like the heart. To date, no global multi-omics study has examined the discrete hepatic response to prophylactic creatine supplementation in a multi-modal rat model of T2D-associated MASLD.
Thus, 8-week-old male Sprague-Dawley rats were fed a Western diet with or without creatine supplementation (20 g/kg) for 8 weeks. Animals were injected with low-dose streptozotocin after 4 weeks of feeding to generate T2D-like hyperglycaemia. Body weight and adiposity were quantified. After the 8-week period, terminal procedures were performed, and acquired sections of the left hepatic lobe underwent paired histological, proteomic, and metabolomic assessment. Hepatic steatosis was quantified by artificial intelligence-assisted analysis of haematoxylin and eosin-stained sections in QuPath (n≥6/group). Integrative multi-omics was performed in parallel, with global proteomics and targeted metabolomics using matched liver samples (n=9/group), chloroform-methanol phase separation, and liquid chromatography-tandem mass spectrometry.
Our findings revealed that, although body weight, adiposity, and mixed hepatic steatosis were not improved by creatine supplementation, the cellular response was markedly different. Multi-omics identified that 331 proteins and 37 metabolites were significantly altered (two-sample t-test, Benjamini-Hochberg procedure, p<0.05). Creatine was found to affect autophagy and mitophagy in the diabetic liver, leading to increased abundance of proteins involved in protein transcription, translation and one carbon metabolism. This study provides the first comprehensive multi-omics comparison of hepatic adaptations to dietary creatine supplementation in T2D rats with MASLD. Our work suggests that creatine induces early multi-omics changes in the liver prior to histological or macroscopic improvements, informing future prophylactic interventions for T2D-associated MASLD.