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Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared with high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD- and HStD-fed mice had more and larger LDs than Chow-fed animals. We observed striking differences in liver LD proteomes of HFD- and HStD-fed mice compared with Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver LD proteome consisting of proteins common in HFD- and HStD-fed mice, as well as a proteome associated with glucose tolerance that included proteins shared in Chow and HStD but not HFD-fed mice. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase to perilipin 5 in the LD proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome. NEW & NOTEWORTHY: This study identified a fatty liver lipid droplet proteome and one associated with glucose tolerance. Notably, glucose intolerance was linked with changes in the ratio of adipose triglyceride lipase to perilipin 5 that is indicative of dysregulation of neutral lipid homeostasis. [ABSTRACT FROM AUTHOR]

Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared with high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD- and HStD-fed mice had more and larger LDs than Chow-fed animals. We observed striking differences in liver LD proteomes of HFD- and HStD-fed mice compared with Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver LD proteome consisting of proteins common in HFD- and HStD-fed mice, as well as a proteome associated with glucose tolerance that included proteins shared in Chow and HStD but not HFD-fed mice. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase to perilipin 5 in the LD proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome. NEW & NOTEWORTHY This study identified a fatty liver lipid droplet proteome and one associated with glucose tolerance. Notably, glucose intolerance was linked with changes in the ratio of adipose triglyceride lipase to perilipin 5 that is indicative of dysregulation of neutral lipid homeostasis.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress per se can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 μm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)