Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic mitochondrial dysfunction characterized by reduced ATP synthesis. We applied the 2H2O-metabolic labeling approach to test the hypothesis that the reduced stability of oxidative phosphorylation proteins contributes to mitochondrial dysfunction in a diet-induced mouse model of NAFLD. A high fat diet containing cholesterol (a so-called Western diet (WD)) led to hepatic oxidative stress, steatosis, inflammation and mild fibrosis, all markers of NAFLD, in low density cholesterol (LDL) receptor deficient (LDLR-/-) mice. In addition, compared to controls (LDLR-/- mice on normal diet), livers from NAFLD mice had reduced citrate synthase activity and ATP content, suggesting mitochondrial impairment. Proteome dynamics study revealed that mitochondrial defects are associated with reduced average half-lives of mitochondrial proteins in NAFLD mice (5.41±0.46 vs. 5.15±0.49 day, P<0.05). In particular, the WD reduced stability of oxidative phosphorylation subunits, including cytochrome b-c1 complex subunit 1 (5.9 ± 0.1 vs 3.4 ± 0.8 day), ATP synthase subunit α (6.3±0.4 vs. 5.5±0.4 day) and ATP synthase F(0) complex subunit B1 of complex V (8.5±0.6 vs. 6.5±0.2 day) (P<0.05). These changes were associated with impaired complex III and F0F1-ATP synthase activities. Markers of mitophagy were increased, but markers of proteasomal degradation activity were reduced in NAFLD mice liver, suggesting that ATP deficiency due to reduced stability of oxidative phosphorylation complex subunits contributed to inhibition of ubiquitin-proteasome and activation of mitophagy. In conclusion, the 2H2O-metabolic labeling approach shows that increased degradation of hepatic oxidative phosphorylation subunits contributed to mitochondrial impairment in NAFLD mice.

Context Hyperglycemia plays a key role in the pathogenesis of cardiovascular complications of diabetes. T2DM is associated with high density lipoprotein (HDL) dysfunction and increased degradation of ApoAI. The mechanism(s) of these changes are largely unknown. Objective To study the role of hyperglycemia-induced glycation on ApoAI kinetics and stability in patients with diet-controlled T2DM. Design ²H₂O-metabolic labeling approach was used to study ApoAI turnover in patients with diet-controlled T2DM (N= 9 (5F), 59.3±8.5 yr.) and matched healthy controls (N= 8(4F), 50.7±11.6 yr.). The effect of Amadori glycation on in vivo ApoAI stability and HDL’s anti-oxidant and cholesterol efflux properties were assessed using proteomics approach and in vitro assays. Results T2DM patients had increased turnover of ApoAI, and impaired cholesterol efflux and anti-oxidant properties of HDL. HbA_1c was negatively correlated with the half-live of ApoAI, and cholesterol efflux function of HDL. Proteomic analysis identified several non-enzymatic early (Amadori) glycation of ApoAI at lysine sites. The kinetic analysis of glycated and native ApoAI peptides in T2DM patients revealed that glycation resulted in 3-fold shorter ApoAI half-life. Conclusions The ²H₂O method allows the detection of early in vivo impairments in HDL metabolism and function that are related to hyperglycemia-induced glycation of ApoAI in T2DM.

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS ( n = 7), NASH ( n = 8), and healthy controls ( n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher ( P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio ( P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL⁻¹·min⁻¹) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins’ turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.