High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

Abstract

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal approximately 4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Figure 1. Effect of HFD on liver histology, triacylglycerol measurements and CYP2E1 protein levels

Representative liver sections from mice maintained for 16 weeks on control (A, 20×) or a HFD (B, 20× and C, 40×). (C) Ballooned hepatocytes (three solid arrows) and Mallory bodies (one broken arrow) were observed in zone 3 of HFD-fed mouse livers. (D) Quantification of liver triacylglycerol content. P=0.07, compared with control at 8 weeks; *P<0.05, compared with control at 16 weeks. (E) Quantification of CYP2E1 protein levels. Representative Western blots from each group are shown above the bar graph in (E). Levels of immunoreactive CYP2E1 protein were normalized to total protein run on duplicate gels stained with SyproRuby protein stain (Invitrogen). *P<0.05, compared with control at 8 weeks; **P<0.01, compared with control at 8 weeks; ***P<0.01, compared with HFD at 8 weeks. (F) Quantification of total protein on duplicate protein-stained gels used for CYP2E1 Western blots. Note there were no differences in total protein between control and HFD groups. Values represent the means±S.E.M. for 4–6 pairs of mice.

Figure 2. Effect of HFD on liver hypoxia

(A) Representative photomicrographs depicting patterns of pimonidazole adduct formation (brown) against a haematoxylin-nuclear counterstain (blue) in liver sections of mice fed either a control diet or HFD for 16 weeks. Increased staining for the pimonidazole adducts demonstrates increased tissue hypoxia in the HFD group as compared with control. Image analysis demonstrated increased area (B) and intensity (C) of pimonidazole adducts in liver from HFD group compared with control. Note: PP, periportal or zone 1 region of liver lobule; PC, pericentral or zone 3 region of liver lobule. Values represent the means±S.E.M. for three pairs of mice. *P<0.05, **P<0.01, compared with control.

Figure 3. Effect of HFD on respiratory rates, RCR and respiratory complex activities in liver mitochondria

State 3 and 4 respiration was measured and the RCR was determined using either glutamate/malate (A and B) or succinate (C and D) as oxidizable substrates in freshly isolated mitochondria. (E) Complex IV activity was measured by monitoring the rate of oxidation of fully reduced cytochrome c at 550 nm. (F) Complex I activity was assessed by measuring the rotenone-sensitive rate of oxidation of NADH initiated by coenzyme Q_1. Complex I and IV activities were measured at the 16 week feeding time point. Values are expressed as the means±S.E.M. for six pairs of mice. *P<0.05, **P<0.01, compared with control.

Figure 4. Effect of HFD on uncoupler-stimulated respiration and the membrane potential in liver mitochondria

(A) Oxygen consumption was measured in freshly isolated mitochondria in the presence of succinate/rotenone (15 mM/5 μM) and the uncoupler FCCP (1 μM). (B) Membrane potential was reported by the red/green fluorescence ratio of JC-1 in freshly isolated coupled (−FCCP) and uncoupled (+FCCP) mitochondria. Both uncoupled respiration and membrane potential are shown for the 16 week feeding time point. Values are expressed as the means±S.E.M. of six pairs of mice. *P<0.05, compared with control. AU, absorbance units.

Figure 5. Effect of HFD on NO-dependent inhibition of mitochondrial respiration

(A) A representative respiration experiment using mitochondria (0.5 mg/ml) from liver of control (black lines) and HFD (grey lines) mice in the presence of succinate/rotenone (15 mM/5 μM) and ADP (0.5 mM). Oxygen traces are shown in the presence of PAPA NONOate (5.0 μM, solid lines), added to the chamber at 80% O₂ (at the arrow) and in its absence (dotted lines). Note that there was no difference in the rate and amount of NO produced between groups (results not shown). (B) Representative traces of respiration rate, % of maximum, as a function of NO concentration using mitochondria from liver of control (black) and HFD (grey) mice. Both O₂ and NO concentrations are measured simultaneously for the entire duration of each experimental run. (C) The concentration of NO that causes a 50% decrease in mitochondrial respiration rate (i.e. IC₅₀) for control and HFD groups is shown. Results obtained from traces shown in (B) (control, black line, IC₅₀=solid line and HFD, grey line, IC₅₀=dashed line). Values are expressed as the means±S.E.M. for six pairs of mice. *P<0.05, compared with control.

Figure 6. Effect of HFD on iNOS protein levels in liver

(A) Representative photomicrographs demonstrating the extent of iNOS staining (red) against a DAPI-nuclear counterstain (blue) in liver sections of mice fed either a control or HFD for 16 weeks. Increased red staining shows elevated tissue levels of iNOS protein in the HFD group as compared with the control. Image analysis demonstrated increased intensity (B) of iNOS staining in liver from HFD groups compared with control. There was no statistically significant difference in the area of iNOS staining between control and HFD groups (results not shown). Values represent the means±S.E.M. for four pairs of mice. *P<0.05, compared with control.

Figure 7. Effect of HFD on 3-NT levels in liver

(A) Representative photomicrographs depicting patterns of 3-NT staining (brown) against a haematoxylin nuclear counterstain (blue) in liver sections of mice fed either a control diet or HFD for 16 weeks. Increased brown staining demonstrates increased tissue 3-NT levels in the HFD group as compared with control. Image analysis demonstrated increased area (B) and intensity (C) of 3-NT staining in liver from the HFD group compared with control. Values represent the means±S.E.M. for five pairs of mice. *P<0.05, compared with control.

Figure 8. Effect of HFD on 3-NT levels in liver mitochondrial proteins

(A) Representative Western blots depicting the extent of nitration in mitochondrial proteins from mice fed either a control or HFD for 16 weeks. Representative Western blots for two separate pairs of mice are shown along with their duplicate stained total protein gels. (B) Quantification of 3-NT levels in mitochondrial proteins. Levels of nitrated proteins were normalized to total protein. (C) Quantification of total protein was performed on duplicate gels, stained with SyproRuby protein stain, used for 3-NT Western blots. Note there were no differences in total protein between control and HFD groups. Values represent the means±S.E.M. for three pairs of mice. **P<0.01, compared with control.