Impaired hepatic mitochondrial function during early lactation in dairy cows: Association with protein lysine acetylation

Abstract

Early lactation is an energy-deming period for dairy cows which may lead to negative energy balance, threatening animal health and consequently productivity. Herein we studied hepatic mitochondrial function in Holstein-Friesian multiparous dairy cows during lactation, under two different feeding strategies. During the first 180 days postpartum the cows were fed a total mixed ration (70% forage: 30% concentrate) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Mendicago sativa plus supplementation (grazing group, G1). From 180 to 250 days postpartum, all cows grazed Festuca arundinacea were supplemented with total mixed ration. Mitochondrial function was assessed measuring oxygen consumption rate in liver biopsies revealed that maximum respiratory rate decreased significantly in grazing cows during early lactation, yet was unchanged in non-grazing cows during the lactation curve. While no differences could be found in mitochondrial content or oxidative stress markers, a significant increase in protein lysine acetylation was found in grazing cows during early lactation but not in cows from the non-grazing group. Mitochondrial acetylation positively correlated with liver triglycerides β-hydroxybutyrate plasma levels, well-known markers of negative energy balance, while a negative correlation was found with the maximum respiratory rate sirtuin 3 levels. To our knowledge this is the first report of mitochondrial function in liver biopsies of dairy cows during lactation. On the whole our results indicate that mitochondrial function is impaired during early lactation in grazing cows that acetylation may account for changes in mitochondrial function in this period. Additionally, our results suggest that feeding total mixed ration during early lactation may be an efficient protective strategy.

Fig 1. Metabolic parameters during lactation.

Liver biopsies and blood samples were obtained at 35 and 250 DPP from cows in the G0 (white) and G1 (grey) groups. Graphs show the concentrations of (A) liver triglyceride, (B) plasma β-hydroxybutyrate and (C) plasma NEFA. Results are shown with box plots, the box extends from the 25th to 75th percentile, the line in the middle of the box is the median, the cross is the mean and the whiskers represent the minimum and maximum values (N = 8–12), * P < 0.05, *** P <0.001, **** P <0.0001. In graphs (D) and (E) the correlations between β-hydroxybutyrate, triglycerides and NEFA are shown (N = 8–12). G0: Cows were fed TMR ad libitum from calving to 180 DPP. G1: Cows grazed Festuca arundinacea plus a commercial concentrate or Medicago supplemented with TMR (50% of G0 offer), depending on heat stress conditions, from calving to 180 DPP. From 180 to 250 DPP both groups grazed Medicago sativa and were supplemented with TMR (50% of G0 offer at 180 DPP).

Fig 2. Mitochondrial function decreases in pasture-fed dairy cows during early lactation.

Oxygen consumption rates were measured in liver biopsies before and after the sequential addition of 10 mM glutamate and 5 mM malate (Glu/Mal), 4 μM ADP, 2 μM oligomycin (Oligo), up to 4 μM FCCP and 0.5 μM rotenone (Rot). (A and B) Show representative traces of oxygen consumption rates obtained for liver biopsies of cows in the G0 group (A) and G1 group (B) at 35 DPP (grey) and 250 DPP (black). (C) Maximum respiratory rate, obtained from oxygen consumption rate measurements performed as described in A and B, of liver biopsies from cows in the G0 (white) and G1 (grey) groups. The box extends from the 25th to 75th percentile, the line in the middle of the box is the median, the cross is the mean and the whiskers represent the minimum and maximum values (N = 9–10), ** P < 0.01, and *** P < 0.001. (D) Maximum respiratory rate of liver biopsies obtained at different points during the lactation curve for both G0 (empty squares) and G1 (grey squares) cows. Data represent least square means ± SEM (N = 9–10). Different letters denote differences between dates (P < 0.05) and * denotes a difference between treatments (P < 0.05) according to Tukey-Kramer test. (G) and (H) show the correlation between maximum respiratory rate and liver triglyceride and plasma β-hydroxybutyrate, respectively (N = 8–12). G0: Cows were fed TMR ad libitum from calving to 180 DPP. G1: Cows grazed Festuca arundinacea plus a commercial concentrate or Medicago supplemented with TMR (50% of G0 offer), depending on heat stress conditions, from calving to 180 DPP. From 180 to 250 DPP both groups grazed Medicago sativa and were supplemented with TMR (50% of G0 offer at 180 DPP).

Fig 3. Evaluation of 4-HNE-protein adducts formation in liver homogenates and mitochondria.

(A and C) Representative western blots of 4-HNE-protein adducts in liver homogenates (A) and isolated mitochondria (C) from cows in the G0 and G1 groups at 35 and 250 DPP; β-actin and SDHA were used as loading controls, respectively. (B and D) Quantification by densitometry of 4-HNE-protein adduct levels normalized by protein levels of loading control and expressed in relation to the average value of the G0 group at 35 DPP. In box plots the box extends from the 25th to 75th percentile, the line in the middle of the box is the median, the cross is the mean and the whiskers represent the minimum and maximum values (N = 8–10). G0: Cows were fed TMR ad libitum from calving to 180 DPP. G1: Cows grazed Festuca arundinacea plus a commercial concentrate or Medicago supplemented with TMR (50% of G0 offer), depending on heat stress conditions, from calving to 180 DPP. From 180 to 250 DPP both groups grazed Medicago sativa and were supplemented with TMR (50% of G0 offer at 180 DPP).

Fig 4. Protein acetylation increases in liver mitochondria from pasture-fed dairy cows during early lactation.

(A) Representative western blots for AcK and SDHA (loading control) in liver subcellular fractions enriched in mitochondria from cows of both G0 and G1 groups at 35 and 250 DPP. (B) Independent western blots were quantified by densitometry. AcK levels were normalized with the loading control and expressed in relation to the average value of the G0 group at 35 DPP. The box extends from the 25th to 75th percentile, the line in the middle of the box is the median, the cross is the mean and the whiskers represent the minimum and maximum values (N = 10), *** P <0.001. (C), (D) and (E) show the correlations between AcK levels and liver triglyceride, plasma β-hydroxybutyrate and maximum respiratory rate, respectively (N = 8–10). G0: Cows were fed TMR ad libitum from calving to 180 DPP. G1: Cows grazed Festuca arundinacea plus a commercial concentrate or Medicago supplemented with TMR (50% of G0 offer), depending on heat stress conditions, from calving to 180 DPP. From 180 to 250 DPP both groups grazed Medicago sativa and were supplemented with TMR (50% of G0 offer at 180 DPP).

Fig 5. Protein lysine acetylation in liver homogenates.

(A) Representative Western blot of AcK levels in liver homogenates from cows of both G0 and G1 groups at 35 and 250 DPP; tubulin was used as loading control. (B) Quantification by densitometry of total AcK levels normalized with the loading control and expressed in relation to the average value of the G0 group at 35 DPP. The box extends from the 25th to 75th percentile, the line in the middle of the box is the median, the cross is the mean and the whiskers represent the minimum and maximum values (N = 10). ** P < 0.01. G0: Cows were fed TMR ad libitum from calving to 180 DPP. G1: Cows grazed Festuca arundinacea plus a commercial concentrate or Medicago supplemented with TMR (50% of G0 offer), depending on heat stress conditions, from calving to 180 DPP. From 180 to 250 DPP both groups grazed Medicago sativa and were supplemented with TMR (50% of G0 offer at 180 DPP).

Fig 6. Mitochondrial protein acetylation correlates with a decrease in sirtuin 3 levels.

(A and C). Representative western blots of sirtuin 5 (SIRT5) and sirtuin 3 (SIRT3) in liver homogenates from cows of both G0 and G1 groups at 35 and 250 DPP, β-actin was used as loading control. (B and D) Independent western blots of sirtuin 5 and sirtuin 3 were quantified by densitometry, normalized with the loading control and expressed in relation to the average value of the G0 group at 35 DPP. (E) Shows the correlation between mitochondrial AcK levels and sirtuin 3. (F) Shows the correlation between mitochondrial maximum respiratory rate and sirtuin 3. In box plots the box extends from the 25th to 75th percentile, the line in the middle of the box is the median, the cross is the mean and the whiskers represent the minimum and maximum values (N = 8). * P < 0.05. G0: Cows were fed TMR ad libitum from calving to 180 DPP. G1: Cows grazed Festuca arundinacea plus a commercial concentrate or Medicago supplemented with TMR (50% of G0 offer), depending on heat stress conditions, from calving to 180 DPP. From 180 to 250 DPP both groups grazed Medicago sativa and were supplemented with TMR (50% of G0 offer at 180 DPP).

Fig 7. Plausible mechanism behind metabolic changes during early lactation.

Lipid reserves are mobilized during early lactation, NEFA reach the blood stream and enter the hepatocyte where they are oxidized to acetyl-CoA in the β-oxidation pathway. The increase in Acetyl-CoA levels leads to acetylation of protein lysine residues. Lower levels of sirtuin 3 (SIRT3) in the liver of G1 cows may contribute to the increase in protein acetylation (AcK-Prot) in the G1 group with respect to the G0 group. Protein lysine acetylation impacts negatively on electron transport chain activity (ETC), Krebs cycle and β-oxidation resulting in an impaired oxidation of NEFA that are re-esterified to triglycerides (TG), giving rise to fatty liver.