Here, we exploited an experimental system in which acute Vhl disruption in mature hepatocytes caused death within days. Vhl inactivation blocked fatty acid oxidation causing liver steatosis and the accumulation of triglycerides in both the liver and plasma. Using liver perfusion experiments we determined that Vhl loss inhibited the production of glucose and ketone bodies likely explaining the reduced circulating ketone and, eventually, glucose levels, leading thereby to the death of mice. Most importantly, Vhl inactivation suppressed mitochondrial respiration and mice were rescued from the lethal effects of Vhl loss by simultaneous inactivation of Hif-1β.
-deficient livers used approximately 50% less oxygen than their wild-type counterparts indicating a profound inhibition of mitochondrial respiration. Loss of Vhl
did not affect all mitochondrial functions however, and urea production was normal. Compellingly, the reduction in O2
extraction in perfused Vhl
-deficient livers was associated with a corresponding increase of a similar magnitude in liver pO2
in live mice. These data show that Vhl
loss blocks mitochondrial respiration in the liver in vivo
. This defect likely underlies the metabolic and death phenotypes. Specifically, gluconeogenesis is strongly dependent on the TCA cycle and mitochondrial respiration (Burgess et al 2006
, Burgess et al 2007
). Even in mice with otherwise normal gluconeogenic gene expression, impaired hepatic TCA cycle and oxidative phosphorylation disrupts gluconeogenesis (Burgess et al 2006
To our knowledge, this is the first study to show that Vhl
loss and constitutive Hif activation is sufficient to block oxygen utilization and mitochondrial respiration in live animals. Despite oxygen availability, its utilization was blocked by Hif. We conclude that there is no other pathway in the liver that can thwart the effects of Hif on oxygen disposition. Incidentally, the adverse effects of sustained Hif activation in the liver may explain why, even under conditions of prolonged hypoxia, Hif-1α upregulation in the liver is short-lived (Stroka et al 2001
These results are in contrast to those from Rankin et al.
who using isolated mitochondria from Alb-Cre; VhlF/F
livers and succinate or glutamate as substrates reported that Vhl
loss in hepatocytes did not affect oxygen consumption (Rankin et al 2009
). We speculate that the differences observed between our study and that by Rankin et al.
arose because in organello
measurements do not fully reproduce the fine metabolic autoregulation that occurs in the closed metabolic pathways in intact organs. Our findings highlight the importance of ex vivo
perfusion experiments and in vivo
The particular roles of Hif-1α and Hif-2α in the metabolic derangement observed are unknown. Rankin and colleagues showed that inactivation of Hif-2α (but not Hif-1α) prevented steatosis in Alb-Cre; VhlF/F
livers (Rankin et al 2009
). However, Kim et al.
showed that overexpression of a constitutively active form of Hif-2α did not result in liver steatosis and that by contrast, liver steatosis was caused by a stable form of Hif-1α (Kim et al 2006b
). In the studies by Kim et al.
, the severe steatosis observed in Alb-Cre; VhlF/F
livers was most closely recapitulated by simultaneous activation of both Hif-1α and Hif-2α proteins and both Hif-1α and Hif-2α proteins may be similarly implicated in Ad-Cre VhlF/F
Extensive changes in gene expression were observed following Vhl
inactivation and it seems unlikely that changes in a single gene would account for the profound suppression in mitochondrial respiration observed. Mitochondrial biogenesis (Zhang et al 2007
, Zhang et al 2008
) and autophagy (Lei et al 2008
, Zhang et al 2008
) are regulated by Hif-1. However, since urea production was unaffected, it is improbable that the decrease in oxygen consumption observed resulted from decreased mitochondrial mass. One mechanism may involve the Fe-S cluster assembly proteins IscU1/2, which are regulated by pVhl in a miR-210-dependent manner (Chan et al 2009
), and which we found to be downregulated in Ad-Cre VhlF/F
livers. Low IscU1/2 levels have been implicated in reducing aconitase and complex I activities (Chan et al 2009
) and may also directly affect β-oxidation, which requires Fe-S cluster containing electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Another factor contributing to decreased mitochondrial respiration may be Pdk1
, a Hif-1 target gene (Kim et al 2006a
, Papandreou et al 2006
) which was induced in Vhl
-deficient livers, and which would downregulate the amount of pyruvate-derived acetyl-CoA available for the TCA cycle.
The phenotype of Ad-Cre VhlF/F
animals resembled human inborn fatty acid oxidation defects which are associated with liver steatosis and hypoketotic hypoglycemia (Wood 1999
). In patients, death may be precipitated by a viral illness, and in mice, death may have been hastened by the adenoviral infection. Humans with inborn errors in FAO are treated with glucose (Saudubray et al 1999
), and exogenous glucose administration prolonged the survival of mice. However, whereas in humans supportive treatment during an acute illness may avert death, glucose administration to mice delayed survival only by a short period of time indicating that the underlying deficit may be more severe.
The finding that Vhl
disruption is sufficient to suppress mitochondrial respiration may be amenable to therapeutic exploitation as it would suggest that VHL
-deficient tumors may be exquisitely dependent on glycolysis for ATP generation. While the effects of Vhl
loss on mitochondrial respiration may be tissue-specific, a similar suppression of mitochondrial functions may also occur in ccRCC. Indeed, ccRCCs have low respiratory chain protein content (Simonnet et al 2002
) and impaired mitochondrial function may contribute to the accumulation of TG in this tumor type. In fact, miR-210 is one of the most upregulated microRNAs in ccRCC (Chow et al 2010
, Juan et al 2010
, Jung et al 2009
, Liu et al 2010
) and this would be expected to downregulate IscU1/2 proteins. Thus inhibitors of glycolysis may be particularly efficacious against this tumor type.
In summary, our data show that proper Hif regulation is essential for oxygen homeostasis in the liver. Unchecked, Hif is sufficient to suppress mitochondrial respiration in vivo leading to impaired fatty acid oxidation and reduced ketone and glucose production ultimately causing death. Despite oxygen availability, Hif blocks oxygen utilization. Thus, no other oxygen sensing pathway exists in hepatocytes that can substantially modulate the effects of Hif.