Science - USA (2021-12-03)

driven by an increase in fumarate reduction,
we calculated the ratio of the absolute con-
centration of^13 C 3 -succinate to that of^13 C 3 -
fumarate, revealing that the heart and WAT,
but not the kidney, liver, pancreas, and gas-
trocnemius, increase fumarate reduction upon
exercise (Fig. 4D). Notably, labeling is not in
the steady state in this experiment, and there-
fore, the net directionality of the SDH complex
upon exercise challenge cannot be determined
by using this protocol. Nevertheless, these data
clearly demonstrate that fumarate reduction
increases in a subset of tissues upon exercise
challenge.
Because net reversal of the SDH complex
supports DHODH activity in cultured cells
when O 2 reduction is blocked (Fig. 3), we tested
whether this was also true in mouse tissues. To
do so, we measured DHODH activity in tissues
that are capable (the liver and kidney) or in-
capable (the heart and gastrocnemius muscle)
of net reversing the SDH complex when O 2 re-
duction is limited. Antimycin treatment ablated
O 2 consumption in the liver, kidney, heart, and
gastrocnemiusmuscleexvivo,butonlythe
liver and kidney maintained net reversal of
the SDH complex (fig. S12, A and B). DHODH
activity was assessed via^13 C 4 -aspartate incor-
poration into^13 C 4 -orotate because labeling
of the^13 C 3 -UTP pool was undetectable in most
tissues. Antimycin treatment or hypoxia expo-
sure reduced the levels of^13 C 4 -orotate in the
heart and gastrocnemius muscle, whereas
they remained unchanged in the liver and
kidney, which net reverse the SDH complex
(Fig. 4E and fig. S12C). These data suggest
that the ability to maintain DHODH activity
correlates with the ability of a tissue to net
reverse the SDH complex upon inhibition of
O 2 reduction.

Discussion

Here, we have elucidated a circuit of electron
flow in the ETC of mammalian mitochondria
that does not require O 2 as a TEA. Although
the O 2 consumption rate is classically used as a
metric for electron flow through the ETC, our
study suggests caveats in directly equating
O 2 consumption with ETC flux and overall
mitochondrial function. In adapting to O 2
limitation, mammalian mitochondria use fu-
marate as a TEA. The accumulation of UQH 2
in hypoxia or upon inhibition of complexes III
or IV drives the SDH complex in reverse to
enable electron deposition onto fumarate.
Fumarate reduction sustains the input of
electrons into the ETC by complex I and
DHODH, enabling NADH reoxidation and
de novo pyrimidine biosynthesis. Although
in vivo, all tested tissues perform fumarate
reduction upon hypoxia exposure, only a sub-
set net reverse the SDH reaction, and only
these can maintain electron inputs into the
ETC (Fig. 4F).

The surprising differences among tissues in

their ability to reduce fumarate likely come

from the distinctive roles of mitochondria in

each tissue. For example, the heart and skeletal

muscle could favor the forward SDH activity

over the reverse SDH activity to maximize ATP

production, whereas the kidney and brain

may reduce fumarate to minimize the elec-

tron leakage out of complex III that gen-

erates ROS. Moreover, similar to the retina

within the eye ( 45 ), we expect interesting dif-

ferences in fumarate reduction among cell

types within a tissue, such as the thymus,

that on a bulk tissue scale, display some de-

gree of both the forward and reverse SDH

activities at physiological O 2 concentrations.

The full extent to which fumarate reduction

contributes to normal physiology and the ways

in which fumarate reduction is regulated re-

main to be understood.

Our data also provide clarity to the long-

standing observation that mtDNA-deficient

cells require uridine to proliferate ( 52 ). The

lack of DHODH activity to support pyrimidine

biosynthesis in these cells has always been

attributed to a deficiency in their ability to

transfer electrons to oxygen as a TEA. Notably,

fumarate reduction by the SDH complex, which

is encoded by the nuclear genome, can support

pyrimidine biosynthesis, albeit to a lesser extent

than oxygen does (Fig. 1E). This is likely caused

by less efficient electron transfer from ubiquinol

onto fumarate compared with cytochrome c.

Thus, mtDNA-deficient cells have the poten-

tial to use fumarate as a TEA to support py-

rimidine biosynthesis. However, given their

dependence on exogenous uridine for rapid

proliferation, these cells are likely missing a

different component of this pathway, and this

warrants further investigation.

Beyond the fundamental role of fumarate

reduction in mammalian mitochondria, there

are many disease contexts to which fuma-

rate reduction likely contributes. Fumarate

reduction is likely important in diseases that

cause tissue hypoxia such as ischemia, diabe-

tes, obesity, and cancer. Cancer is an area of

particular interest, given that some tumors

have mutations in the SDH complex ( 53 ),

the tumor microenvironment is hypoxic, and

the ETC supports tumor growth in multi-

faceted ways ( 18 , 54 – 58 ). For example, given

the importance of DHODH-mediated pyrim-

idine biosynthesis for tumor growth ( 21 ),

fumarate reduction might sustain this pro-

cess in the hypoxic tumor microenvironment.

However, it is also possible that the nutrient

limitations in the tumor microenvironment,

particularly those of glucose and aspartate

( 23 , 24 , 59 ), may limit de novo pyrimidine

biosynthesis. It will be critical for future

work to investigate the role of fumarate re-

duction in the context of diseases such as

cancer and to dissociate the forward and

reverse activities of the SDH complex in each

of these systems.

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