Discussion
This study demonstrates that cartilaginous tissue formation can be
significantly upregulated by controlling nutrient metabolism and that
this response appears to be mediated through resultant changes in HIF-1α
signalling. Chondrocytes, although characteristically described as
anaerobic cells (Khan et al. , 2009), appear to switch their
metabolism in response to nutrient availability. Under
low-to-intermediate volumes of media (< 2
mL/106 cells), chondrocytes display an anaerobic
phenotype. At higher media volumes (> 2
mL/106 cells), glucose uptake increases and metabolism
switches to a mixed aerobic-anaerobic phenotype with the aerobic pathway
appearing to be more utilized with further increases in media volume.
This response is similar to the Crabtree effect — the phenomenon where
aerobic cells also excrete fermentation products (e.g. ethanol or
lactate) in the presence of oxygen and high external glucose (De Deken,
1966). While several potential mechanisms have been postulated to
explain the Crabtree effect (e.g. catabolite repression, catabolite
inactivation, limited respiratory capacity), recently it has been
explained by the overflow of glucose metabolites caused by the
saturation of TCA cycle capacity (Pronk et al. , 1996). Here, we
postulate an inverse response (“inverse Crabtree effect” ) where
under high glucose availability, increased glucose uptake leads to the
saturation of the fermentation pathway causing metabolite overflow to
the TCA cycle (Figure 5). However, it should be noted that additional
studies to quantify metabolic pathway flux (e.g. 13C
flux analyses) are required to confirm this notion.
Previous studies have also shown that increased glucose uptake occurs
under elevated media volumes (Heywood et al. , 2006; Suitset al. , 2008) and that metabolic switching can occur in response
to glucose availability (Otte, 1991; Lee & Urban, 1997; Heywoodet al. , 2010; Heywood et al. , 2014). Under low glucose,
increases in oxidative phosphorylation have been observed, providing
evidence for another Crabtree-like effect under such conditions (Otte,
1991; Lee & Urban, 1997; Heywood et al. , 2010; Heywood et
al. , 2014). Taken together, this suggests that chondrocytes may have
several different metabolic phenotypes depending on the availability of
glucose (in the presence of oxygen): predominantly anaerobic under
intermediate glucose levels (Warburg-like effect) and increased aerobic
activity under either low or high glucose levels (Crabtree-like
effects).
One important consequence of changes in metabolic phenotype was the
increased biosynthetic response (2.2- to 3.5-fold) observed at the
transition between metabolic states (~ 2
mL/106 cells). Previous studies have demonstrated that
increased nutrient availability (by media volume) can improve
cartilaginous tissue growth (Mauck et al. , 2003; Heywood et
al. , 2006; Khan et al. , 2009; Oze et al. , 2012) as well
as that chondrocytes are more synthetically active under mixed
aerobic-anaerobic metabolism (Lane et al. , 1977; Obradovicet al. , 1999; Khan et al. , 2009). When cultured at, or
near, the point of transition between anaerobic to mixed
aerobic-anaerobic metabolism, a state of pseudo-hypoxia occurs resulting
in the initiation of HIF-1 signaling leading to increased cartilaginous
tissue formation. This response is believed to manifest as a result of
the pooling of intracellular metabolites during the transition to
different metabolic states, which in turn, can stabilize HIF-1α by
interfering with PHD2 (Lu et al. , 2002; Kim et al. , 2010;
Ren et al. , 2011; De Saedeleer et al. , 2012; Bailey &
Nathan, 2018). Of the 14 metabolites investigated, only intracellular
lactate and succinate were correlated with PHD2 activity. Both lactate
(De Saedeleer et al. , 2012) and succinate (Bailey & Nathan,
2018) can affect PHD2 enzymatic activity; however, by different means.
Lactate inhibits PHD2 by competing with its substrate α-ketoglutarate
(De Saedeleer et al. , 2012) whereas succinate affects PHD2
activity through product inhibition at high concentrations (Bailey &
Nathan, 2018). While additional work is needed to determine the relative
contributions of intracellular lactate and succinate pools on PHD2
activity, most likely this affect can be attributed to lactate due to
observation that succinate was only present in trace amounts.
HIF-1α stabilization occurred primarily at intermediate volumes, leading
to the regulation of hypoxia-induced gene expression (Lu et al. ,
2002; Kim et al. , 2010; Ren et al. , 2011; De Saedeleeret al. , 2012; Bailey & Nathan, 2018). Although HIF-1 has many
target genes, several support chondrogenesis and regulate cartilage
homeostasis (Kim et al. , 2010), including glucose uptake (GLUT1),
TCA cycle suppression (PDK1), and chondrogenic differentiation (SOX9);
each of which was upregulated (by 2.0- to 2.7-fold) under these
conditions. Lastly, loss of function experiments using YC-1 (to degrade
HIF-1α) confirmed the involvement of the HIF-1α pathway in these
studies. While HIF mediated gene transcription can be induced by other
factors, they most likely do not play as prominent a role in the current
study. HIF-1α can also be regulated by FIH-1 (factor inhibiting HIF)
(Masoud & Li, 2015). Similarly, in the presence of oxygen, FIH-1
hydroxylates HIF-1α to prevent interaction with p300 and blocks
transcriptional activation (Masoud & Li, 2015); however, intracellular
pyruvate does not affect FIH-1 expression (Dalgard et al. , 2004)
or its activity (Hewitson et al. , 2007). Additionally, the other
known HIF transcription factors (HIF‑2α, HIF‑3α) are also probably not
involved as intracellular pyruvate does not affect HIF-2α (Ren et
al. , 2011) and HIF‑3α has multiple variants with different and opposing
functions (Duan, 2016). Lastly, while direct hypoxia has been
investigated as an anabolic stimulus for chondrocytes (Coyle et
al. , 2009; Yodmuang et al. , 2013), conflicting results have been
observed, most likely due to the fact that chondrocytes require oxygen
to a certain degree and prolonged hypoxia has been definitively shown to
inhibit ECM synthesis (Gibson et al. , 2008).
It is also recognized that other factors could potentially contribute to
the observed response. To account for changes in hydrostatic pressure
between cultures of different media volumes, these studies were
conducted using different sized culture plates (i.e. 24-, 12- and 6-well
plates). Estimates of the maximum hydrostatic pressure difference
between conditions were relatively small (≤ 50 Pa) due to the minimal
changes in media height above the cultures (≤ 5 mm). Previous studies
have shown that hydrostatic pressures of several orders of magnitude
higher (kPa to MPa range) are required to elicit changes in ECM
synthesis (anabolic or catabolic) (Elder & Athanasiou, 2009) indicating
that the potential influences were negligible. Limitations in oxygen
delivery to the cultures can also be a concern with varying culture
volumes (Place et al. , 2017). However, the distance from the
cells to the media surface was also accounted for by using different
sized culture plates and held relatively constant across groups (within
5 mm). In addition, as media buffering capacity changes proportionally
with volume, there may have been an influence of extracellular pH.
Chondrocytes are sensitive to pH with relatively small changes
influencing ECM synthesis (Wilkins & Hall, 1995). However, observed
differences in extracellular pH did not correlate with the changes in
ECM deposition, which was maximal at intermediate media volumes. Lastly,
as the cultures were only evaluated after a 4-week culture period (or
the last 48-hour media exchange cycle), additional studies are required
to determine whether these effects manifest throughout the culture
period.