The first part of our results demonstrate that enterocytic apoptosis is in part
responsible for triggering intestinal barrier dysfunction upon I/R challenge, and glucose
uptake mediated by SGLT1 attenuated the loss of intestinal barrier function partly via
anti-apoptotic PI3K/Akt signaling (Figure. 11).
The histological findings suggest that mesenteric I/R triggers epithelial apoptosis
that accompanies villous disintegration and decreased crypt cell proliferation. Changes
in intestinal permeability in I/R rats were demonstrated by the transmural HRP flux rate
ex vivo and by the lumen-to-blood passage of enterally-administered FD4 and
gadodiamide in vivo. The increase of epithelial permeability paralleled the augmentation
of BT in I/R intestines, indicating gut barrier damage. These pathological changes were
attenuated by enteral instillation of ZVAD, providing direct evidence that epithelial
apoptosis is in part responsible for triggering intestinal barrier damage and bacterial
influx. This study is thus the first to demonstrate that modification of epithelial caspase
activity reduces intestinal permeability and BT in animals. We also showed that
pretreatment with ZVAD diminished the increase of MPO activity and the production of
TNFα and MIP-1α in ischemic intestines, suggesting that epithelial apoptosis and
barrier dysfunction preceded mucosal inflammation.
The intestinal permeability was evaluated using ex vivo and in vivo assays. A
twofold increase of the transmural HRP flux was observed in the intestinal tissues in I/R
rats compared with sham controls. However, the increased HRP flux was only evident at
30-60 and 60-90 minutes after the addition of the probe to Ussing chambers; the
delayed observation and the extracorporeal, oxygenated setting may produce artificial
results of the extent of I/R injury. In light of these technical limitations, we also
evaluated gut permeability in vivo by measuring the luminal-to-blood passage of
enterally administered FD4 or gadodiamide during the reperfusion period. The plasma
level of FD4 was four times higher in I/R rats than in sham controls, indicating
compromised intestinal barrier function after ischemic challenge. A novel MRI-based
assay developed previously from our laboratory was used to detect the real-time
permeability changes in vivo. This more sensitive assay showed a five- to tenfold
increase of SNR in liver and kidney in ischemic rats at the early phase (<15 minutes) of
reperfusion, supporting the hypothesis that it is an acute break of the epithelial barrier
that leads to augmented portal drainage of luminal probes. The kidney SNR peaked at
15 minutes post-reperfusion and declined afterwards, which was likely due to the high
concentration of gadodiamide in the renal medulla and calyces leading to a greater T2
shortening effect than T1 shortening effect. These results are in agreement with others
showing increased gut permeability upon I/R challenge (Sun et al., 1998; Khanna et al.,
2001). In comparison to the HRP flux assay and the fluorescence intensity test, this
MRI-based method is fast and sensitive, and allows us to rapidly detect changes in gut
permeability and provides a means for further studies of targeted pharmacological
intervention.
Physiological cytoprotective mechanisms contributing to resistance against
apoptosis include upregulation of glucose transporters (e.g. SGLT1, GLUT1, and
GLUT4) and maintenance of high intracellular glucose concentration (Sun et al., 1994;
Hall et al., 2001; Russo et al., 2004; Yu et al., 2005; Yu et al., 2006; Wofford et al., 2008;
Yu et al., 2008). The phenomenon of glucose protection has been documented in a
number of cell types, including neurons, leukocytes, myocardiocytes, and vascular
smooth muscle cells (Sun et al., 1994; Hall et al., 2001; Russo et al., 2004; Wofford et
al., 2008). Previous in vitro studies utilizing human intestinal epithelial Caco-2 cells
have also demonstrated that SGLT1 glucose uptake inhibits cell apoptosis and barrier
impairment caused by bacterial and parasitic products (Yu et al., 2005; Yu et al., 2006;
Yu et al., 2008). Based on our finding that epithelial apoptosis is partly responsible for
intestinal barrier dysfunction upon I/R challenge, we sought to investigate the use of
glucose supplementation to correct excessive cell death and to improve gut barrier
integrity. In I/R rats, glucose uptake mediated by the phloridzin-sensitive SGLT1
protected the intestinal epithelium from apoptosis and attenuated the increase in
permeability and BT, leading to diminished inflammatory responses. Taken together
with previous studies (Yu et al., 2005; Yu et al., 2006; Yu et al., 2008), our finding
suggests that SGLT1-mediated cytoprotection may operate under a number of metabolic
and microbial stress conditions.
The signal transduction pathways responsible for promoting cell survival were also
investigated. Membrane translocation and increased phosphorylation of Akt in villous
epithelial cells were paralleled by enhanced Akt kinase activity in the gut mucosa
following glucose uptake by SGLT1. This finding is consistent with previous studies
showing that Akt is a downstream step of a signaling pathway induced by Na+-glucose
cotransport (Zhao et al., 2004; Shiue et al., 2005), which triggered the activation of
Na+-H+ exchanger (NHE). Our pharmacological blockade studies with LY294002 and
wortmannin confirmed that PI3K/Akt activation is involved in the glucose-mediated
protection against epithelial apoptosis, mucosal pathology and gut barrier damage
induced by I/R. Previous reports documented that PI3K/Akt signaling inhibits cell
apoptosis by phosphorylating Bad, either directly or indirectly via mTOR (Chang et al.,
2003; LoPiccolo et al., 2008). A number of other downstream targets of Akt, such as
IκBα/NFκBα, FoxO1/3a and GSK3, are also involved in promoting cell survival and
cell cycle progression (Liang et al., 2003; Urbich et al., 2005; Dan et al., 2008; Bai et al.,
2009). We showed here that SGLT1 glucose uptake induced the phosphorylation of Akt
and downstream targets such as mTOR, Bad and FoxO1/3a in intestinal mucosa. These
results provide an explanation of the anti-apoptotic effects of enteral glucose, which
may contribute to cytoprotective mechanisms in intestinal I/R. It is noteworthy that
recent evidence points to an unanticipated role of Bad in linking pathways of glucose
metabolism and cell apoptosis (Danial et al., 2003; Danial, 2008). Danial et al.
demonstrated that Bad resides in a mitochondrial complex with glucokinase and
participates in mitochondrial respiration in response to glucose (Danial et al., 2003;
Danial, 2008). The phosphorylation status of Bad is associated with glucokinase activity;
glucose deprivation results in dephosphorylation of Bad and Bad-dependent cell death
(Danial et al., 2003; Danial, 2008). These findings highlight the role that mitochondrial
Bad plays in coordinating glucose metabolism and the apoptotic machinery. Whether
the phosphorylation status of mitochondrial Bad affects the mode of glucose
metabolism in ischemic intestines requires further investigation.
Recent reports indicated that the IκBα/NFκB pathway is involved in anti-apoptotic
events in intestinal epithelial cells against various pathogenic stimuli (Potoka et al.,
2000; Egan et al., 2004; Nenci et al., 2007). However, our results demonstrated that
instillation of enteric glucose decreased the levels of phosphorylated IκBα in ischemic
guts. Despite links between Akt and IκBα/NFκB pathways in the mechanism of cell
survival (Chang et al., 2003; Dan et al., 2008; Bai et al., 2009), the IκBα/NFκB pathway
is not involved in the glucose-mediated anti-apoptotic signaling in intestinal epithelium.
The upregulation of phospho-IκBα may be partly responsible for proinflammatory
cytokine production after ischemic challenge. Overall, we demonstrated that SGLT1
glucose uptake in intestinal epithelial cells activated anti-apoptotic pathways that
involved PI3K/Akt signaling and increased phosphorylation of mTOR, Bad and
FoxO1/3a.
Accumulating data supports the notion that SGLT1 orchestrates a number of
fundamental cellular processes besides its canonical absorptive function. Previous
reports showed that activation of SGLT1 induced the recruitment of GLUT2 to the
brush border membrane for diffusive glucose transport via a PKCβ-dependent pathway
(Kellett et al., 2000; Kellett, 2001). Others documented that cotransport of Na+ with
glucose triggered the activation of Akt and phosphorylation of cytoskeleton-associated
ezrin, leading to recruitment of NHE to apical membrane that facilitated absorption of
sodium and hydrogen in intestines (Zhao et al., 2004; Shiue et al., 2005). A recent study
by Palazzo et al. demonstrated that activation of SGLT1 suppressed bacterial
LPS-induced NFκB signaling in intestinal epithelial cells, and suggested that SGLT1
plays a novel immunomodulatory role (Palazzo et al., 2008). Moreover, oral ingestion
but not intraperitoneal administration of glucose attenuated proinflammatory cytokine
production and protected endotoxemic mice from lethal septic shock (Palazzo et al.,
2008). Clinically, oral rehydration therapy that targets SGLT1 to drive passive diffusion
of water is a widely-used supportive therapy for diarrheal patients. Early enteral
nutrition (EN) is advocated for patients with multiple pathologies for its known benefit
in lowering the risk of septic complications compared to parenteral supplementation.
One area that has gained much attention in nutrition therapy nowadays is preoperative
oral carbohydrate loading (CHO) (Fearon et al., 2005; Martindale et al., 2006). This
novel concept involves giving patients an isotonic carbohydrate solution at midnight the
day before surgery and 2-3 hours preoperatively, in contrast to the general routine of
overnight fasting before surgery. Patients given CHO showed reduced postoperative
insulin resistance and shorter hospital stay after colorectal resection (Soop et al., 2004).
The advantage of early EN and CHO may also involve SGLT1-mediated nutritive and
non-nutritive functions.
The second part of our study demonstrates a novel mechanism through which
glycolytic pyruvate confers resistance to RIP-dependent necroptosis in hypoxic
colorectal carcinoma via mitochondrial superoxide scavenging. Despite long-standing
observation of cell necrosis in the hypoxic core of colorectal tumors, there is an
apparent lack of knowledge on its molecular mechanisms. In our study, we
demonstrated that oxygen and glucose deprivation induced RIP-1/3 signaling and
morphological necrotic features, i.e. rupture of plasma membrane, 8 to 24 hours after
the onset of challenge in colorectal carcinoma cells. Recent studies from other
laboratories have also showed necroptosis in intestinal epithelial cells in models of
chronic intestinal inflammation in gene-deficient mice (Gunther et al., 2011; Welz et al.,
2011). Although there is no data on the timing of necrotic death caused by hypoxic
stress in normal human epithelial cells, rapid villous necrosis in jejunum and colon was
found in rats after 60 minutes of hemorrhagic shock or 40 minutes of mesenteric
ischemia (Chang et al., 2005; Higa et al., 2007), showing that normal cells are more
sensitive to oxygen and nutrient deprivation compared to cancer cells. It is noteworthy
that normoxic controls in glucose-free media displayed no sign of necroptosis,
indicating that glucose deprivation alone did not trigger necrotic death.
Mitochondrial dysfunctions and free radical generation have also been implicated
in the necrotic process caused by cytotoxic agents.(Goossens et al., 1995; Temkin et al.,
2006; Zhang et al., 2009). The transient mitochondrial hyperpolarization observed in
our study may reflect a temporal reversal of electron transport chain ATP synthase
activity due to a decline in intracellular oxygen, leading to adverse proton pumping
against the electrochemical gradient to intermembranous spaces. Moreover, the final
mitochondrial potential collapse correlated well with organelle swelling at later time
points (16-24 hrs) of hypoxia. Interestingly, we identified high levels of mitochondrial
superoxide production prior to plasma membrane disintegration in hypoxic colonic
carcinoma cells. This seemingly paradoxical situation of ROS emission in hypoxia has
also been previously documented in cardiomyocytes after infarction (Becker et al., 1999;
Camara et al., 2007). The generation of mitochondrial ROS has been suggested to be
caused by electron leak to oxygen in the respiratory chain complexes whereby low
levels of oxygen remain inside cells at the early stage of hypoxic challenge (Turrens,
2003). By using antioxidants, we demonstrated that mitochondria-derived superoxide
plays a critical role in hypoxia-induced necrotic mechanisms and is upstream of RIP
signals.
We further showed that GLUT-dependent glucose uptake and glycolytic
metabolism inhibits hypoxia-induced RIP signaling and necrotic features in colorectal
cancer cells. Several studies have implicated that glycolytically generated ATP may
restore energy supply in cancer cells and act as a key factor for glucose-mediated death
resistance against hypoxia and genotoxic agents (Xu et al., 2005; Zhou et al., 2012).
Recent data in activated macrophages and B lymphocytes have demonstrated that
glycolytic ATP promotes cell survival via maintenance of transmembrane potential of
defective mitochondria that are unable to generate ATP (Dufort et al., 2007; Garedew et
al., 2010). Here, we focused on the role of glycolytic pyruvate, and examined if
cell-permeable derivative may lead to anti-necrotic effects. We demonstrated that
hypoxia-induced necroptosis (i.e. LDH leakage, RIP activation, and morphological
damage) was reverted by pyruvate, however, the resistance was uncoupled with
restoration of cellular energy and mitochondrial potential. Another line of evidence
showed that glucose addition reduced the mitochondrial ROS levels but did not alter the
redox enzyme activity. The aforementioned data indicate that a non-enzymatic free
radical scavenging mechanism, such as those reported with pyruvate, may contribute to
death resistance. Indeed, our results showed that supplementation of pyruvate
significantly reduced the mitochondrial superoxide levels in hypoxic cells in an
ATP-independent manner. It is noteworthy that glucose-mediated recovery of
mitochondrial transmembrane potential in hypoxic cells is not dependent on pyruvate
but may rely on ATP. Taken together, glycolytic metabolism may promote hypoxic cell
survival by a two-fold mechanism including scavenging of mitochondrial superoxide by
pyruvate and maintenance of mitochondrial potential by ATP.
We confirmed that presence of glucose during hypoxic challenge did not ablate
oxygen deprivation stress as evidenced by the nuclear translocation of HIF1α. The
HIF1-targeted upregulation of GLUTs led to enhanced glucose uptake and glycolytic
metabolism, serving as a positive cycle to promote cell survival. Normal human small
intestinal epithelial cells express SGLT1 on apical membrane and GLUT2 on basolateral
membrane for luminal-to-serosal flux of dietary glucose absorption (Yu et al., 2005; Yu
et al., 2008). In contrast to normal colonocytes, human colorectal carcinoma specimens
abnormally display SGLT1 and GLUT isoforms 1-4 on the apical membrane or
cytoplasm of tumor cells (Godoy et al., 2006; Guo et al., 2011; de Wit et al., 2012).
Previous studies have indicated that transcriptional regulation of GLUT1 is mediated by
HIF1 binding to a nucleotide sequence corresponding to a hypoxia-responsive element
in promoter regions of GLUT1 gene, following oxygen deprivation (Ouiddir et al., 1999;
Hayashi et al., 2004).
Other than glucose transporters (GLUT1 and GLUT3), a large number of genes
encoding for glycolytic enzymes, angiogenic growth factors, cell survival and
proliferative signals, and prolyl hydroxylases (PHD) are also upregulated by HIF1
(Denko, 2008; Chiacchiera et al., 2009; Marin-Hernandez et al., 2009). In our study, a
transient drop of GLUTs expression was observed after 16 hrs but recovered after 24 hrs
of hypoxia. The temporary reduction of GLUTs expression may be due to the regulatory
loop of HIF1α/PHD. Under normoxic conditions, PHD-mediated hydroxylation of
proline residues on HIF1α permits its ubiquitination followed by proteosomal
degradation. Hypoxia sensing downregulates PHD activity and thus, stabilizes HIF1α
levels, leading to its nuclear translocation and downstream transcription of target genes
(Jaakkola et al., 2001; Kondo et al., 2006). Paradoxically, PHDs are themselves target
genes of HIF1 transcription, indicating a negative feedback mechanism (Marxsen et al.,
2004; Stiehl et al., 2006). In addition, HIF1α degrades within 8-12 hrs without de novo
synthesis (Millonig et al., 2009). Therefore, we speculate that the fluctuation of GLUTs
expression after the onset of hypoxia may reflect the HIF1α/PHD loop. Experiments to
address the temporal relationship, cellular distribution, and pathophysiological
significance of various isoforms of glucose transporters by HIF1 in colonic carcinoma
are currently under progress.
Angiogenic pathways and mediators have been extensively studied as potential
anti-cancer drugs for several decades. In some cases, patients fail to respond to
antiangiogenic agents or simply develop drug resistance. Tumors may display invasive
metastatic transition in response to the increasing hypoxic microenvironment by cancer
treatment (Zeng et al., 2010; Rapisarda et al., 2012). Here, our data show a critical role
of glycolytic pyruvate in death resistance and underscore site-specific glucose
transporters and metabolic pathways as potential candidates for the development of
novel cancer killing strategies. The resistance to antiangiogesis or hypoxic stress may be
overcome by combinational strategies with glucose- or pyruvate-targeted therapy. In
summary, the second part of the studies demonstrated glycolytic metabolism inhibits
hypoxia-induced RIP-dependentnecroptosis in colonic cancer cells by suppression of
mitochondrial ROS (Figure 24).