Human gut mucosa serves as a barrier between external environment and internal milieu.
The mucosal barrier includes a single layer of columnar epithelial cells connected by
apical tight junctional complex, a hydrated gel layer formed by mucin covering the
epithelial cells, and immune cells in lamina propria (Turner, 2009). The epithelial cells
that prevents abnormal influx of noxious luminal contents is also responsible for
nutrient uptake through apically expressed transporters (Laukoetter et al., 2006). In
physiological conditions, commensal bacteria (~100 trillions) are restricted in the gut
lumen and separated from the lamina propria by epithelial cells and mucus layers.
However, abnormal translocation of bacteria to sterile extraintestinal visceral organs
may occur under inflammatory situation and metabolic stress, resulting in sepsis and
life-threatening complications(Yu et al., 2012).
1.1 Physiological hypoxia in the gut
The gut epithelium is anatomically positioned between an anaerobic lumen and a highly
vascularized lamina propria. Despite rich blood supply to the intestine, a steep oxygen
gradient was found along the crypt-villus axis where low oxygen concentration was
seen at the villous tips. The term “physiological hypoxia” was used to describe gut
epithelial cells under an extremely low level of oxygenation acquire regulatory
mechanisms to adapt to the shifted oxygen gradient (Colgan et al., 2010).
1.2 Pathological hypoxia in the gut
Mesenteric ischemia/reperfusion (I/R) is seen in patients with mesenteric artery
embolism, strangulated hernias, and in neonatal necrotizing enterocolitis. It also seen in
the case of major abdominal and vascular surgery or collapse of systemic circulation
occurred by traumatic or hemorrhage shock (Mallick et al., 2004; Yasuhara, 2005; Yu,
2010). The disruption of blood supply during ischemia reduces oxygen and nutrient
supply to mucosal cells. The production of free radicals stimulated by subsequent
restoration of blood flow causes further oxidative stress, which increases inflammation
and aggravates tissue damage (Nilsson et al., 1994; Mallick et al., 2004; Yu, 2010). One
of the major consequences of mesenteric I/R is heightened mucosal cell death and gut
barrier damage, leading to enteric bacterial translocation and septic complications. Both
modes of cell death, apoptosis and necrosis, were observed in the gut mucosa upon
mesenteric I/R injury (Wu et al., 2004; Aban et al., 2005; Chang et al., 2005).
1.3 Hypoxia in colorectal cancer
Rapid cell growth and poorly formed vasculature characterized in malignant cancers are
often accompanied by a hypoxic microenvironment in the tumor core (Brown and
Giaccia 1998; Milosevic, Fyles et al. 2004). Necrotic cell death has been reported in the
hypoxic core in colorectal tumors due to depletion of oxygen and nutrients, However,
malignant cells may develop adaptive mechanisms by metabolic reprogramming to
survive under extremely deprived conditions, which is also related to tumor resistance
to anticancer drugs (Hawley et al., 1992).
1.4 Modes of cell death
Cell apoptotis (programmed cell death) is characterized by caspase-dependent signaling
pathways and nuclear DNA fragmentation. The signaling pathway of apoptosis may be
triggered extrinsically via Fas or TNF receptors, or intrinsically via mitochondrial
pathways, leading to caspase-3 cleavage and ultimately, endonuclease activation for
oligonucleosome formation (Ramachandran et al., 2000). The Bcl-2 family (e.g.,
anti-apoptotic Bcl-2, Bcl-XL, and pro-apoptotic Bax) are important regulators of
mitochondrial-dependent apoptosis (Mayer et al., 2003). The mitochondrial undergo an
increase in membrane permeability and a reduction of membrane potential accompanied
by cytosolic release of cytochrome c. The release of cytochrome c from mitochondria
can be blocked by anti-apoptotic protein Bcl-2, while the translocation of pro-apoptotic
Bax from cytosol bound to mitochondria induces cytochrome c release during apoptosis.
The released cytochrome c binding to an apoptosis binding factor-1 forms apoptosomes
to activate caspase-9. (Shimizu et al., 1999; Zheng et al., 2004). Morphological changes,
such as cell shrinkage, membrane blebbing, and chromatin condensation, are seen in
cells that undergo apoptosis (Ramachandran et al., 2000).
On the other hand, necrotic cell death has been traditionally regarded as an
uncontrolled form of cell death. Cell necrosis is characterized by morphological
alteration, i.e. cytoplasmic swelling, subcellular organelle breakdown, loss of plasma
membrane integrity and release of cellular contents. Most research of necrotic death in
enterocytes is based on morphological and biochemical analysis (Chakrabarti et al.,
2003; Jilling et al., 2006; Higa et al., 2007; Kalischuk et al., 2007; Hunter et al., 2008;
Vincenti et al., 2010). Recent data from human intestinal epithelial HT-29 cells,
lymphocytes, monocytes and fibroblasts have indicated that signaling molecules such as
receptor-interacting proteins (RIPs) are involved in necrotic mechanisms triggered by
cytotoxic agents such as tumor necrosis factor (TNF) (Meurette et al., 2007; Declercq et
al., 2009; He et al., 2009). The formation of RIP1-RIP3 complex followed by auto- or
mutual phosphorylation of RIP proteins is associated with mitochondrial bioenergetic
alterations and intracellular ATP decline in cells undergoing programmed necrosis
(Temkin et al., 2006; Cho et al., 2009; Zhang et al., 2009; Berghe et al., 2010). Reactive
oxygen species (ROS) derived from the mitochondrial respiratory chain has also been
implicated in necrotic mechanisms (Declercq et al., 2009; Zhang et al., 2009). To date,
the involvement and cascading order of these necrotic effectors in ischemic or hypoxic
enterocytes remain unclear.
1.5 Role of glucose in death resistance
Several lines of evidence indicated that presence of glucose may inhibit cell death in
intestinal epithelial cells. Previous studies demonstrated that sodium-dependent glucose
uptake protects intestinal cells against apoptosis caused by bacterial lipopolysaccharide
and parasitic products (Yu et al., 2005; Yu et al., 2006; Yu et al., 2008). Increased levels
of anti-apoptotic Bcl-2 and Bcl-xL were noted following activation of apically
expressed sodium-glucose transporter (SGLT)-1 (Yu et al., 2005). A more recent report
showed that initiation of Na+-glucose cotransport by SGLT1 triggered downstream Akt
signaling for regulation of cellular functions (Shiue et al., 2005). A critical role of
PI3K/Akt signaling in resistance to apoptosis has been reported in multiple cell types
(Chang et al., 2003; Bouchard et al., 2008). Studies have shown that PI3K/Akt
phosphorylates and thereby inactivates Bad (a pro-apoptotic mitochondrial Bcl-2 family
protein) or phosphorylates mTOR that then activates p70S6K to phosphorylate Bad
(Chang et al., 2003; Bouchard et al., 2008). Other downstream targets of Akt that are
involved in regulating cell survival and cell cycle progression include IκBα/NFκB,
glycogen synthase kinase 3 (GSK3), and Forkhead family of transcription factors
(FoxOs) (Liang et al., 2003; Urbich et al., 2005; Dan et al., 2008; Bai et al., 2009).
One of the pro-survival mechanisms in tumor cells is by metabolic reprogramming
for high levels of glycolysis. The term “Warburg’s effect” describes the phenomenon
that over fifty percent of cellular energy is produced by glycolysis in the tumor in
compared to that ninety percent of energy is produced by mitochondrial oxidative
phosphorylation in normal tissues (Warburg, 1956). Moreover, a large body of evidence
shows that upregulation of glycolytic enzymes and glucose transporters (GLUTs) are
linked to transcription activity of hypoxia inducible factor (HIF) 1α triggered by low
oxygen condition (Yeh et al., 2008; Chiacchiera et al., 2009; Marin-Hernandez et al.,
2009). Recent reports documented that HIF1α and GLUT-1 colocalize at peri-necrotic
regions in human colorectal tumors (Greijer et al., 2008; Airley et al., 2010), suggesting
that glucose metabolism may confer anti-necrotic resistance to hypoxic stress. Glucose
is catalyzed to ATP and pyruvate by a cascade of glycolytic enzymes, such as
glucokinase and glyceraldehyde-3-phosphate dehydrogenase (GPD)(Fleming et al.,
1997). The final glycolytic product pyruvate is also the starting substrate for
tricarboxylic acid (TCA) cycle after being transported across inner mitochondrial
membrane by mitochondrial pyruvate carrier (MPC) (Hildyard et al., 2005; Herzig et al.,
2012). Aside from its critical role as the link between glycolysis and mitochondrial
respiration, pyruvate also scavenges ROS through a non-enzymatic reaction (Kao et al.,
2010). Numerous studies have suggested that chemoresistance may be due in part to
glycolytic ATP as a preferential energy source for promoting cancer cell survival (Xu et
al., 2005), (Zhou et al., 2012).
To date, whether enteric glucose uptake prevents epithelial cell death caused by
mesenteric I/R or hypoxic stress remains unclear. Moreover, molecular mechanisms of
glucose-mediated resistance to cell apoptosis and necrosis have yet to be explored.
1.6. Aim of the study
In the current study, we explored whether enteral glucose confer anti-death signaling
against hypoxic stress in rat intestinal I/R or in human colorectal cancer line.