Because autophagy is distinguished by the formation of
double-membraned autophagosomes or vacuoles, the investigation of autophagy is
mainly based on the detection of autophagosomes or autophagy vacuoles. Another
important method to demonstrate autophagy is through the detection of
autolysosomes, which are formed by the fusion of autophagosomes and lysosomes
and represent one type of acidic vesicular organelle (AVO).
Listed below are some of the most common methods for detecting autophagy (Chen
et al., 2010).
Autophagy was first
described by transmission electron microscopy (TEM) and TEM remains one of the
most widely used and sensitive techniques to visualize the existence of
autophagic vesicles (Eskelinen, 2008a).
autophagy qualitatively, as early autophagic compartments (autophagosomes)
containing morphologically intact cytosol or organelles, or as late,
degradative autophagic structures (autolysosomes) containing partially degraded
cytoplasmic as well as organelle material (Eskelinen, 2008b).
The second widely used
method for autophagy verification is the detection and quantification of green
fluorescent protein-LC3 puncta (GFP-LC3) by fluorescence microscopy. Under
normal conditions, LC3 is distributed evenly throughout the cytoplasm. During
autophagy, LC3 is triggered to autophagosome membranes. In this detection method, cDNA vectors
containing GFP either alone or combined with LC3 (GFP-LC3) are transfected into
cells by using lipofectamine-based techniques. Next, the transfected cells are
treated with a stress for several hours to several days. Because GFP-LC3
protein conjugates are triggered to autophagosome membranes during autophagy, a
characteristic punctuate distribution results. When the cells are observed
under a fluorescence microscope, the occurrence of GFP-LC3 puncta can be
quantified (Chen et al., 2010).
The possibly the most
reliable method for detection of autophagy is to measure LC3-II protein. Based
on the importance of LC3 processing for autophagosome formation and function,
antibody to LC3-I and LC3-II is used in western blotting techniques to detect
autophagy. LC3-I and LC3-II can be easily distinguished based on their distinctive
mobility in SDS–PAGE. In spite of increased molecular weight than LC3-I, LC3-II
migrates more rapidly in SDS–PAGE compared to LC3-I, likely due to higher
hydrophobicity associated with the PE group (Tanida et al., 2008).
tends to be more sensitive than LC3?I in immunoblotting, simple comparison of LC3?I and LC3?II, or summation of LC3?I and LC3?II, is not convenient. Rather,
comparison of the amount of LC3?II among samples is likely to be a more accurate method. It should
be noted that LC3 is expressed as three isoforms in mammalian cells, LC3A, LC3B
and LC3C, but only LC3B correlates with increased levels of autophagic
vesicles, and therefore it is recommended to use anti-LC3B antibodies for
analysis (Barth et al., 2010).
Because of the
importance of autophagy in mammalian physiology, it is therefore reasonable to
assume that autophagy impairment could contribute to human diseases. Defects in
autophagy have been associated with numerous neurodegenerative diseases and
have also been reported in liver and muscle diseases. Other pathological
situations such as T2DM and obesity, as well as inflammatory pathologies
have also been correlated with defective autophagy (Jing and Lim, 2012).
Because ?-cell loss in
human T2DM characterized by several histological features which are similar to
those in neurodegenerative diseases (Matveyenko et al., 2009) and
dysregulated autophagy has been supposed as one of the potential mechanisms of
the neurodegenerative disorders such as Alzheimer’s disease or Parkinson’s
disease, autophagy may also be contributed to the development of ?-cell changes
associated with T2DM (Winslow and Rubinsztein, 2008).
Autophagy and ?-Cell Homeostasis:
Even under normoglycemic
state, ?-cells are exposed to both oxidative and ER stress. Autophagy
plays an important role in maintaining normal ?-cell function by opposing these
stressors via a number of different pathways.
The main function of
pancreatic ?-cells is the controlling of glucose homeostasis. In response to
high glucose, exocytosis of insulin-containing secretory granules happens in
?-cells. To replenish the insulin lack, insulin translation should be stimulated.
Even under non-stimulatory glucose concentrations, insulin synthesis continues,
showing that constant protein synthesis and degradation happen in ?-cells. To
maintain secretory granules of ?-cells in an optimal state, aged ?-cell
granules should undergo degradation through autophagy (Uchizono et al.,
2007). The lysosomal degradation of
secretory granules is exerted in a specialized form of autophagy, termed
crinophagy (Orci et al., 1984). The crinophagic activity is
dependent on glucose levels; at low levels, crinophagy elevates and
intracellular insulin levels decrease, whereas at high glucose levels, insulin
degradation is blocked (Landstrom et al., 1988). Interestingly,
crinophagy is targeted specifically toward aged ? granules and acts as a
relatively long-term mechanism for keeping insulin stores at optimal levels,
while maintaining the secretory capacity (Uchizono et al., 2007).
The ongoing requirement of ?-cells
to synthesize large amounts of insulin to maintain euglycemia places a heavy
demand for protein folding on the ER. If allowed to accumulate, misfolded
proteins cause ER stress and will eventually aggregate within the cytosol
disrupting cell function. The UPR aims to counteract this by pausing protein
translation, degrading misfolded proteins and increasing generation of
molecular chaperones involved in protein folding. There is a
direct link between the UPR and autophagy, which plays a key role in the
degradation and clearance of misfolded proteins from the cytosol of ?-cells,
maintaining normal function (Lee and Ozcan, 2014).
Autophagy also maintains ?-cell
homeostasis by eliminating damaged or dysfunctional organelles. Normal
oxidative mitochondrial metabolism is central to glucose-stimulated insulin
secretion, as insulin secretion is dependent on a rise in the intracellular
ATP/ADP ratio (Barlow and Thomas, 2015). Therefore, mitochondrial
dysfunction, specifically abnormal respiratory chain activity, results in
defective insulin secretion. A specific form of autophagy, termed mitophagy,
acts to prevent accumulation of depolarized mitochondria and maintains optimal ?-cell
mitochondrial function (Twig et al., 2008).
Autophagy can induce cell
survival but under certain conditions it might contribute to the cell death. Most
evidence shows that autophagy is primarily a pro-survival rather than a
pro-death mechanism. However, it can also mediate cellular death depending on
cell context and specific circumstances (Kim and Lee, 2010).
Autophagy and ?-Cell Dysfunction:
A physiological role of
autophagy in mammalian biology has been showed in various animal models and impaired
autophagy probably plays an important role in the pathophysiology of a variety
of diseases including T2DM. An expected
consequence of dysfunctional autophagy is the accumulation of non-functional
organelles, such as mitochondria, which are the primary site of the production
of ROS (Gonzalez et al., 2011).
dysfunction, associated with increased ROS production, has been implicated as
mechanisms contributing to IR. Therefore, it is possible that deteriorated
autophagy may contribute to IR. Insulin and intracellular molecules such as
mTOR are well-known inhibitors of autophagy, whereas glucagon, a counter-regulatory
hormone of insulin, promotes autophagy. These observations support the possibility
that autophagy is involved in diabetes via its role in affecting hormone action
and organelle function. Other factors
such as lipotoxicity glucotoxicity may also be relevant in the induction of cell
stress and affect the physiological function of autophagy (Gonzalez et al.,
the pathogenesis of T2DM several events including glucotoxicity, lipotoxicity, increased
FFAs, islet amyloid polypeptide (IAPP), chronic low-grade ongoing inflammation
and oxidative stress can promote protein misfolding, mTORC1 hyperactivation and
reduced autophagy flux (Figure 1.17). Thereafter, protein misfolding
(for instance, proinsulin) promotes ER stress and eventually ?-cell death
occurs via apoptosis