Role of Protein Kinase C in Diabetic Complications

Diabetes, a chronic metabolic disorder, is a major threat nowadays worldwide (Shaw et al., 2010). The resultant hyperglycemia has been shown to be responsible for major diabetic complications like myopathy, retinopathy, nephropathy, and neuropathy which occur after a few years of onset of diabetes (Geraldes and King, 2010). It is believed that intra and extracellular changes by hyperglycemia can induce the signal transduction pathways which lead to the dysfunction of gene expression and proteins (Peppa et al., 2002). One of the most studied pathways; the diacylglycerol-Protein kinase C (DAG-PKC) pathway is activated by hyperglycemia in diabetes (Giacco and Brownlee, 2010). In this review, the functions of PKC and interconnected signaling changes in the growth of diabetic complications have been demonstrated.


Introduction
Diabetes, a chronic metabolic disorder, is a major threat nowadays worldwide (Shaw et al., 2010). The resultant hyperglycemia has been shown to be responsible for major diabetic complications like myopathy, retinopathy, nephropathy, and neuropathy which occur after a few years of onset of diabetes (Geraldes and King, 2010). It is believed that intra and extracellular changes by hyperglycemia can induce the signal transduction pathways which lead to the dysfunction of gene expression and proteins (Peppa et al., 2002). One of the most studied pathways; the diacylglycerol-Protein kinase C (DAG-PKC) pathway is activated by hyperglycemia in diabetes (Giacco and Brownlee, 2010). In this review, the functions of PKC and interconnected signaling changes in the growth of diabetic complications have been demonstrated.

Protein Kinase C (PKC)
Serine/threonine kinases are ancestors of Protein kinase C that include 12 isoforms and are differentiated according to their domains that connect calcium or DAG; mutually they control the kinase activity (Schramm et al., 2012). On the basis of biochemical properties, PKC is divided into three types: conventional PKC, novel PKC, atypical PKC (Spiegel et al., 1996). Conventional PKC connects Ca 2+ and DAG, novel PKC binds only DAG but not Ca 2+ , and atypical PKC binds none of them (Spiegel et al., 1996). For phosphorylation, the correct isoforms are required for the activation of conventional and novel PKC and the existence of cofactors such as DAG and Ca 2+ (Schramm et al., 2012). Conventional and novel PKC are elevated by diacylglycerol (DAG), which in turn is amplified by the hyperglycemia in diabetes (Spiegel et al., 1996). When they are correctly phosphorylated, an increase in Ca 2+ or DAG will rapidly allow cells to translocate (Gonçalves, 2013). Quick and temporary increases of Ca 2+ levels and DAG levels are frequently induced by cytokines during the activation of phospholipase C. Activation of PKC requires a sustained decrease of DAG, which involves the activated phospholipase D/C or DAGs de novo synthesis. All the above-mentioned pathways possibly participate in the activation of the DAG-PKC cascade in hyperglycemic and diabetic conditions (Michael et al., 2017).

Hyperglycemia Activates PKC
Diabetic state or hyperglycemia is responsible for the synthesis of a higher level of DAG via an elevation in the formation of glycolytic intermediate (dihydroxyacetone phosphate), which activates PKC (Tripathi and Srivastav, 2006). The most studied pathway via which PKC induces diabetes is the DAG-PKC pathway (Nilsson, 2016). DAG is induced by hyperglycemia and leads to activation of different isoforms of PKC (Tripathi and Srivastav, 2006). de novo DAG synthesis can also be increased when glucose concentration is high during different metabolic pathways (Noh and King, 2007). Another mechanism by which DAG synthesis can be increased is by inhibiting the glyceraldehyde-3-phosphate dehydrogenase which is a glycolytic enzyme and results in upregulation of dihydroxyacetone phosphate to DAG by triggering higher metabolites from glycolysis into pathways of glucose (Nilsson, 2016). In diabetes, no changes in DAG levels were seen in a peripheral nervous system and central nervous system, but the disturbance in DAG levels was reported in vascular tissues and nonvascular tissues (Adibhatla et al., 2008).

Activation of PKC Leads to Microvascular Complications
Hyperglycemia leads to diabetic complications and shows harmful effects on the human body. These complications are also due to higher levels of DAG-PKC, oxidative stress and an increased amount of glycation. Microvascular complications include retinopathy, nephropathy, neuropathy, and myopathy (Sheetz and King, 2002).

Retinopathy
Nowadays, many mechanistic approaches have been found by which vascular tissue damage is induced by hyperglycemia. But the major pathway which leads to microvascular changes is due to elevation in levels of DAG-PKC (Karasu, 2010). The most significant regulatory system for activation and deactivation of the receptor pathway is pursued by adding or subtracting the phosphate group in intracellular protein, via phosphatases and kinases (Carri, 2014). Phosphorylation of many proteins at threonine or serine activates the physiological responses of the cascade which are mediated by PKC. Activation of PKC induces some changes i.e retinal leakage, permeability in endothelial, loss of capillary pericytes (Carri, 2014). In the initial stages of diabetic retinopathy, there is a loss of pericyte in retina around the capillaries followed by the weakening of capillary walls and which cause the leakage of fluid due to the high permeability of walls (Nassar et al., 2007). Recent studies demonstrate that two isoforms of PKC were activated i.e PKCβ and PKCδ through hyperglycemia. Further, these isoforms follow two different approaches; PKCβ will lead to cellular growth whereas PKCδ will give rise to cellular apoptosis (Nagpal et al., 2007). PKCβ after activation affects the vascular endothelial growth factor due to which the permeability of capillary walls increases and blood flow decreases which leads to the dysfunctioning of endothelial and cause macular edema (Dirks et al., 2006). But PKCδ after activation shows two distinct pathways which ultimately lead to cellular apoptosis: 1) increase in production of reactive oxygen species and NF-κB activity which in turn activate the caspase 2) by upregulating the SHP-1 (protein tyrosine phosphatase) which will decrease the survival signaling pathway of platelet-derived growth factor both of these different approaches results in loss of pericyte as shown below in (Nassar et al., 2007)

Nephropathy
All the causes for risk factor and cardiovascular mortality rate worldwide are due to diabetic nephropathy which is the foremost reason for the end-stage renal disease (Dirks et al., 2006). Due to hyperglycemia, there is an increase in glomerular filtration rate in the kidney which elevates the glomerular filtration pressure (Tonneijck et al., 2017). Several approaches have been studied about the elevation of glomerular rate & pressure together with improved production of prostaglandin and angiotensin II. But the most important pathway leading to an increase in angiotensin II and in vasodilatory prostaglandin is via the DAG-PKC (Kaschina and Unger, 2003). In diabetes, hyperglycemia results in the formation of prostaglandin II which could be a cause of the activation of PKC. Another factor that results in the enhancement of the filtration rate is a higher level of nitric oxide. In diabetes, high amount of nitric oxide metabolites i.e. NO 2 and NO 3 is excreted through urine due to a high level of nitric oxide present in mesangial cell and in inducible nitric oxide synthase (Williamson et al., 1993). But in hyperglycemia both i.e. formation of nitric oxide and inducible nitric oxide synthase can be initiated by an agonist of PKC and blocked by its inhibitor which shows that in PKC induced diabetes there is an increase in levels of NO synthase (Caldwell et al ., 2003). Previous studies demonstrated that there was a decrease in the production of nitric oxide and glomerular cGMP in glomeruli of diabetic rats which was restored by PKC inhibitor. Thus, this shows that possibly it might be due to an increase in the level of glucose-induced activated PKC which may supervise renal hemodynamics by elevating or declining the levels of nitric oxide production based on the sort of cells and period of hyperglycemia (Kaschina and Unger, 2003). Nephropathic studies also show that in glomerular mesangium there was an accumulation of extracellular matrix. This showed that there was an elevation in the oxidative stress due to hyperglycemia. In renal glomeruli, PKC activities were increased due to hyperglycemia which in turn upregulates many isoforms of the NADPH oxidases to generate too many oxidants (Marsigliante et al., 2001). This increased level of PKC leading to the activation of MAPK which upregulates the fibrotic growth factors. Several studies show that growth factors play a major function in the accumulation of ECM (Khera, 2006).
Current studies have shown that there is an increase in vascular endothelial growth factor (VEGF) in the glomerulus. PKC is known to regulate activator protein-1 (AP-1), which promotes the binding of AP-1 to the promoter region of the VEGF gene. AGEs are also able to activate PKC, which further increase the expression of VEGF and this activated PKC is also responsible for many natural effects of VEGF (Prabhakar, 2004).

Neuropathy
Diabetes can cause nerve injury. Burning, numbness, pain in the feets all are the symptoms of diabetic neuropathy ( Figure 2) (Argoff et al., 2006). It has been demonstrated that in nerves there is an existence of PKC-α, PKC-β1, PKC-β2, PKC-γ, PKC-δ and PKC-ε isoforms which were confirmed by immunochemical analysis (Cameron et al., 2005). Whereas the exact mechanisms of their action are not clear, but currently it is believed that these factors lead to reduced Na + -K + -ATPase activity and vasoconstriction, reduced endoneural blood flow (Martin et al., 2003). Diabetic rats have shown a decline in the activity of PKC in sciatic nerve tissue. There was also a reduction of Na + -K + -ATPase levels due to the involvement of PKC which could be due to a decline in nerve regeneration and conduction (Evcimen and King, 2007). It was also demonstrated that in diabetic mice there was a reduction in membrane-associated PKC activity and when treated with PKC inhibitor sustained Na + -K + -ATPase action was observed (Marsigliante et al., 2001). Preclinical study also suggested that specific PKC inhibitors were used to recover the neural function i.e inhibition of PKC in nerves boost the blood flow (Brooks et al., 2008) (Figure 2).

Cardiovascular Disease
The diabetic patient suffers from diastolic dysfunction, interstitial fibrosis, hypertension, and congestive heart failure (Naito et al., 2001). In hyperglycemia, activated PKC gives rise to cardiomyopathy via inhibiting metabolic actions of insulin. In the myocardium, insulin loss is coupled with lower basal expression factor-1 of hypoxia, which strikes VEGF actions in the myocardium (Valko et al., 2007). In comparison to normal patients other than diabetes, diabetic patients with cardiomyopathy have shown declined expression of VEGF. Immunoblotting showed an increase in levels of PKC-β1 and PKC-β2 in the failed heart of humans as compared to a healthy live heart (Geraldes and King, 2010). Previous studies show that when the human heart was treated with LY333531 which is a PKC-β inhibitor then it leads to the inactivation of PKC (Sakata et al., 2015). In hyperglycemic condition, there is an increase in levels of PKC-α, PKC-β1 and PKC-β2 isoforms in the heart (Arimura et al., 2004). In diabetic heart, both PKC-β inhibitor and ACE improve the gene profile & PKC activity (Naito et al., 2001).The isoforms of PKC i.e. PKC-α and PKC-ε also have important functions in cardiomyopathy and elevate the contractility of the heart leading to less vulnerability to heart attacks (Arimura et al., 2004).

PKC Inhibitors
The role of PKC in the development of diabetic complications has become an area of interest for research (Gennas et al., 2009). There are many specific and non-specific compounds that have inhibitory action for PKC (Khera, 2006). Nonspecific inhibitors are not in use because clinically they have been proven as toxic while specific inhibitors have shown great therapeutic use (Khera, 2006). The function of different PKC isoforms was demonstrated by observing the binding of proteins to specific sites after activation and translocation (Aielleo, 2002). Several PKC inhibitors that are being studied are Midostaurin (PKC412, CGS41251, N-benzoyl staurosporine), UCN-01 (KW-2401, NSC-638850), Lestaurtinib (CEP-701, KT-5555), Ro-31-8220 and Go6976 (Shen et al., 2003;Wang et al., 2008;Wang et al., 2009. Ro-32-0432, PKC-α inhibitor, was found to be effective in AGE mediated damage in diabetic nephropathy. Ruboxistaurin mesylate (LY333531), a potent PKCβII inhibitor, has been evaluated in clinical studies for diabetic complications (Vinik, 2005;Joy et al., 2005;Aiello, et al., 2006). Several clinical benefits such as reduced retinal vascular leakage and improved visual acuity in diabetic macular edema, however, phase III clinical endpoints did not reproduce the desired results (American Diabetes Association, 2005). Vitamin E, a selective inhibitor, also has an inhibitory effect on the DAG-PKC but does not inhibit PKC directly (Aielleo, 2002). Vitamin E has shown a great effect in nephropathy and retinopathy (Kizub et al., 2014). (Table 1) Modulation of PKC activity for clinical drug development is vital as isozyme specific disruptions in PKC activity have been identified in human disease states such as diabetes (Nishikawa et al., 2000, Geraldes et al., 2010. Therefore, understanding the basic biology of the disease and multiple pathways involved in the progression of the disease is essential for therapeutic progress in this field. As shown in table 1, various preclinical studies conducted on PKC inhibitors in diabetes and related complications have been summarised. The role of protein kinase C activation and the vascular complications of diabetes.

Preclinical Studies Exploring The Role of PKC in Diabetes
Evcimen and King studied that activated PKC results in diabetic retinopathy, nephropathy, and cardiovascular disease. So, to cure these complications, use of PKC inhibitors has been introduced with great therapeutics approaches (Evcimen and King, 2007).

The role of protein kinase C activation in diabetic nephropathy
Noh and king concluded that PKC plays a major role in diabetes nephropathy (Noh and king, 2007) 4

Ruboxistaurin: PKC-β inhibition for complications of diabetes
Danis and Sheetz demonstrated that activated PKC plays a major role in the development of diabetic complications and they concluded the beneficial effect of PKC-β inhibitor against complications (Danis and Sheetz, 2009).

Activation of Protein Kinase C Isoforms and Its Impact on Diabetic Complications
Geraldes and King presented that one of the chronic adverse effects of hyperglycemia is the activation of PKC which cause vascular dysfunction and ultimately worsen the diabetic complications (Geraldes and King, 2010 et al., 2002).

Clinical Perspective of PKC
In leukemia patients, phase I studies were conducted to see the positive outcomes of paramethoxymethyamphetamine (PMA) and additionally PMA in combination with 25-dihydroxy vitamin D3, or sodium butyrate was also tested on patients. The results suggested that drugs given in combination were more effective as compared to PMA alone (Diaz et al., 2015). Bryostatin 1 has been used in clinical trials which is one of the PKC modulators and downregulates some PKC isozymes. The anti-tumour activity was shown by Bryostatin 1 in phase I studies but Phase II studies have not yielded clinically usefull results as it was expected. Phase II studies combining bryrostatin 1 with other cytotoxic agents have not shown any efficacy and so it has failed to move in further trials (Koya, 2014). As the same case with safingol which is a specific PKC inhibitor and has shown cytotoxic effects when given in combination with conventional chemotherapy agents where as safingol alone has a nominal effect on tumor cell growth (   (Chua et al., 2005)

Conclusion
In this review, we studied hyperglycemia is a key factor responsible for diabetic complications which shows harmful effects on the human body and leads to activation of PKC. Microvascular complications like retinopathy, nephropathy, neuropathy, and myopathy occur due to chronic pathological condition leading to a higher level of DAG-PKC, oxidative stress and an increased amount of glycation. Hence, it is concluded that PKC is a therapeutic approach for treating vascular diabetic complications as clinical trials have shown beneficial effects of selective PKC inhibitors in diabetic complications.