**3.3. Activation of protein kinase C (PKC)**

PKCs are a family of at least 11 isoforms that are widely distributed in mammalian tissues. The enzyme phosphorylates various target proteins. The activity of the classic isoforms is depend‐ ent on both Ca2+ ions and phosphatidylserine and is greatly enhanced by diacylglycerol (DAG) [53]. Intracellular hyperglycaemia increases the *de novo* synthesis of DAG from the glycolytic intermediatedihydroxyacetonephosphatebyreducingittoglycerol-3-phosphate andstepwise acylation[66].HyperglycaemiamayalsoactivatePKCisoforms indirectlythroughbothligation of AGE receptors[67] and increased activity of the polyol pathway [68], presumably by increasingreactiveoxygenspecies.OnesignificanteffectofPKCactivationisseeninthedecrease in the vasodilator producing endothelial nitric oxide (NO) synthase (eNOS), while the vasocon‐ strictor endothelin-1 is increased. Transforming growth factor- β and plasminogen activator inhibitor-1 are also increased [69].Abnormal activation of PKC has been implicated in the decreased glomerular production of nitric oxide induced by experimental diabetes [70], and in the decreased production of nitric oxide in smooth muscle cells that is induced by hyperglycae‐ mia [71]. Activation of PKC also contributes to increased microvascular matrix protein accumulation by inducing expression of TGF-b1, fibronectin and type IV collagen both in cultured mesangial cells [72] and in glomeruli of diabetic rats [66].

#### **3.4. Increased flux through the hexosamine pathway**

Glucose is one of the most largely used energy substrate in living cells. A fraction (2–3%) of the glucose entering the cell is converted into UDP–N-Acetyl Glucosamine (UDP-GlcNAc), through the hexosamine biosynthetic pathway (HBP). The level of UDPGlcNAc in the cell thus reflects the flux of glucose through this pathway [73]. In this pathway, fructose-6-phosphate is diverted from glycolysis to provide substrates for reactions that require UDP-*N* acetylglu‐ cosamine, such as proteoglycan synthesis and the formation of *O*-linked glycoproteins [23]. O-GlcNAcylation may affect the phosphorylation status of a protein, by regulating the phosphorylation of adjacent residues or by competing for the same serine or threonine residue (the so-called Yin-Yang mechanism), in which modification of a serine or threonine residue by either phosphorylation or O-GlcNAcylation differently affects the protein's function [73]. O-GlcNAcylations also regulate insulin signaling and seem to play an important part in the development of diabetes and its complications [74,75]. Inhibition of the rate-limiting enzyme in the conversion of glucose to glucosamine, glutamine:fructose-6-phosphate amidotransfer‐ ase (GFAT), blocks hyperglycaemia-induced increases in the transcription of TGF-α, TGF-β1. This pathway has been shown to play a role both in hyperglycaemia-induced abnormalities of glomerular cell gene expression and in hyperglycemia-induced cardiomyocyte dysfunction in cell culture [25,76].
