br Acknowledgements br Insulin is

Acknowledgements
Insulin is a vital peptide hormone with the exclusive capacity to regulate the blood glucose that entirely is secreting from pancreatic β cells (Kasai et al., 2014). The human insulin comprises 51 amino c-met residues, with a molecular mass of 5.808 kDa. INS, responsible for expression of human insulin is located on chromosome 11p15.5 (Fu et al., 2013; Komatsu et al., 2013). Insulin is synthesized as preproinsulin, which undergoes successive courses of processing, folding/unfolding and other modifications as well as packaging in the secretory vesicles to form mature insulin ready for releasing from pancreatic β cells (Weiss, 2009; Weiss et al., 2014) (Fig. 1).
Processing, folding and post-translational modifications of newly-translated preproinsulin to form insulin The primary product of INS translation by the ribosomes on rough endoplasmic reticulum (rER) is preproinsulin. During co-translational insertion into the lumen of ER, preproinsulin is converted to proinsulin through the removal of its N-terminal portion termed “signal peptide”(Weiss et al., 2014; Sun et al., 2015). Generally, efficacies of processing, folding and packaging of insulin in β cells are more significant than the other proteins. Usually, around 30% of the newly translated proteins in majority of the cells are degraded due to the errors in translation or post-translational modifications (e.g. folding and processing), whereas only less than 1% of the newly synthesized preproinsulin peptides do not successfully pass the checkpoints to yield the functional insulin and hence they undergo degradation (Gehart and Ricci, 2013; Schubert et al., 2000). Unfolded proinsulin refolds and gets three disulfide bounds (Fig. 1). However, our understanding about the folding mechanisms in the lumen of ER and Golgi apparatus are not so much (Weiss, 2009; Weiss et al., 2015). At the next step, two proinsulin peptides join and form homodimer through non-covalent interactions, ready to be packaging into transport vesicles to cis-Golgi (CG) destination.
Transportation from ER to Golgi apparatus Insulin peptides pass through various steps that are essential for insulin maturation from ER to plasma membrane. As mentioned, transport vesicles are responsible to deliver insulin peptides from ER to CG. Generally, trafficking of the transport vesicles between ER and Golgi apparatus is governing through two types of vesicles with different coating proteins termed as coatomers. Transport of vesicles from ER toward CG, termed as anterograde transport is mediated by COPII coated vesicles whereas transport from CG toward ER termed as the retrograde transport is mediated by COPI coated vesicles (Fig. 2, Fig. 3) (Cruz-Garcia et al., 2013; Fang et al., 2015). COPII-dependent ER exported vesicles play a vital role in insulin secretion (Fang et al., 2015). Translocation from cis to trans face of each cisternae and stacks of Golgi complex, is followed by developing secretory granules through budding at trans-Golgi network (TGN) face, and subsequent microtubule- and actin-based transport, vesicle priming, tethering, docking and eventually fusion with the plasma membrane (exocytosis) (Gehart and Ricci, 2013; Park and Loh, 2008). However, lesser portions of the secretory vesicles may also undergo an endocytosis, for another turn of exocytosis (re-exocytosis), which is commonly termed as kiss and run processing (Kasai et al., 2014). Within each cisternal stack, selective enzymatic modifications are carrying out on proinsulin molecules, during passing from cis-entry-face to trans-exit-face of Golgi complex apparatus. Consequently, proteolytically processed proinsulins converts to insulin, through of excising C-peptide and two extra dipeptides (Sun et al., 2015) (Fig. 1). C-peptide and insulin are both stored in secretory vesicles and co-released in equimolar concentrations, in response to increased glucose levels (Venugopal and Jialal, 2018). Each insulin monomer is composed of A and B chains that are linked by two disulfide bonds (Fig. 1) (Weiss, 2009). Accordingly, maturation stages take place during traveling of insulin containing vesicles from CG to TGN, and then toward plasma membrane for insulin secretion (Weiss, 2009; Weiss et al., 2014). Pancreatic β cells possess extraordinary amount of zinc cation (Zn2+), which is required for process of insulin biosynthesis, storage and maturation of insulin secretory granules and insulin secretion (Bosco et al., 2014). Six insulin chains (arranged as three heterodimers) are joined with two Zn2+ ions (or four Zn2+ in the presence of high chloride concentrations) to form rhombohedral hexameric insulin cubic crystals (Fu et al., 2013; Weiss et al., 2014) (Fig. 7). Mitochondria, ER and Golgi serve as intracellular Zn2+ reservoir compartments. However, the free Zn2+ concentration in the post-Golgi insulin granules is much higher than ER, presumably due to buffering influence of calreticulin proteins with multiple zinc binding sites that are abundantly in ER (Weiss et al., 2014; Tan et al., 2006; Lu et al., 2016). In the vesicles, zinc transporter member 8 (ZnT8 or solute carrier family 30/SLC30A8) is responsible for accumulation of Zn2+ (Davidson and Wenzlau, 2014). Indeed, inside of the secretory granules at the presence of Zn2+, insulin concentration reaches to approximately 40 mM as insoluble crystalline hexameric form while it is at micromolar concentration or lower after secretion, in portal vein or blood circulation (Fu et al., 2013).