Wednesday, 13 January 2010

CMB2002: PKA, PDE, HSL, cAMP, phosphorylation and perilipin

One of the questions I had during the CMB2002 feedback session was on PKA, hormone sensitive lipase (HSL), phosphorylation and perilipin, and how this all relates to the breakdown of triglycerides.

PKA phosphorylates HSL and perlipin. However, for PKA to do that it has to be active, that is, cAMP needs to be available to activate the kinase (see figure 1).

If the PDE is active it hydrolyses cAMP, this will reduce cAMP levels, and this will reduce the level of active PKA. Net result, less HSL phosphorylated, less perilipin phosphorylated, therefore less triglyceride broken down. That is, HSL has to be phosphorylated to be active, and to remove the perilipin from blocking HSL access to the fat droplet in the adipocytes it also has to be phosphorylated.

Hsl2

Figure 1: Regulation of lipolysis. Activation of a G-protein coupled receptor (GPCR) by a hormone (A) (e.g. glucagon) or catecolamine (e.g. epinephrine) causes an exchange of GTP for GDP on the α subunit of a heterotrimeric Gs protein. The active GTP bound α subunit activates adenylyl cylcase (AC), which converts ATP to cAMP. cAMP activates protein kinase A (PKA), which in turn phosphorylates, and therefore activates, hormone sensitive lipase (HSL), and phosphorylates perilipin and removes its 'block' on the fat droplet. Net result: increased lipolysis and hence production of fatty acids and glycerol. The activation of the insulin receptor (IR) by insulin (I), via insulin receptor substrate (IRS) and a number of other proteins, results in the phosphorylation of protein kinase B (PKB), which in turn phosphorylates and activates phosphodiesterase 3B (PDE3B). The active PDE3B hydrolyses cAMP to AMP, and therefore effectively lowers intracellular cAMP levels, therefore reducing PKA activity. As phosphatases (P) are removing phosphates from HSL and perilipin there will be a decrease in phosphorylated HSL and perilipin as PKA is not replacing the phosphates. The net result is less HSL and perilipin is phosphorylated, so lipolysis is reduced.

If you were to examine the intracellular cAMP levels of adipocytes that been stimulated with a catecolamine, or with insulin, or a combination of insulin and catecolamine you may see the type of result shown in figure 2. Basically, in the absences of insulin or a catecolamine cAMP levels are at a basal level. Upon the addition of a catecolamine levels are raised 2 fold. If insulin alone is used then levels of cAMP are reduced below that of basal, and in the presence of insulin and catecolamine the levels of cAMP may be higher than basal, but not as high as catecolamine only as PDE3B is active (see figure 2).

C amp

Figure 2: Hypothetical levels of cAMP upon ligand challenge. Stimulation of adipocytes with a catecolamine causes an increase in intracellular levels of cAMP. Stimulation of adipocytes with insulin causes cAMP levels to fall below basal levels as PDE3B is activated. The stimulation of the cells with catecolamine and insulin dampens the response of the catecolamine as PDE3B is activated by insulin.

What you have to remember is these systems are dynamic and in a state of equilibrium. cAMP is being made (adenylyl cyclase) and destroyed (PDEs). Kinases will be putting phosphates on to proteins, and phosphatases will be removing them. All that is happening is this equilibrium is being changed and pushed in one direction or the other, active or inactive, phosphorylated or not phosphorylated.