Triacylglycerol dephosphorylation

Decreased triacylglycerol degradation Elevated insulin favors the dephosphorylated (inactive) state of hormone-sensitive lipase (see p. 187). Triacylglycerol degradation is thus inhibited in the well-fed state. 

Regulation The concentration of free fatty acids in the blood is controlled by the rate at which hormone-sensitive triacylglycerol lipase hydrolyzes the triacylglycerols stored in adipose tissue. Glucagon, epinephrine and norepinephrine cause an increase in the intracellular level of cAMP which allosterically activates cAMP-dependent protein kinase. The kinase in turn phosphorylates hormone-sensitive lipase, activating it, and leading to the release of fatty acids into the blood. Insulin has the opposite effect it decreases the level of cAMP which leads to the dephosphorylation and inactivation of hormone-sensitive lipase. 

The breakdown of fatty acids in (3-oxidation (see Topic K2) is controlled mainly by the concentration of free fatty acids in the blood, which is, in turn, controlled by the hydrolysis rate of triacylglycerols in adipose tissue by hormone-sensitive triacylglycerol lipase. This enzyme is regulated by phosphorylation and dephosphorylation (Fig. 5) in response to hormonally controlled levels of the intracellular second messenger cAMP (see Topic E5). The catabolic hormones glucagon, epinephrine and norepinephrine bind to receptor proteins on the cell surface and increase the levels of cAMP in adipose cells through activation of adenylate cyclase (see Topic E5). The cAMP allosterically activates... 

The anabolic hormone insulin has the opposite effect to glucagon and epinephrine. It stimulates the formation of triacylglycerols through decreasing the level of cAMP, which promotes the dephosphorylation and inactivation of hormone-sensitive lipase (Fig. 5). Insulin also stimulates the dephosphorylation of acetyl CoA carboxylase, thereby activating fatty acid synthesis (see Topic K3). Thus fatty acid synthesis and degradation are coordinately controlled so as to prevent a futile cycle. 

A major effect of insulin is on the adipose tissue, both in regard to glucose uptake and intracellular metabolic effects. The major metabolic conversion in response to insulin is a dephosphorylation of hormonesensitive lipase to convert this enzyme into the inactive form. A second important metabolic aspect is an increase in synthesis and secretion to the blood-vessel surface of lipoprotein lipase. This enzyme is responsible for breaking down circulating triacylglycerols, particularly from VLDL as well as from chylomicrons, into fatty acids and glycerol. 

Insulin causes activation of a protein tyrosine kinase in many cells. Insulin also causes increased glucose transport in selected tissues. Insulin will stimulate dephosphorylation of many proteins phosphorylated in response to cAMP. Insulin is an anabolic hormone and in many tissues causes increases in protein and glycogen synthesis. Insulin also causes an increase in adipose lipoprotein lipase and triacylglycerol storage in the adipose tissue. 

Triacylglycerols are formed by reaction of two molecules of fatty acyl-CoA with glycerol 3-phosphate to form phosphatidic acid this product is dephosphorylated to a diacylglycerol, then acylated by a third molecule of fatty acyl-CoA to yield a triacylglycerol. 

Fig. 9. Reciprocal regulation of fatty acid synthesis and oxidation. Malonyl-CoA, the product of the ACC reaction, inhibits CPT-1, which is localized at the outer mitochondrial membrane and catalyzes the conversion of fatty acyl-CoA to fatty acyl-camitine for mitochondrial fatty acid import and oxidation. At the inner mitochondrial membrane, fatty acyl moieties are converted to CoA thioesters by CPT-II before undergoing -oxidation. ACC is activated by citrate and inhibited by fatty acyl-CoA. AMPK is activated by AMP and the high AMP level reflects the low energy state of the cell. Activation of AMPK in response to increases in AMP involves phosphorylation by an upstream AMPK kinase (AMPKK), the tumor suppressor LKB1, and AMPK is inactivated/dephosphory-lated by protein phosphatase 2A (PP2A), which is first activated by insulin via PI3K/Akt pathway. ACC is dephosphorylated/activated by PP2A and is inactivated upon phosphorylation by AMPK. ACC can also be phos-phorylated/inactivated by PKA. TAG, triacylglycerol FA, fatty acid.

In both adipose tissue and liver, triacylglycerols are produced by a pathway in which glycerol 3-phosphate reacts with fatty acyl CoA to form phosphatidic acid. Dephosphorylation of phosphatidic acid produces diacylglycerol. Another fatty acyl CoA reacts with the diacylglycerol to form a triacylglycerol (see Fig. 33.20). 

At present, little is known about the phosphatidate phosphohydrolase step in the biosynthetic pathway to triacylglycerols. The enzyme was specifically measured by Moore et al, (1973) in subcellular fractions of castor bean endosperm and found to be located mainly in the endoplasmic reticulum, with some activity in the soluble fraction. In leaves, the highest specific activity was found in an undefined particulate fraction, whereas the m or part of the activity was soluble (Heinz, 1977). Other evidence for a specific phosphatidate phosphohydrolase activity in chloroplast membranes was indirect (Joyard and Douce, 1977). Care must always be taken to ensure that the activity is not due to a nonspecific phosphatase in view of the report by Blank and Snyder (1970) that wheat germ contains an acid phosphatase capable of dephosphorylating phosphatidic acid. The enzyme described by Moore et al. 

GC, generally chromatography with a carrier gas (hydrogen, helium or nitrogen) as the mobile phase. Useful for any volatile lipid compound, such as fatty acid methyl esters, triacylglycerols and sterol esters, or compounds that can be made volatile, such as phospholipids after (enzymic) dephosphorylation. 

Phosphatidate is dephosphorylated to yield 1,2-diacylglycerol which is subsequently esterified at the sn-3 position to yield a triacylglycerol. 

---