Metabolic Impact of Adult-Onset, Isolated, Growth Hormone Deficiency (AOiGHD) Due to Destruction of Pituitary Somatotropes

Growth hormone (GH) inhibits fat accumulation and promotes protein accretion, therefore the fall in GH observed with weight gain and normal aging may contribute to metabolic dysfunction. To directly test this hypothesis a novel mouse model of adult onset-isolated GH deficiency (AOiGHD) was generated by cross breeding rat GH promoter-driven Cre recombinase mice (Cre) with inducible diphtheria toxin receptor mice (iDTR) and treating adult Cre+/−,iDTR+/− offspring with DT to selectively destroy the somatotrope population of the anterior pituitary gland, leading to a reduction in circulating GH and IGF-I levels. DT-treated Cre−/−,iDTR+/− mice were used as GH-intact controls. AOiGHD improved whole body insulin sensitivity in both low-fat and high-fat fed mice. Consistent with improved insulin sensitivity, indirect calorimetry revealed AOiGHD mice preferentially utilized carbohydrates for energy metabolism, as compared to GH-intact controls. In high-fat, but not low-fat fed AOiGHD mice, fat mass increased, hepatic lipids decreased and glucose clearance and insulin output were impaired. These results suggest the age-related decline in GH helps to preserve systemic insulin sensitivity, and in the context of moderate caloric intake, prevents the deterioration in metabolic function. However, in the context of excess caloric intake, low GH leads to impaired insulin output, and thereby could contribute to the development of diabetes

Role of the interfacial binding domain in the oxidative susceptibility of lecithin:cholesterol acyltransferase.

We had previously shown that the cholesterol esterification activity of lecithin:cholesterol acyltransferase (LCAT) is destroyed by oxidation, but still it retains the ability to hydrolyse water-soluble substrates. This suggested that the inactivation of the enzyme is not due to its catalytic function, but due to a loss of its hydrophobic binding. Since recent studies have shown that a tryptophan residue in the putative interfacial domain (Trp(61)) is critical for the activity, we determined the possible role of this residue in the oxidative susceptibility and substrate specificity of LCAT by site-directed mutagenesis. Deletion of Trp(61) resulted in a 56% loss of cholesterol esterification (LCAT) activity, but the phospholipase A(2) (PLA(2)) and the esterase activities of the enzyme were stimulated slightly. Replacing Trp(61) with another aromatic residue [Trp(61)-->Tyr (W61Y)] resulted in an increase in all activities (14-157%), whereas replacing it with an aliphatic residue [Trp(61)-->Gly (W61G)] caused a dramatic loss of LCAT (-90%) and PLA(2) (-82%) activities, but not the esterase activity (-5%). W61Y was the most sensitive to oxidation, whereas W61G was the most resistant, with respect to the LCAT and PLA(2) activities. However, the activities which do not involve interfacial binding, namely the esterase activity and the transesterification of short-chain phospholipids, were more resistant to oxidation in all LCATs, indicating a selective loss of the interfacial binding by oxidation. Furthermore, replacing the two cysteines (Cys(31) and Cys(184)) in the Trp(61) deletion mutant caused additional resistance of the enzyme to oxidizing agents, showing that both domains of the enzyme contribute independently to its oxidative susceptibility. Since the hydrolysis of truncated phospholipids, generated during the oxidation of low-density lipoproteins, does not require the interfacial-binding domain, our results suggest that LCAT may take part in the detoxification of these compounds even after the loss of its cholesterol esterification function

Rate of acyl migration in lysophosphatidylcholine (LPC) is dependent upon the nature of the acyl group. Greater stability of sn-2 docosahexaenoyl LPC compared to the more saturated LPC species.

Several previous studies reported that sn-2 acyl lysophosphatidylcholines (LPCs) undergo rapid isomerization due to acyl migration, especially at physiological pH and temperature. However, these studies have been carried out using mostly sn-2 palmitoyl LPC, whereas the naturally occurring sn-2 LPCs are predominantly unsaturated. In this study, we investigated the acyl migration in four naturally occurring sn-2 acyl LPCs (sn-2 16:0, sn-2 18:1, sn-2 20:4, and sn-2 22:6) stored at various temperatures in aqueous or organic solvents, employing LC/MS to analyze the isomer composition. At 37°C and pH 7.4, the order of acyl migration rates (from sn-2 to sn-1) in aqueous buffer was 16:0 LPC> 18:1 LPC> 20:4 LPC> 22:6 LPC. The rate of isomerization of sn-2 16:0 LPC was 2-5 times greater than that of sn-2 22:6 under these conditions. Complexing the LPCs to serum albumin accelerated the acyl migration of all species, but sn-2 22:6 LPC was least affected by the presence of albumin. The migration rates were lower at lower temperatures (22°C, 4°C, and -20°C), but the differences between the LPC species persisted. All the sn-2 acyl LPCs were more stable in organic solvent (chloroform: methanol, 2:1 v/v), but the effect of the acyl groups on acyl migration was evident in the solvent also, at all temperatures. Storage of sn-2 22:6 LPC at -20°C for 4 weeks in the organic solvent resulted in about 10% isomerization, compared to 55% isomerization for sn-2 16:0. These results show that the sn-2 polyunsaturated LPCs can be stored at -20°C or below for several days without appreciable isomerization. Furthermore, they demonstrate that the sn-2 polyunsaturated LPCs generated in vivo are much more stable under physiological conditions than previously assumed

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with (32)P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([(14)C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([(14)C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of (32)P-labelled phosphatidylcholine or (32)P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and P(i) in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and P(i). The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway

Stability of sn-2 acyl LPCs in organic solvent.

<p>The sn-2 acyl LPCs were dissolved in chloroform: methanol (2:1 v/v) at a concentration of 1 mg/ml, and stored at the indicated temperature for various periods. Aliquots were taken out at the indicated time points, brought to room temperature (22°C), and subjected to LC/MS, as described in Methods. The percentage of the sn-2 acyl isomer remaining was calculated from the area counts of the two isomers. The values shown are mean ± SD of 3 separate experiments.</p

Time course of isomerization of albumin-bound sn-2 acyl LPCs at 37°C.

<p>Freshly prepared sn-2 acyl LPCs were complexed with 0.1% BSA in PBS buffer at pH 7.4, and incubated at 37°C for the indicated periods of time. The percent of the sn-2 acyl isomer remaining after each time point was determined by LC/MS/MS. The values shown are mean ± SD of 3 separate analyses. Note that the error bars too small to be visible at some points.</p

Identification of the regioisomers of LPC from the product ion spectra.

<p>The product ion spectra were obtained for the early eluting (peak 1) and late eluting (peak 2) isomers of each LPC as described in Methods. The intensity of the molecular ion as percentage of that of phosphocholine ion (m/z 184) differentiates the two regioisomers, with the sn-1 acyl isomer showing much higher percent of molecular ion [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187826#pone.0187826.ref017" target="_blank">17</a>]. The relative intensities of the molecular ion, expressed as percent of phosphoryl choline ion (m/z 184.1) for the various LPCs are as follows: 16:0 LPC (4.7% for peak 1 and 65.5% for peak 2); 18:1 LPC (3.7% for peak 1 and 44.8% for peak 2); .20:4 LPC (10.2% for peak 1 and 60.5% for peak 2); 22:6 LPC (15.3% for peak 1, and,172% for peak 2). In addition, the choline fragment ion (m/z 104.1) is completely absent from the sn-2 acyl isomers, but was prominently present in all the sn-1 acyl isomers.</p

Selected LC/MS chromatograms of sn-2 acyl LPC species incubated at 37°C in Tris-HCl buffer, pH 7.4.

<p>The aqueous solution of each sn-2 acyl LPC (2 mg/ml) was incubated at 37°C, and aliquots were taken out at the indicated time. The samples were diluted with equal volume of methanol, and analyzed by LC/MS/MS in the MRM mode, as described in Methods.</p

Stability of aqueous dispersions of sn-2 acyl LPCs at various temperatures.

<p>The sn-2 acyl LPCs were dispersed in Tris-HCl buffer, pH 7.4 at a concentration of 2 mg/ml and incubated under nitrogen in the dark at the indicated temperature for 24 h. Aliquots were taken out at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h, brought to room temperature, diluted with equal volume of methanol and subjected to LC/MS/MS analysis as described in Methods. The percentage of the sn-2 acyl isomer remaining was calculated from the area counts of the two isomers. The values shown are mean ± SD of 3 separate experiments.</p

Effect of serum albumin on the isomerization of sn-2 acyl LPC at 37°C.

<p>Freshly prepared sn-2 acyl LPCs were complexed with BSA in PBS, pH 7.4, and incubated at 37°C for the indicated periods. The isomer composition was then determined by LC/MS/MS as described in Methods. Chromatograms of only selected time periods are shown. The time course of the isomerization of each LPC is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187826#pone.0187826.g006" target="_blank">Fig 6</a>.</p