Effects of levonorgestrel on enzymes responsible for synthesis of triacylglycerols in rat liver

The effects of levonorgestrel treatment (4 micrograms/day per kg body weight 0.75 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.     

Hormones and triacylglycerol metabolism under normoxic and ischemic conditions

Summary Fatty acids, the preferred substrate in normoxic myocardium, are derived from either exogenous or endogenous triacylglycerols. The supply of exogenous fatty acids is dependent of the rate of lipolysis in adipose tissue and of the lipoprotein lipase activity at the coronary vascular endothelium. A large part of the liberated fatty acids is reesterified with glycerol-3-phosphate and converted to triacylglycerols. Endogenous lipolysis and lipogenesis are intracellular compartmentalized multienzyme processes of which individual hormone-sensitive steps have been demonstrated in adipose tissue. The triacylglycerol lipase is the rate-limiting enzyme of lipolysis and glycerol-3-phosphate acyltransferase and possibly phosphatidate phosphohydrolase are the rate-limiting enzymes of lipogenesis. The hormonal regulation of both processes in heart is still a matter of dispute. Triacylglycerol lipase activity in myocardial tissue has two intracellular sources: 1, the endoplasmic reticular and soluble neutral lipase, and 2. the lysosomal acid lipase. Studies in our laboratory have indicated that whereas lipolysis is enhanced during global ischemia and anoxia, overall lipolytic enzyme activities in heart homogenates were not altered. In addition we were unable to demonstrate alterations in tissue triacylglycerol content and glycerol-3-phosphate acyltransferase activity under these conditions. Lipolysis, is subject to feedback inhibition by product fatty acids. Therefore all processes leading to an increased removal of fatty acids from the catalytic site of the lipase will stimulate lipolysis. These studies will be reviewed. In addition, studies from our department have demonstrated the capacity of myocardial lysosomes to take up and degrade added triacylglycerol-particles in vitro. Such a process, stimulated by Ca²⁺ and stimulated by acidosis, offers another physiological target for hormone actions.     

Effect of chemical modificationin situ onl-glycerol-3-phosphate dehydrogenase in brown adipose tissue mitochondria

Mitochondriall-glycerol-3-phosphate dehydrogenase (E.C. 1.1.99.5.) was studied by chemical modificationin situ with different amino acid side chain specific reagents in mitochondria isolated from hamster brown adipose tissue. The SH-modifying reagents have only slight effect on the enzyme activity. The most effective chemicals were tetranitromethane and diazobenzene sulfonic acid. The enzyme activity can be abolished completely by both of them. In the presence of Ca²⁺ and/or glycerol-3-phosphate inhibition was greater at the same electrophilic reagent concentration. The effect of Ca²⁺ and glycerol-3-phosphate is nonadditive on inhibition by these reagents.     

Lipolysis in heart and adipose tissue: effects of inhibition of glycogenolysis and uncoupling of oxidative phosphorylation.

In the Langendorff heart, lipolysis is arrested when glycogenolysis is inhibited by the addition of 5-gluconolactone. Glucose partially overcomes the inhibition as well as uncoupling of oxidative phosphorylation by dinitrophenol. In isolated fat cells hormone-sensitive lipolysis is also inhibited by glycogenolysis inhibition and in these cells also, glucose addition overcomes the inhibition. In fat cells, uncoupling of oxidative phosphorylation does not stimulate lipolysis, probably because of the relatively low concentration of mitochondria in white adipose tissue. The data are interpreted that both in heart and adipose tissue cells, the removal of fatty acids produced by the endogenous lipase is the main stimulus for lipolysis. Attempts to generate in fat cells glycerol-3-phosphate by glycerogenesis from pyruvate or lactate led to the observation that not only these latter anions, but also propionate and acetate strongly stimulate lipolysis. It suggests that long-chain fatty acid removal from fat cells may be stimulated by anion exchange.     

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins1

Liver and intestinal cytosol contain abundant levels of long chain fatty acyl-CoA binding proteins such as liver fatty acid binding protein (L-FABP) and acyl-CoA binding protein (ACBP). However, the relative function and specificity of these proteins in microsomal utilization of long chain fatty acyl-CoAs (LCFA-CoAs) for sequential transacylation of glycerol-3-phosphate to form phosphatidic acid is not known. The results showed for the first time that L-FABP and ACBP both stimulated microsomal incorporation of the monounsaturated oleoyl-CoA and polyunsaturated arachidonoyl-CoA 8–10-fold and 2–3-fold, respectively. In contrast, these proteins inhibited microsomal utilization of the saturated palmitoyl-CoA by 69% and 62%, respectively. These similar effects of L-FABP and ACBP on microsomal phosphatidic acid biosynthesis were mediated primarily through the activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting step, rather than by protecting the long chain acyl-CoAs from microsomal hydrolase activity. In fact, ACBP but not L-FABP protected long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity in the order: palmitoyl-CoA>oleoyl-CoA>arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Relationship between glycerol-3-phosphate dehydrogenase,fatty acid synthase and fatty acid binding proteins in developing human placenta

The activities of the enzymes glycerol-3-phosphate dehydrogenase and fatty acid synthase are inhibited by palmitoyl-coenzyme A and oleate. The two isoforms of fatty acid binding proteins (PI 6.9 and PI 5.4) enhance the activities of glycerol-3-phosphate dehydrogenase and fatty acid synthase in the absence of palmitoyl-coenzyme A or oleate and also protect them against palmitoyl-coenzyme A or oleate inhibition. Levels of fatty acid binding proteins, the activities of the enzymes fatty acid synthase and glycerol-3-phosphate dehydrogenase increase with gestation showing a peak at term. However, the activity of fatty acid synthase showed the same trend up to the 30th week of gestation and then declined slightly at term. With the advancement of pregnancy when more lipids are required for the developing placenta, fatty acid binding proteins supply more fatty acids and glycerol-3-phosphate for the synthesis of lipids. Thus a correlation exists between glycerol-3-phosphate dehydrogenase, fatty acid synthase and fatty acid binding proteins in developing human placenta.     

Gylcerol-3-phosphate shuttle and its function in intermediary metabolism of hamster brown-adipose tissue.

1. Brown adipose tissue of the hamster possesses high specific activities of soluble, cytoplasmic NAD-linked, as well as mitochondrial flavin-coupled, glycerol-3-phosphate dehydrogenases. The ratio of the two enzyme activities is high (close to 1), when compared with other tissues of the hamster. 2. In the presence of rotenone, NADH is oxidised very poorly by homogenates of brown adipose tissue. A high rate of oxidation is obtained upon further addition of dihydroxyacetone phosphate, which itself is negligible oxidised. When followed fluorimetrically glycerol 3-phosphate can also be observed to induce NADH oxidation, but only after a significant lag time. Similar results are obtained with isolated mitochondria plus high-speed supernatant. With high-speed supernatant alone, only dihydroxyacetone phosphate has any effect, whereas with isolated mitochondria neither dihydroxyacetone phosphate nor glycerol 3-phosphate induce any NADH disappearance. 3. Respiration induced by NADH plus dihydroxyacetone phosphate in homogenates equals 56% of the respiration induced by glycerol 3-phosphate alone. 4. Respiration induced by NADH plus dihydroxyacetone phosphate, as well as that induced by glycerol 3-phosphate, is inhibited by the same concentrations of inhibitors as are required for inhibition of the mitochondrial dehydrogenase i.e. EDTA, long-chain unsaturated fatty acids, long-chain fatty acyl CoA esters. 5. In isolated brown adipocytes in the presence of rotenone, norepinephrine significantly inhibits respiration induced by glycerol 3-phosphate. 6. The results obtained are discussed with respect to the role of glycerol 3-phosphate as an electron sink for cytosolic reducing equivalents to maintain a low level of extramitochondrial NADH. A means of maintaining a level of glycerol 3-phosphate adequate for triglyceride synthesis is also considered.     

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.     

Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue.

1. Adipose tissue from Angus and Brahman steers incubated with [1-14C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 min) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 min). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diacylglycerol acyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.     

Triglyceride synthesis by small-intestinal epithelium of the pig, sheep and chicken

1. A comparative study was made of triglyceride synthesis by the intestinal epithelium of pigs, sheep and chickens. In pig and chicken tissue both the glycerol 3-phosphate and the monoglyceride pathway of triglyceride synthesis were operative, but the former pathway predominated in sheep tissue. 2. The fatty acid specificity of the glycerol 3-phosphate pathway was studied in pig and sheep total-homogenate preparations. Maximum incorporation was obtained with myristic acid and palmitic acid under optimum conditions for each fatty acid. Lauric acid, myristic acid, oleic acid, linoleic acid and linolenic acid were inhibitory at concentrations above their optimum, but octanoic acid, decanoic acid, palmitic acid and stearic acid did not show this effect. 3. Subcellular fractionation located the glycerol 3-phosphate and monoglyceride pathways of triglyceride synthesis in the microsomes in all instances. Phosphatidate phosphohydrolase was associated with both the microsomes and the particle-free supernatant. 4. Glycerol 1-mono-oleate was incorporated into triglycerides to a greater extent than glycerol 1-mono-palmitate or glycerol 1-monostearate by microsomal preparations from pig and chicken. 5. A lipase specific for monoglycerides was detected in the particle-free supernatant of all the species examined.     

Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.     

Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction.

The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.     

A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats

The amount of triacylglycerol (TAG) that accumulates in adipose tissue depends on 2 opposing processes: lipogenesis and lipolysis. We have previously shown that the weight and lipid content of epididymal (EPI) adipose tissue increases in growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The aim of this work was to study the pathways involved in lipogenesis and lipolysis, which ultimately regulate lipid accumulation in the tissue. De novo fatty acid synthesis was evaluated in vivo and was similar for rats fed an LPHC diet or a control diet; however, the LPHC-fed rats had decreased lipoprotein lipase activity in the EPI adipose tissue, which suggests that there was a decreased uptake of fatty acids from the circulating lipoproteins. The LPHC diet did not affect synthesis of glycerol-3-phosphate (G3P) via glycolysis or glyceroneogenesis. Glycerokinase activity - i.e., the phosphorylation of glycerol from the hydrolysis of endogenous TAG to form G3P - was also not affected in LPHC-fed rats. In contrast, adipocytes from LPHC animals had a reduced lipolytic response when stimulated by norepinephrine, even though the basal adipocyte lipolytic rate was similar for both of the groups. Thus, the results suggest that the reduction of lipolytic activity stimulated by norepinephrine seems essential for the TAG increase observed in the EPI adipose tissue of LPHC animals, probably by impairment of the process of activation of lipolysis by norepinephrine.     

Randomly interesterified triacylglycerol containing medium- and long-chain fatty acids stimulates fatty acid metabolism in white adipose tissue of rats

We have reported previously that randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule (MLCT) resulted in significantly lower body fat accumulation and higher hepatic fatty acid oxidation than from long-chain triacylglycerol (LCT) in rats. To understand the metabolic changes occurring in white adipose tissue, the fatty acid oxidation and synthesis, and the adipocytokine level were measured in rats fed with MLCT or LCT for 2 weeks. In comparison with LCT, MLCT lowered not only the fatty acid synthase and glycerol-3-phosphate dehydrogenase activities in perirenal adipose tissue, but also the serum insulin and leptin levels, in addition to significantly reducing the body fat accumulation. In contrast, fatty acid oxidation measured as the carnitine palmitoyltransferase activity in the tissue was significantly higher in the MLCT-fed rats than in the LCT-fed rats. It seems that the altered fatty acid metabolism in adipose tissue per se was also responsible for the lower adiposity by dietary MLCT.     

Metabolism of D- and L-glyceraldehyde in adipose tissue: a stereochemical probe for glycerokinase activity

Distributions of (14)C have been determined in free glycerol, in glycerol from triglycerides, in glucose from glycogen, and in lactate after incubation of d-glyceraldehyde-3-(14)C and l-glyceraldehyde-3-(14)C with rat adipose tissue. The distributions are interpreted in terms of presently accepted possible reactions for the initial metabolism of glyceraldehyde. Formation of glycerol-1-(14)C from d-glyceraldehyde-3-(14)C indicates that in adipose tissue glyceraldehyde is reduced to glycerol. Incorporation of (14)C from d-glyceraldehyde-3-(14)C into carbon 3 of the glycerol of triglyceride indicates that d-glyceraldehyde is either phosphorylated or oxidized to d-glyceric acid, or both, in its initial metabolism. Incorporation of (14)C from l-glyceraldehyde-3-(14)C into carbon 3 of glycerol indicates that l-glyceraldehyde is reduced to glycerol, which is phosphorylated and (or) converted to d-glyceric acid via l-glyceric acid. Some (14)C from l-glyceraldehyde-3-(14)C is incorporated into carbon 1 of glycerol of triglycerides and carbon 4 of glycogen; the explanation for this incorporation is uncertain.     

Competition of electrons to enter the respiratory chain: a new regulatory mechanism of oxidative metabolism in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are the external NADH dehydrogenases (Nde1p and Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p. Subsequently, glycerol 3-phosphate donates electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p). At saturating concentrations of NADH, the activation of external NADH dehydrogenases completely inhibits glycerol 3-phosphate oxidation. Studies on the functionally isolated enzymes demonstrated that neither Nde1p nor Nde2p directly inhibits Gut2p. Thus, the inhibition of glycerol 3-phosphate oxidation may be caused by competition for the entrance of electrons into the respiratory chain. Using single deletion mutants of Nde1p or Nde2p, we have shown that glycerol 3-phosphate oxidation via Gut2p is inhibited fully when NADH is oxidized via Nde1p, whereas only 50% of glycerol 3-phosphate oxidation is inhibited when Nde2p is functioning. By comparing respiratory rates with different respiratory substrates, we show that electrons from Nde1p are favored over electrons coming from Ndip (internal NADH dehydrogenase) and that when electrons come from either Nde1p or Nde2p and succinodehydrogenase, their use by the respiratory chain is shared to a comparable extent. This suggests a very specific competition for electron entrance into the respiratory chain, which may be caused by the supramolecular organization of the respiratory chain. The physiological consequences of such regulation are discussed.