Human rectal absorption of short- and medium-chain C2-C10 fatty acids

PURPOSE: To study the effects of running suture adjustment for reduction of astigmatism after penetrating keratoplasty. Suture adjustments performed during surgery and during the early postoperative and late postoperative periods were retrospectively compared. METHODS: We studied 53 patients who received running suture adjustment after penetrating keratoplasty, either intraoperatively (ISA group, n = 18), early (< 2 weeks) postoperatively (EPSA group, n = 19), or late (> 1 month) postoperatively (LPSA group, n = 16). Refractive and topographic astigmatism and corneal topography were examined at 1, 3, and 6 months after surgery. RESULTS: Overall mean refractive astigmatism and topographic astigmatism at 6 months were 2.55 +/- 1.61 D and 3.12 +/- 1.89 D, respectively (mean +/- SD). The mean refractive astigmatism and topographic astigmatism were 1.88 +/- 1.04 D and 2.35 +/- 1.35 D in the ISA group, 2.32 +/- 1.17 D and 2.70 +/- 1.21 D in the EPSA group, and 3.01 +/- 1.62 D and 4.62 +/- 2.51 D in the LPSA group, respectively (mean +/- SD). The LPSA group demonstrated significantly increased topographic astigmatism compared to the ISA group (p = 0.0048) and the EPSA group (p = 0.015). Although 31.6 and 25.0% of the EPSA and LPSA groups, respectively, did not require postoperative suture adjustments, more eyes (10/18 eyes, 55.6%) in the ISA group did not require the procedure. CONCLUSIONS: Early postoperative suture adjustment was more effective than late postoperative adjustment. Intraoperative suture adjustment may further reduce final astigmatism and the necessity for postoperative suture manipulation.     

Protein kinase signaling pathway triggered by cell swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in isolated rat hepatocytes

Incubation of isolated hepatocytes with glutamine or proline or in hypotonic media is known to activate glycogen synthase and acetyl-CoA carboxylase as a result of cell swelling. We report here that the same experimental conditions caused an activation of phosphatidylinositol 3-kinase and p70 ribosomal protein S6 kinase (p70 S6 kinase) but did not modify the activity of p42 mitogen-activated protein kinase. In addition, rapamycin, an inhibitor of p70 S6 kinase activation, prevented the amino acid- and hypotonicity-induced activation of p70 S6 kinase but did not block the activation of glycogen synthase and acetyl-CoA carboxylase, thus ruling out p70 S6 kinase as a necessary component in the activation pathway. By contrast, wortmannin or LY294002, inhibitors of phosphatidylinositol 3-kinase, completely blocked the activation of phosphatidylinositol 3-kinase and p70 S6 kinase and partly blocked the activation of glycogen synthase and acetyl-CoA carboxylase. Therefore, phosphatidylinositol 3-kinase might be a component of the signaling pathway that is triggered by cell swelling and is responsible, at least in part, for the activation of glycogen synthase and acetyl-CoA carboxylase. Incubation of hepatocytes with 0.1 microM epidermal growth factor doubled the activity of p42 mitogen-activated protein kinase without activating glycogen synthase.     

Effects of fatty acids and hormones on fatty acid metabolism and gluconeogenesis in bovine hepatocytes

Primary cultures of hepatocytes were used to study the effects of extracellular oleate concentration and hormones on fatty acid metabolism and gluconeogenesis. Rates of oleate uptake and oxidation to acid-soluble products varied linearly as oleate concentrations increased (0.1 to 2 mM), but rates of triglyceride accumulation varied quadratically. Insulin increased the proportion of oleate that was esterified by 22% without affecting the formation of acid-soluble products. Cells incubated with 2 mM [1-(14)C]oleate for 24 h eliminated 9.6% of the labeled intracellular lipid as acid-soluble products in the following 24 h when no oleate was present during depletion and eliminated 7.7% when 2 mM oleate was present. Insulin reduced labeled triglyceride depletion by 49%. Gluconeogenesis from [2-(14)C] propionate was depressed by 24%, and formation of acid-soluble products was increased by 46% in cells infiltrated with lipid because of previous exposure to 2 mM oleate for 45 h. Rates of gluconeogenesis from propionate were reduced 23% when 2 mM oleate was present during the 3-h period that gluconeogenesis was measured, and the effect was not modified by lipid infiltration. Lipid infiltration influenced hepatic function, and insulin regulated hepatic triglyceride concentration.     

Overexpression and kinetic characterization of the carboxyltransferase component of acetyl-CoA carboxylase

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K)biocytin parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K)biocytin profile and 7. 5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1' proton of biocytin.     

Effects of dicarboxylic acids on fatty acid-metabolizing enzymes in cultured rat hepatocytes

A clear chain-length dependent effect was observed for peroxisomal fatty acid beta-oxidation and carnitine acetyltransferase and also for mitochondrial carnitine palmitoyltransferase in primary cultures of rat hepatocytes. The extent of modulation of peroxisomal beta-oxidation was higher with even-carbon numbered dicarboxylic acids than with odd-carbon numbered ones, although such a tendency was not detected in the mitochondrial reactions. These results indicate difference in the effect of fatty acid-derived dicarboxylates on peroxisomal and mitochondrial functions.     

Identification of human acetyl-CoA carboxylase isozymes in tissue and in breast cancer cells

1. In the rat, acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid metabolism, exists as at least two different isozymes (M(r) 265,000 and 280,000) that display distinct tissue-specific distribution and regulation. 2. Based on the study of human tissue and human-derived breast cancer cell lines by enzyme isolation and protein blotting techniques, we have now identified two human isoforms of M(r) 265,000 (HACC 265) and 275,000 (HACC 275), each of which is homologous to one of the rat isozymes. 3. Human breast carcinoma cell lines show variable expression of these two isoforms, mirrored in the estimation of ACC acetyl-CoA kinetics.     

Effects of medium-chain fatty acids and their acylglycerols on the transport of penicillin V across Caco-2 cell monolayers

The transport-enhancing effects of medium-chain fatty acids (caproic, caprylic, and capric acids) and their acylglycerols (mono-, di-, and triacylglycerols) were investigated by using Caco-2 cell monolayers as a model of the human intestinal epithelium. Penicillin V was used as a model for a hydrophilic bioactive compound. Among the fatty acids and acylglycerols tested, 1,2-dicaproin, monocaprin, monocaprylin, and capric acid sodium salt effectively enhanced the transport rate, whereas other substances enhanced the rate only slightly or not at all. With each of these four substances, the rate of enhancement was proportional to the concentration at low concentrations, but leveled off at high concentrations. The transport-enhancing effects were well correlated with the reduction in surface tension and with a physico-chemical parameter, denoted by the surface energy-lowering coefficient, characterizing the surface activity of a substance.     

Rapid induction of hepatic acetyl-CoA carboxylase activity after estradiol benzoate injection in the quail

The time course of hepatic Acetyl-CoA carboxylase activity as well as hepatic and plasmatic fatty acids concentrations following a single injection of estradiol benzoate (EB, 0.2 mg/kg) was studied in the quail. Acetyl-CoA carboxylase activity increases rapidly and reaches its peak 3 h after the injection of EB. Similarly, hepatic and plasmatic fatty acids concentrations are significantly increased 6 h after the hormonal injection and attain their highest level 18 h later. These results suggest that estrogen affects the hepatic fatty acids biosynthesis by regulating the conversion of acetyl-CoA to malonyl-CoA.     

Trans unsaturated fatty acids inhibit lecithin: cholesterol acyltransferase and alter its positional specificity

Although dietary trans unsaturated fatty acids (TUFA) are known to decrease plasma HDL, the underlying mechanisms for this effect are unclear. We tested the hypothesis that the decreased HDL is due to an inhibition of lecithin:cholesterol acyltransferase (LCAT), the enzyme essential for the formation of HDL, by determining the activity of purified LCAT in the presence of synthetic phosphatidylcholine (PC) substrates containing TUFA. Both human and rat LCATs exhibited significantly lower activity (-37% to -50%) with PCs containing 18:1t or 18:2t, when compared with the PCs containing corresponding cis isomers. TUFA-containing PCs also inhibited the enzyme activity competitively, when added to egg PC substrate. The inhibition of LCAT activity was not due to changes in the fluidity of the substrate particle. However, the inhibition depended on the position occupied by TUFA in the PC, as well as on the paired fatty acid. Thus, for human LCAT, 18:1t was more inhibitory when present at sn-2 position of PC, than at sn-1, when paired with 16:0. In contrast, when paired with 20:4, 18:1t was more inhibitory at sn-1 position of PC. Both human and rat LCATs, which are normally specific for the sn-2 acyl group of PC, exhibited an alteration in their positional specificity when 16:0-18:1t PC or 16:1t-20:4 PC was used as substrate, deriving 26-86% of the total acyl groups for cholesterol esterification from the sn-1 position. These results show that the trans fatty acids decrease high density lipoprotein through their inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, and also alter LCAT's positional specificity, inducing the formation of more saturated cholesteryl esters, which are more atherogenic.     

Postexercise recovery of skeletal muscle malonyl-CoA, acetyl-CoA carboxylase, and AMP-activated protein kinase

Previous studies have demonstrated that oxygen consumption and fat oxidation remain elevated in the postexercise period. The purpose of this study was to determine whether malonyl-CoA, an inhibitor of fatty acid oxidation, remains depressed in muscle after exercise. Rats were sprinted for 5 min (40 m/min, 5% grade) or run for 30 min (21 m/min, 15% grade). Red quadriceps malonyl-CoA returned to resting values by 90 min postexercise in the sprinting rats and remained significantly lower at least 90 min postexercise in the 30-min exercise group. AMP-activated protein kinase activity remained significantly elevated (P < 0.05) for 10 min after exercise in both groups. The most rapid rate of glycogen repletion was in the first 30 min postexercise. The respiratory exchange ratio decreased from a nonexercise value of 0.87 +/- 0.01 to an average 0.82 +/- 0.01 during the 90-min period after 30 min of exercise. Thus muscle malonyl-CoA remains depressed and fat oxidation is elevated for relatively prolonged periods after a single bout of exercise. This may allow fat oxidation to contribute more to muscle energy requirements, thus leaving more glucose for replenishment of muscle glycogen.