Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion.

Isolated mouse islets were used to compare the effects of three saturated (myristate, palmitate and stearate) and three unsaturated (oleate, linoleate and linolenate) long-chain fatty acids on insulin secretion. By varying the concentrations of fatty acid (250-1250 micromol/l) and albumin simultaneously or independently, we also investigated whether the insulinotropic effect is determined by the unbound or total concentration of the fatty acids. Only palmitate and stearate slightly increased basal insulin secretion (3 mmol/l glucose). All tested fatty acids potentiated glucose-induced insulin secretion (10-15 mmol/l), and the following rank order of potency was obtained when they were compared at the same total concentrations: palmitate approximately = stearate > myristate > or = oleate > or = linoleate approximately = linolenate. The effect of a given fatty acid varied with the fatty acid to albumin molar ratio, in a way which indicated that the unbound fraction is the important one for the stimulation of beta cells. When the potentiation of insulin secretion was expressed as a function of the unbound concentrations, the following rank order emerged: palmitate > myristate > stearate approximately = oleate > linoleate approximately = linolenate. In conclusion, the acute and direct effects of long-chain fatty acids on insulin secretion are due to their unbound fraction. They are observed only at fatty acid/albumin ratios higher than those normally occurring in plasma. Saturated fatty acids are stronger insulin secretagogues than unsaturated fatty acids. Unbound palmitate is by far the most potent of the six common long-chain fatty acids.     

Studies on long chain fatty acid:CoA ligase from human small intestine.

Long chain fatty acid:CoA ligase (EC 6.2.1.3.) was examined in human small intestinal mucosa using the hydroxamate-trapping method. With optimal assay requirements using palmitate as substrate a significant difference of specific activities could be detected in the total homogenate from duodenum, 40.9 +/- 11.6 nmol/min per mg protein versus upper jejunum, 51.9 +/- 13.7 (P less than 0.05). The enzyme activity of the microsomal fraction of upper jejunum was 101.8 +/- 44 nmol/min per mg protein. ATP, CoA, and Mg2+ were essential constituents of the reaction. A broad pH-optimum was observed between 6.75 and 7.75 with a maximum at a pH of 7.25. Whereas palmitate in the presence of albumin revealed a wide range of optimal concentration in supporting maximal enzyme activity, oleate was found to strongly inhibit the reaction. Where substrate specificity with both the total homogenate and the microsomal fraction was concerned, maximal reaction rates were obtained with palmitate for the long chain saturated fatty acids C12:0' C14:0' C16:0' and C18:0' and with oleate for the long chain unsaturated fatty acids C18:1 C18:2' and C18:3' respectively. The highest specific activity of the enzyme was localised in the microsomal fraction. The kinetic data and properties of the long chain fatty acid: CoA ligase from human intestine are discussed with respect to the intestinal enzyme from other species.     

Metabolic fate of fatty acids in type II cells isolated from adult rat lung

The present study is concerned with the metabolic fate of palmitate, oleate and linoleate in isolated rat lung type II cells. The cells readily oxidize the exogenously supplied fatty acids to CO₂ and incorporate them into lipids. The distribution between the pathways of oxidation and esterification is similar for saturated and unsaturated fatty acids. The majority of the fatty acids taken up by the cells is utilized for lipid synthesis. The fatty acids are incorporated preferentially into phospholipids, particularly into phosphatidylcholine. Addition of unsaturated fatty acids decreases the utilization of palmitate by type II cells. The distribution of palmitate between oxidation and esterification is not altered in the presence of unsaturated fatty acids. Addition of carnitine stimulates the fatty acid oxidation and decreases the esterification of fatty acids.     

Physiological free fatty acid concentrations do not increase free estradiol in plasma

Consumption of dietary fat has been linked to the high incidence of breast cancer. The in vitro effects of palmitate, oleate, linoleate, and eicosapentaenoate at supraphysiological concentrations [1.8-4.8 mM; endogenous (0.8) plus added (1-4)] on the binding of 17 beta-estradiol (E2) to sex hormone-binding globulin and albumin were evaluated in plasma, using centrifugal ultrafiltration. The E2-sex hormone-binding globulin complex was significantly dissociated by oleate, linoleate, eicosapentaenoate, and palmitate at 1.8 to 2.8-mM concentrations. The same FFA caused a significant increase in E2 binding to albumin at similar concentrations. However, only the unsaturated fatty acids at 3.8 to 4.8-mM concentrations (i.e. oleate, linoleate, and eicosapentaenoate) significantly increased the nonprotein-bound free E2 fraction. Thus, these results suggest that free E2 concentrations are not affected by physiological concentrations of FFA.     

Prolactin-induced protein kinase C activity in a mouse mammary cell line (NOG-8).

The normal mouse mammary epithelial cells, NOG-8, respond to the mitogenic signal of prolactin with a 2.5-fold increase in cell number within 3 days in vitro. When prolactin is added to subconfluent cells for 5-15 min, there is a 5-fold increase in protein kinase C activity. Upon longer exposure (24 h) to the hormone, the enzyme activity returns to that of control. The potent protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), blocks both the prolactin-induced enzyme activity and subsequent increase in cell number. Prior to prolactin treatment, 90% of the protein kinase C activity resides in the cytosol with only 10% associated with the membranes. After only 5 min of prolactin treatment, 70% of the enzyme activity is now localized to the membranes. These data suggest that prolactin uses the protein kinase C pathway for signal transduction in NOG-8 cells thus leading to enhanced cell growth.     

Fatty acids modulate protein kinase C activation in porcine vascular smooth muscle cells independently of their effect on de novo diacylglycerol synthesis

Aims/hypothesis. Diabetes-induced activation of protein kinase C has been associated with the development of vascular complications. Elevated de novo diacylglycerol synthesis has been postulated to underlie this protein kinase C activation. Diabetes also increases the circulating concentrations of non-esterified fatty acids, which are immediate precursors of diacylglycerol through the de novo pathway. We hypothesized that increased fatty acids contribute to de novo diacylglycerol synthesis and activation of protein kinase C in vascular cells. Methods. Primary cultures of porcine carotid smooth muscle cells were exposed to fatty acids, bound to albumin in physiologic ratios. Diacylglycerol and triacylglycerol were measured in extracts of these cells. Protein kinase C activation was measured as membrane translocation with isoform-specific antibodies. Results. Saturated fatty acids caused considerable accumulation of diacylglycerol through de novo synthesis. Unsaturated fatty acids increased triacylglycerol, but not diacylglycerol. Platelet-derived growth factor activated the α, ɛ and ζ protein kinase C isoforms. Activation of the α and ζ isoforms was amplified by oleate pretreatment but inhibited by palmitate. In the absence of growth factor stimulation, neither palmitate nor oleate had any effect on the membrane/cytosol distribution of any protein kinase C isoform. Conclusion/interpretation. Saturated fatty acids elicited de novo diacylglycerol synthesis in vascular smooth muscle cells without activating protein kinase C. Effects of fatty acids on protein kinase C activation by platelet-derived growth factor did not correlate with the effects on de novo diacylglycerol synthesis. These results indicate that de novo diacylglycerol synthesis is, by itself, insufficient to activate protein kinase C. [Diabetologia (2000) 43: 1136–1143] Received: 3 April 2000 and in revised form: 15 May 2000     

In vivo absorption of medium-chain fatty acids by the rat colon exceeds that of short-chain fatty acids

BACKGROUND AND AIMS: Short-chain fatty acids (SCFAs) are main fuels of the colonic epithelium, and are avidly absorbed by the colon of animal and man. The current knowledge on colonic metabolism and absorption of medium-chain fatty acids (MCFAs) is limited. In some clinical situations, colonic absorption of high-energy substances could compensate for reduced absorptive capacity because of a shortened or malfunctioning small bowel. We evaluated and compared colonic absorption and metabolism of MCFAs (octanoate, decanoate, and dodecanoate), SCFAs (acetate and butyrate), and long-chain fatty acids (LCFAs) (oleate). METHODS: Rats were surgically operated on to cannulate a 7-cm segment of proximal colon, isolate the vasculature, and cannulate the right colic vein draining this segment. The lumen was perfused with (14)C-labeled substrates for 100 minutes. Right colic vein blood was analyzed for total (14)C, (14)CO(2), and metabolites by scintillation counting and high-performance liquid chromatography. RESULTS: The transport from the colonic lumen to mesenteric blood of substrate carbon from MCFAs exceeded by 2-13-fold that of SCFAs and LCFAs. The CO(2) production from the oxidation of MCFAs was as high as or higher than that from SCFAs. CO(2) produced from the LCFA, oleate, was lower than from SCFAs or MCFAs. In addition to CO(2), ketone bodies were major metabolites of SCFAs and MCFAs. Ketogenesis from butyrate and the MCFAs was significantly higher than from acetate and oleate. A substantial proportion (50%-90%) of all substrates was absorbed without being metabolized. CONCLUSIONS: The colonic epithelium serves to absorb and partially metabolize MCFAs. For patients with a compromised small-bowel function, colonic absorption of MCFAs could represent an important way of receiving calories.     

Evidence for adipose-muscle cross talk: opposing regulation of muscle proteolysis by adiponectin and Fatty acids

Illnesses associated with insulin resistance exhibit increases in whole-body protein degradation and amino acid oxidation. However, the mechanisms stimulating muscle catabolism under these conditions are not clear. Because insulin resistance is associated with accumulation of lipids in muscle, we measured protein degradation in muscles of mice fed a high-fat diet. Muscle protein catabolism was accelerated on the high-fat diet, and this was associated with an increase in plasma free fatty acid and a decrease in plasma levels of the adipocyte-derived cytokine adiponectin. To evaluate how free fatty acids influence adiponectin-mediated changes in muscle protein breakdown we examined C2C12 skeletal muscle cells exposed to free fatty acids. Both saturated fatty acids (palmitate) and unsaturated fatty acids (oleate) increased protein degradation (25 and 18%, respectively) in part by activating the E3 ubiquitin ligases. Adenovirus-mediated overexpression of adiponectin blocked fatty acid-induced protein degradation in C2C12 cells. Palmitate activated the E3 ubiquitin ligases by suppressing insulin receptor substrate-1/Akt signaling in the C2C12 muscle cells, whereas adiponectin attenuated the E3 ubiquitin ligase activation by increasing both insulin receptor substrate-1 tyrosine phosphorylation and Akt Ser473 phosphorylation. In related experiments, adiponectin overexpression decreased TNFalpha and IL-6 expression in 3T3-L1 adipocytes, whereas exposure to free fatty acids had the opposite effect. We conclude that the balance between free fatty acids and adiponectin impacts muscle proteolysis in insulin-resistant conditions and suggest a role for adipose tissue-muscle cross talk in diabetes and obesity.     

Human platelets have cholesteryl ester hydrolytic activity toward plasma high density lipoproteins

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward high density lipoprotein (HDL) labelled with cholesteryl [1-(14)C] oleate resulting in esterilkation of [l-(14)C] oleate to platelet phospholipid. The observed CEH activity was enhanced by 100 nM prostacyclin (PGI(2)), inhibited by 500 μM 2', 3' dideoxyadenosine (DDA), but unaffected by 100 mM chloroquine diphosphate. The CEH activity may represent a mechanism for delivery of other unsaturated fatty acids from HDL to platelets with subsequent modification of the fatty acid composition of platelet phospholipids and potential modification of platelet reactivity.     

Palmitate induces insulin resistance without significant intracellular triglyceride accumulation in HepG2 cells

Previous studies showed that increased release of free fatty acids from adipocytes leads to insulin resistance and triglyceride (TG) accumulation in the liver, which may progress into hepatic steatohepatitis. We and other investigators have previously reported that palmitate induces endoplasmic reticulum stress-mediated toxicity in several tissues. This work investigated whether palmitate could induce insulin resistance and steatosis in HepG2 cells. We treated cells with either saturated fatty acid (palmitate) or unsaturated fatty acid (oleate), and observed that palmitate significantly activated c-jun N-terminal kinase and inactivated protein kinase B. Both 4-phenylbutyric acid and glycerol significantly activated protein kinase B, confirming the involvement of endoplasmic reticulum stress in palmitate-mediated insulin resistance. Oleate, but not palmitate, significantly induced intracellular TG deposition and activated sterol regulatory element binding protein-1. Instead, diacylglycerol level and protein kinase C ? activity were significantly increased by palmitate, suggesting the possible role of diacylglycerol in palmitate-mediated lipotoxicity. Therefore, the present study clearly showed that palmitate impairs insulin resistance, but does not induce significant TG accumulation in HepG2 cells.     

Absorption and metabolism of octanoate by the rat colon in vivo: concentration dependency and influence of alternative fuels

BACKGROUND: Compared with short and long chain fatty acids, medium chain fatty acids (MCFAs) have been shown to provide the highest colonic absorption of substrate carbon. Moreover, colonic epithelial cells fulfil their basic energy requirements as easily from MCFAs as from short chain fatty acids. AIMS: To further characterise octanoate as a colonic luminal substrate, we determined in vivo the influence of (i) substrate concentration and (ii) alternative luminal fuels, on rat colonic transport and metabolism. METHODS: Segments of rat proximal colon (8 cm) were cannulated and perfused for 100 min with (14)C labelled octanoate. The right colic vein was also cannulated and venous blood analysed for total (14)C, (14)CO(2), and metabolites by scintillation counting and high performance liquid chromatography. RESULTS: Tracer appearance in mesenteric blood stabilised after 20-40 minutes of perfusion. Increasing luminal octanoate concentrations from 2 to 40 mM resulted in linear increases in total carbon absorption. Maximum CO(2) production was reached near 10 mM. A substantial proportion of octanoate was absorbed without being metabolised (59-94%). The luminal presence of a mixture of alternative fuels had no influence on either octanoate transport or metabolism. CONCLUSIONS: This study demonstrated substantial concentration dependent colonic absorption of octanoate, rendering this MCFA a potential and much needed high energy substrate for patients with compromised small bowel function. Moreover, octanoate meets the basic energy requirements of colonic epithelial cells in vivo as well as butyrate. This study also demonstrates the divergence of in vitro and in vivo data regarding fatty acid absorption and metabolism in the colonic epithelium.     

Stimulation of human platelet guanylate cyclase by unsaturated fatty acid peroxides.

Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity of human platelet homogenates was stimulated by the addition of phospholipase A2 or unsaturated fatty acids such as oleic, vaccenic, linoleic, linolenic, eicosenoic, eicosadienoic, and arachidonic acids. The addition of lipoxidase potentiated the fatty acid-induced stimulation of guanylate cyclase purified by DEAE-cellulose column chromatography. The extent of the stimulation was dependent on the concentration of the oxidized form of these fatty acids (peroxides). Saturated fatty acids such as stearic and arachidic acids had no effect on the guanylate cyclase activity in the presence or absence of lipoxidase, indicating that human plateletguanylate cyclase is stimulated by unsaturated fatty acid peroxides rather than by fatty acids.Hemoglobin prevented the enzyme stimulation produced by low concentrations of fatty acid peroxides, but enhanced stimulation of the enzyme activity with high concentrations of fatty acid peroxides. 2-Mercaptoethanol, dithiothreitol, and N-ethylmaleimide inhibited the guanylate cyclase activities both in the presence and absence of unsaturated fatty acidperoxide. The stimulation of guanylate cyclase activity by unsaturated fatty acid peroxidesis attributed to oxidation of sulfhydryl residues of the enzyme protein.     

Chronic effects of different non-esterified fatty acids on pancreatic islets of rats

Aims: The aim of this study was to examine the chronic effects of different non-esterified fatty acids (NEFA) on insulin secretion by pancreatic islets of normal Wistar rats in vitro. Methods: Pancreatic islets were isolated from normal Wistar rats, and were incubated with 0.2, 0.4, or 0.8 mmol/L palmitate (C16:0), stearate (C18:0), oleate (C18:1), or linoleate (C18:2) for 24 h, then the insulin secretion and pyruvate dehydrogenase (PHD) activity were examined. Results: Neither islet insulin content nor islet DNA content differed among islets incubated with each kind of NEFA. Compared with control, linoleate significantly inhibited glucose-stimulated insulin secretion (GSIS) and PDH activity at each concentration (p<0.05), while others inhibited GSIS and PDH activity significantly only at 0.4 and 0.8 mmol/L (p<0.05). There was no significant difference in GSIS and PDH activity among islets pretreated by palmitate, stearate, and oleate at the same concentration (p>0.05). However, linoleate decreased GSIS more than others at the same concentration (p<0.05), while linoleate (0.4 or 0.8 mmol/L) inhibited PDH activity more than others at the same concentration (p<0.05). Conclusions: Elevation of palmitate, stearate, oleate or linoleate decreases the β-cell secretory response to glucose, through inhibiting PDH activity. Linoleate exerts more negative effect on GSIS than other NEFA.     

Giardia lamblia: phospholipid analysis of human isolates.

Thin layer chromatograms for phospholipids obtained from 11 human Giardia lamblia isolates and their culture media have shown that phosphatidylcholine and sphingomyelin are the predominant phospholipid classes in all samples. A decrease in the relative percentage of the different classes, especially of phosphatidylcholine, was noticed in the medium after Giardia growth. Fatty acid analysis of the parasite phosphatidylcholine demonstrated that while oleate and palmitate were the major fatty acids in most isolates, arachidonate predominated in two of those studied. Some isolates contained small amounts of myristate, which was not present in the phosphatidylcholine of the culture medium. Moreover, stearate and linoleate predominated in phosphatidylcholine obtained from both media types. The saturated/unsaturated fatty acid ratio also varied for the different isolates. These results appear to suggest heterogeneity in the metabolic activity and utilization of lipid molecules between Giardia isolates.     

Liver fatty acid-binding protein: a marker for studying cellular differentiation in gut epithelial neoplasms.

Human liver fatty acid binding protein is a 127 residue cytoplasmic protein synthesized in liver and in the intestinal epithelium. Previous studies of normal and transgenic mice indicated that the liver fatty acid-binding protein gene is a sensitive marker of enterocytic differentiation. This study shows the use of immunohistochemical methods to examine liver fatty acid-binding protein gene expression in normal human colonic epithelium, colonic villoglandular adenomas, nonmucinous and mucinous adenocarcinomas, and several types of noncolonic epithelial neoplasms. Cells containing liver fatty acid-binding protein were found in normal colonic epithelium, in two thirds of colorectal villoglandular adenomas and nonmucinous adenocarcinomas, and in one third of mucinous adenocarcinomas but not in noncolonic, nonhepatic carcinomas. All liver fatty acid-binding protein-positive colonic adenomas and adenocarcinomas contained patches of immunoreactive cells distributed among histologically identical patches of cells without liver fatty acid-binding protein immunoreactivity. This "mosaicism" was also found in metastases from liver fatty acid-binding protein-positive colonic adenocarcinomas. Immunostaining of these liver fatty acid-binding protein-positive tissues for carcinoembryonic antigen did not show a mosaic cellular pattern in its expression. These data suggest that within a given neoplasm, differences exist in the differentiation programs of monoclonally-derived, malignant colonic epithelial cells and that liver fatty acid-binding protein is a useful marker for operationally defining these subpopulations. Liver fatty acid-binding protein is also a potentially useful diagnostic marker for colorectal and hepatic carcinomas.     

Actions of sulfasalazine and 5-aminosalicylic acid as reactive oxygen scavengers in the suppression of bile acid-induced increases in colonic epithelial cell loss and proliferative activity

Sulfasalazine suppresses mucosal injury in patients with ulcerative colitis, but the mechanism of its therapeutic action is uncertain. In the present study, we examined the mechanism of the protective action of sulfasalazine in a rat model in which colonic epithelial cell loss and subsequent increases in epithelial proliferative activity were induced by intracolonic instillation of sodium deoxycholate. Sulfasalazine or its therapeutically active metabolite 5-aminosalicylic acid suppressed the loss of deoxyribonucleic acid into the colonic lumen and the subsequent increases in mucosal ornithine decarboxylase activity and tritiated thymidine incorporation into deoxyribonucleic acid induced by sodium deoxycholate. Sulfasalazine and 5-aminosalicylic acid also blocked xanthine-xanthine oxidase-induced loss of deoxyribonucleic acid and the subsequent proliferative response. In vitro sodium deoxycholate increased reactive oxygen formation by colonic mucosal scrapings or isolated crypt epithelium. These actions of sodium deoxycholate on reactive oxygen formation were blocked by sulfasalazine or 5-aminosalicylic acid. Sulfapyridine, a therapeutically inactive metabolite of sulfasalazine, had no effect on sodium deoxycholate-induced increases in surface cell sloughing, ornithine decarboxylase, tritiated thymidine incorporation into deoxyribonucleic acid, chemiluminescence, or superoxide production. The ability of sulfasalazine and 5-aminosalicylic acid to scavenge reactive oxygen may play a role in their therapeutic effects of inflammatory bowel disease.     

Isolation and characterization of an unsaturated fatty acid-requiring mutant of cultured mammalian cells.

An unsaturated fatty acid-requiring mutant derived from Chinese hamster ovary (CHO) cells has been isolated and characterized. This mutant grows normally when oleate or other unsaturated fatty acids are supplemented in the growth medium. Unlike the wild-type CHO cells, growth stops when medium is deprived of unsaturated fatty acid. Whole cell pulse experiments with [14C]acetate or [14C]stearate indicate that the mutant is defective in unsaturated fatty acid synthesis. Enzyme assays in vitro show that the enzymatic defect of the mutant is localized to the microsomal stearoyl-CoA desaturase.     

Inhibition of human platelet phospholipase A2 activity by unsaturated fatty acids.

Phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity from human platelets increases significantly when the enzyme is separated from an endogenous inhibitor(s). The inhibitor, associated mainly with a particulate fraction, has now been identified as a mixture of unsaturated fatty acids. Treatment of the inhibitor with trypsin, RNase, DNase, or heat did not diminish its inhibitory activity, which was extractable by organic solvents. Incubation of PLA2 with phospholipids or various neutral lipids, including saturated fatty acids, had little or no effect on enzymatic activity. In contrast, unsaturated fatty acids such as palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), all of which were detected in the particulate fraction, or longer chained unsaturated fatty acids inhibited PLA2 activity by 50% at approximately equal to 5 X 10(-7) M. The level of unsaturated fatty acids in the inhibitor fraction was equivalent to approximately equal to 10(-4) M, apparently sufficient to effectively inhibit PLA2 activity. Methylation of unsaturated fatty acids caused a complete loss of inhibitory activity, and subsequent demethylation restored the activity, suggesting that a free carboxyl group was necessary. Inhibition of PLA2 by unsaturated fatty acids appeared to be noncompetitive. PLA2 absolutely required Ca2+ for activity; the inhibition by unsaturated fatty acids was not reversed by Ca2+. The finding that unsaturated fatty acids are potent inhibitors of PLA2 would explain its generally low activity in human platelet extracts and its marked increase of activity during the course of enzyme purification.