Effects of dietary fat on the saturated and monounsaturated fatty acid metabolism in growing pigs

The effect of dietary fats differing in fatty acid (FA) composition on the metabolism of saturated FA (SFA) and monounsaturated FA (MUFA) in growing pigs was investigated. The deposition of FA in the body and the fate of individual dietary FA were assessed after slaughter. Gilts with an initial body weight (BW) of 60 kg were used as experimental animals. Six pigs were slaughtered at 60 kg BW, while further 18 pigs received three isoenergetic and isonitrogen experimental diets containing linseed oil, rapeseed oil or beef tallow at 50 g/kg diet until they reached 105 kg (six pigs per group). The chemical composition and the content of FA in the whole body were determined and compared across groups. Regardless of dietary treatment, the whole body contained similar amounts of protein, fat and total FA. The total accumulation (percentage of net intake and de novo production) of SFA and MUFA was similar in all groups, but the processes of elongation and desaturation of SFA and MUFA depended upon the type of FA added to the diet. A high dietary content and intake of MUFA inhibits desaturation compared to SFA- and PUFA-rich diets, whereas a high SFA content and intake lowers elongation rate. The increasing net intake of total SFA and MUFA was associated with a lower total de novo production of these FA in the whole body of pigs.     

Composition of the saturated and monounsaturated fatty acids of Mycobacterium phlei

Combined gas-liquid chromatography-mass spectrometry has been used to identify unusual fatty acids of Mycobacterium phlei. In addition to many normal saturated and monounsaturated fatty acids, series of iso, anteiso, 10-methyl, and (n-8)-methyl substituted fatty acids have been found and identified. These mid-chain branched acids may arise by methylation of monounsaturated acids followed (if necessary) by chain elongation.     

Neutral amino acid transport in isolated rat pancreatic islets

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.     

Intrahepatic transplantation of pancreatic islets in the rat.

Islets of Langerhans from isogeneic donor rats were transplanted directly into the hepatic parenchyma of recipients which had been made severely diabetic by streptozotocin (glycaemia ranging between 400 and 1090 mg%). Complete control lasting up to 13 months was achieved in 65% of recipients by using 600-800 islets. Following intravenous glucose administration, each rat responded similarly to normal rats with a rapid but reduced release of insulin. Cytoimmunofluorescence and electron microscopic studies demonstrated the presence of both functional insulin and glucagon cells, within the transplanted islets. It is suggested that for various reasons direct intrahepatic transplantation might become the preferred method for islets.     

Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets

Pyruvate dehydrogenase (PDH) converts pyruvate to acetyl-CoA, links glycolysis to the Krebs cycle, and plays an important role in glucose metabolism and insulin secretion in pancreatic beta cells. In beta cells from obese and Type 2 diabetic animals, PDH activity is significantly reduced. PDH is negatively regulated by multiple pyruvate dehydrogenase kinase (PDK) isotypes (PDK subtypes 1-4). However, we do not know whether fatty acids or high glucose modulate PDKs in islets. To test this we determined PDH and PDK activities and PDK gene and protein expression in C57BL/6 mouse islets. Both high palmitate and high glucose reduced active PDH activity and increased PDK activity. The gene and protein for PDK3 were not expressed in islets. Palmitate up-regulated mRNA expression of PDK1 (2.9-fold), PDK2 (1.9-fold), and PDK4 (3.1-fold). High glucose increased PDK1 (1.8-fold) and PDK2 (2.7-fold) mRNA expression but reduced PDK4 mRNA expression by 40 percent in cultured islets. Changed PDK expression was confirmed by Western blotting. These results demonstrate that in islet cells both fat and glucose regulate PDK gene and protein expression and indicate that hyperglycemia and hyperlipidemia contribute to the decline in diabetic islet PDH activity by increasing mRNA and protein expression of PDK.     

Hexose metabolism in pancreatic islets. Effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets.

Anaplerotic reactions leading to the de novo synthesis of fatty acids, were recently proposed to participate in the coupling of metabolic to secretory events in the process of glucose-stimulated insulin release. In an attempt to validate such a proposal, the effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release was investigated in glucose-stimulated rat pancreatic islets. The inhibitor of ATP citrate-lyase, when tested in the 1.0-2.0 mM range, failed to affect glucose-stimulated insulin release, but also failed to inhibit the incorporation of 14C-labelled acetyl residues derived from L-[U-14C]leucine into islet lipids. A partial inhibition of fatty acid labelling by 3H2O was only observed in islets incubated for 120 min in the presence of 5.0 mM (-)-hydroxycitrate and absence of CaCl2. These findings suggest that (-)-hydroxycitrate is not, under the present experimental conditions, a useful tool to abolish fatty acid synthesis in intact pancreatic islets.     

Differential regulation by fatty acids of protein histidine phosphorylation in rat pancreatic islets

Long-chain fatty acids (e.g. arachidonic acid) have been implicated in physiological control of insulin secretion. We previously reported histidine phosphorylation of at least two islet proteins (e.g., NDP kinase and the beta subunit of trimeric G-proteins), and suggested that such a signalling step may have regulatory roles in beta cell signal transduction, specifically at the level of G-protein activation. Since our earlier findings also indicated potential regulation by long-chain fatty acids of islet G-proteins, we undertook the current study to verify putative regulation, by fatty acids, of protein histidine phosphorylation of NDP kinase and Gbeta subunit in normal rat islets. The phosphoenzyme formation of NDP kinase was stimulated by various fatty acids in the following rank order: linoleic acid > arachidonic acid > oleic acid > palmitic acid = stearic acid = control. Furthermore, the catalytic activity of NDP kinase was stimulated by these fatty acids in the rank order of: oleic acid > arachidonic acid > linoleic acid > palmitic acid = stearic acid = control. Arachidonic acid methyl ester, an inactive analog of arachidonic acid, did not significantly affect either the phosphoenzyme formation or the catalytic activity of NDP kinase. Interestingly, arachidonic acid exerted dual effects on the histidine phosphorylation of beta subunit; it significantly stimulated the phosphorylation at 33 microM beyond which it was inhibitory. Together, these findings identify additional loci (e.g., NDP kinase and Gbeta subunit) at which unsaturated, but not saturated, fatty acids could exert their intracellular effects leading to exocytotic secretion of insulin.     

Failure of streptozotocin tetraacetate to undergo extensive hydrolysis and to inhibit D-glucose metabolism and insulinotropic action in rat pancreatic islets

The esterification of several monosaccharides, such as D -glucose, D -mannoheptulose and 2-deoxy-D -glucose was recently reported to increase their biological efficiency as either nutrient or antimetabolic agent. In the present study, however, the tetraacetate ester of streptozotocin was unexpectedly found to be less potent than unesterified streptozotocin in inhibiting D -glucose metabolism and insulinotropic action in isolated rat pancreatic islets. This coincided with a much lower rate for the hydrolysis of streptozotocin tetraacetate than D -glucose pentaacetate in islet homogenates. These findings document that the esterification of single sugars is not always a successful procedure to enhance their biological potency, for instance because of too low a rate for the intracellular hydrolysis of the ester. To the extent that the activity of the concerned esterase(s) may differ in distinct cell types, as suggested by a prior observation, advantage could be taken of such a situation to target selected esters towards specific, e.g. tumoural cells. Copyright © 1998 John Wiley & Sons, Ltd.     

Active transport of myo-inositol in rat pancreatic islets.

myo-Inositol transport by isolated pancreatic islets was measured with a dual isotope technique. Uptake was saturable with a half-maximal response at approx. 75 microM. With 50 microM-inositol, uptake was linear for at least 2 h during which time the free intracellular concentration rose to double that of the incubation medium. Inositol transport is therefore active and probably energized by electrogenic co-transport of Na+ down its concentration gradient as uptake was inhibited by ouabain, Na+ removal or depolarizing K+ concentrations. Inositol transport was abolished by cytochalasin B which binds to hexose carriers, but not by carbamoylcholine or Li+ which respectively stimulate or inhibit phosphoinositide turnover. Uptake of inositol was not affected by 3-O-methylglucose or L-glucose (both 100 mM) nor by physiological concentrations of D-glucose. The results suggest that most intracellular inositol in pancreatic islets would be derived from the extracellular medium. Since the transport mechanism is distinct from that of glucose, inositol uptake would not be inhibited during periods of hyperglycaemia.     

Hyaluronidase-induced inhibition of the insulinotropic action of sulfonylureas, leucine, and glucagon in rodent isolated pancreatic islets.

Exposure of hamster pancreatic islets to hyaluronidase during isolation by means of collagenase inhibits the insulinotropic action of several chemically different sulfonylureas, leucine, and glucagon without affecting glucose-stimulated insulin secretion. This inhibition is reversible for tolbutamide and leucine but irreversible for glucagon. Hyaluronidase inhibits reversibly the insulinotropic action of tolbutamide without affecting that of glucose also in mouse and rat isolated pancreatic islets . These findings suggest the existence of functionally related pancreatic beta cell receptors for tolbutamide and leucine different from those for glucose and glucagon and illustrate the potential usefulness of hyaluronidase as an enzymatic probe applicable toward investigating the cellular mechanism of action of key insulinotropic agents.     

Lipid polymorphism of mixtures of dioleoylphosphatidylethanolamine and saturated and monounsaturated phosphatidylcholines of various chain lengths

The L alpha-HII phase transition behavior of many lipid-water liquid crystals is dominated by the competition between the tendency to curl the lipid layers to an intrinsic radius of curvature and opposing hydrocarbon packing constraints. In particular, packing constraints can increase the free energy of the inverted hexagonal (HII) phase as compared to that of the lamellar (L alpha) phase. This is especially true where the lipid molecule is not long enough to reach into the corners of the lattice in large hexagonal structures necessitated by a large intrinsic radius of curvature. In this paper it is shown that the addition of a minor fraction long-chain lipid to a system of otherwise uniform chain composition can also relax packing constraints, thereby lowering the lamellar to hexagonal transition temperature. For the specific systems used, dioleoylphosphatidylethanolamine (di-18:1c-PE) with minor fractions of 1,2-diacyl-sn-glycero-3-phosphocholines [di-n:1c-PC (n = 14, 18, 22, and 24)], the observed HII lattices systematically increased in size with increasing chain length, suggesting that the chain length also may affect the intrinsic curvature of the mixture. These experiments demonstrate that the lipid "shape concept", which is a qualitative expression of the concept quantitatively described by the intrinsic radius of curvature, is insufficient to understand the L alpha-HII transition. It is necessary to, at least, consider the competition between curvature and packing.     

Hexose metabolism in pancreatic islets. Insignificance of D-glucose futile cycling in rat islets.

When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e. about 1% of the rate of exogenous D-[5-3H]glucose utilization. Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s). It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.     

Inositol cycling and phosphoinositide metabolism in rat pancreatic islets.

The effects of extracellular inositol and LiCl on intra-islet inositol cycling were investigated in isolated rat islets. Islets were cultured for 7 days in inositol-free RPMI 1640 containing 11.1 mM glucose and labeled with 3.7 MBq myo-[2-3H] inositol for the final 3 days. The labeled islets were then perifused under various conditions. There was a persistent increase in [3H] efflux from labeled islets stimulated with 16.7 mM glucose for 60 min. Addition of 5 mM inositol resulted in marked release of [3H] from islets and a decrease in radioactive inositol-lipid. When islets were perifused with 5 mM LiCl, the glucose-induced efflux of [3H] was greatly inhibited. The inhibitory effect of LiCl on [3H] efflux was partially corrected by the addition of 5 mM inositol. A prominent effect of LiCl was an increase in inositol monophosphate, indicating increased phospholipase C activity. This was detected within 5 min after glucose stimulation. The present data suggest that there is always very active intra-islet inositol cycling and that glucose can augument inositol-lipid metabolism.     

SCD1 mediates the influence of exogenous saturated and monounsaturated fatty acids in adipocytes: Effects on cellular stress,inflammatory markers and fatty acid elongation

Palmitate (PA), stearate (SA), palmitoleate (PMA) and oleate (OA) are among the most abundant fatty acids (FAs) in adipose tissue (AT). These FAs differentially regulate AT inflammation by altering adipocyte signalling pathways and the secretion of proinflammatory cytokines. Intracellular levels of these FAs are controlled, in part, by stearoyl-CoA desaturase 1 (SCD1). Therefore, SCD1 may have an important role mediating FA-regulation of adipocyte inflammation. Given this, we hypothesized that the influence of PA, SA, PMA and OA on inflammation and cellular stress, as well as FA metabolism, would be exacerbated with reduced SCD1 activity. Real-time RT-PCR, immunoassays, gas chromatography and western blotting were used to examine the expression and secretion of common inflammatory markers, as well as FA profiles and markers of cellular stress, in 3T3-L1 adipocytes. FA treatments differentially affected inflammatory markers and FA profiles in SCD1-inhibited adipocytes. Specifically, SA significantly increased the expression of Ccl5 (5.3-fold) and Mcp-1 (3.2-fold), and the secretion of IL-6 (17.8-fold) and MCP-1 (4.0-fold) in SCD1-inhibited adipocytes compared to controls. The proinflammatory effects observed with SA are particularly notable given that SCD1-inhibited adipocytes increased elongation of PA to SA, as determined using U-¹³C-PA. The effects of PA, PMA and OA were not as substantial as those of SA, although PA did significantly increase Ccl5 (2.7-fold) and Mcp-1 (1.2-fold) expression in SCD1-inhibited adipocytes. None of the FAs altered markers of cellular stress. Collectively, these results emphasize the differential effects of individual FAs and highlight how SCD1 influences their regulation of adipocyte inflammation.     

Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species

Enhanced production of monounsaturated fatty acids (FA) derived from carbohydrate-enriched diets has been implicated in the development of obesity and insulin resistance. The FA elongases Elovl-5 and Elovl-6 are regulated by nutrient and hormone status, and have been shown using intact yeast and mammalian microsome fractions to be involved in the synthesis of monounsaturated FAs (MUFA). Herein, targeted knockdown and overexpression of Elovl-5 or Elovl-6 was used to determine their roles in de novo synthesis of specific MUFA species in mammalian cells. Treatment of rat insulinoma (INS)-1 cells with elevated glucose increased de novo FA synthesis and the ratio of MUFAs to saturated FAs. Elovl-5 knockdown decreased elongation of 16:1,n-7. Elovl-5 overexpression increased synthesis of 18:1,n-7; however, this increase was dependent on stearoyl-CoA desaturase–driven 16:1,n-7 availability. Knockdown of Elovl-6 decreased elongation of 16:0 and 16:1,n-7, resulting in accumulation of 16:1,n-7. Elovl-6 overexpression preferentially drove synthesis of 16:0 elongation products 18:0 and 18:1,n-9 but not 18:1,n-7. These findings demonstrate that coordinated induction of FA elongase and desaturase activity is required for balanced synthesis of specific n-7 versus n-9 MUFA species. Given the relative abundance of 16:0 to 16:1,n-7 and the specificity of Elovl-6 for 16:0, Elovl-6 is a major elongase for 18:1,n-9 production.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

Dietary saturated fatty acid content affects lymph lipoproteins: studies in the rat

We examined effects on intestinal absorption of cholesterol and triglycerides and intestinal lipoprotein formation by feeding rats diets in which saturated fatty acids (palmitic plus stearic) comprised 78%, 68%, 48%, or 38% of triglyceride fatty acids. Absorption into lymph of radiolabeled cholesterol was proportional to triglyceride absorption. The rates of absorption of these lipids were related inversely to the % saturated fatty acids fed. The distribution of newly absorbed cholesterol and triglyceride into intestinal lipoproteins differed. With increasing cholesterol absorption more was recovered in very low density lipoproteins in contrast to the appearance preferentially in chylomicrons of larger quantities of fatty acid. Lymph lipid content did not reflect a consistent pattern in relation to the experimental diet fed. The fatty acid composition of triglyceride-rich lymph lipoproteins resembled the diet closely. One-quarter of the intestinal lymph particles from rats fed the highly saturated diets was flattened and polygonal as judged by electron microscopy if cooled to room temperature; whereas with the same diets, particles collected and isolated at 37 degrees C were round. Proportions of A-I and C apolipoproteins in triglyceride-rich intestinal particles varied inversely; apoA-I increased as fat/cholesterol absorption was greater. Diet-induced alterations in plasma lipoproteins and increased circulating triglycerides in this study in rats were unrelated to the variations in intestinal absorption or lymph lipoprotein formation.     

Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.