Site Of Fatty Acid Synthesis Research Articles

Proteomic analysis of liver tissue between fat and lean broiler lines

ABSTRACT 1. The liver is the major site of fatty acid synthesis in chickens. Lipid metabolism in the liver correlates with the deposition of triglycerides in adipose tissue. Northeast Agricultural University broilers lines divergently selected for abdominal fat content (NEAUHLF) provide a unique model to study the mechanisms of fat deposition. 2. In previous studies, differentially expressed genes (DEGs) in the livers of fat and lean broilers were evaluated across different developmental stages. Whether protein expression differences exist between the livers of fat and lean broilers is largely unknown. The current experiment used 2D fluorescence difference gel electrophoresis (2D-DIGE) to screen expressed protein (DEP) spots in the liver tissues of NEAUHLF at one, four and seven weeks of age. 3. Twenty-two DEPs were identified by MALDI-TOF-MS that were involved in lipid, energy, protein and amino acid metabolism, oxidative stress, cytoskeleton, and transport. 4. These data furthered the understanding of the fat and lean phenotypes of broiler chickens.

A Stargardt disease-3 mutation in the mouse Elovl4 gene causes retinal deficiency of C32–C36 acyl phosphatidylcholines

Stargardt disease-3 (STGD3) is a juvenile dominant macular degeneration caused by mutations in elongase of very long chain fatty acid-4. All identified mutations produce a truncated protein which lacks a motif for protein retention in endoplasmic reticulum, the site of fatty acid synthesis. In these studies of Stgd3-knockin mice carrying a human pathogenic mutation, we examined two potential pathogenic mechanisms: truncated protein-induced cellular stress and lipid product deficiency. Analysis of mutant retinas detected no cellular stress but demonstrated selective deficiency of C32–C36 acyl phosphatidylcholines. We conclude that this deficit leads to the human STGD3 pathology.

Competition between 24:5n-3 and ALA for Δ6 desaturase may limit the accumulation of DHA in HepG2 cell membranes

The use of Delta 6 desaturase (D6D) twice in the conversion of alpha-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; 22:6n-3) suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 microM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of >18 microM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes.

Comparative Aspects of Lipid Metabolism: Impact on Contemporary Research and Use of Animal Models

The emerging obesity crisis and consequent concerns for corrective measures and appropriate public policy have stimulated research into causes, prevention, remediation, and health consequences of obesity and associated maladies. Such research areas include eating behavior, appetite control, and food intake regulation as well as the regulation of lipid metabolism, cardiovascular function, endocrine function, and dyslipidemia states utilizing various animal models and cell culture systems. Although the liver has a central role in lipid/fatty acid synthesis and glucose is the precursor for de novo fatty acid synthesis in rodents and humans, in many other species, adipose tissues are the primary sites of lipogenesis. In addition, many species utilize acetic acid as a precursor for fatty acid synthesis. This fundamental difference in the site of fatty acid synthesis and the pattern of consequent lipid trafficking influences overall animal lipid metabolism and the role of regulatory hormones and transcription factors. Researchers utilizing various animal species in targeted biomedical research should be aware of these species differences when interpreting their data. In addition, many animal species are used for food production, recreational, and companion purposes. Understanding the lipid metabolism regulatory mechanisms of such species from a comparative perspective is important for the proper nutrition and health of these animals.

ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species

Fatty acid synthase (FAS), a key lipogenic enzyme, is expressed in the two major sites of fatty acid production in the body, that is, the liver and the adipose tissue. Surprisingly, the relative contribution of these sites to lipogenesis is highly variable among species. For example, besides the situation in rodents, where liver and fat are equally active, lipogenesis in some mammals such as the pig occurs principally in adipose tissue, whereas in avian species, the liver is the main lipogenic site. We addressed the question concerning the factors determining the site of fatty acid synthesis. We show that the expression of adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1) mRNA, but not SREBP-2, is linked to FAS protein content or activity in adipose tissues and livers of pig, chicken, and rabbit. Tissue differences in ADD-1/SREBP-1 mRNA expression between species were paralleled by commensurate variations in the nuclear concentration of SREBP-1 protein. Moreover, overexpression of ADD-1/SREBP-1 by adenoviral gene transfer induces FAS in chicken adipocytes, where lipogenesis is normally low. Conversely, the expression of a dominant negative form of ADD-1/SREBP-1 in pig adipocytes downregulates FAS expression. These results reinforce the role of ADD-1/SREBP-1 as a key regulator of lipogenesis, by extending its importance to nonrodent mammals and birds. Furthermore, they establish that differential expression of ADD-1/SREBP-1 is a key determinant of the site of fatty acid synthesis in the body.—Gondret, F., P. Ferré, and I. Dugail. ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. J. Lipid Res. 2001. 42: 106–113.

Comparative study of the lipogenic potential of human and rat adipose tissue

The reported low activity of lipogenic enzymes (especially adenosine triphosphate [ATP]-citrate lyase) in human adipose tissue led to the general conclusion that in humans lipogenesis occurs primarily in the liver. However, recent studies indicate that the liver plays a minor role in de novo lipogenesis and suggest that adipose tissue may be the principal lipogenic human tissue. In an attempt to resolve these contradictions we reinvestigated the lipogenic potential of human adipose tissue and compared with adipose tissue of rats fed a high-fat diet for 2 weeks and fasted overnight before death. These conditions mimic the nutritional state of patients at the moment of tissue sampling. We found that overnight fasting of the rats maintained previously for 12 days on a high-fat diet caused a decrease of ATP-citrate lyase of about 7-fold. Thus, in human adipose tissue, the mean activity of ATP-citrate lyase was approximately 8 times lower than in rats fed a high-fat diet and fasted overnight, and about 50 times lower than in rats maintained on normal laboratory diet. Unlike ATP-citrate lyase, fatty acid synthase (FAS) activity was only slightly lower in human adipose tissue than in rats maintained on a normal laboratory diet. Comparable FAS activity was found when rats were fed a high-fat diet and fasted overnight. The average activities of human adipose tissue acetyl-coenzyme A carboxylase, malic enzyme, and glucose-6-phosphate dehydrogenase were approximately 3-, 4-, and 6-fold lower than in adipose tissue from rats fed a high-fat diet and fasted overnight before tissue sampling, while the activity of 6-phosphogluconate dehydrogenase in humans was higher than in rat adipose tissue. No significant differences in lipogenic enzyme activities were found between male and female and between lean and obese patients. The rate of fatty acid synthesis in intact pieces of human adipose tissue was approximately 5 times lower than in adipose tissue pieces of rats fed a high-fat diet and fasted overnight before tissue samples were taken. The comparison of the lipogenic potential of humans and rats (maintained on the diet to mimic the nutritional state of patients at the time of tissue sampling) suggests that human adipose tissue is an important site of fatty acid synthesis.

Differences in activity of hepatic microsomal triglyceride transfer protein among species

Five sows, five cows, five hens, six guinea pigs, six rabbits, and six rats were used in a study to determine if hepatic microsomal triglyceride transfer protein activity differed among species that varied in site of fatty acid synthesis and rate of hepatic triglyceride export. No differences in plasma nonesterified fatty acids were seen among species. Plasma concentrations of glucose were highest in the hen, intermediate in the rat, guinea pig, and rabbit and lowest in the sow and cow. Liver triglyceride was low in all species with the only significant difference being between the hen and the guinea pig (4.7 and 1.1%, DM basis, respectively). No microsomal triglyceride transfer protein activity was found in muscle. The cow, rat, and guinea pig had the lowest levels and the hen and rabbit the highest levels of duodenal microsomal triglyceride transfer protein activity. Hepatic microsomal triglyceride transfer protein activity was significantly higher in the sow than the other species. Hepatic microsomal triglyceride transfer protein activity was 1.51, 1.63, 2.36, 2.72, 2.95, and 6.70 nmole triolein transferred/h/mg microsomal protein for the guinea pig, rabbit, cow, rat, hen, and sow, respectively. Microsomal triglyceride transfer protein activity in duodenal tissue was 18.0, 18.6, 19.2, 33.4, 113, and 161% of hepatic microsomal triglyceride transfer protein activity for the sow, cow, rat, guinea pig, hen, and rabbit, respectively. Hepatic microsomal triglyceride transfer protein activity scaled to liver weight and metabolic body size was 2.69, 3.36, 4.58, 5.83, 7.49, and 22.3 nmole triolein transferred in the liver/min/kg body weight 0.75 for the rabbit, guinea pig, rat, hen, cow, and sow, respectively. There was little relationship between previously published rates for triglyceride export and hepatic microsomal triglyceride transfer protein activity measured in this experiment.

"Spot 14" protein: a metabolic integrator in normal and neoplastic cells.

"Spot 14" (S14) was originally identified as a mRNA from rat liver that responded rapidly to thyroid hormone, and has now been shown to play a key role in the tissue-specific regulation of lipid metabolism. In addition to its responsiveness to thyroid hormone, S14 gene transcription is controlled by dietary substrates, such as glucose and polyunsaturated fatty acids, and by fuel-related hormones including insulin and glucagon. The S14 protein forms homodimers via a carboxyl-terminal "zipper" domain. The protein is located primarily in the cell nucleus, and its expression in liver is limited to the perivenous portion of the hepatic lobule, the site of fatty acid synthesis. S14 protein is critical for the induction of key enzymes involved in the switching of hepatic metabolism from the fasted to the fed state. S14 antisense oligonucleotides inhibit both the intracellular production of lipids and their export as very low-density lipoprotein (VLDL) particles. S14 acts at the level of transcription to regulate expression of genes encoding key metabolic enzymes, including those required for long-chain fatty acid synthesis. The human S14 gene is located at 11q13.5, a region that is amplified in a subset of aggressive breast cancers. S14 mRNA is expressed in most breast cancer-derived cell lines, and the protein is found in the nuclei of two thirds of human breast cancer specimens, but not in normal nonlactating mammary glands. S14 expression in breast tumors is highly concordant with overabundance of a key lipogenic enzyme. This indicates the association of S14 with enhanced tumor lipogenesis, an established marker of poor prognosis. In addition to the utility of S14 as a model system for elucidation of the mechanism of thyroid hormone action, studies of its regulation and function have provided insights into tissue-specific metabolic control by hormones and dietary substrates in both normal and neoplastic tissues.

Structure-Function Relationships of the Saccharomyces cerevisiae Fatty Acid Synthase: THREE-DIMENSIONAL STRUCTURE

The three-dimensional structure of the Saccharomyces cerevisie fatty acid synthase was computed from electron microscopy of stain images. The barrel-shaped structure (point group symmetry 32) has major and minor axes of approximately 245 x 220 A, respectively, and consists of two different subunits organized in an alpha6beta6 complex (Mr = 2.5 x 10(6)). Two sets of three beta subunits form triangle-shaped caps that enclose the ends of the barrel. The wall of the barrel appears to consist of three N-shaped alpha subunit pairs each with an over and underlying arch-shaped beta subunit. Inside the molecule there are three major interconnected cavities that are tilted approximately 20 degrees with respect to its major axis. An axle-shaped structure extends the length of the cavity on the 3-fold axis and is connected to the two ends of the barrel. The cavities are partially divided on the equator of the molecule by three spokes that extend from the axle on the 2-fold axis to the exterior wall. We propose that these six cavities constitute the six equivalent sites of fatty acid synthesis resulting in an extraordinary structure-function relationship with the 42 catalytic sites involved in fatty acid synthesis inside the molecule. The six cavities each have two funnel-shaped openings ( approximately 20 A in diameter) which may serve to permit the diffusion of substrates and products in and out of these functional units. The subunits appear to be arranged in a manner that affords extensive intermolecular interactions contributing to the stability of this macromolecular complex.

Expression of cutaneous fatty acid-binding protein and its mRNA in rat skin.

Cutaneous fatty acid-binding protein (C-FABP) has been purified from rat skin. Since there was little information about the role of C-FABP in the skin, we investigated the expression of C-FABP and its mRNA in normal rat skin using an immunohistochemical technique and in situ hybridization. In the epidermis, C-FABP mRNA was found to be expressed in basal cells and highly in prickle cells, while C-FABP itself was strongly expressed in the upper prickle and the granular cell layers. In sebaceous glands, both C-FABP and its mRNA were expressed in both peripheral and differentiating cells, although the expression of C-FABP mRNA gradually reduced during differentiation of sebocytes. Since epidermis and sebaceous glands are active sites of fatty acid synthesis, these results suggest that C-FABP may have important roles in the transport and synthesis of fatty acids.

Ulrtrastructural analysis of satellite cells in skeletal muscle of obese and lean LA/N-facp rats

The biochemical phenomena associated with obesity have been studied extensively. Hollands and Cawthorn have reported that the back postural muscle in the ob/ob obese mouse model are responsible for nearly as much fatty acid production as the liver. The hindlimb musculature has been shown to produce higher levels of fatty acids in venous return blood when compared to the arterial blood. These studies have shown the ability of obese muscle to serve as major sites of fatty acid synthesis in the obese animals. This study has sought to identify any ultrastructural variations in red skeletal muscle from the hindlimb and back postural muscles. These studies have revealed aberrations in the satellite cells between the obese and lean phenotypes as well as the presence of lipid droplets in the capillaries of the obese animals’ psoas major muscle.Samples were taken from the bellies of the psoas major representing the back postural muscles and the gastrocnemeius and soleus muscles representing the hindlimb muscle.

The Compartmentation of Acetyl-Coenzyme A Carboxylase in Plants.

Although the biochemical pathways for fatty acid synthesis are more or less similar in plants and animals (Harwood, 1988), there is a major cell biological difference between these two groups of eukaryotes. In plants, the major site of fatty acid synthesis is the plastid, an organelle absent from the animal cell. Many aspects of plastid biology, including fatty acid synthesis, reflect the organelle's origins as a prokaryotic symbiont. The synthesis of fatty acids, such as palmitic acid, the prototype 16-carbon fatty acid, requires one molecule of acetyl-CoA and seven molecules of malonyl-CoA, which are added sequentially with the addition of two carbons to the growing fatty acid chain and the release of CO2 at each step. These reactions are catalyzed by fatty acid synthase, an enzyme complex known to exist in a prokaryotic and a eukaryotic form (Wakil et al., 1983; Harwood, 1988). The prokaryotic form (type II) of fatty acid synthase is found in plants. The synthase is composed of several dissociable proteins, whereas the eukaryotic form (type I) found in animals and yeasts is composed of one or two large multifunctional, nondissociable proteins. For either form, the synthesis requires malonyl-CoA, which is supplied by ACCase in the following reaction:

Proposed mechanisms for the regulation of growth hormone action in poultry: metabolic effects.

Growth hormone (GH) administration to nonruminant red meat animals markedly alters carcass composition so that dramatic reductions in adipose tissue accretion and enhancement of lean tissue growth occur. These repartitioning effects of GH in the pig are reported to reflect antagonism of the lipogenic effect of insulin on adipose tissue, the primary site of fatty acid synthesis in this species, so that glucose disposal and utilization by adipose tissue are markedly reduced, and substrate availability to muscle is increased. In poultry, a significant positive response to GH administration is not consistently achieved, and factors such as posthatch period of development and the pattern of tissue exposure to GH are important determinants of the response to GH enhancement. This may relate to the status of target tissue GH receptors that appear subject to down regulation in the adult chicken. Sensitivity of the bird to the appetite-suppressive effects of GH and interaction between this effect and energy intake have recently been demonstrated and need to be further explored. Growth hormone clearly influences hepatic lipogenesis and net lipid deposition in the broiler chicken. However, future research emphasis on the regulation of GH receptor binding activity and gene expression and their relationship to GH action, as well as on newer components of the GH axis such as GH-binding proteins, will help to clarify controlling mechanisms in poultry.

Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis.

Porcine Fatty Acid Synthase: Cloning of a Complementary DNA, Tissue Distribution of Its mRNA and Suppression of Expression by Somatotropin and Dietary Protein

A cDNA for porcine fatty acid synthase was isolated and used to examine the tissue distribution of fatty acid synthase mRNA within the pig and to determine the impact of recombinant porcine somatotropin (rpSt) and the level of dietary protein on fatty acid synthase mRNA abundance in pig liver and adipose tissue. A 1.5-kb cDNA representing the thioesterase domain of porcine fatty acid synthase was isolated from a lambda gt 11 liver cDNA library. Northern analysis with total RNA extracted from adipose tissue, liver, heart, lung, kidney and intestine revealed a single major fatty acid synthase mRNA species of 8-9 kb. The amount of fatty acid synthase mRNA in hepatic tissue was 25% of the amount in adipose tissue, which suggests that the liver may be a significant site of fatty acid synthesis in the pig. Fatty acid synthase mRNA abundance was significantly reduced in the adipose tissue (P less than 0.01) and the liver (P less than 0.1) by chronic daily administration (60 micrograms/kg) of rpSt. In addition, increasing the amount of dietary protein decreased (P less than 0.1) the abundance of fatty acid synthase mRNA in adipose tissue but had no effect on liver fatty acid synthase expression. In contrast, the abundance of adipose fatty acid binding protein mRNA was unaffected by rpSt or dietary protein. These data indicate that the reduction in the level of fatty acid synthase mRNA is a factor in the pSt-mediated suppression of fatty acid synthesis in porcine adipose tissue.

Metabolism of leucine by bovine adipose tissue: Effects of media supplementation and insulin

Leucine metabolism in comparison to glucose, and with substrate and insulin supplementation, were studied in bovine adipose tissue slices obtained from the tailhead region. In addition, leucine metabolism by isolated adipocytes in a Krebs-Ringer bicarbonate (KRB) buffer was compared to metabolism in Medium-199. Slices oxidized leucine and incorporated the amino acid into cellular protein and lipid, though at much lower rates than for glucose. Glucose addition increased leucine oxidation and its incorporation into lipid but did not affect protein synthesis. Insulin, up to 100 ng/ml, had no effect. Isolated cells convened a higher proportion of the leucine utilized to lipid, and less to protein, than did slices. Absolute rates of oxidation and lipid synthesis were lower, and protein synthesis higher, for Medium-199 than for KRB. Of the total leucine utilized, conversion to lipid represented the largest percentage in both buffers. Insulin had no effect in either buffer system. Bovine adipose tissue, the major site of fatty acid synthesis in this species, was found to both oxidize leucine, and utilize the amino acid for synthesis of cellular components. The isolated adipocyte, free of connective tissue, directs this “ketogenic” amino acid primarily towards lipid synthesis, by mechanisms which appear to be insulin insensitive in the adult bovine, as studied under short-term, in-vitro conditions.

Relative contribution of the main tissues and organs to body fatty acid synthesis in the rat

Tritiated water was used to measure the rate of fatty acid synthesis in the main tissues and organs of 7-week old Wistar male rats in order to determine the relative contribution of each tissue to body fatty acid synthesis. We reached the following conclusions: (a) the liver is the main site of fatty acid synthesis, it alone synthesizes 42% of the newly synthesized fatty acids in the body. (b) The dissectable white adipose tissues synthesize 27% of the fatty acids in the body. This group of tissues is heterogeneous because the mesenteric adipose tissue alone contains 40% of the labeled fatty acids present in the white adipose tissues. (c) Besides the intestines, organs other than the liver play a negligible role (2% of the total) in fatty acid synthesis. (d) The skin contributes 7% of the body fatty acid synthesis. (e) The rest of the carcass, essentially composed of the musculature and the skeleton, contributes 18% of body fatty acid synthesis and accounts for 33% of the extrahepatic tissue fatty acid synthesis.

Fatty acid synthesis in mouse brown adipose tissue the influence of environmental temperature on the proportion of whole-body fatty acid synthesis in brown adipose tissue and the liver

Fatty acid synthesis has been measured in vivo with 3H 2O in mice acclimated at different environmental temperatures (33, 22, 4°C), and the importance of brown adipose tissue and the liver to whole-body fatty acid synthesis at each temperature assessed. At 33°C, when non-shivering thermogenesis is minimal, the rate of fatty acid synthesis in interscapular brown adipose tissue was lower than in the liver, but higher than in white adipose tissue and the carcass. At 4°C, when non-shivering thermogenesis is maximal, the fatty acid synthesis rate in interscapular brown adipose tissue was many times greater than in any other tissue. High fatty acid synthesis rates were also found in other brown adipose tissue depots — subscapular, dorsocervical and axillary — of cold-acclimated mice. In mice maintained at 22°C the rate of fatty acid synthesis was also higher in brown adipose tissue than in other tissues. Overall, the relative importance of brown adipose tissue as a site of fatty acid synthesis increased with lower environmental temperatures, while that of the liver decreased. It was calculated that brown adipose tissue in total accounted for approx. 5% of whole-body fatty acid synthesis at 33°C, 10% at 22°C and 30% at 4°C. In contrast, hepatic synthesis amounted to 32% of whole-body fatty acid synthesis at 33°C, 16% at 22°C and only 11% at 4°C. An estimate of the contribution that de novo synthesis makes to total fatty acid utilization by interscapular brown adipose tissue suggests that fatty acid synthesis and breakdown constitutes a significant heat-dissipating ‘cycle’ in brown adipose tissue of cold-acclimated mice. Such a cycle is not evident in suckling animals since fatty acid synthesis in brown adipose tissue is very low during early development.

Important sites of lipogenesis in the mouse other than liver and white adipose tissue.

The musculature of the shoulders and back has been identified as a major site of fatty acid synthesis in mice.

Evidence that fatty acid synthesis in the interscapular brown adipose tissue of cold-adapted rats is increased in vivo by insulin by mechanisms involving parallel activation of pyruvate dehydrogenase and acetyl-coenzyme A carboxylase.

Plasma insulin concentrations in cold-adapted rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in interscapular brown adipose tissue were determined from the incorporation of 3H from 3H2O into tissue lipid. Rates of synthesis were greatly elevated after glucose administration and markedly decreased after injection with anti-insulin serum. Parallel changes in the initial activities of both acetyl-CoA carboxylase and pyruvate dehydrogenase were observed under these conditions, but no changes in total activities were evident. The results suggest that this tissue is an important site of fatty acid synthesis in the cold-adapted rat and that this feature of the tissue is sensitive to changes in plasma insulin concentrations.