Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters

Male rats with thoracic duct cannulae were intubated with corn oil or fatty acid methyl esters and the lymph was collected over the next 2-72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate - polyacrylamide - glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas--liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phosphatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B-100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from corn oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.     

Diglyceride kinase from Escherichia coli. Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

Diglyceride kinase from Escherichia coli: Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

Recombination of human erythrocyte apoprotein and lipid. I. Interaction of apoprotein and lipid at the air-water interface

The formation and stabilization of a complex between total erythrocyte apoprotein and monolayers of total erythrocyte lipid as measured by changes of surface pressure (Δπ) and rate of change of surface pressure (dπ/dt) was studied as a function of pH, ionic strength, and lipid surface pressure. Penetration of apoprotein into lipid monolayers was favored by conditions in which lipid and apoprotein were oppositely charged. Once the interaction was completed, the resultant surface complex was resistant to large changes in subphase pH and ionic strength as shown by the insensitivity of Δπ to these parameters. The dπ/dt, however, showed strong dependence on pH and ionic strength, but not on lipid surface pressure. A sharp decrease in dπ/dt around pH 3.5–4.5 is associated with the change in apoprotein charge from (+) to (?). Comparison of complex formation between apoprotein and bovine serum albumin, cytochrome c, and human hemoglobin suggests that erythrocyte apoprotein was specialized in its interaction with erythrocyte lipids. The data show that formation of an apoprotein-lipid complex at the air-water interface has both electrostatic and hydrophobic components. This contradicts results from other laboratories studying erythrocyte membrane recombination by bulk methods.     

Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1

To study the assembly of newly synthesized lipids with apoprotein A1, we administered [2-3H]glycerol to young chickens and determined the hepatic intracellular sites of lipid synthesis and association of nascent lipids with apoprotein A1. [2-3H]glycerol was rapidly incorporated into hepatic lipids, reaching maximal levels at 5 min, and this preceded the appearance of lipid radioactivity in the plasma. The liver was fractionated into rough and smooth endoplasmic reticulum and Golgi cell fractions. The isolated cell fractions were further subfractionated into membrane and soluble (content) fractions by treatment with 0.1 M Na2CO3, pH 11.3. At various times, the lipid radioactivity was measured in each of the intracellular organelles, in immunoprecipitable apoprotein A1, and in materials that floated at buoyant densities similar to those of plasma lipoproteins. Maximal incorporation occurred at 1 min in the rough endoplasmic reticulum, at 3-5 min in the smooth endoplasmic reticulum, and at 5 min in the Golgi cell fractions. The majority (66-93%) of radioactive glycerol was incorporated into triglycerides with smaller (4-27%) amounts into phospholipids. About 80% of the lipid radioactivity in the endoplasmic reticulum and 70% of that in the Golgi cell fractions was in the membranes. The radioactive lipids in the content subfraction were distributed in various density classes with most nascent lipids floating at a density less than or equal to 1.063 g/ml. Apoprotein A1 from the Golgi apparatus, obtained by immunoprecipitation, contained sixfold more nascent lipids than did that from the endoplasmic reticulum. These data indicate that [2-3H]glycerol is quickly incorporated into lipids of the endoplasmic reticulum and the Golgi cell fractions, that most of the nascent lipids are conjugated with apoproteins A1 in the Golgi apparatus, and that very little association of nascent lipid to apoprotein A1 occurs in the endoplasmic reticulum.     

Reduced hepatic release of apoprotein B after enteral nutrition in rats

To investigate the effects of enteral and parenteral alimentation on VLDL release from the liver, a lipid-free liquid nutriment was continuously administered to free-moving rats via the oral cavity (oral group), stomach (enteral group) or superior caval vein (parenteral group). After 1-week of nutrition, the plasma VLDL concentrations were significantly lowered in the enterally-fed group. By immunoblotting assay using a specific antiserum, plasma contents of both apoprotein B-100 and B-48, the major components of rat apoprotein B, were found to be decreased in the enteral group, whereas only that of apoprotein B-48 was reduced in the parenteral group as compared with the oral group. Sucrose gradient centrifugation of the lipid droplets in the liver from the enteral group showed an increase of the free-triacylglycerol fraction with a concomitant increase of the apoprotein B-48-rich triacylglycerol fraction. These results suggest that enteral nutrition causes triacylglycerol accumulation in the liver, at least in part by impairment of lipoprotein release from the liver.     

Oxidative modification of glycated low density lipoprotein in the presence of iron

This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8% modification of lysine residues) and 84% of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. Alpha-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+.     

Interaction between hepatic microsomal membrane lipids and apolipoprotein A-I

Incubation of apoprotein A-I (apo-A-I), the major protein component of human high density lipoprotein, with rat liver microsomal membranes under conditions of elevated pH and ionic strength leads to the production of a soluble protein:lipid complex (A-I/MM complex). The A-I/MM complex, as purified by density gradient centrifugation and agarose column chromatography, possesses a lipid composition similar to the hepatic microsomal membrane and a protein/lipid ratio similar to that of plasma high density lipoproteins, but markedly different from that of recombinant particles prepared with synthetic lipids. The A-I/MM complex constitutes a more physiological recombinant particle than can be formed using synthetic lipids and may be a suitable model for the newly assembled intracellular high density lipoproteins. Incubation of the erythrocyte plasma membranes with apo-A-I under the same conditions as used with microsomal membranes fails to generate any lipid:apoprotein complexes. This membrane specificity for forming soluble lipoprotein complexes suggests that the microsomal membranes possess a unique feature, possibly their lipid composition, which render them particularly suitable to serve as lipid donors to the apoproteins which are undergoing assembly within the endoplasmic reticulum/Golgi organelles.     

Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6-30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 microm triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.     

Loss of apoB-100 secondary structure and conformation in hydroperoxide rich, electronegative LDL(-).

A subpopulation of low-density lipoproteins (LDL) is present in human plasma that contains lipid hydroperoxides and is more negatively charged (LDL(-)) than normal native LDL. By circular dichroism and tryptophan lifetime measurements we found that apoB-100 secondary structure is markedly decreased and its conformation is severely altered in LDL(-). The low tryptophan fluorescence intensity confirms the oxidative degradation of the lipoprotein, and the very long lifetime value of one of its decay components indicates a low polarity environment for the remaining unbleached residues. Either a peculiar folding or, most likely, a sinking of the apoB-100 into the lipid core can account for the observed long lifetime component. Oxidation in vitro produces a similar unfolding of the apolipoprotein but the lifetime of tryptophan fluorescence is shifted to lower values, indicating that the denatured apoprotein remains at the hydrophilic surface of the lipoprotein particle. A disordering and an increased polarity of the LDL(-) surface lipids was demonstrated by measuring the generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). The looser monolayer packing apparently favors the new conformation of apoB-100 and its sinking into a more hydrophobic environment, possibly accounting for it reduced receptor binding properties.     

Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts

Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30-40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties.     

HDL-induced cardiac prostacyclin synthesis: relative contribution of HDL apoprotein and lipid

The objective of this study was to determine if the apoprotein or lipid constituents of high density lipoproteins (HDL) mediate HDL-induced prostacyclin synthesis in the Langendorff-perfused rabbit heart. Acetylation, acetoacetylation, or partial removal by trypsin digestion of HDL apoprotein did not reduce the ability of the lipoprotein to stimulate cardiac prostacyclin synthesis. Delipidated apoproteins were less effective in stimulating cardiac prostacyclin synthesis in comparison to intact HDL. In contrast, protein-free lipid vesicles, made from HDL lipids, caused a pronounced stimulation of cardiac prostacyclin synthesis. These results suggest that HDL apoproteins, in their native state, are not essential for HDL-induced cardiac prostacyclin synthesis. The stimulation of cardiac prostacyclin synthesis by HDL may depend on the lipoprotein's lipid rather than on its apoprotein constituents.     

Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of TNF to the culture medium for 24-48 h resulted in an increase of [3H]oleic acid uptake and esterification into lipids. The effect could be seen already with 0.3 ng/ml and was maximal with 30 ng/ml. The effect of TNF was mainly on the incorporation of [3H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3H]cholesteryl ester. In the human SMC there was a 40% increase in [3H]oleic acid into total lipids, while the rise in [3H]triacylglycerol ranged between 60-90%. TNF did not modulate cellular triacyglycerol synthesis in cultured mouse peritoneal macrophages. Since TNF was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triacylglycerol accumulation.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

Apolipoprotein AIMilano. Accelerated binding and dissociation from lipids of a human apolipoprotein variant

The lipid binding properties of apolipoprotein (apo) AIMilano, a molecular variant of human apolipoprotein AI, characterized by the Arg173----Cys substitution, was investigated by the use of dimyristoylphosphatidylcholine liposomes. Both the variant AIMilano and normal AI are incorporated to the same extent in stable complexes isolated by gel filtration, showing similar dimensions and stoichiometries. A higher affinity of apo-AIMilano for dimyristoylphosphatidylcholine is suggested by the faster association rate of the variant apoprotein compared to normal AI; similarly, apo-AIMilano is more readily displaced by guanidine hydrochloride from the isolated dimyristoylphosphatidylcholine-apoprotein complexes. When the secondary structure of apo-AIMilano was investigated by spectrofluoroscopy and circular dichroism, a higher fluorescence peak wavelength and a lower alpha-helical content were detected in the variant apoprotein compared to normal AI. The substitution Arg173----Cys in the AIMilano dramatically alters the amphipathic nature of the modified alpha-helical fragment of apoprotein AI. The association rate with lipids is accelerated by an increased exposure of hydrophobic residues. The reduced stability of the lipid-apoprotein particles is possibly mediated by a reduction in the number of helical segments involved in lipid association. The high flexibility of the AIMilano apolipoprotein in the interaction with lipids may explain its accelerated catabolism and the possibly improved uptake capacities for tissue lipids.     

Proteolipid in bovine lung surfactant: its role in surfactant function

The chemical and biophysical properties of the proteins in the lipid extracts of lung surfactant have not clearly been determined. These proteins were isolated from lung surfactant lipids by Sephadex LH-20 chromatography and purified with silicic acid chromatography followed by dialysis against organic solvents. The proteolipid thus obtained had a protein to phospholipid ratio of 3 to 1 (w/w). The proteolipid apoprotein had a nominal molecular weight of ca. 5 kDa. We evaluated the functional role of this proteolipid by combining it with proteolipid-depleted surfactant lipids or synthetic dipalmitoylphosphatidylcholine (DPPC) and then measuring with a pulsating bubble surfactometer. The proteolipid and DPPC recombinant reproduced the surface activity of natural lung surfactant. We conclude that this 5 kDa proteolipid apoprotein is a functionally important constituent of lung surfactant.     

Lipids,lipid turnover,and phospholipase D in plant suspension culture cells (Daucus carota)

The lipid pattern of Daucus carota L. suspension culture cells and other plant cell strains was analyzed. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol were the main components. The characteristic plastidal and mitochondrial lipids could also be identified. All strains tested exhibited a phospholipase D activity. Several lipid precursors were found to be well utilized by the cells and to be special markers for certain lipids or parts of the lipid molecules. The half life times of the major lipids ranged at about half a generation time of the cells.     

Effects of dietary protein type on the response of lipid metabolism to orotic acid in rats

The effects of orotic acid supplementation to casein, egg protein, soy protein and wheat gluten diets on the lipids of liver and serum were compared. When orotic acid was added, the contents of total lipids and triacylglycerol in the liver of the casein group were significantly higher or tended to be higher than those of the other three dietary groups. Dietary orotic acid had no effect on the food intake. The liver weight, and liver total lipids, triacylglycerol, cholesterol and phospholipids were increased or tended to be increased by the addition of orotic acid. The serum triacylglycerol level was decreased by the addition of orotic acid to either the casein or soy protein diet. Thus, the response to liver lipid accumulation induced by orotic acid feeding depended on the dietary protein type.     

Characterization of the seed and leaf lipids of high and low linolenic acid flax genotypes

The total seed lipids of four flax (Linum usitatissimum) genotypes, differing markedly in their acyl composition, were extracted and fractionated using column, preparative, and thin-layer chromatography. In the total lipid extract of seeds, the lower linolenate content of the cultivar Glenelg (39.1% compared to that of cv. Croxton (50.5%) was associated with a higher oleate content. Further reductions in linolenate content in the induced mutants of cv. Glenelg, M1722 (17.2%) and "Zero" (1.9%) were accompanied by equivalent increases in linoleate but only minor increases in oleate. Similar changes were observed in the major triacylglycerol fraction of the simple lipids (fatty acid esters of glycerol and sterols), but there was considerable heterogeneity for acyl composition in the minor simple lipid components, including both diacylglycerols and sterol esters, and the complex lipids (glycolipids and phospholipids). The induced mutations substantially reduced linolenate content of all lipid fractions but in no case was it eliminated. Maturation of "Zero" seed at 15/10 degrees C (compared to 24/19 degrees C) increased linoleate and decreased stearate and oleate contents in all lipid fractions. In contrast to seed lipids, the acyl composition of the leaf lipids of the mutant genotypes was the same as those of their parent.     

AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the alpha subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPKalpha or a kinase-inactive AMPKalpha, respectively. An insulin-resistant state was induced by exposing cells to 30 mm glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3alpha/beta. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPKalpha. The kinase-inactive AMPKalpha increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPKalpha negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.