Dissociation of Systemic Glucose Homeostasis from Triacylglyceride Accumulation by Reduced <Emphasis Type="Italic">Acsl6</Emphasis> Expression in Skeletal Muscle

Long chain acyl-CoA synthetase (ACSL) is an enzyme that activates fatty acids before they are further metabolized. ACSL6 is the one of main ACSL isoforms exclusively expressed in skeletal muscle, but the consequences of the suppression of this gene in systemic glucose homeostasis has yet to be reported. Hence, we investigated the roles of ACSL6 gene in glucose tolerance and TAG distribution in physiological conditions. Eight-week-old male C57BL/6J mice were administered with control or Acsl6 siRNAs and then fed with either AIN-93 control diet or high fat diet. At seven days after the first siRNA injection, oral glucose tolerance tests and TAG quantification were performed. In vivo administration of Acsl6 siRNA decreased Acsl6 expression only in skeletal muscle under AIN-93 or a high fat diet. However Acsl6 siRNA injection to animals increased TAG accumulation in the liver without the change of Acsl6 expression. Atelocollagen mediated Acsl6 suppression enhanced whole-body glucose tolerance coinciding with decreased TAG accumulation in skeletal muscle of mice fed an AIN-93 diet. However, the improved glucose tolerance by Acsl6 reduction was ablated by high fat diet. Moreover reduced Acsl6 did not alter the phosphorylation of insulin signaling proteins in skeletal muscle. These results suggest that Acsl6 reduction in skeletal muscle enhances glucose homeostasis and dissociates the insulin responses from TAG accumulation in skeletal muscle.     

Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction,but pathologic hypertrophy is reversed by rapamycin

In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1^H −/−), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy and increased phosphorylated S6 kinase (S6K), a substrate of the mechanistic target of rapamycin, mTOR. Doppler echocardiography revealed evidence of significant diastolic dysfunction, indicated by a reduced E/A ratio and increased mean performance index, although the deceleration time and the expression of sarco/endoplasmic reticulum calcium ATPase and phospholamban showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1^H −/− mice with rapamycin. Six to eight week old Acsl1^H −/− mice and their littermate controls were given i.p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10 weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1^H −/− hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1^H −/− hearts exhibited an 8-fold higher uptake of 2-deoxy[1-¹⁴C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-¹⁴C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1^H −/− mice.     

Diminished Acyl-CoA Synthetase Isoform 4 Activity in INS 832/13 Cells Reduces Cellular Epoxyeicosatrienoic Acid Levels and Results in Impaired Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells is potentiated by fatty acids (FA). The initial step in the metabolism of intracellular FA is the conversion to acyl-CoA by long chain acyl-CoA synthetases (Acsls). Because the predominantly expressed Acsl isoforms in INS 832/13 cells are Acsl4 and -5, we characterized the role of these Acsls in beta-cell function by using siRNA to knock down Acsl4 or Acsl5. Compared with control cells, an 80% suppression of Acsl4 decreased GSIS and FA-potentiated GSIS by 32 and 54%, respectively. Knockdown of Acsl5 did not alter GSIS. Acsl4 knockdown did not alter FA oxidation or long chain acyl-CoA levels. With Acsl4 knockdown, incubation with 17 mm glucose increased media epoxyeicosatrienoic acids (EETs) and reduced cell membrane levels of EETs. Further, exogenous EETs reduced GSIS in INS 832/13 cells, and in Acsl4 knockdown cells, an EET receptor antagonist partially rescued GSIS. These results strongly suggest that Acsl4 activates EETs to form EET-CoAs that are incorporated into glycerophospholipids, thereby sequestering EETs. Exposing INS 832/13 cells to arachidonate or linoleate reduced Acsl4 mRNA and protein expression and reduced GSIS. These data indicate that Acsl4 modulates GSIS by regulating the levels of unesterified EETs and that arachidonate controls the expression of its activator Acsl4.     

Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites.

Consumption of a Western diet rich in saturated fats is associated with obesity and insulin resistance. In some insulin-resistant phenotypes this is associated with accumulation of skeletal muscle fatty acids. We examined the effects of diets high in saturated fatty acids (Sat) or n-6 polyunsaturated fatty acids (PUFA) on skeletal muscle fatty acid metabolite accumulation and whole-body insulin sensitivity. Male Sprague-Dawley rats were fed a chow diet (16% calories from fat, Con) or a diet high (53%) in Sat or PUFA for 8 wk. Insulin sensitivity was assessed by fasting plasma glucose and insulin and glucose tolerance via an oral glucose tolerance test. Muscle ceramide and diacylglycerol (DAG) levels and triacylglycerol (TAG) fatty acids were also measured. Both high-fat diets increased plasma free fatty acid levels by 30%. Compared with Con, Sat-fed rats were insulin resistant, whereas PUFA-treated rats showed improved insulin sensitivity. Sat caused a 125% increase in muscle DAG and a small increase in TAG. Although PUFA also resulted in a small increase in DAG, the excess fatty acids were primarily directed toward TAG storage (105% above Con). Ceramide content was unaffected by either high-fat diet. To examine the effects of fatty acids on cellular lipid storage and glucose uptake in vitro, rat L6 myotubes were incubated for 5 h with saturated and polyunsaturated fatty acids. After treatment of L6 myotubes with palmitate (C16:0), the ceramide and DAG content were increased by two- and fivefold, respectively, concomitant with reduced insulin-stimulated glucose uptake. In contrast, treatment of these cells with linoleate (C18:2) did not alter DAG, ceramide levels, and glucose uptake compared with controls (no added fatty acids). Both 16:0 and 18:2 treatments increased myotube TAG levels (C18:2 vs. C16:0, P < 0.05). These results indicate that increasing dietary Sat induces insulin resistance with concomitant increases in muscle DAG. Diets rich in n-6 PUFA appear to prevent insulin resistance by directing fat into TAG, rather than other lipid metabolites.     

Lipid Mixtures Containing a Very High Proportion of Saturated Fatty Acids Only Modestly Impair Insulin Signaling in Cultured Muscle Cells

In vitro examinations of the effect of saturated fatty acids on skeletal muscle insulin action often use only one or two different fatty acid species, which does not resemble the human plasma fatty acid profile. We compared graded concentrations (0.1-0.8mM) of 3 different lipid mixtures: 1) a physiologic fatty acid mixture (NORM; 40% saturated fatty acids), 2) a physiologic mixture high in saturated fatty acids (HSFA; 60% saturated fatty acids), and 3) 100% palmitate (PALM) on insulin signaling and fatty acid partitioning into triacylglycerol (TAG) and diacylglycerol (DAG) in cultured muscle cells. As expected, PALM readily impaired insulin-stimulated pAkt^Thr308/Akt and markedly increased intracellular DAG content. In contrast, the fatty acid mixtures only modestly impaired insulin-stimulated pAkt^Thr308M/Akt, and we found no differences between NORM and HSFA. Importantly, NORM and HSFA did not increase DAG content, but instead dose-dependently increased TAG accumulation. Therefore, the robust impairment in insulin signaling found with palmitate exposure was attenuated with physiologic mixtures of fatty acids, even with a very high proportion of saturated fatty acids. This may be explained in part by selective partitioning of fatty acids into neutral lipid (i.e., TAG) when muscle cells were exposed to physiologic lipid mixtures.     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

Dexamethasone facilitates lipid accumulation and mild feed restriction improves fatty acids oxidation in skeletal muscle of broiler chicks (Gallus gallus domesticus)

Effects of dexamethasone (DEX) and mild feed restriction on the uptake and utilization of fatty acids in skeletal muscle of broiler chicks (Gallus gallus domesticus) were investigated. Male Arbor Acres chicks (7-days old, n = 30) were injected with DEX or saline for 3 days, and a feed restriction group was included. DEX enhanced circulating very low density lipoprotein (VLDL) level and the lipid accumulation in both adipose and skeletal muscle tissues. Compared with the control, liver-carnitine palmitoyltransferase 1 (L-CPT1) and AMP-activated protein kinase (AMPK) α2 mRNA level of M. biceps femoris (BF) were down-regulated significantly by DEX, while mRNA expression of lipoprotein lipase (LPL), fatty acid transport protein 1 (FATP1), heart-fatty acid binding protein (H-FABP), long-chain acyl-CoA dehydrogenase (LCAD), activities of LPL and AMPK in both skeletal muscles were not obviously affected. Feed restriction increased the mRNA expression of LPL, L-CPT1 and LCAD of M. pectoralis major (PM), and FATP1, H-FABP, L-CPT1 and LCAD of BF. In conclusion, DEX retards the growth of body mass but facilitates lipid accumulation in both adipose and skeletal muscle tissues. In contrast to the favorable effect of mild feed restriction, DEX did not alter the uptake of fatty acids in the skeletal muscle. The result suggests that DEX may promote intramyocellular lipid accumulation by suppressed fatty acid oxidation while mild feed restriction improved fatty acid oxidation in skeletal muscle, especially in red muscle. Glucocorticoids (GCs) regulated muscle fatty acid metabolism in a different way from energy deficit caused by mild feed restriction.     

Role of triacylglycerols in leaves

Triacylglycerol (TAG) levels of up to 5 mg (g fresh weight)⁻¹ were identified in leaves of 13 plants. The fatty acid composition of the leaf TAG was distinct from the total leaf fatty acids that predominately arose from galactolipids in the thylakoid membranes and was very similar to the TAG found in the seeds. The exception was in senescent crabapple leaves where the TAG composition was more similar to that of the total leaf suggesting that this TAG might arise from fatty acids released from membrane lipids. In crabapple leaves the TAG was metabolically active with higher levels at the end of the photoperiod than the beginning. ¹⁴C-acetate was also incorporated into leaf TAG more rapidly in the light than dark. The rate of TAG accumulation in the light was about 6% of the net photosynthetic rate. These observations suggest that TAG serves along with carbohydrates as a diurnal photosynthetic store in these plants.     

Biosynthesis of triacylglycerol in Thunbergia alata: Additional evidence for involvement of phosphatidylcholine in unusual monoenoic oil production

The seed oil of Thunbergia alata has an unusual fatty acid composition which consists of more than 80 % 16:1^Δ6. This fatty acid is produced in the plastid by the action of a Δ6 palmitoyl (16:0)-ACP desaturase. To examine the biosynthesis of triacylglycerol (TAG) containing high concentrations of this unusual monoenoic fatty acid, endosperm dissected from developing T. alata seeds was labeled with [1-¹⁴C]-acetate. At early time points (5–15 min), the predominant labeled lipid was PC whereas at later time points (greater than 30 min) TAG became the major labeled lipid. Analysis of the acyl group composition of each lipid revealed that radiolabeled 16:1^Δ6 was highest at early time points in PC while at later time points, it was found to be highest in TAG. Further analysis of the distribution of labeled acyl groups within PC indicated that 16:1^Δ6 at the sn-2 position comprised the majority (55–78 %) of total labeled acyl groups whereas 16:1^Δ6 at the sn-1 position constituted only a small fraction (12–15 %) of the total labeled acyl groups. In contrast, unlabeled PC contained lower amounts of 16:1^Δ6 (16 %) at the sn-2 position. These results are consistent with previous studies suggesting a flux of novel monoenoic acids through PC during TAG biosynthesis, and furthermore imply a stereospecific flux through the sn-2 position of PC.     

The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41

Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gα_i/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes.     

N − 3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells

Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N − 3 polyunsaturated fatty acids (n − 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n − 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 μM of PAL without or with 50 μM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase Cθ activation. EPA and DHA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n − 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n − 3PUFA, but only EPA and DHA increased PAL β-oxidation, decreased PAL incorporation into DG and reduced protein kinase Cθ activation. EPA and DHA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial β-oxidation.     

Regulation of cardiolipin biosynthesis by fatty acid transport protein-1 IN HEK 293 cells

Cardiolipin (CL) is a major phospholipid involved in energy metabolism mammalian mitochondria and fatty acid transport protein-1 (FATP-1) is a fatty acid transport protein that may regulate the intracellular level of fatty acyl-Coenzyme A's. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we examined the effect of altering FATP-1 levels on CL biosynthesis. HEK-293 mock- and FATP-1 siRNA transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleic acid bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-³H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-³H]glycerol into CL (2-fold, p < 0.05) compared to controls indicating elevation in de novo CL biosynthesis. The reason for this was an increase in [1,3-³H]glycerol uptake and increase in activity and mRNA expression of the CL biosynthetic enzymes. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-³H]glycerol into CL (65%, p < 0.05) indicating reduced CL synthesis. [1,3-³H]Glycerol uptake was unaltered whereas activity of cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) and CDS-2 mRNA expression were reduced in FATP-1 expressing cells compared to control. In addition, in vitro CDS activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.     

Acsl1 is essential for skin barrier function through the activation of linoleic acid and biosynthesis of ω-O-acylceramide in mice

The long-chain acyl-CoA synthase1 (Acsl1) is a major enzyme that converts long-chain fatty acids to acyl-CoAs. The role of Acsl1 in energy metabolism has been elucidated in the adipose tissue, heart, and skeletal muscle. Here, we demonstrate that systemic deficiency of Acsl1 caused severe skin barrier defects, leading to embryonic lethality. Acsl1 mRNA and protein are expressed in the Acsl1^+/+ epidermis, which are absent in Acsl1^?/? mice. In Acsl1^?/? mice, epidermal ceramide [EOS] (Cer[EOS]) containing ω-O-esterified linoleic acid, a lipid essential for the skin barrier, was significantly reduced. Conversely, ω-hydroxy ceramide (Cer[OS]), a precursor of Cer[EOS], was increased. Moreover, the levels of triglyceride (TG) species containing linoleic acids were lower in Acsl1^?/? mice, whereas those not containing linoleic acid were comparable to Acsl1^+/+ mice. As TG is considered to work as a reservoir of linoleic acid for the biosynthesis of Cer[EOS] from Cer[OS], our results suggest that Acsl1 plays an essential role in ω-O-acylceramide synthesis by providing linoleic acid for ω-O-esterification. Therefore, our findings identified a new biological role of Acsl1 as a regulator of the skin barrier.     

Domain-dependent modulation of insulin-induced AS160 phosphorylation and glucose uptake by Ca2+/calmodulin-dependent protein kinase II in L6 myotubes

In skeletal muscle, the molecular mechanisms by which insulin stimulates glucose transport remains incompletely understood. Our study investigated the cellular dynamics of intracellular Ca²⁺ mobilisation and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation on insulin-induced skeletal muscle glucose transport. L6 myotubes were treated without or with insulin [100 nM] for 15 min and subsequently monitored for glucose uptake using isotope-labelled 2-deoxyglucose (I-2DOG), intracellular Ca²⁺ (Ca_i²⁺) release using Fluo-4AM and protein phosphorylation using Western blotting. Acute exposure of myotubes to insulin increased both Akt substrate-160 kDa (AS160) phosphorylation and I-2DOG uptake. Insulin concurrently increased Ca_i²⁺ and activated CaMKII. Exposing myotubes to either BAPTA/AM to sequester Ca_i²⁺ or KN-93 to inhibit CaMKII activity, decreased insulin-induced glucose uptake without affecting AS160 phosphorylation. On the other hand, blocking either calmodulin or the autoregulatory domain of CaMKII blocked the effect of insulin on both AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished insulin-mediated glucose uptake. These results illustrate impairments in Ca_i²⁺ mobilisation and CaMKII activation are sufficient to negatively influence insulin-dependent glucose transport by L6 myotubes. Additionally, our results show for the first time that Ca_i²⁺ and domain-dependent CaMKII signalling differentially affect insulin-induced AS160 phosphorylation, and establish that Ca²⁺ and CaMKII are components of the insulin signalling pathway in L6 myotubes.     

Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [³H]d-chiro-inositol (DCI) by 159-fold. [³H]myo-Inositol uptake increased by 37-fold. In contrast, [¹⁴C]d-glucose, [¹⁴C]2-deoxy-d-glucose, or [¹⁴C]3-O-methyl-d-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K_m of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a K_i of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 h) increased [³H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.     

Design and synthesis of lupeol analogues and their glucose uptake stimulatory effect in L6 skeletal muscle cells

Structure modifications of lupeol at the isopropylene moiety have been described via allylic oxidation using selenium dioxide. The antidiabetic efficacy of lupeol analogues were evaluated in vitro as glucose uptake stimulatory effect in L6 skeletal muscle cells. From all tested compounds, 2, 3, 4b and 6b showed significant stimulation of glucose uptake with respective percent stimulation of 173.1 (p <0.001), 114.1 (p <0.001), 98.3 (p <0.001) and 107.3 (p <0.001) at 10 μM concentration. Stimulation of glucose uptake by these compounds is associated with enhanced translocation of glucose transporter 4 (GLUT4) and activation of IRS-1/PI3-K/AKT-dependent signaling pathway in L6 cells. Structure–activity relationship analysis of these analogues demonstrated that the integrity of α,β-unsaturated carbonyl and acetyl moieties were important in the retention of glucose uptake stimulatory effect. It is therefore proposed that naturally occurring lupeol and their analogues might reduce blood glucose, at least in part, through stimulating glucose utilization by skeletal muscles.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.