Loss of regulation of lipogenesis in the Zucker diabetic rat. II. Changes in stearate and oleate synthesis

De novo lipogenesis and dietary fat uptake are two major sources of fatty acid deposits in fat of obese animals. To determine the relative contribution of fatty acids from these two sources in obesity, we have determined the distribution of c16 and c18 fatty acids of triglycerides in plasma, liver, and epididymal fat pad of Zucker diabetic fatty (ZDF) rats and their lean littermates (ZL) under two isocaloric dietary fat conditions. Lipogenesis was also determined using the deuterated water method. Conversion of palmitate to stearate and stearate to oleate was calculated from the deuterium incorporation by use of the tracer dilution principle. In the ZL rat, lipogenesis was suppressed from 70 to 24%, conversion of palmitate to stearate from 86 to 78%, and conversion of stearate to oleate from 56 to 7% in response to an increase in the dietary fat-to-carbohydrate ratio. The results suggest that suppression of fatty acid synthase and stearoyl-CoA desaturase activities is a normal adaptive mechanism to a high-fat diet. In contrast, de novo lipogenesis, chain elongation, and desaturation were not suppressed by dietary fat in the ZDF rat. The lack of ability to adapt to a high-fat diet resulted in a higher plasma triglyceride concentration and excessive fat accumulation from both diet and de novo synthesis in the ZDF rat.     

In vitro effects of sterculic acid on lipid biosynthesis in Rhodococcus opacus strain PD630 and isolation of mutants defective in fatty acid desaturation

The in vivo effects of sterculic acid methyl ester on triacylglycerol fatty acid composition in the oleaginous, hydrocarbon-degrading bacterium R. opacus strain PD630 was investigated. Sterculic acid, a cyclopropene fatty acid and an inhibitor of the stearoyl-CoA desaturase system, strongly inhibited the synthesis of monoenic fatty acids, of saturated fatty acids with more than 16 carbon atoms and of odd-numbered fatty acids when added to the culture medium. In addition, chemical mutagenesis and the application of the penicillin enrichment technique provided mutants, which were more or less completely impaired in the desaturation of long-chain fatty acids and exhibited in some cases a similar fatty acid composition like the wild-type in the presence of sterculic acid methyl ester. The implications of these findings for fatty acid metabolism in R. opacus strain PD630 are discussed.     

Effects of sterculic acid on stearoyl-CoA desaturase in differentiating 3T3-L1 adipocytes

The effects of sterculic acid on cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100 microM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shows that de novo synthesis and elongation of fatty acids, as well as cell differentiation, were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells, the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation, scd gene expression or SCD protein translation.     

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

Temperature-induced Changes in the Synthesis of Unsaturated Fatty Acids by Acanthamoeba castellanii

ABSTRACT. Major fatty acid components of Acanthamoeba castellanii lipids extracted after growth at 30°C include myristate, palmitate, stearate and the polyunsaturates linoleate, eicosadienoate, eicosatrienoate and arachidonate, with oleate as the sole major monounsaturated fatty acid. By comparison, growth at 15°C gave increased linoleate, eicosatrienoate and arachidonate, but decreased oleate and palmitate. When the growth temperature was shifted downwards from 30°C to 15°C, increased lipid unsaturation occurred over a period of 24 h; thus decreases of oleate and eicosadienoate were accompanied by increases in linoleate, eicosatrienoate, arachidonate and eicosapentaenoate. An upwards shift from 15°C to 30°C gave negligible alterations in fatty acid composition over a similar period. At 15°C organisms rapidly use [1-¹⁴C] acetate for de novo fatty acid synthesis; stearate is converted via oleate to further desaturation and chain elongation products. Similar short term experiments at 30°C indicate only de novo synthesis and Δ9-desaturation; synthesis of polyunsaturates was a much slower process. Rapid incorporation of [1-¹⁴C] oleate at 30°C was not accompanied by metabolic conversion over two hours, whereas at 15°C n-6 desaturation to linoleate was observed. Temperature shift of organisms from 15°C to 30°C in the presence of [1-¹⁴C] acetate revealed that over half of the fatty acids in newly-synthesised lipids were saturated, but the proportions of unsaturated fatty acids increased with time until the total polyenoate components reached 17% after 22 h. A shift of temperature in the reverse direction gave a corresponding figure of 60% for polyunsaturated fatty acids. These results emphasize the importance of n-6 desaturation in the low temperature adaptation of Acanthamoeba castellanii .     

Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Fatty-acid desaturation and microsomal lipid fatty-acid composition in experimental hyperthyroidism.

We have studied the influence of experimental hyperthyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty-acid composition. Tri-iodothyronine treatment (25 micrograms/100 g body weight) daily for 3 weeks caused no significant changes in delta 9 (stearate) desaturation but a 24% decrease in delta 6 (linoleate) desaturation. Much larger doses of tri-iodothyronine increased delta 9 desaturation. Liver microsomal fatty-acid composition in hyperthyroidism is altered with significantly increased proportions of stearate and arachidonate and decreased proportions of palmitate, palmitoleate, linoleate (C18:2) and eicosa-8,11,14-trienoate (C20:3). These changes, other than the decreases proportion of C20:3 fatty acid, which may be due to the diminished delta 6 desaturase activity, cannot be attributed to changes in fatty-acid desaturation. Most of these changes were also found to be due not simply to the decreased weight gain or the increased food intake of the hyperthyroid animals. Only the decreased C18:2 fatty-acid proportions could be mimicked by restricting food intake of control animals and none of the changes were prevented by restricting food intake of hyperthyroid animals. Thus most of the changes in microsomal lipid fatty-acid composition are likely to be due to a thyroid hormone effect on peripheral lipid mobilization or lipid degradation.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Coordination of peroxisomal beta-oxidation and fatty acid elongation in HepG2 cells

A major product of mitochondrial and peroxisomal beta-oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal beta-oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of beta-oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-(13)C(18)]stearate or [U-(13)C(16)] palmitate. Using mass isotopomer analysis we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the tricarboxylic acid cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mm [U-(13)C(18)]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mm [U-(13)C(16)]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that 1) chain elongation activity is a useful and novel probe for peroxisomal beta-oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.     

The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.     

Increased availability of endogenous and dietary oleic acid contributes to the upregulation of cardiac fatty acid oxidation

In the present study, we tested the hypothesis that cardiac substrate utilization is affected by oleic acid originating from the endogenous conversion of stearate by stearoyl-CoA desaturase and from the diet. Here, we show that the cardiac oleate content is increased in tristearate (TS)- and trioleate (TO)-fed rats when compared with chow-fed rats. TS or TO feeding increases mitochondrial fatty acid oxidation via activation of expression of the oxidative genes, activation of the AMP-activated protein kinase pathway, and a decrease in glucose uptake. These results suggest that oleic acid, both dietary and de novo synthesized, affects substrate utilization in the heart. Furthermore, our data show that the endogenous synthesis of oleate in the heart can compensate for a deficiency of this fatty acid in the diet.     

Control of acyl lipid desaturation in the yeast Rhodotorula gracilis via the use of the cyclopropenoid fatty acid,sterculate

Summary The effect of the cyclopropene fatty acid, sterculic acid, on fatty acid metabolism in the oleaginous yeast Rhodotorula gracilis (CBS 3043) has been studied. Sterculate caused approximately 90% inhibition of [1-¹⁴C]sterate desaturation but only about 25% inhibition when [1-¹⁴C]acetate was used as precursor. Measurement of acyl-coenzyme A (CoA) pool labelling and the pattern of distribution of radioactivity within lipid classes suggested that the high rate of inhibition of stearate desaturation was due to sterculate reducing the formation of stearoyl-CoA. In agreement with previous suggestions for plant and animal systems, experiments with a post-mitochondrial (20 000 g × 30 min) supernatant suggested that sterculoyl-CoA was the active inhibitor. The action of sterculate on endogenous lipid composition was most marked in stationary-phase cells. In such cells an increase in stearate was seen especially in the triacylglycerol pools, while -linolenate was increased predominantly in the phospholipid fractions. The increased flux of carbon to -linolenate at the same time that stearate desaturation appeared to be inhibited by sterculate, indicated that that two separate pathways for desaturation, employing different substrates, may operate in R. gracilis. We suggest that sterculate inhibits the stearoyl-(acyl-)CoA-dependent pathway but has little effect when phospholipids are used as substrates for acyl chain desaturation. Offprint requests to: J. L. Harwood     

Loss of regulation of lipogenesis in the Zucker diabetic (ZDF) rat

We present here a study on the role of leptin in the regulation of lipogenesis by examining the effect of dietary macronutrient composition on lipogenesis in the leptin receptor-defective Zucker diabetic fatty rat (ZDF) and its lean litter mate (ZL). Animals were pair fed two isocaloric diets differing in their fat-to-carbohydrate ratio providing 10 and 30% energy as fat. Lipogenesis was measured in the rats using deuterated water and isotopomer analysis. From the deuterium incorporation into plasma palmitate, stearate, and oleate, we determined de novo synthesis of palmitate and synthesis of stearate by chain elongation and of oleate by desaturation. Because the macronutrient composition and the caloric density were controlled, changes in de novo lipogenesis under these dietary conditions represent adaptation to changes in the fat-to-carbohydrate ratio of the diet. De novo lipogenesis was normally suppressed in response to the high-fat diet in the ZL rat to maintain a relatively constant amount of lipids transported. The ZDF rat had a higher rate of lipogenesis, which was not suppressed by the high-fat diet. The results suggest an important hormonal role of leptin in the feedback regulation of lipogenesis.     

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.