Ferroptosis, Acyl Starvation, and Breast Cancer.

To maintain their growth rate, cancer cells must secure a supply of fatty acids, which are necessary for building cell membranes and maintaining energy processes. This is one of the reasons why tissues with intensive fatty acid metabolism, such as the mammary gland, are more likely to develop tumors. One natural or induced defense process against cancer is ferroptosis, which interferes with normal fatty acid metabolism. This leads to the oxidation of polyunsaturated fatty acids, which causes a rearrangement of the metabolism and damages cell membranes. As a consequence of this oxidation, there is a shortage of normal polyunsaturated fatty acids, which disturbs the complicated metabolism of fatty acids. This imbalance in metabolism, resulting from the deficiency of properly structured fatty acids, is called, by these authors, "acyl starvation." When cancer cells are exposed to alternating hypoxia and reoxygenation, they often develop resistance to neoadjuvant therapies. Blocking the stearoyl-CoA desaturase - fatty acid-binding protein 4 - fatty acid translocase axis appears to be a promising pathway in the treatment of breast cancer. On the one hand, the inhibition of desaturase leads to the formation of toxic phospholipid hydroperoxides in ferroptosis, whereas on the other hand, the inhibition of fatty acid-binding protein 4 and translocase leads to a reduced uptake of fatty acids and disruption of the cellular transport of fatty acids, resulting in intracellular acyl starvation. The disruption in the metabolism of fatty acids in cancer cells may augment the effectiveness of neoadjuvant therapy. SIGNIFICANCE STATEMENT: Regulation of the metabolism of fatty acids in cancer cells seems to be a promising therapeutic direction. Studies show that the induction of ferroptosis in cancer cells, combined with use of neoadjuvant therapies, effectively inhibits the proliferation of these cells. We link the process of ferroptosis with apoptosis and SCD1-FABP4-CD36 axis and propose the term "acyl starvation" for the processes leading to FA deficiency, dysregulation of FA metabolism in cancer cells, and, most importantly, the appearance of incorrect proportions FAs.

BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments.

The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

Drug discovery for X-linked adrenoleukodystrophy: An unbiased screen for compounds that lower very long-chain fatty acids.

X-linked adrenoleukodystrophy (XALD) is a genetic neurologic disorder with multiple phenotypic presentations and limited therapeutic options. The childhood cerebral phenotype (CCALD), a fatal demyelinating disorder affecting about 35% of patients, and the adult-onset adrenomyeloneuropathy (AMN), a peripheral neuropathy affecting 40%-45% of patients, are both caused by mutations in the ABCD1 gene. Both phenotypes are characterized biochemically by elevated tissue and plasma levels of saturated very long-chain fatty acids (VLCFA), and an increase in plasma cerotic acid (C26:0), along with the clinical presentation, is diagnostic. Administration of oils containing monounsaturated fatty acids, for example, Lorenzo's oil, lowers patient VLCFA levels and reduced the frequency of development of CCALD in presymptomatic boys. However, this therapy is not currently available. Hematopoietic stem cell transplant and gene therapy remain viable therapies for boys with early progressive cerebral disease. We asked whether any existing approved drugs can lower VLCFA and thus open new therapeutic possibilities for XALD. Using SV40-transformed and telomerase-immortalized skin fibroblasts from an XALD patient, we conducted an unbiased screen of a library of approved drugs and natural products for their ability to decrease VLCFA, using measurement of C26:0 in lysophosphatidyl choline (C26-LPC) by tandem mass spectrometry as the readout. While several candidate drugs were initially identified, further testing in primary fibroblast cell lines from multiple CCALD and AMN patients narrowed the list to one drug, the anti-hypertensive drug irbesartan. In addition to lowering C26-LPC, levels of C26:0 and C28:0 in total fibroblast lipids were reduced. The effect of irbesartan was dose dependent between 2 and 10 µM. When male XALD mice received orally administered irbesartan at a dose of 10 mg/kg/day, there was no reduction in plasma C26-LPC. However, irbesartan failed to lower mouse fibroblast C26-LPC consistently. The results of these studies indicate a potential therapeutic benefit of irbesartan in XALD that should be validated by further study.

Murine glomerular leukotriene B4 synthesis. Manipulation by (n-6) fatty acid deprivation and cellular origin.

Leukotriene (LT) B4 is an important pro-inflammatory autocoid. In order to investigate the potential role of this eicosanoid in renal inflammation, in this study we determined the capability of glomeruli to synthesize this mediator. Glomeruli were able to synthesize LTB4 when provided with exogenous substrate in a dose-dependent fashion in the presence of ionophore A23187. Ionophore, although by itself a weak agonist for LTB4 formation, was required for LTB4 production from exogenous arachidonate. The identity of LTB4 was confirmed by specific radioimmunoassay, high pressure liquid chromatography, and gas chromatography/mass spectrometry. The synthesis of LTB4 was inhibited by BW755C (a lipoxygenase/cyclooxygenase inhibitor) but not indomethacin. Essential fatty acid (EFA) deficiency, obtained by the deprivation of (n-6) fatty acids, is known to exert a protective effect in renal inflammatory states. This dietary manipulation markedly attenuated the ability of glomeruli to synthesize LTB4. In contrast, the synthesis of cyclooxygenase products from exogenous arachidonate was increased by EFA deficiency. Because EFA deficiency has been shown to deplete glomeruli of resident mesangial macrophages, it was hypothesized that this effect accounted for the diminished LTB4 synthesis. To test this hypothesis, glomeruli were depleted of macrophages using x-irradiation. Glomeruli from these animals exhibited a marked decrease in LTB4 synthesis. Glomerular synthesis of cyclooxygenase products was unaffected by irradiation. In sum, glomeruli have the capability to synthesize LTB4, and this capacity is correlated with the presence of glomerular macrophages. EFA deficiency attenuates the ability of glomeruli to synthesize LTB4 by depleting them of macrophages.

Effects of fatty acid deficiency on the lipid composition and physical properties of guinea pig rough endoplasmic reticulum.

Twenty-six days of fat deficiency brought about a decrease of linoleic and an increase of oleic acid in rough endoplasmic reticulum (RER) of guinea pig liver. Arachidonic acid was only slightly decreased in some phospholipids whereas eicose-5,8,11-trienoic acid was not enhanced except in phosphatidyl-inositol. All these changes were relevant specifically in phosphatidylinositol molecules and less important in phosphatidylcholine and phosphatidylethanolamine. Fat deficiency did not modify the relative proportion of phospholipids and cholesterol. Therefore, fat deficient guinea pig microsomes are a good model to study the effect of unsaturated fatty acids on membrane properties. Fluorescent anisotropy of RER membranes, lipids and phospholipids labeled with diphenylhexatriene, was increased by the fat deficiency. The most important increase was observed in liposomes of a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin. A small change was found in phosphatidylcholine and phosphatidylethanolamine dispersions at 37 degrees C. The modification of the lipid unsaturation evoked fluorescent anisotropy changes. Temperature-dependent fluorescent polarization curves of RER membranes labeled with trans-parinaric acid did not show inflections in the temperature range from 5 to 45 degrees C but, RER lipids and phospholipids presented a phase separation at about 20 degrees C. This inflection point was not modified by the fat deficient diet. In those liposomes prepared with a mixture of RER phosphatidylinositol, phosphatidylserine and sphingomyelin, the inflection point was produced at about 37 degrees C.

Octanoic acidemia and octanoylcarnitine excretion with dicarboxylic aciduria due to defective oxidation of medium-chain fatty acids.

Five patients aged 7 to 21 months are described who developed attacks of coma after a short prodromal illness with diarrhea or vomiting or both. Four had concomitant hypoglycemia, and all had hypoketonemia, with excessive urinary excretion of medium-chain dicarboxylic acids, medium-chain (omega-1)-hydroxyacids, suberylglycine, hexanoylglycine, and octanoylcarnitine. All patients accumulated octanoic acid, decanoic acid, and cis-4-decenoic acid in plasma. Fibroblasts from three patients showed a decreased rate of octanoate oxidation (10%, 12%, and 29% of control values, respectively). These findings suggest a deficiency of medium-chain acyl-CoA dehydrogenase, most probably an autosomal recessive inherited metabolic disorder. Two of the patients died during an acute attack, and a third had severe neurologic sequelae; the two remaining patients recovered. Plasma free carnitine levels were low, but total carnitine was normal. The three surviving patients underwent a fasting test, which did not lead to hypoglycemia, although hypoketonemia, dicarboxylic aciduria, and excessive mobilization of fatty acids did occur. The surviving patients were maintained on frequent carbohydrate-enriched meals.

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant.

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.

The potential role of thromboxane and prostacyclin in endotoxic and septic shock.

The potential role of thromboxane (TxA2), a platelet aggregator and vasoconstrictor, and prostacyclin (PGI2) a platelet anti-aggregator and vasodilator, in endotoxic and septic shock was investigated. Early endotoxic shock in the rat is associated with marked elevations of plasma TxB2 (the stable metabolite of TxA2) and lesser increases in plasma 6-keto-PGF1 alpha (the stable metabolite of PGI2). Selective inhibition of TxA2 synthesis by several different chemical classes of Tx synthetase inhibitors was beneficial in endotoxic shock. In contrast, shock induced by acute intra-abdominal sepsis in the rat was characterized by high levels of plasma 6-keto-PGF1 alpha, which exceeded plasma TxA2 six- to eight fold at most time intervals studied. Tx synthetase inhibitors were not protective in this model of acute sepsis, but treatment with fatty acid cyclo-oxygenase inhibitors, an antibiotic (gentamicin), or reduction in arachidonic acid metabolism by essential fatty acid (EFA) deficiency significantly prolonged survival time. An important aspect of the latter study is that decreased arachidonic acid metabolism was an effective adjunct to antibiotic therapy. Conjoint administration of gentamicin in EFA-deficient rats or with indomethacin synergistically improved long-term survival, a result that was not evident with single treatment interventions. In addition to experimental studies, plasma TxB2 levels were measured during clinical sepsis. These studies demonstrated that plasma TxB2 levels were elevated tenfold in patients dying of septic shock compared with septic survivors or nonseptic controls. These composite experimental and clinical observations suggest that arachidonic acid metabolites play a role in the pathogenesis of endotoxic and septic shock.

Essential fatty acid deficiency: effects of cross-fostering mice at birth on myelin levels and composition.

Female mice were assigned an essential fatty acid (EFA)-deficient diet or a control diet at mating, and litters were cross-fostered at birth to produce three groups of animals: pups fed a control diet prenatally and deficient diet postnatally (C leads to D); a deficient diet prenatally and a control diet postnatally (D leads to C); and a control diet throughout life (C leads to C). The yield of myelin, the developmental pattern of the major proteins, and the proportion of major lipids were examined in the purified myelin of the three groups at 3, 6, and 9 weeks. Myelin yield was lower at 9 weeks in both the (C leads to D) and (D leads to C) groups compared to controls. There was an alteration in the ratios of the proteins and major lipid classes in myelin from the (C leads to D) animals at 9 weeks, whereas the ratios of these components were normal in the (D leads to C) animals at this age. However, at three weeks the lipid composition of the myelin isolated from (D leads to C) animals was abnormal. The results suggest that postnatal EFA deficiency results in hypomyelination in mice and that the myelin formed is of abnormal composition during early postnatal brain development. Prenatal EFA deficiency results in less severe hypomyelination with only the earliest myelin formed being of abnormal lipid composition.

Effect of fatty acid deficiency on microsomal membrane fluidity and cooperativity of the UDP-glucuronyltransferase.

The effect of fat deprivation on microsomal membrane fluidity of guinea-pig livers and the kinetic cooperativity of UDP-glucuronyl transferase towards its natural substrate, the UDP-glucuronic acid, were studied. Fat deprivation in the diet of weanling guinea-pigs evoked a typical essential fatty-acid-deficient pattern in the composition of the microsomal membrane. The unsaturated:saturated fatty acid ratio progressively declined in the membrane during the 21-day period tested. This decline determined a gradual increase in the fluorescence anisotropy (rs) of the membrane labeled with diphenylhexatriene and the apparent microviscosity of the lipid bilayer calculated from these values increased from 1.1 to 1.8 poise. In addition, when the infinitely slow decaying fluorescence anisotropy ((r infinity), which is proportional to the square of the lipid order parameter, was calculated from rs data, a significant increase in these parameters was also obtained. Furthermore, this decrease of the double bond index:saturated acid ratio of the membrane was associated with a parallel increase in Hill coefficients of the UDP-glucuronyl transferase that gradually lost the negative homotropic effect and cooperativity of UDP-glucuronic acid. The Hill coefficient varied from 0.39 to 0.98 during the 21-day period studied. Our observations indicate on one side that changes in the fat composition of the diet are accompanied by modifications in the lipid composition and fluidity of the microsomal membrane, and the apparent cooperativity of the enzyme. On the other side, they suggest that the evaluation of Hill coefficients of UDP-glucuronyl transferase might be used as a sensitive test to investigate conformational changes in the microsomal membrane of the liver.

[Formula diets in gastroenterology]. / Formeldiäten in der Gastroenterologie.

Elemental diets are in use for therapy of intestinal fistulas, diarrhoe of unknown etiology, short-bowel-syndrom and chronic inflammatory bowel diseases.

Essential fatty acid deficiency in surgical patients.

Parenteral nutrition may protect patients unable to eat from malnutrition almost indefinitely. If fat is not also given EFAD will occur. This outlines a prospective study of 28 surgical patients on total intravenous fat-free nutrition to determine the developmental course of EFAD and the response to therapy. Twenty-eight patients ranging from newborn to 66 years receiving parenteral nutrition without fat had regular determinations of the composition of total plasma fatty acids and the triene/tetraene ratio using gas liquid chromatography. Physical signs of EFAD were looked for also. Patients found to have evidence of EFAD were treated with 10% Intralipid. Topical safflower oil was used in three infants. Total plasma fatty acid composition was restudied following therapy. In general, infants on fat-free intravenous nutrition developed biochemical EFAD within two weeks, but dermatitis took longer to become evident. Older individuals took over four weeks to develop a diagnostic triene/tetraene ratio (greater than 0.4; range 0.4 to 3.75). Therapeutic correction of biochemical EFAD took 7 to 10 days but dermatitis took longer to correct. Cutaneous application of safflower oil alleviated the cutaneous manifestations but did not correct the triene/tetraene ratio of total plasma fatty acids. These studies indicate that surgical patients who are unable to eat for two to four weeks, depending upon age and expected fat stores, should receive fat as a part of their intravenous regimen.

In vitro biosynthesis of prostaglandin E2 by kidney medulla of essential fatty acid deficient rats.

Weanling rats were fed either a semisynthetic diet with no fat, with 28% by wt partially hydrogenated fish oil, or with 28% by wt arachis oil(control diet) for 6 or 7 1/2 months. The in vitro conversion of arachidonic acid to prostaglandin E2 by homogenates of the rat kidney medulla was measured by gaschromatography with electron capture detection. The kidney medulla of essential fatty acid deficient animals showed increased activity for the in vitro conversion of exogenous arachidonic acid to prostaglandin E2 when compared to the controls. The change of the enzymatic activity in the essential fatty acid deficient animals was reversible, as shown by refeeding. Inhibition of the prostaglandin synthetase was found at exogenous substrate concentrations higher than 50-100 muM.

Total parenteral nutrition in pediatrics: the Borden award address.

Research in parenteral nutrition in infants has proceeded rapidly over the past few years, thanks in large part to the perfection of safe central venous delivery of hypertonic nutritive infusates. At present, there are clear definitions of indications and expectations of results for this method of therapy in two well-defined groups of patients--i.e., selected surgical neonates and infants with chronic intractable diarrhea. In addition, we have suggestive evidence of another potentially valuable application in the nutritional management of very low birthweight infants. However, in this group, a controlled study will be necessary before the role of total parenteral nutrition (TPN) in neonatal care of such infants can be determined precisely. Results obtained with TPN in adults with inflammatory bowel disease or acute renal failure suggest that trials of this technique in pediatric patients with these disorders should be carried out. As a result of the research in TPN carried out thus far, we have learned how to minimize or to treat many of the complications of the technique and we have identified at least the ways by which still others can be prevented. The future holds many new advances not only in the refinement of existing parenteral nutritional solutions but also, and perhaps of even greater importance; in the perfection of individualized total nutritional therapy for specific patients using the enteral route for those discrete components of intake for which digestive and/or absorptive mechanisms are unimpaired and using the parenteral route for the remainder.