Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.     

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.     

Effects of Simvastatin on Lipid Metabolism in Wild-Type Mice and Mice with Muscle PGC-1α Overexpression

Previous studies suggest that statins may disturb skeletal muscle lipid metabolism potentially causing lipotoxicity with insulin resistance. We investigated this possibility in wild-type mice (WT) and mice with skeletal muscle PGC-1α overexpression (PGC-1α OE mice). In WT mice, simvastatin had only minor effects on skeletal muscle lipid metabolism but reduced glucose uptake, indicating impaired insulin sensitivity. Muscle PGC-1α overexpression caused lipid droplet accumulation in skeletal muscle with increased expression of the fatty acid transporter CD36, fatty acid binding protein 4, perilipin 5 and CPT1b but without significant impairment of muscle glucose uptake. Simvastatin further increased the lipid droplet accumulation in PGC-1α OE mice and stimulated muscle glucose uptake. In conclusion, the impaired muscle glucose uptake in WT mice treated with simvastatin cannot be explained by lipotoxicity. PGC-1α OE mice are protected from lipotoxicity of fatty acids and triglycerides by increased the expression of FABP4, formation of lipid droplets and increased expression of CPT1b.     

Liquid Chromatography–Mass Spectrometric Analysis of Lipids Present in Human Meibomian Gland Secretions

The purpose of the study was to qualitatively characterize the major lipid species present in human meibomian gland secretions (MGS) by means of high-performance liquid chromatography with atmospheric pressure ionization mass spectrometric detection of the analytes (NP HPLC-MS). Two different NP HPLC-MS methods have been developed to analyze lipid species that were expected to be present in MGS. The first method was optimized for the analysis of relatively nonpolar lipids [wax esters (WE), di- and triacyl glycerols (DAG and TAG), cholesterol (Chl) and its esters (Chl-E), and ceramides (Cer)], while the second method was designed to separate and detect phospholipids. The major lipid species in MGS were found to be WE, Chl-E, and TAG. A minor amount of free Chl (less then 0.5% of the Chl-E fraction) was detected in MGS. No appreciable amounts of DAG and Cer were found in MGS. The second NP HPLC-MS method, capable of analyzing model mixtures of authentic phospholipids (e.g. phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin) in submicrogram/mL concentrations, showed little or no presence of these species in the MGS samples. These observations suggest that MGS are a major source of the nonpolar lipids of the WE and Chl-E families for the tear film lipid layer (TFLL), but not of the previously reported phospholipid components of the TFLL.     

Effects of (R)- and (S)-α-Hydroxylation of Acyl Chains in Sphingosine,Dihydrosphingosine, and Phytosphingosine Ceramides on Phase Behavior and Permeability of Skin Lipid Models

Ceramides (Cers) with α-hydroxylated acyl chains comprise about a third of all extractable skin Cers and are required for permeability barrier homeostasis. We have probed here the effects of Cer hydroxylation on their behavior in lipid models comprising the major SC lipids, Cer/free fatty acids (C ₁₆-C ₂₄)/cholesterol, and a minor component, cholesteryl sulfate. Namely, Cers with (R)-α-hydroxy lignoceroyl chains attached to sphingosine (Cer AS), dihydrosphingosine (Cer AdS), and phytosphingosine (Cer AP) were compared to their unnatural (S)-diastereomers and to Cers with non-hydroxylated lignoceroyl chains attached to sphingosine (Cer NS), dihydrosphingosine (Cer NdS), and phytosphingosine (Cer NP). By comparing several biophysical parameters (lamellar organization by X-ray diffraction, chain order, lateral packing, phase transitions, and lipid mixing by infrared spectroscopy using deuterated lipids) and the permeabilities of these models (water loss and two permeability markers), we conclude that there is no general or common consequence of Cer α-hydroxylation. Instead, we found a rich mix of effects, highly dependent on the sphingoid base chain, configuration at the α-carbon, and permeability marker used. We found that the model membranes with unnatural Cer (S)-AS have fewer orthorhombically packed lipid chains than those based on the (R)-diastereomer. In addition, physiological (R)-configuration decreases the permeability of membranes, with Cer (R)-AdS to theophylline, and increases the lipid chain order in model systems with natural Cer (R)-AP. Thus, each Cer subclass makes a distinct contribution to the structural organization and function of the skin lipid barrier.     

Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs

Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide‐linked to two different ω‐hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester‐linked to linoleic acid (LNA; 18:2n‐6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5 % LNA and 23.5 % γ‐linolenic acid (GLA; 18:3n‐6)], in essential fatty acid (EFA)‐deficient guinea pigs, we further investigated the effects of BO on the substitution of ester‐linked GLA for LNA in these two epidermal Cer1 species by LC–MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA‐deficient guinea pigs increased LNA ester‐linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester‐linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester‐linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20‐Metabolized fatty acids of LNA or GLA were not ester‐linked to these Cer1 species. Dietary BO induced GLA ester‐linked to C32wh:1/d20:1 of epidermal Cer1.     

Changes in muscle lipid composition and resistance adaptation to temperature in the freshwater crayfish,Austropotamobius pallipes

The phospholipid and fatty acid composition of muscle lipid extracts from crayfish acclimated to 4 C and 25 C (18 hr-light photoperiod) were analyzed. The phospholipid content and class distribution, and cholesterol content were unaffected by the acclimation treatment. Unsaturation of muscle phosphoglycerides was higher in cold acclimated crayfish. Serine/inositol phosphoglycerides from cold-acclimated animals showed somewhat higher proportions of mono-and polyunsaturated fatty acids, whereas choline and ethanolamine phosphoglycerides were less affected. This was correlated with a decreased resistance of cold-acclimated crayfish to lethal high temperature. Acclimation at 4 C under an 8 hr-light photoperiod caused an increased fatty acid unsaturation of the total phospholipid fraction compared to the 4 C, 18 hr-light photoperiod acclimated animals. The resistance of 4 C acclimated crayfish to lethal high temperature, however, was unaffected by daylength treatment. The resistance of freshwater crayfish to lethal high temperature is not simply related to the degree of saturation of the muscle phospholipids. It is suggested that a breakdown in the integrity of a bulk-lipid bilayer is not involved in the process of heat death; rather, that a membrane-bound protein factor, whose thermal sensitivity is modified by changes in its phospholipid environment during temperature adaptation but not during photoperiod adaptation, is the primary site of heat injury.     

Increased Insulin Sensitivity and Distorted Mitochondrial Adaptations during Muscle Unloading

We aimed to further investigate mitochondrial adaptations to muscle disuse and the consequent metabolic disorders. Male rats were submitted to hindlimb unloading (HU) for three weeks. Interestingly, HU increased insulin sensitivity index (ISI) and decreased blood level of triglyceride and insulin. In skeletal muscle, HU decreased expression of pyruvate dehydrogenase kinase 4 (PDK4) and its protein level in mitochondria. HU decreased mtDNA content and mitochondrial biogenesis biomarkers. Dynamin-related protein (Drp1) in mitochondria and Mfn2 mRNA level were decreased significantly by HU. Our findings provide more extensive insight into mitochondrial adaptations to muscle disuse, involving the shift of fuel utilization towards glucose, the decreased mitochondrial biogenesis and the distorted mitochondrial dynamics.     

Mitochondrial Bioenergetics and Turnover during Chronic Muscle Disuse

Periods of muscle disuse promote marked mitochondrial alterations that contribute to the impaired metabolic health and degree of atrophy in the muscle. Thus, understanding the molecular underpinnings of muscle mitochondrial decline with prolonged inactivity is of considerable interest. There are translational applications to patients subjected to limb immobilization following injury, illness-induced bed rest, neuropathies, and even microgravity. Studies in these patients, as well as on various pre-clinical rodent models have elucidated the pathways involved in mitochondrial quality control, such as mitochondrial biogenesis, mitophagy, fission and fusion, and the corresponding mitochondrial derangements that underlie the muscle atrophy that ensues from inactivity. Defective organelles display altered respiratory function concurrent with increased accumulation of reactive oxygen species, which exacerbate myofiber atrophy via degradative pathways. The preservation of muscle quality and function is critical for maintaining mobility throughout the lifespan, and for the prevention of inactivity-related diseases. Exercise training is effective in preserving muscle mass by promoting favourable mitochondrial adaptations that offset the mitochondrial dysfunction, which contributes to the declines in muscle and whole-body metabolic health. This highlights the need for further investigation of the mechanisms in which mitochondria contribute to disuse-induced atrophy, as well as the specific molecular targets that can be exploited therapeutically.     

Effect of a diet rich in sunflower oil on aspects of lipid metabolism in the genetically-obese rat

Aspects of the lipid metabolism of male, obese and lean Zucker rats were compared using animals which had been fed ad libitum for 32 days on a diet (HS) which contained 200 g sunflowerseed oil/kg or one (LS) which contained 50 g/kg of the oil. When compared with the LS diet, the HS diet decreased the characteristic lipid accretion in the liver of obese rats from 126 mg (LS) to 81 mg (HS)/g wet weight; corresponding values for the lean rats were 39 mg and 56 mg/g wet weight of liver, respectively. The HS diet depressed lipid synthesis de novo by liver homogenates and decreased the Δ9-desaturase activity of liver microsomes from obese and clean rats by about 50%. Δ9-Desaturase activity in vitro was also depressed by the addition of linoleic acid to liver microsomes from both obese and lean rats fed ad libitum on a standard laboratory diet. Depressed Δ9-desaturase activity, due to ingestion of the HS diet, was reflected in lower ratios of 16∶1/16∶0 and 18∶1/18∶0 fatty acids in tissue lipids from obese and lean rats. Ingestion of the HS compared with the LS diet resulted in increased proportions of 18∶2ω6 in liver lipids and adipose tissue triacylglycerols of obese and lean rats. The HS diet also increased the proportions of 20∶4ω6 in adipose triacylglycerols of obese and lean rats and in liver lipids of obese animals but not in their lean littermates.     

Novel Insight in Idiopathic Normal Pressure Hydrocephalus (iNPH) Biomarker Discovery in CSF

Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological disease, causing motor and cognitive dysfunction and dementia. iNPH and Alzheimer’s disease (AD) share similar molecular characteristics, including amyloid deposition, t-tau and p-tau dysregulation; however, the disease is under-diagnosed and under-treated. The aim was to identify a panel of sphingolipids and proteins in CSF to diagnose iNPH at onset compared to aged subjects with cognitive integrity (C) and AD patients by adopting multiple reaction monitoring mass spectrometry (MRM-MS) for sphingolipid quantitative assessment and advanced high-resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS) for proteomic analysis. The results indicated that iNPH are characterized by an increase in very long chains Cer C22:0, Cer C24:0 and Cer C24:1 and of acute-phase proteins, immunoglobulins and complement component fragments. Proteins involved in synaptic signaling, axogenesis, including BACE1, APP, SEZ6L and SEZ6L2; secretory proteins (CHGA, SCG3 and VGF); glycosylation proteins (POMGNT1 and DAG1); and proteins involved in lipid metabolism (APOH and LCAT) were statistically lower in iNPH. In conclusion, at the disease onset, several factors contribute to maintaining cell homeostasis, and the protective role of very long chains sphingolipids counteract overexpression of amyloidogenic and neurotoxic proteins. Monitoring specific very long chain Cers will improve the early diagnosis and can promote patient follow-up.     

Insulin Resistance Is Not Sustained Following Denervation in Glycolytic Skeletal Muscle

Denervation rapidly induces insulin resistance (i.e., impairments in insulin-stimulated glucose uptake and signaling proteins) in skeletal muscle. Surprisingly, whether this metabolic derangement is long-lasting is presently not clear. The main goal of this study was to determine if insulin resistance is sustained in both oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles following long-term (28 days) denervation. Mouse hindlimb muscles were denervated via unilateral sciatic nerve resection. Both soleus and EDL muscles atrophied ~40%. Strikingly, while denervation impaired submaximal insulin-stimulated [³H]-2-deoxyglucose uptake ~30% in the soleus, it enhanced submaximal (~120%) and maximal (~160%) insulin-stimulated glucose uptake in the EDL. To assess possible mechanism(s), immunoblots were performed. Denervation did not consistently alter insulin signaling (e.g., p-Akt (Thr308):Akt; p-TBC1D1 [phospho-Akt substrate (PAS)]:TBC1D1; or p-TBC1D4 (PAS):TBC1D4) in either muscle. However, denervation decreased glucose transporter 4 (GLUT4) levels ~65% in the soleus but increased them ~90% in the EDL. To assess the contribution of GLUT4 to the enhanced EDL muscle glucose uptake, muscle-specific GLUT4 knockout mice were examined. Loss of GLUT4 prevented the denervation-induced increase in insulin-stimulated glucose uptake. In conclusion, the denervation results sustained insulin resistance in the soleus but enhanced insulin sensitivity in the EDL due to increased GLUT4 protein levels.     

The Antipsychotic Risperidone Alters Dihydroceramide and Ceramide Composition and Plasma Membrane Function in Leukocytes In Vitro and In Vivo

Atypical or second-generation antipsychotics are used in the treatment of psychosis and behavioral problems in older persons with dementia. However, these pharmaceutical drugs are associated with an increased risk of stroke in such patients. In this study, we evaluated the effects of risperidone treatment on phospholipid and sphingolipid composition and lipid raft function in peripheral blood mononuclear cells (PBMCs) of older patients (mean age >88 years). The results showed that the levels of dihydroceramides, very-long-chain ceramides, and lysophosphatidylcholines decreased in PBMCs of the risperidone-treated group compared with untreated controls. These findings were confirmed by in vitro assays using human THP-1 monocytes. The reduction in the levels of very-long-chain ceramides and dihydroceramides could be due to the decrease in the expression of fatty acid elongase 3, as observed in THP-1 monocytes. Moreover, risperidone disrupted lipid raft domains in the plasma membrane of PBMCs. These results indicated that risperidone alters phospholipid and sphingolipid composition and lipid raft domains in PBMCs of older patients, potentially affecting multiple signaling pathways associated with these membrane domains.     

High-Fat Diet Impairs Muscle Function and Increases the Risk of Environmental Heatstroke in Mice

Environmental heat-stroke (HS) is a life-threatening response often triggered by hot and humid weather. Several lines of evidence indicate that HS is caused by excessive heat production in skeletal muscle, which in turn is the result of abnormal Ca²⁺ leak from the sarcoplasmic reticulum (SR) and excessive production of oxidative species of oxygen and nitrogen. As a high fat diet is known to increase oxidative stress, the objective of the present study was to investigate the effects of 3 months of high-fat diet (HFD) on the HS susceptibility of wild type (WT) mice. HS susceptibility was tested in an environmental chamber where 4 months old WT mice were exposed to heat stress (41 °C for 1 h). In comparison with mice fed with a regular diet, mice fed with HFD showed: (a) increased body weight and accumulation of adipose tissue; (b) elevated oxidative stress in skeletal muscles; (c) increased heat generation and oxygen consumption during exposure to heat stress; and finally, (d) enhanced sensitivity to both temperature and caffeine of isolated muscles during in-vitro contracture test. These data (a) suggest that HFD predisposes WT mice to heat stress and (b) could have implications for guidelines regarding food intake during periods of intense environmental heat.     

Dietary phospholipids are more efficient than neutral lipids for long-chain polyunsaturated fatty acid supply in European sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> larval development

We evaluated the effects of dietary lipid class (phospholipid vs. neutral lipid) and level of n−3 long-chain PUFA (LC-PUFA) on the growth, digestive enzymatic activity, and histological organization of the intestine and liver in European sea bass larvae. Fish were fed from the onset of exogenous feeding at 7 to 37 d post-hatch with five isoproteic and isolipidic compound diets with different levels of EPA and DHA. Diet names indicated the percentage of EPA and DHA contained in the phospholipids (PL) and neutral lipids (NL), as follows: PL5, PL3, PL1, NL1, and NL3. Histological observations showed different patterns of lipid absorption and accumulation in the intestinal mucosa depending on the level and nature of the dietary lipid fraction. Fish fed high levels of neutral lipids (11%, NL3 diet: 2.6% of EPA+DHA in the NL fraction) showed large intracellular and intercellular lipid deposits in the anterior intestine, but no such lipid accumulation was detected when larvae were fed with low and moderate levels of EPA and DHA in the phospholipid and neutral lipid fractions of the diet (PL and NL1 diets). PL were preferentially absorbed in the postvalvular intestine, and the accumulation of marine PL was inversely correlated to their dietary level. The postvalvular intestinal mucosa and liver showed signs of steatosis; large lipid vacuoles were observed in this region of the intestine and in the liver and were inversely correlated with the level of dietary neutral lipids. The best results in terms of growth, survival, and development (maturation of the digestive system and histological organization of the liver and intestinal mucosa) were obtained in the group fed with 2.3% of EPA and DHA in the PL fraction of the diet (PL3 diet), revealing that European sea bass larvae use the LC-PUFa contained in the PL fraction more efficiently than those from the NL fraction of the diet.     

Assessment of a Small Molecule Synthetic Lignan in Enhancing Oxidative Balance and Decreasing Lipid Accumulation in Human Retinal Pigment Epithelia

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 β release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 β release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.     

The effect of antioxidant deficiency on tissue lipid composition in the rat. I. Gastrocnemius and quadriceps muscle

The gastrocnemius and quadriceps muscle phospholipids of the antioxidant-deficient rat fed a source of both linoleate and linolenate showed a progressive net increase in arachidonate, a progressive net decrease in all other polyunsaturated fatty acids, and there was a concomitant accumulation of fluorescent pigment of the lipofuscin or ceroid type in the tissue. An increased incorporation of intraperitoneally injected, isotopically labeled acetate into not only arachidonate but also the other higher polyunsaturated fatty acids, was observed. The net loss of the higher polyunsaturated fatty acids from the membrane lipids (presumably via lipid peroxidation) apparently was partially compensated by a homeostatic mechanism which involved conversion of the available precursors, linoleate and linolenate, to the higher polyunsaturated fatty acids. The rates of decrease of the polyunsaturated fatty acids in the muscle phospholipids and accumulation of fluorescent pigment in the tissue were correlated with the rate of production of creatinuria.     

Butyrate Alters Pyruvate Flux and Induces Lipid Accumulation in Cultured Colonocytes

Butyrate is considered the primary energy source of colonocytes and has received wide attention due to its unique health benefits. Insight into the mechanistic effects of butyrate on cellular and metabolic function relies mainly on research in in-vitro-cultured cells. However, cells in culture differ from those in vivo in terms of metabolic phenotype and nutrient availability. For translation, it is therefore important to understand the impact of different nutrients on the effects of butyrate. We investigated the metabolic consequences of butyrate exposure under various culturing conditions, with a focus on the interaction between butyrate and glucose. To investigate whether the effects of butyrate were different between cells with high and low mitochondrial capacity, we cultured HT29 cells under either low- (0.5 mM) or high- (25 mM) glucose conditions. Low-glucose culturing increased the mitochondrial capacity of HT29 cells compared to high-glucose (25 mM) cultured HT29 cells. Long-term exposure to butyrate did not alter mitochondrial bioenergetics, but it decreased glycolytic function, regardless of glucose availability. In addition, both high- and low-glucose-grown HT29 cells showed increased lipid droplet accumulation following long-term butyrate exposure. Acute exposure of cultured cells (HT29 and Caco-2) to butyrate increased their oxygen consumption rate (OCR). A simultaneous decrease in extracellular acidification rate (ECAR) was observed. Furthermore, in the absence of glucose, OCR did not increase in response to butyrate. These results lead us to believe that butyrate itself was not responsible for the observed increase in OCR, but, instead, butyrate stimulated pyruvate flux into mitochondria. Indeed, blocking of the mitochondrial pyruvate carrier prevented a butyrate-induced increase in oxygen consumption. Taken together, our results indicate that butyrate itself is not oxidized in cultured cells but instead alters pyruvate flux and induces lipid accumulation.     

Multifunctional Role of Lipids in Modulating the Tumorigenic Properties of 4T1 Breast Cancer Cells

Tumor growth and progression are linked to an altered lipid metabolism in the tumor microenvironment (TME), including tumor cells and tumor-associated macrophages (TAMs). A growing number of lipid metabolism targeting drugs have shown efficacy in anti-tumor therapy. In addition, exogenously applied lipids and lipid analogues have demonstrated anti-tumor activities in several cancers, including breast cancer. In this study, we investigated the anti-tumor efficacies of the natural lipids palmitic acid (PA), sphingomyelin (SM), ceramide (Cer) and docosahexaenoic acid (DHA) on breast cancer cells. All tested lipids reduced the malignancy of breast cancer cells in vitro by impairing cell proliferation, migration and invasiveness. PA showed superior anti-tumor properties, as it additionally impaired cancer cell viability by inducing apoptosis, without affecting healthy cells. Co-culture experiments further demonstrated that Cer and PA reduced the immunosuppressive phenotype of M2 macrophages and the M2 macrophage-promoted the epithelial–mesenchymal transition (EMT) and migration of breast cancer cells. At the molecular level, this coincided with the up-regulation of E-cadherin. Our results highlight a powerful role for exogenously applied PA and Cer in reducing breast cancer tumorigenicity by simultaneously targeting cancer cells and M2 macrophages. Our findings support the notion that lipids represent alternative biocompatible therapeutic agents for breast cancer.