The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

Preserving cell shape under environmental stress.

Maintaining cell shape and tone is crucial for the function and survival of cells and tissues. Mechanotransduction relies on the transformation of minuscule mechanical forces into high-fidelity electrical responses. When mechanoreceptors are stimulated, mechanically sensitive cation channels open and produce an inward transduction current that depolarizes the cell. For this process to operate effectively, the transduction machinery has to retain integrity and remain unfailingly independent of environmental changes. This is particularly challenging for poikilothermic organisms, where changes in temperature in the environment may impact the function of mechanoreceptor neurons. Thus, we wondered how insects whose habitat might quickly vary over several tens of degrees of temperature manage to maintain highly effective mechanical senses. We screened for Drosophila mutants with defective mechanical responses at elevated ambient temperatures, and identified a gene, spam, whose role is to protect the mechanosensory organ from massive cellular deformation caused by heat-induced osmotic imbalance. Here we show that Spam protein forms an extracellular shield that guards mechanosensory neurons from environmental insult. Remarkably, heterologously expressed Spam protein also endowed other cells with superb defence against physically and chemically induced deformation. We studied the mechanical impact of Spam coating and show that spam-coated cells are up to ten times stiffer than uncoated controls. Together, these results help explain how poikilothermic organisms preserve the architecture of critical cells during environmental stress, and illustrate an elegant and simple solution to such challenge.

Finding a new generation of spies and manuscript reviewers.

This material was presented as a poster at the Seventh International Congress on Peer Review and Biomedical Publication, 8-10 September 2013, Chicago, IL, USA.

Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes, and neutrophils, and the oxidative burst from human blood.

Peripheral blood mononuclear cells and platelets have long been recognized as having the potential to act as sensitive markers for mitochondrial dysfunction in a broad range of pathological conditions. However, the bioenergetic function of these cells has not been examined from the same donors, yet this is important for the selection of cell types for translational studies. Here, we demonstrate the measurement of cellular bioenergetics in isolated human monocytes, lymphocytes, and platelets, including the oxidative burst from neutrophils and monocytes from individual donors. With the exception of neutrophils, all cell types tested exhibited oxygen consumption that could be ascribed to oxidative phosphorylation with each having a distinct bioenergetic profile and distribution of respiratory chain proteins. In marked contrast, neutrophils were essentially unresponsive to mitochondrial respiratory inhibitors indicating that they have a minimal requirement for oxidative phosphorylation. In monocytes and neutrophils, we demonstrate the stimulation of the oxidative burst using phorbol 12-myristate 13-acetate and its validation in normal human subjects. Taken together, these data suggest that selection of cell type from blood cells is critical for assessing bioenergetic dysfunction and redox biology in translational research.

Injury-induced insulin resistance in adipose tissue.

Hyperglycemia and insulin resistance are common findings in critical illness. Patients in the surgical ICU are frequently treated for this 'critical illness diabetes' with intensive insulin therapy, resulting in a substantial reduction in morbidity and mortality. Adipose tissue is an important insulin target tissue, but it is not known whether adipose tissue is affected by critical illness diabetes. In the present study, a rodent model of critical illness diabetes was used to determine whether adipose tissue becomes acutely insulin resistant and how insulin signaling pathways are being affected. There was a reduction in insulin-induced phosphorylation of IR, IRS-1, Akt and GSK-3ß. Since insulin resistance occurs rapidly in adipose tissue, but before the insulin resistance in skeletal muscle, it may play a role in the initial development of critical illness diabetes.

Transforming the architecture of compound eyes.

Eyes differ markedly in the animal kingdom, and are an extreme example of the evolution of multiple anatomical solutions to light detection and image formation. A salient feature of all photoreceptor cells is the presence of a specialized compartment (disc outer segments in vertebrates, and microvillar rhabdomeres in insects), whose primary role is to accommodate the millions of light receptor molecules required for efficient photon collection. In insects, compound eyes can have very different inner architectures. Fruitflies and houseflies have an open rhabdom system, in which the seven rhabdomeres of each ommatidium are separated from each other and function as independent light guides. In contrast, bees and various mosquitoes and beetle species have a closed system, in which rhabdomeres within each ommatidium are fused to each other, thus sharing the same visual axis. To understand the transition between open and closed rhabdom systems, we isolated and characterized the role of Drosophila genes involved in rhabdomere assembly. Here we show that Spacemaker, a secreted protein expressed only in the eyes of insects with open rhabdom systems, acts together with Prominin and the cell adhesion molecule Chaoptin to choreograph the partitioning of rhabdomeres into an open system. Furthermore, the complete loss of spacemaker (spam) converts an open rhabdom system to a closed one, whereas its targeted expression to photoreceptors of a closed system markedly reorganizes the architecture of the compound eyes to resemble an open system. Our results provide a molecular atlas for the construction of microvillar assemblies and illustrate the critical effect of differences in a single structural protein in morphogenesis.

Prevention of carcinogenesis and inhibition of breast cancer tumor burden by dietary stearate.

Previous studies have shown that stearate (C18:0), a dietary long-chain saturated fatty acid, inhibits breast cancer cell neoplastic progression; however, little is known about the mechanism modulating these processes. We demonstrate that stearate, at physiological concentrations, inhibits cell cycle progression in human breast cancer cells at both the G(1) and G(2) phases. Stearate also increases cell cycle inhibitor p21(CIP1/WAF1) and p27(KIP1) levels and concomitantly decreases cyclin-dependent kinase 2 (Cdk2) phosphorylation. Our data also show that stearate induces Ras- guanosine triphosphate formation and causes increased phosphorylation of extracellular signal-regulated kinase (pERK). The MEK1 inhibitor, PD98059, reversed stearate-induced p21(CIP1/WAF1) upregulation, but only partially restored stearate-induced dephosphorylation of Cdk2. The Ras/mitogen-activated protein kinase/ERK pathway has been linked to cell cycle regulation but generally in a positive way. Interestingly, we found that stearate inhibits both Rho activation and expression in vitro. In addition, constitutively active RhoC reversed stearate-induced upregulation of p27(KIP1), providing further evidence of Rho involvement. To test the effect of stearate in vivo, we used the N-Nitroso-N-methylurea rat breast cancer carcinogen model. We found that dietary stearate reduces the incidence of carcinogen-induced mammary cancer and reduces tumor burden. Importantly, mammary tumor cells from rats on a stearate diet had reduced expression of RhoA and B as well as total Rho compared with a low-fat diet. Overall, these data indicate that stearate inhibits breast cancer cell proliferation by inhibiting key check points in the cell cycle as well as Rho expression in vitro and in vivo and inhibits tumor burden and carcinogen-induced mammary cancer in vivo.

Transgenic expression of n-3 fatty acid desaturase (fat-1) in C57/BL6 mice: Effects on glucose homeostasis and body weight.

The fat-1 gene, derived from Caenorhabditis elegans, encodes for a fatty acid n-3 desaturase. In order to study the potential metabolic benefits of n-3 fatty acids, independent of dietary fatty acids, we developed seven lines of fat-1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein-2 (aP2) gene for adipocyte-specific expression (AP-lines). We were unable to obtain homozygous fat-1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat-1 transgenic mice (AP-3) fed a high n-6 unsaturated fat (HUSF) diet had an n-6/n-3 fatty acid ratio reduced by 23% (P < 0.025) and the n-3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (P < 0.020). Docosahexaenoic acid (DHA) was increased by 19% (P < 0.015) in white adipose tissue. Male AP-3-fat-1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high-carbohydrate (HC) diet. In contrast, the female AP-3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat-1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat-1 mice have reduced glucose tolerance compared to wild-type mice. Finally the inability of fat-1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n-3 fatty acids may be detrimental to reproduction.

Free fatty acids enhance breast cancer cell migration through plasminogen activator inhibitor-1 and SMAD4.

Obesity is a risk factor for breast cancer and is associated with increased plasma concentrations of free fatty acids (FFAs). We and others have demonstrated that FFA induces plasminogen activator inhibitor-1 (PAI-1) expression in a variety of cells. Emerging evidence supports elevation of PAI-1 as a prognostic marker for breast cancer. Therefore, we hypothesized that FFAs might increase expression of PAI-1 in breast cancer cells and facilitate breast cancer progression. Secreted PAI-1 was higher in invasive and metastatic MDA-MB-231 cells compared with less invasive and non-metastatic Hs578T cells. Utilizing FFAs with different saturation and chain lengths, we demonstrated that linoleic acid induced expression of PAI-1 in MDA-MB-231 cells. Linoleic acid also induced in vitro migration of MDA-MB-231. By contrast, other FFAs tested had little or no effect on PAI-1 expression or migration. Linoleic acid-induced breast cancer cell migration was completely inhibited by virally expressed antisense PAI-1 RNA. Furthermore, increased expression of PAI-1 by FFAs was not detected in the SMAD4-deficient MDA-MB-468 breast carcinoma cells. Electrophoretic mobility-shift assay confirmed that linoleic acid-induced expression of PAI-1 was mediated, at least in part, by SMAD4 in MDA-MB-231 cells. That linoleic acid induces PAI-1 expression in breast cancer cells through SMAD4 provides a novel insight into understanding the relationships between two migration-associated molecules, FFAs, and PAI-1.

WASp is required for the correct temporal morphogenesis of rhabdomere microvilli.

Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation is largely undefined. Here, we report that the Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. We show that WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres.

Stearate preferentially induces apoptosis in human breast cancer cells.

Stearic acid (stearate) is an 18-carbon saturated fatty acid that has been shown to inhibit invasion and proliferation and induce apoptosis in various human cell types. The specificity of stearate-induced apoptosis for cancerous versus noncancerous breast cells has not been examined, and the mechanism underlying stearate-induced apoptosis is unknown. Morphological analysis, cell viability, and caspase-3 activity assays demonstrated that stearate activated apoptosis preferentially in cancerous breast cells in a time- and dose-dependent manner. Inhibition of de novo diacylgycerol synthesis or protein kinase C (PKC) blocked stearate-induced caspase-3 activity, indicating the involvement of a novel or classical PKC isozyme. To our knowledge this is the first study showing that stearate induces apoptosis preferentially in breast cancer cells and implicates protein kinase C in the signaling cascade. These results raise the possibility of dietary stearate having a beneficial role in the prevention or treatment of breast cancer.

Performance of the Sebia CAPILLARYS 2 for detection and immunotyping of serum monoclonal paraproteins.

We evaluated the performance of the CAPILLARYS 2 (Sebia, Norcross, GA) capillary electrophoresis system for detection and identification of monoclonal proteins in serum samples. We analyzed 104 serum specimens by Sebia Hydragel serum protein electrophoresis (agarose gel electrophoresis [AGE]/immunofixation electrophoresis [IFE]) and CAPILLARYS 2 capillary zone electrophoresis (CZE)/immunosubtraction. AGE and CZE had sensitivities of 90% and 81%, respectively, based on IFE as the "gold standard," and all bands detected were confirmed by IFE (100% specificity). AGE and CZE had an overall agreement of 91% on serum protein electrophoresis. In the population tested, IgG was detected in 29% of samples by IFE and 30% using immunosubtraction. Similarly IgA was detected in 9% of cases by IFE and 8% by immunosubtraction. IgM and light chains were detected in 6% and 3% of cases, respectively, by IFE, whereas CZE/immunosubtraction did not detect any IgM or light chains. In our hands, AGE and CZE had the same specificity for detection of monoclonal proteins; however, CZE/immunosubtraction seems to be less sensitive than IFE for the detection of IgM and, possibly, serum light chains.

Gonadal Mosaicism Induced by Chemical Treatment of Sperm in Drosophila melanogaster.

Accurate interpretation of forward genetic screens of chromosomes exposed in mature spermatozoa to a mutagenic chemical requires understanding-incomplete to date-of how exposed chromosomes and their replicas proceed through early development stages from the fertilized ovum to establishment of the germline of the treated male's offspring. We describe a model for early embryonic development and establishment of the germline of Drosophila melanogaster and a model-validating experiment. Our model proposes that, barring repair, DNA strands modified by treatment with alkylating agents are stable and mutagenic. Each replication of an alkylated strand can result in misreplication and a mutant-bearing daughter nucleus. Daughter nuclei thenceforth replicate faithfully and their descendants comprise the embryonic syncytium. Of the 256 nuclei present after the eighth division, several migrate into the polar plasm at the posterior end of the embryo to found the germline. Based upon distribution of descendants of the alkylated strands, the misreplication rate, and the number of nuclei selected as germline progenitors, the frequency of gonadal mosaicism is predictable. Experimentally, we tracked chromosomes 2 and 3 from EMS-treated sperm through a number of generations, to characterize autosomal recessive lethal mutations and infer gonadal genetic content of the sons of treated males. Over 50% of 106 sons bore germlines that were singly, doubly, or triply mosaic for chromosome 2 or chromosome 3. These findings were consistent with our model, assuming a rate of misreplication between 0.65 and 0.80 at each replication of an alkylated strand. Crossing treated males to mismatch-repair-deficient females had no apparent effect on mutation rate.

The Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster.

The majority of genes of multicellular organisms encode proteins with functions that are not required for viability but contribute to important physiological functions such as behavior and reproduction. It is estimated that 75% of the genes of Drosophila melanogaster are nonessential. Here we report on a strategy used to establish a large collection of stocks that is suitable for the recovery of mutations in such genes. From approximately 72,000 F(3) cultures segregating for autosomes heavily treated with ethyl methanesulfonate (EMS), approximately 12,000 lines in which the treated second or third chromosome survived in homozygous condition were selected. The dose of EMS induced an estimated rate of 1.2-1.5 x 10(-3) mutations/gene and predicts five to six nonessential gene mutations per chromosome and seven to nine alleles per locus in the samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic mutations induced in the initial exposure to the mutagen, many of the lines are segregating or are now fixed for lethal mutations on the mutagenized chromosome. The features of this collection, known as the Zuker collection, make it a valuable resource for forward and reverse genetic screens for mutations affecting a wide array of biological functions.

Long-chain saturated fatty acids induce annexin II translocation to detergent-resistant membranes.

DRM (detergent-resistant membranes), which are resistant to solublization by non-ionic detergents, have been demonstrated to be involved in many key cell functions such as signal transduction, endocytosis and cholesterol trafficking. Covalent modification of proteins by fatty acylation has been proposed to be an important protein-targeting mechanism for DRM association. However, little is known concerning the effects of LCSFA (long-chain saturated fatty acids) on protein composition of DRM in human cancer cells. In the present study, we found that, in Hs578T human breast cancer cells, the major protein increased in DRM in response to the LCSFA stearate (C18:0) was annexin II. Our results demonstrated that annexin II accumulated in DRM specifically in response to physiological concentrations of stearate and palmitate (C16:0), but not long-chain unsaturated fatty acids, in a time- and concentration-dependent manner. This process was reversible and dependent on cholesterol and intracellular calcium. Although calcium was necessary for this translocation, it was not sufficient to induce the annexin II translocation to DRM. We also demonstrate that stearate induced the acylation of caveolin but not that of annexin II. Association of annexin II with caveolin, although not necessarily direct, specifically occurs in DRM in response to stearate. Finally, bromostearate, a stearate analogue that effectively blocks protein acylation, does not induce annexin II translocation to DRM. We conclude that exogenously added LCSFA strongly induces the translocation of annexin II to DRM in Hs578T human breast cancer cells at least partially by association with acylated caveolin.

Polydrug fatality involving metaxalone.

A 29-year old female with a history of depression was found dead in a hotel room. The death scene investigation found empty pill bottles and an empty liter bottle of wine. Metaxalone, a centrally acting muscle relaxant, along with citalopram, ethanol, and chlorpheniramine were identified in the postmortem samples and quantitated by gas chromatography-mass spectrometry. The concentration of metaxalone in femoral vein blood was 39 mg/L. The heart blood concentration was 54 mg/L. Femoral vein blood concentrations of citalopram and chlorpheniramine were 0.77 mg/L and 0.04 mg/L, respectively. Ethanol levels were 0.13 g/dL in vitreous and 0.08 g/dL in heart blood. Other tissue samples were also analyzed. The authors consider the metaxalone concentrations toxic and potentially fatal. The citalopram concentrations were lower than those reported in fatal cases for this drug alone. Death was ascribed to polydrug abuse/overdose with metaxalone a major contributor. This represents the first reported case to our knowledge in which a metaxalone overdose significantly contributed to death.

Dietary stearic acid leads to a reduction of visceral adipose tissue in athymic nude mice.

Stearic acid (C18:0) is a long chain dietary saturated fatty acid that has been shown to reduce metastatic tumor burden. Based on preliminary observations and the growing evidence that visceral fat is related to metastasis and decreased survival, we hypothesized that dietary stearic acid may reduce visceral fat. Athymic nude mice, which are used in models of human breast cancer metastasis, were fed a stearic acid, linoleic acid (safflower oil), or oleic acid (corn oil) enriched diet or a low fat diet ad libitum. Total body weight did not differ significantly between dietary groups over the course of the experiment. However visceral fat was reduced by ∼70% in the stearic acid fed group compared to other diets. In contrast total body fat was only slightly reduced in the stearic acid diet fed mice when measured by dual-energy x-ray absorptiometry and quantitative magnetic resonance. Lean body mass was increased in the stearic acid fed group compared to all other groups by dual-energy x-ray absorptiometry. Dietary stearic acid significantly reduced serum glucose compared to all other diets and increased monocyte chemotactic protein-1 (MCP-1) compared to the low fat control. The low fat control diet had increased serum leptin compared to all other diets. To investigate possible mechanisms whereby stearic acid reduced visceral fat we used 3T3L1 fibroblasts/preadipocytes. Stearic acid had no direct effects on the process of differentiation or on the viability of mature adipocytes. However, unlike oleic acid and linoleic acid, stearic acid caused increased apoptosis (programmed cell death) and cytotoxicity in preadipocytes. The apoptosis was, at least in part, due to increased caspase-3 activity and was associated with decreased cellular inhibitor of apoptosis protein-2 (cIAP2) and increased Bax gene expression. In conclusion, dietary stearic acid leads to dramatically reduced visceral fat likely by causing the apoptosis of preadipocytes.

Diet modulation is an effective complementary agent in preventing and treating breast cancer lung metastasis.

A significant percentage of breast cancer victims will suffer from metastases indicating that new approaches to preventing breast cancer metastasis are thus needed. Dietary stearate (ST) and chemotherapy have been shown to reduce breast cancer metastasis. We tested the complementary use of dietary ST with a taxol-based chemotherapy which work through separate mechanisms to reduce breast cancer metastasis. We therefore carried out a prevention study in which diets were initiated prior to human MDA-MB-435 cancer cells being injected into the host and a treatment study in which diets were combined with paclitaxel (PTX). Using an orthotopic athymic nude mouse model and three diets [corn oil (CO) control diet, low fat (LF) or ST] the prevention study demonstrated that the ST diet decreased the incidence of lung metastasis by 50 % compared to both the LF and CO diets. The ST diet also reduced the number and size of metastatic lung nodules compared to the LF diet. Results of the treatment study indicated that both the CO and ST diets decreased the number of mice with lung metastasis compared to the LF diet. Both CO and ST also decreased the number of lung metastases per mouse compared to the LF diet however only the ST diet cohort was significant. Histomorphometric analysis of the lung tumor tissue indicated that the ST diet plus PTX decreased angiogenesis compared to the LF diet plus PTX. In conclusion these results support combining diet with chemotherapy in both treatment and prevention settings.

Prostatic alpha-linolenic acid (ALA) is positively associated with aggressive prostate cancer: a relationship which may depend on genetic variation in ALA metabolism.

Previous observational studies have reported associations between prostate cancer and alpha-linolenic acid (ALA). However, few investigations have been able to study this relationship prospectively and in well-controlled settings. Moreover, no studies have determined whether single nucleotide polymorphisms (SNPs) that influence ALA metabolism are associated with this common cancer. The purpose of this study was to explore associations between prostatic levels of ALA, SNPs and prostate cancer-specific biomarkers in samples collected from a previous randomized clinical trial conducted using a presurgical model and which tested the effects of flaxseed supplementation, a rich source of ALA, prior to prostatectomy (n = 134). Serum prostate-specific antigen (PSA) was determined and immunohistochemistry was used to assess tumor proliferation rate (Ki67). Prostatic ALA was determined with gas chromatography. Seven previously identified SNPs associated with delta-6 desaturase activity (rs99780, rs174537, rs174545, rs174572, rs498793, rs3834458 and rs968567) were tested for associations with prostatic ALA, PSA and Ki67. Despite consuming seven times more ALA per day, men in the flaxseed arm had similar amounts of prostatic ALA relative to men not consuming flaxseed. In unadjusted analysis, there were significant positive associations between prostatic ALA and PSA (ρ = 0.191, p = 0.028) and Ki67 (ρ = 0.186, p = 0.037). After adjusting for covariates (flaxseed, age, race, BMI and statin-use) the association between ALA and PSA remained (p = 0.004) but was slightly attenuated for Ki67 (p = 0.051). We did not observe associations between any of the SNPs studied and prostatic ALA; however, in models for PSA there was a significant interaction between rs498793 and ALA and for Ki67 there were significant interactions with ALA and rs99780 and rs174545. Independent and inverse associations were observed between rs174572 and Ki67. This study provides evidence that prostatic ALA, independent of the amount of ALA consumed, is positively associated with biomarkers of aggressive prostate cancer and that genetic variation may modify this relationship.