Long-chain omega-3 polyunsaturated fatty acids (LC-O3PUFAs) exhibit
therapeutic potential for the treatment and prevention of the neurological
deficits associated with spinal cord injury (SCI). However, the mechanisms
implicated in these protective responses remain unclear. The objective of the
present functional metabolomics study was to identify and define the dominant
metabolic pathways targeted by dietary LC-O3PUFAs. Sprague-Dawley rats were fed
rodent purified chows containing menhaden fish oil-derived LC-O3PUFAs for 8
weeks before being subjected to sham or spinal cord contusion surgeries. We
show, through untargeted metabolomics, that dietary LC-O3PUFAs regulate
important biochemical signatures associated with amino acid metabolism and free
radical scavenging in both the injured and sham-operated spinal cord. Of
particular significance, the spinal cord metabolome of animals fed with
LC-O3PUFAs exhibited reduced glucose levels (−48%) and polar
uncharged/hydrophobic amino acids (<−20%) while showing
significant increases in the levels of antioxidant/anti-inflammatory amino acids
and peptides metabolites, including β-alanine (+24%),
carnosine (+33%), homocarnosine (+27%),
kynurenine (+88%), when compared to animals receiving control
diets (p < 0.05). Further, we found that dietary LC-O3PUFAs
impacted the levels of neurotransmitters and the mitochondrial metabolism, as
evidenced by significant increases in the levels of N-acetylglutamate
(+43%) and acetyl-CoA levels (+27%),
respectively. Interestingly, this dietary intervention resulted in a global
correction of the pro-oxidant metabolic profile that characterized the
SCI-mediated sensorimotor dysfunction. In summary, the significant benefits of
metabolic homeostasis and increased antioxidant defenses unlock important
neurorestorative pathways of dietary LC-O3PUFAs against SCI.
Metabolomics; DHA metabolome; Spinal cord injury; ROS; Neuroprotection; Pain
Chronic neuropathic pain is a frequent comorbidity following spinal cord injury (SCI) and often fails to respond to conventional pain management strategies. Preventive administration of docosahexaenoic acid (DHA) or consumption of a diet rich in omega-3 polyunsaturated fatty acids (O3PUFAs) confers potent prophylaxis against SCI and improves functional recovery. The present study examines whether this novel dietary strategy provides significant antinociceptive benefits in rats experiencing SCI-induced pain. Rats were fed control chow or chow enriched with O3PUFAs for 8 weeks before being subjected to sham or cord contusion surgeries, continuing the same diets after surgery for another 8 more weeks. The paw sensitivity to noxious heat was quantified for at least 8 weeks post-SCI using the Hargreaves test. We found that SCI rats consuming the preventive O3PUFA-enriched diet exhibited a significant reduction in thermal hyperalgesia compared to those consuming the normal diet. Functional neurometabolomic profiling revealed a distinctive deregulation in the metabolism of endocannabinoids (eCB) and related N-acyl ethanolamines (NAEs) at 8 weeks post-SCI. We found that O3PUFAs consumption led to a robust accumulation of novel NAE precursors, including the glycerophospho-containing docosahexaenoyl ethanolamine (DHEA), docosapentaenoyl ethanolamine (DPEA), and eicosapentaenoyl ethanolamine (EPEA). The tissue levels of these metabolites were significantly correlated with the antihyperalgesic phenotype. In addition, rats consuming the O3PUFA-rich diet showed reduced sprouting of nociceptive fibers containing CGRP and dorsal horn neuron p38 MAPK expression, well-established biomarkers of pain. The spinal cord levels of inositols were positively correlated with thermal hyperalgesia, supporting their role as biomarkers of chronic neuropathic pain. Notably, the O3PUFA-rich dietary intervention reduced the levels of these metabolites. Collectively, these results demonstrate the prophylactic value of dietary O3PUFA against SCI-mediated chronic pain.
DHA; EPA; dietary fatty acids; endocannabinoid metabolome; spinal cord injury; chronic pain
An urgent need exists for graduate and professional schools to establish evidence-based STEM (science, technology, engineering, and math) pipeline programs to increase the diversity of the biomedical workforce. An untapped yet promising pool of willing participants are capable high school students that have a strong STEM interest but may lack the skills and the guided mentoring needed to succeed in competitive STEM fields. This study evaluates and compares the impact of the Loma Linda University (LLU) Summer Health Disparities Research Program on high school (HS) and undergraduate (UG) student participants. The primary focus of our summer research experience (SRE) is to enhance the research self-efficacy of the participants by actively involving them in a research project and by providing the students with personalized mentoring and targeted career development activities, including education on health disparities. The results of our study show that our SRE influenced terminal degree intent and increased participant willingness to incorporate research into future careers for both the HS and the UG groups. The quantitative data shows that both the HS and the UG participants reported large, statistically significant gains in self-assessed research skills and research self-efficacy. Both participant groups identified the hands-on research and the mentor experience as the most valuable aspects of our SRE and reported increased science skills, increased confidence in science ability and increased motivation and affirmation to pursue a science career. The follow-up data indicates that 67% of the HS participants and 90% of the UG participants graduated from college with a STEM degree; for those who enrolled in graduate education, 61% and 43% enrolled in LLU, respectively. We conclude that structured SREs can be highly effective STEM strengthening interventions for both UG and HS students and may be a way to measurably increase institutional and biomedical workforce diversity.
Apoptotic cell death has been proposed to play a role in the neuronal loss observed following traumatic injury in the CNS and PNS. The present study uses an in vitro tissue culture model to investigate whether free fatty acids (FFAs), at concentrations comparable to those found following traumatic brain injury, trigger cell death. Nerve growth factor (NGF)-differentiated PC12 cells exposed to oleic and arachidonic acids (2 : 1 ratio FFA/BSA) showed normal cell survival. However, when cells were exposed to stearic and palmitic acids, there was a dramatic loss of cell viability after 24 h of treatment. The cell death induced by stearic acid and palmitic acid was apoptotic as assessed by morphological analysis, and activation of caspase-8 and caspase-3-like activities. Western blotting showed that differentiated PC12 cells exposed to stearic and palmitic acids exhibited the signature apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP). Interestingly, blockade of caspase activities with the pan-caspase inhibitor z-VAD-fmk failed to prevent the cell death observed induced by palmitic or stearic acid. RT-PCR and RNA blot experiments showed an up-regulation of the Fas receptor and ligand mRNA. These findings are consistent with our hypothesis that FFAs may play a role in the cell death associated with trauma in the CNS and PNS.
caspases; Fas ligand; Fas receptor; fatty acid; neuronal injury
We evaluated the changes in apolipoproteins, glycemic status, and body composition after 3 months using a culturally sensitive diabetes education program, En Balance, in diabetic Hispanics.
Thirty-four (9 males, 25 females) Hispanic diabetics participated in the En Balance program over three months. Body composition was determined by dual energy X-ray absorptiometry (DXA), fasting plasma glucose (FPG), A1c, and apolipoproteins (Apo) measured after 3 months participation. Differences were analyzed using paired t testing and relationships between changes in Apo, A1c, total cholesterol, body mass index and body composition by Spearman correlations.
Completion of En Balance resulted in a significant reduction in weight (80.31 ± 1.97 kg vs 81.25 ± 17.97 kg, P=.015), FPG (143.21 ± 57.8 mg/dL vs 166.41 ± 65.9 mg/dL P=.003), and A1c (7.08 ± 1.6% vs 7.87 ± 2.0%, P=<.001). DXA demonstrated reduction in total fat (29.54 ± 10.0 kg vs 30.24 ± 11.80 kg, P=<.001) and trunk fat (15.09 ± 5.6 kg vs 16.87 ± 5.4 kg, P=.001). High density lipoprotein significantly increased (48.85 ± 11.4 vs 44.65 ± 8.8, P=.002) and total serum cholesterol/high density lipoprotein ratio decreased (3.87 ± .98 vs 4.35 ± 1.0, P=.001). There were significant correlations at three months between changes in Apo A1 and A2 (r=.559, P<.001), Apo E and total cholesterol (r=.746, P<.001), between A1c and FPG (r=.563, P=.001) and BMI and body weight (r=.732, P<.001).
The En Balance program improved body composition, A1c, FPG, total cholesterol/HDL ratio and HDL. If these trends can be sustained, En Balance may serve as a unique educational paradigm for improving type 2 diabetes in Hispanics.
Hispanics; Type 2 Diabetes; Diabetes Education Programs
This study was designed to assess the feasibility of culturally and language-sensitive diabetes education as a way to increase physical activity and to improve health/diabetes management in a group of Spanish-speaking Hispanics in the Inland Empire region of Southern California.
En Balance is a culturally sensitive diabetes education program designed for Spanish-speaking Hispanic adults. The 3-month educational intervention assessed 16 males and 23 females living in Riverside and San Bernardino counties of Southern California. Baseline and 3-month evaluations of physical activity were assessed using the validated Arizona Activity Frequency Questionnaire.
After 3 months on the En Balance program, there was a significant increase in moderate intensity physical activity energy expenditure (M = 368 ± 894 kcal/day, P < 0.01) and high intensity physical activity energy expenditure (M = 405 ± 2569 kcal/day, P = 0.05) compared to baseline and significant reductions in A1C (−0.90%, P = 0.01), total cholesterol (−13.44 mg/dl, P = 0.01), LDL cholesterol (−10.28 mg/dl, P = 0.03), and waist circumference (−1.52 cm, P = 0.04).
En Balance program resulted in significant mean increases in both moderate and high intensity physical activity energy expenditure among this group of Hispanic diabetic participants, indicating that despite a general pattern of low physical activity in this group, an intervention that stresses both nutrition and exercise in culturally sensitive ways can positively impact participant’s physical activity levels as well as impact nutritional changes.
Hispanic; diabetes; education; physical activity; glucose control
Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) confer multiple health benefits and decrease the risk of neurological disorders. Studies are needed, however, to identify promising cellular targets and to assess their prophylactic value against neurodegeneration. The present study (1) examined the efficacy of a preventive diet enriched with ω-3 PUFAs to reduce dysfunction in a well-established spinal cord injury (SCI) animal model and (2) used a novel metabolomics data analysis to identify potential neurolipidomic targets. Rats were fed with either control chow or chow enriched with ω-3 PUFAs (750 mg/kg/day) for 8 weeks before being subjected to a sham or a contusion SCI operation. We report new evidence showing that rats subjected to SCI after being pre-treated with a diet enriched with ω-3 PUFAs exhibit significantly better functional outcomes. Pre-treated animals exhibited lower sensory deficits, autonomic bladder recovery, and early improvements in locomotion that persisted for at least 8 weeks after trauma. We found that SCI triggers a robust alteration in the cord PUFA neurolipidome, which was characterized by a marked docosahexaenoic acid (DHA) deficiency. This DHA deficiency was associated with dysfunction and corrected with the ω-3 PUFA-enriched diet. Multivariate data analyses revealed that the spinal cord of animals consuming the ω-3 PUFA-enriched diet had a fundamentally distinct neurolipidome, particularly increasing the levels of essential and long chain ω-3 fatty acids and lysolipids at the expense of ω-6 fatty acids and its metabolites. Altogether, dietary ω-3 PUFAs prophylaxis confers resiliency to SCI mediated, at least in part, by generating a neuroprotective and restorative neurolipidome.
DHA; functional recovery; prevention; lipidomics
The lens epithelium derived growth factor p75 (LEDGF/p75) is a transcription co-activator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kD (DFS70), and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull down and AlphaScreen® assays, co-immunoprecipitation, and nuclear co-localization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS cells, while this effect was more pronounced in PC3 cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes.
LEDGF/p75; MeCP2; protein-protein interactions; PWWP domain; transcription
Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells.
A “Human oxidative stress and antioxidant defense” qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting.
Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs.
Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death.
LEDGF/p75; prostate cancer; oxidative stress; gene profiling; peroxiredoxin; cytoglobin
Genes for the fatty acid binding protein (FABP) family encode small 14–15 kDa cytosolic proteins and can be regulated during type 2 diabetes mellitus (T2DM) and obesity. This study compared association of single nucleotide polymorphisms (SNPs) in FABP1-5 with T2DM in different ethnic groups. Associations with T2DM of SNPs in these proteins were assessed in African American (AA), non-Hispanic White (NHW), and Hispanic American (HA) individuals. A total of 650 DNA samples were genotyped; control samples were obtained from Coriell’s North American Human Variation Panel Repository (NAVP) of apparently healthy individuals and T2DM cases were taken from the American Diabetes Association GENNID Study. The rs454550 SNP of FABP5 showed a significant association with T2DM in NHW (OR: 9.03, 95% CI: 1.13–71.73, p=0.014). Our analysis also identified a new FABP5 SNP (nSNP) that showed a significant association with T2DM in NHW (OR: 0.44, 95% CI: 0.19–0.99, p=0.045) and AA (OR: 0.17, 95% CI: 0.03–0.80, p=0.016). The Ala54Thr FABP2 polymorphism was significantly associated with T2DM in HA individuals only (OR: 1.85, 95% CI: 1.05–3.27, p=0.032). All other FABP SNPs did not show association with T2DM. These findings suggest a potential distinct role of SNPs in FABP5, 2 genes in T2DM in different populations.
Fatty Acid Binding Protein (FABP); Single Nucleotide Polymorphism (SNP) and Type 2 Diabetes (T2DM)
Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury (SCI). Docosahexaenoic acid (DHA, C22:6 n-3) is neuroprotective when administered following SCI, but its potential as a pretreatment modality has not been addressed. This study used a novel DHA pretreatment experimental paradigm that targets acute cellular and molecular events during the first week after SCI in rats. We found that DHA pretreatment reduced functional deficits during the acute phase of injury, as shown by significant improvements in Basso-Beattie-Bresnahan (BBB) locomotor scores, and the detection of transcranial magnetic motor evoked potentials (tcMMEPs) compared to vehicle-pretreated animals. We demonstrated that, at 7 days post-injury, DHA pretreatment significantly increased the percentage of white matter sparing, and resulted in axonal preservation, compared to the vehicle injections. We found a significant increase in the survival of NG2+, APC+, and NeuN+ cells in the ventrolateral funiculus (VLF), dorsal corticospinal tract (dCST), and ventral horns, respectively. Interestingly, these DHA protective effects were observed despite the lack of inhibition of inflammatory markers for monocytes/macrophages and astrocytes, ED1/OX42 and GFAP, respectively. DHA pretreatment induced levels of Akt and cyclic AMP responsive element binding protein (CREB) mRNA and protein. This study shows for the first time that DHA pretreatment ameliorates functional deficits, and increases tissue sparing and precursor cell survival. Further, our data suggest that DHA-mediated activation of pro-survival/anti-apoptotic pathways may be independent of its anti-inflammatory effects.
CNS injury; inflammation; secondary insult; spinal cord injury; therapeutic approaches for the treatment of neuronal cell death
Lipid overload resulting in lipotoxicity is prominent in a number of chronic diseases and has been associated with cellular dysfunction and cell death. This study characterizes palmitic acid-induced lipotoxicity (PA-LTx) in Schwann cell cultures grown in normal and high glucose concentrations. The study shows for the first time that Schwann cell (SC) cultures exposed to elevated levels of PA exhibit a dose and time –dependent loss in cell viability. Hoescht and Annexin V/7AAD staining confirmed cell death through apoptosis and the lipotoxic effect was more dramatic in SC cultures grown under high glucose conditions. The first indication of cellular dysfunction in treated SC cultures was a decrease in Ca++ levels in the endoplasmic reticulum (ER, [Ca++]ER) observed five minutes following the initial challenge with PA. This decrease in [Ca++]ER was followed by a significant increase in the expression of ER stress signature genes CHOP, Xbp1 and GRP78. The early ER stress response induced by PA-LTx was followed by a strong mitochondrial membrane depolarization. Flow cytometry using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) showed an increase in oxidative stress within three to six hours after PA treatment. Treatment of cultures undergoing PA-LTx with the calcium chelator BAPTA-AM and the antioxidant MC1-186 significantly reversed the lipotoxic effect by decreasing the generation of ROS and significantly increasing cell viability. We conclude that lipotoxicity in Schwann cells results in cellular dysfunction and cell death that involves a robust ER stress response, mitochondrial dysfunction and an augmented stated of cellular oxidative stress (ASCOS).
Lipotoxicity; Schwann Cells; Peripheral neuropathy; ER stress; Type 2 diabetes
The purpose of this study was to assess dietary intake habits of Mexican American Hispanic adults participating in the En Balance diabetes education program.
En Balance is a 3-month culturally sensitive diabetes education intervention for Spanish-speaking Hispanics. Of the 46 participants enrolled, 39 mainly Mexican American Hispanic adults with type 2 diabetes completed the En Balance program. Participants lived in the Riverside and San Bernardino counties of California, and all participants completed the program by June 2008. Dietary intake was assessed at baseline and at 3 months using the validated Southwest Food Frequency Questionnaire.
Clinically important decreases in glycemic control and serum lipid levels were observed at the end of the 3-month program. The baseline diet was characterized by a high intake of energy (2478 ± 1140 kcal), total fat (87 ± 44 g/day), saturated fat (28 ± 15 g/day), dietary cholesterol (338 ± 217 mg/day), and sodium (4236 ± 2055 mg/day). At 3 months, the En Balance group mean intake of dietary fat (P = .045) and dietary cholesterol (P = .033) decreased significantly. Low dietary intakes of docosahexaenoic acid, eicosapentaenoic acid, and vitamin E were also observed in these adults with type 2 diabetes.
The En Balance program improved glycemic control and lipid profiles in a group of Hispanic diabetic participants. En Balance also promoted decreases in dietary fat and dietary cholesterol intake.
Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity.
apoptosis; cathepsin L; docosahexanoic acid; lipotoxicity; lysosomal dysfunction
Diabetes mellitus and obesity are prevalent in the Hispanic community. This group has not benefited greatly from diabetes interventions due to cultural, language and financial constraints. We designed a prospective cohort study to determine the clinical impact on adiposity and glycemic control in Hispanics with type 2 diabetes.
Research design and methods
The program conducted in Spanish by a multidisciplinary team of health care providers focused on improving glycemic control and complications through cultural lifestyle changes. Outcomes were changes in glycemic control by fasting insulin, glucose and HbA1c, body composition and selected adipokines, adiponectin, leptin and ghrelin. Body composition was measured by dual energy x-ray absorptiometry. Changes from baseline at three months were compared using paired t-tests and with Spearman’s correlations.
Glycemic control improved by HbA1c (7.9% ± 2.0% vs 7.1% ± 1.7%; P = <0.001), and fasting glucose (166.4 ± 66.0 mg/dl vs 143.2 ± 57.9 mg/dl; P = 0.003). Body weight (81.3 ± 17.9 kg vs 80.3 ± 18.0 kg; P = 0.002), waist circumference (101.6 ± 13.4 cm vs 99.1 ± 12.7 cm; P = 0.015), and truncal fat (16.5 ± 5.7 kg vs 15.9 ± 5.6 kg; P = 0.001) decreased. Only leptin (19.6 ± 15.0 ng/ml vs 16.3 ± 12.7 ng/ml; P = 0.002) was reduced and related to change in body weight (r = 0.392; P = 0.022).
Our program significantly improved glycemic control and decreased obesity in diabetic Hispanic subjects. The early benefits on glycemic control may be related to reductions in leptin through loss of adipose tissue. Success in impacting diabetes and related complications can occur in a culturally focused and multidisciplinary context.
glycemic control; obesity; leptin; culture
Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.
C20:5n-3 eicosapentaenoic acid; C22:6n-3 docosahexaenoic acid; epidermal fatty acid-binding protein; fatty acid binding; n-3/n-6 polyunsaturated fatty acids; neuronal differentiation
The purpose of this study was to evaluate the effects of a culturally sensitive diabetes education program for Hispanics with type 2 diabetes.
This study is a prospective cohort study to test the impact of a comprehensive diabetes education program on blood glucose control on Hispanics with type 2 diabetes. The educational program focused on maintaining glycemic control and general aspects of managing diabetes and complications. The study participants were recruited by flyers placed in Hispanic markets and in ambulatory care clinics. A total of 34 Hispanic male and female subjects with type 2 diabetes participated in the study. The concentrations of glucose, insulin, hemoglobin A1c (HbA1c), total cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein (HDL) cholesterol were analyzed at baseline and at 3 months.
A significant mean change was observed for HbA1c, fasting plasma glucose, cholesterol/HDL ratio, and HDL after 3 months of education compared with baseline. There were significant reductions in weight, total fat, percent fat, trunk fat, and waist-to-hip ratio compared with baseline. After 3 months, subjects showed a significant positive correlation between changes in body mass index and insulin and weight, total fat, trunk fat, and fat free mass and insulin.
A culturally sensitive program conducted in Spanish had a significant impact on important clinical parameters in Hispanic subjects with diabetes in a relatively short time period. The study demonstrates the importance of designing education intervention studies that are sensitive to cultural diversity, particularly in at-risk diabetic subjects.
Lipotoxicity involves a series of pathological cellular responses after exposure to elevated levels of fatty acids. This process may be detrimental to normal cellular homeostasis and cell viability. The present study shows that nerve growth factor-differentiated PC12 cells (NGFDPC12) and rat cortical cells (RCC) exposed to high levels of palmitic acid (PA) exhibit significant lipotoxicity and death linked to an “augmented state of cellular oxidative stress” (ASCOS). The ASCOS response includes generation of reactive oxygen species (ROS), alterations in the mitochondrial transmembrane potential, and increase in the mRNA levels of key cell death/survival regulatory genes. The observed cell death was apoptotic based on nuclear morphology, caspase-3 activation, and cleavage of lamin B and PARP. Quantitative real-time PCR measurements showed that cells undergoing lipotoxicity exhibited an increase in the expression of the mRNAs encoding the cell death-associated proteins BNIP3 and FAS receptor. Cotreatment of NGFDPC12 and RCC cells undergoing lipotoxicity with docosahexaenoic acid (DHA) and bovine serum albumin (BSA) significantly reduced cell death within the first 2 hr following the initial exposure to PA. The data suggest that lipotoxicity in NGFDPC12 and cortical neurons triggers a strong cell death apoptotic response. Results with NGFDPC12 cells suggest a linkage between induction of ASCOS and the apoptotic process and exhibit a temporal window that is sensitive to DHA and BSA interventions.
ASCOS; DHA; hypoxia/ischemia; lipotoxicity; neurotoxicity; traumatic brain injury
Hormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality.
We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity.
These results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC.
Cyclodextrins (CDs) are used to deliver hydrophobic molecules in aqueous environments. Methyl-β-cyclodextrin (MβCD), a member of this family of molecules, has been proposed to be a good carrier to deliver fatty acids to cells in culture. This report focuses on studying the in vitro effects of MβCD on nerve growth factor-differentiated PC12 (NGFDPC12) cells, a tissue culture model to study neuronal survival and differentiation. The main findings are: (1) NGFDPC12 cells have normal viability when exposed to 0.12% MβCD but showed a significant loss in cell viability at higher concentrations; (2) NGFDPC12 cells exposed to 0.25% MβCD exhibit nuclear condensation, blebbing and apoptotic bodies, and whole cell lysates exhibited an increase in caspase-3-like activity and high levels of Bax and Bcl-XL protein expression compared to control. Cultures treated with 0.25% MβCD also showed cleavage of normal 21-kDa Bax protein into a 18-kDa fragment. (3) Experiments using 0.12% MβCD to deliver oleic acid did not affect cell viability, in contrast NGFDPC12 cultures in which 0.25% MβCD concentration is used exhibited similar loss of cell viability as observed with 0.25% MβCD alone. Treating these cultures with caspase-3 inhibitor z-VAD-fmk did not protect the cells from MβCD toxic effects. (4) Immortalized Schwann cells (iSC) exposed to MβCD 0.12% did not show loss of cell viability while 0.25% MβCD triggered a significant toxicity but with a different dose and time course dynamic than NGFDPC12 cells. Thus, NGDPC12 or iSC cell cultures exposed to 0.12% MβCD exhibits normal viability while higher concentrations increase in cell death and apoptosis.
Methyl-β-cyclodextrin; toxicity; caspase; Bcl-2; Bcl-XL; and Bax
Keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis in alveolar type II cells in vitro. KGF stimulates lipogenic enzymes, including fatty acid synthase and stearyl-CoA desaturase-1, and transcription factors involved in lipogenesis, such as sterol regulatory element binding protein (SREBP)-1c and CCAAT/enhancer binding protein (C/EBP)α and C/EBPδ. To define the role of SREBP-1c on the induction of lipogenic genes and lipogenesis by KGF in primary cultures of rat type II cells, we used adenoviral vectors to alter levels of SREBP-1c. Overexpression of a dominant-negative form of SREBP-1 decreased lipogenesis and decreased the induction of fatty acid synthase and stearyl coenzyme A desaturase–1 by KGF. Conversely, adenovirus-mediated overexpression of a constitutively active form of SREBP-1c mimicked the effect of KGF on lipogenic enzymes and lipogenesis. These data indicate that SREBP-1c is required for the stimulation of lipogenesis by KGF in the alveolar type II cells and is a key regulator of lung lipid metabolism and that expression of SREBP-1c is sufficient to induce lipogenesis in rat type II cells.
adenovirus; fatty acid synthesis; surfactant
Epidermal fatty acid-binding protein (E-FABP/FABP5/DA11) binds and transport long-chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E-FABP protects nerve growth factor-differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM-induced lipotoxicity (PAM-LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E-FABP. Antioxidants MCI-186 and N-acetyl cysteine prevented E-FABP's induction in expression by PAM-LTx, while tert-butyl hydroperoxide increased ROS and E-FABP expression. Non-metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E-FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE-FABP showed reduced E-FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E-FABP cellular levels by pre-loading the cells with recombinant E-FABP diminished the PAM-induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E-FABP expression and enhanced the resistance of NGFDPC12 cells to PAM-LTx. We conclude that E-FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.
Epidermal fatty acid-binding protein (E-FABP) may protect nerve cells from the damaging exposure to high levels of free fatty acids (FA). We show that E-FABP can neutralize the effects of reactive oxygen species (ROS) generated by the high levels of FA in the cell and protect PC12 cells from lipotoxic injuries common in Type 2 diabetes neuropathy. Potentially, E-FABP gene up-regulation may be mediated through the NFkB pathway and future studies are needed to further evaluate this proposition.
antioxidants; FABP; lipotoxicity; PPAR; reactive oxygen species