Exogenous Galactosylceramide as Potential Treatment for CLN3 Disease.

OBJECTIVE: CLN3 disease is the commonest of the neuronal ceroid lipofuscinoses, a group of pediatric neurodegenerative disorders. Functions of the CLN3 protein include antiapoptotic properties and facilitating anterograde transport of galactosylceramide from Golgi to lipid rafts. This study confirms the beneficial effects of long-term exogenous galactosylceramide supplementation on longevity, neurobehavioral parameters, neuronal cell counts, astrogliosis, and diminution in brain and serum ceramide levels in Cln3 Δex7/8 knock-in mice. Additionally, the impact of galactosylceramide on ceramide synthesis enzymes is documented. METHODS: A group of 72 mice received galactosylceramide or vehicle for 40 weeks. The effect of galactosylceramide supplementation on Cln3 Δex7/8 mice was determined by performing behavioral tests, measuring ceramide in brains and serum, and assessing impact on longevity, subunit C storage, astrogliosis, and neuronal cell counts. RESULTS: Galactosylceramide resulted in enhanced grip strength of forelimbs in male and female mice, better balance on the accelerating rotarod in females, and improved motor coordination during pole climbing in male mice. Brain and serum ceramide levels as well as apoptosis rates were lower in galactosylceramide-treated Cln3 Δex7/8 mice. Galactosylceramide also increased neuronal cell counts significantly in male and female mice and tended to decrease subunit C storage in specific brain regions. Astrogliosis dropped in females compared to a slight increase in males after galactosylceramide. Galactosylceramide increased the lifespan of affected mice. INTERPRETATION: Galactosylceramide improved behavioral, neuropathological, and biochemical parameters in Cln3 Δex7/8 mice, paving the way for effective therapy for CLN3 disease and use of serum ceramide as a potential biomarker to track impact of therapies. ANN NEUROL 2019;86:729-742.

Whole-exome screening for primary congenital glaucoma in Lebanon.

PURPOSE: Mutations were previously identified in the CYP1B1 gene in six out of 18 Lebanese families (33%) with primary congenital glaucoma (PCG). The purpose of this study is to determine the frequency and type of pathogenic mutations in other genes and compare to other populations using whole-exome sequencing and perform genotype-phenotype correlations. METHODS: Twelve PCG patients previously negative for CYP1B1/MYOC mutations were subjected to whole-exome sequencing. Targeted screening for glaucoma-associated genes was performed. Candidate variants were verified by Sanger sequencing and evaluated in family members for segregation analysis and in 100 normal controls. Clinical correlations were established as to severity of disease presentation, course, and visual outcomes. RESULTS: Six mutations in known PCG-causing genes were identified in five patients: homozygous mutations in CYP1B1 (p.R368G), LTBP2 (p.E1013G), and TEK (p.T693I), and heterozygous mutations in FOXC1 (p.Q92*), TEK (c.3201-1 G>A), ANGPT1 (p.K186N), and CYP1B1 (p.R368G). Two patients, negative for CYP1B1 in the previous study, were revealed positive in the current study, due to different sets of primers and PCR conditions. Potentially damaging variants were noted in several candidate genes. Except for FOXC1 mutations, all genetic variants described here are novel. Intra-ocular pressure and final optic nerve cup-to-disc ratio were highest in the patient with three mutations in LTBP2/TEK/ANGPT1 genes. CONCLUSION: This study provides new data on the spectrum of mutations of PCG in Lebanon. This highlights the genetic heterogeneity of the Lebanese population, noted for high rates of consanguinity in 50% in this cohort. This study emphasizes the importance of whole-exome sequencing in elucidating new candidate genes for PCG in the Lebanese.

Sex-dimorphism in cardiac nutrigenomics: effect of trans fat and/or monosodium glutamate consumption.

BACKGROUND: A paucity of information on biological sex-specific differences in cardiac gene expression in response to diet has prompted this present nutrigenomics investigation. Sexual dimorphism exists in the physiological and transcriptional response to diet, particularly in response to high-fat feeding. Consumption of Trans-fatty acids (TFA) has been linked to substantially increased risk of heart disease, in which sexual dimorphism is apparent, with males suffering a higher disease rate. Impairment of the cardiovascular system has been noted in animals exposed to Monosodium Glutamate (MSG) during the neonatal period, and sexual dimorphism in the growth axis of MSG-treated animals has previously been noted. Processed foods may contain both TFA and MSG. METHODS: We examined physiological differences and changes in gene expression in response to TFA and/or MSG consumption compared to a control diet, in male and female C57BL/6J mice. RESULTS: Heart and % body weight increases were greater in TFA-fed mice, who also exhibited dyslipidemia (P < 0.05). Hearts from MSG-fed females weighed less than males (P < 0.05). 2-factor ANOVA indicated that the TFA diet induced over twice as many cardiac differentially expressed genes (DEGs) in males compared to females (P < 0.001); and 4 times as many male DEGs were downregulated including Gata4, Mef2d and Srebf2. Enrichment of functional Gene Ontology (GO) categories were related to transcription, phosphorylation and anatomic structure (P < 0.01). A number of genes were upregulated in males and downregulated in females, including pro-apoptotic histone deacetylase-2 (HDAC2). Sexual dimorphism was also observed in cardiac transcription from MSG-fed animals, with both sexes upregulating approximately 100 DEGs exhibiting sex-specific differences in GO categories. A comparison of cardiac gene expression between all diet combinations together identified a subset of 111 DEGs significant only in males, 64 DEGs significant in females only, and 74 transcripts identified as differentially expressed in response to dietary manipulation in both sexes. CONCLUSION: Our model identified major changes in the cardiac transcriptional profile of TFA and/or MSG-fed mice compared to controls, which was reflected by significant differences in the physiological profile within the 4 diet groups. Identification of sexual dimorphism in cardiac transcription may provide the basis for sex-specific medicine in the future.

Y-chromosomal diversity in Lebanon is structured by recent historical events.

Lebanon is an eastern Mediterranean country inhabited by approximately four million people with a wide variety of ethnicities and religions, including Muslim, Christian, and Druze. In the present study, 926 Lebanese men were typed with Y-chromosomal SNP and STR markers, and unusually, male genetic variation within Lebanon was found to be more strongly structured by religious affiliation than by geography. We therefore tested the hypothesis that migrations within historical times could have contributed to this situation. Y-haplogroup J*(xJ2) was more frequent in the putative Muslim source region (the Arabian Peninsula) than in Lebanon, and it was also more frequent in Lebanese Muslims than in Lebanese non-Muslims. Conversely, haplogroup R1b was more frequent in the putative Christian source region (western Europe) than in Lebanon and was also more frequent in Lebanese Christians than in Lebanese non-Christians. The most common R1b STR-haplotype in Lebanese Christians was otherwise highly specific for western Europe and was unlikely to have reached its current frequency in Lebanese Christians without admixture. We therefore suggest that the Islamic expansion from the Arabian Peninsula beginning in the seventh century CE introduced lineages typical of this area into those who subsequently became Lebanese Muslims, whereas the Crusader activity in the 11(th)-13(th) centuries CE introduced western European lineages into Lebanese Christians.

Identifying genetic traces of historical expansions: Phoenician footprints in the Mediterranean.

The Phoenicians were the dominant traders in the Mediterranean Sea two thousand to three thousand years ago and expanded from their homeland in the Levant to establish colonies and trading posts throughout the Mediterranean, but then they disappeared from history. We wished to identify their male genetic traces in modern populations. Therefore, we chose Phoenician-influenced sites on the basis of well-documented historical records and collected new Y-chromosomal data from 1330 men from six such sites, as well as comparative data from the literature. We then developed an analytical strategy to distinguish between lineages specifically associated with the Phoenicians and those spread by geographically similar but historically distinct events, such as the Neolithic, Greek, and Jewish expansions. This involved comparing historically documented Phoenician sites with neighboring non-Phoenician sites for the identification of weak but systematic signatures shared by the Phoenician sites that could not readily be explained by chance or by other expansions. From these comparisons, we found that haplogroup J2, in general, and six Y-STR haplotypes, in particular, exhibited a Phoenician signature that contributed > 6% to the modern Phoenician-influenced populations examined. Our methodology can be applied to any historically documented expansion in which contact and noncontact sites can be identified.

Effect of trans-fat, fructose and monosodium glutamate feeding on feline weight gain, adiposity, insulin sensitivity, adipokine and lipid profile.

The incidence of obesity and type 2 diabetes mellitus (T2DM) is increasing, and new experimental models are required to investigate the diverse aspects of these polygenic diseases, which are intimately linked in terms of aetiology. Feline T2DM has been shown to closely resemble human T2DM in terms of its clinical, pathological and physiological features. Our aim was to develop a feline model of diet-induced weight gain, adiposity and metabolic deregulation, and to examine correlates of weight and body fat change, insulin homeostasis, lipid profile, adipokines and clinical chemistry, in order to study associations which may shed light on the mechanism of diet-induced metabolic dysregulation. We used a combination of partially hydrogenated vegetable shortening and high-fructose corn syrup to generate a high-fat-high-fructose diet. The effects of this diet were compared with an isoenergetic standard chow, either in the presence or absence of 1.125 % dietary monosodium glutamate (MSG). Dual-energy X-ray absorptiometry body imaging and a glucose tolerance test were performed. The present results indicate that dietary MSG increased weight gain and adiposity, and reduced insulin sensitivity (P < 0.05), whereas high-fat-high-fructose feeding resulted in elevated cortisol and markers of liver dysfunction (P < 0.01). The combination of all three dietary constituents resulted in lower insulin levels and elevated serum ß-hydroxybutyrate and cortisol (P < 0.05). This combination also resulted in a lower first-phase insulin release during glucose tolerance testing (P < 0.001). In conclusion, markers of insulin deregulation and metabolic dysfunction associated with adiposity and T2DM can be induced by dietary factors in a feline model.

WFS1 mutations are frequent monogenic causes of juvenile-onset diabetes mellitus in Lebanon.

Most cases of juvenile-onset diabetes (JOD) are diagnosed as type 1 diabetes (T1D), for which genetic studies conducted in outbred Caucasian populations support the concept of multifactorial inheritance. However, this view may be partly challenged in particular population settings. In view of the suggestive evidence for a high prevalence of Wolfram syndrome (WFS) in Lebanon, the phenotypic variability associated with WFS1 mutations, and the high consanguinity rate in Lebanon, we aimed to evaluate the contribution of WFS1 mutations as monogenic determinants to JOD in Lebanon. We performed a family-based genetic study, with linkage analysis followed by systematic mutation screening of WFS1 exons in all JOD probands. The study population consisted of an unbiased recruitment of all juvenile-onset insulin-dependent diabetic patients from a specialized diabetes pediatric clinic in Beirut, Lebanon. Homozygous or compound heterozygous WFS1 mutations were found in 22 of the 399 JOD probands (5.5%), resulting in WFS (17 probands) or in non-syndromic non-autoimmune diabetes mellitus (DM, five probands). These accounted for 12.1% (21/174) of probands in consanguineous families, compared with 0.4% (1/225) in non-consanguineous families. Of the 38 patients identified with homozygous or compound heterozygous WFS1 mutations, 11 (29%) had non-syndromic DM, all of whom carried a particular WFS1 mutation, WFS1(LIB), encoding a protein with an extended C-terminal domain. This mutation resulted in a delayed onset or absence of extrapancreatic features. These results underscore the major impact of population-specific factors, such as population-specific mutations and founder effects, and family structure in the genetic determinism of JOD.

Sex differences in gene expression with galactosylceramide treatment in Cln3Δex7/8 mice.

BACKGROUND: CLN3 disease is caused by mutations in the CLN3 gene. The purpose of this study is to discern global expression patterns reflecting therapeutic targets in CLN3 disease. METHODS: Differential gene expression in vehicle-exposed mouse brain was determined after intraperitoneal vehicle/Galactosylceramide (GalCer) injections for 40 weeks with GeneChip Mouse Genome 430 2.0 arrays. RESULTS: Analysis identified 66 genes in male and 30 in female brains differentially expressed in GalCer-treated versus vehicle-exposed Cln3Δex7/8 mice. Gene ontology revealed aberrations of biological function including developmental, cellular, and behavioral processes. GalCer treatment altered pathways of long-term potentiation/depression, estrogen signaling, synaptic vesicle cycle, ErbB signaling, and prion diseases in males, but prolactin signaling, selenium compound metabolism and steroid biosynthesis in females. Gene-gene network analysis highlighted networks functionally pertinent to GalCer treatment encompassing motor dysfunction, neurodegeneration, memory disorder, inflammation and astrogliosis in males, and, cataracts, inflammation, astrogliosis, and anxiety in females. CONCLUSIONS: This study sheds light on global expression patterns following GalCer treatment of Cln3Δex7/8 mice. Understanding molecular effects of GalCer on mouse brain gene expression, paves the way for personalized strategies for treating this debilitating disease in humans.

Exogenous Flupirtine as Potential Treatment for CLN3 Disease.

CLN3 disease is a fatal neurodegenerative disorder affecting children. Hallmarks include brain atrophy, accelerated neuronal apoptosis, and ceramide elevation. Treatment regimens are supportive, highlighting the importance of novel, disease-modifying drugs. Flupirtine and its new allyl carbamate derivative (compound 6) confer neuroprotective effects in CLN3-deficient cells. This study lays the groundwork for investigating beneficial effects in Cln3Δex7/8 mice. WT/Cln3Δex7/8 mice received flupirtine/compound 6/vehicle for 14 weeks. Short-term effect of flupirtine or compound 6 was tested using a battery of behavioral testing. For flupirtine, gene expression profiles, astrogliosis, and neuronal cell counts were determined. Flupirtine improved neurobehavioral parameters in open field, pole climbing, and Morris water maze tests in Cln3Δex7/8 mice. Several anti-apoptotic markers and ceramide synthesis/degradation enzymes expression was dysregulated in Cln3Δex7/8 mice. Flupirtine reduced astrogliosis in hippocampus and motor cortex of male and female Cln3Δex7/8 mice. Flupirtine increased neuronal cell counts in male mice. The newly synthesized compound 6 showed promising results in open field and pole climbing. In conclusion, flupirtine improved behavioral, neuropathological and biochemical parameters in Cln3Δex7/8 mice, paving the way for potential therapies for CLN3 disease.

Effect of dietary monosodium glutamate on trans fat-induced nonalcoholic fatty liver disease.

The effects of dietary monosodium glutamate (MSG) on trans-fatty acid (TFA)-induced nonalcoholic fatty liver disease (NAFLD) are addressed in an animal model. We used Affymetrix microarray analysis to investigate hepatic gene expression and the contribution of visceral white adipose tissue (WAT) to diet-induced NAFLD. Trans-fat feeding increased serum leptin, FFA, HDL-cholesterol (HDL-C), and total cholesterol (T-CHOL) levels, while robustly elevating the expression of genes involved in hepatic lipogenesis, including the transcription factor sterol-regulatory element binding protein 1c. Histological examination revealed hepatic macrosteatosis in TFA-fed animals. Conversely, dietary MSG at doses similar to human average daily intake caused hepatic microsteatosis and the expression of beta-oxidative genes. Serum triglyceride, FFA, and insulin levels were elevated in MSG-treated animals. The abdominal cavities of TFA- or MSG-treated animals had increased WAT deposition compared with controls. Microarray analysis of WAT gene expression revealed increased lipid biosynthetic gene expression, together with a 50% decrease in the key transcription factor Ppargc1a. A combination of TFA+MSG resulted in the highest levels of serum HDL-C, T-CHOL, and leptin. Microarray analysis of TFA+MSG-treated livers showed elevated expression of markers of hepatic inflammation, lipid storage, cell damage, and cell cycle impairment. TFA+MSG mice also had a high degree of WAT deposition and lipogenic gene expression. Levels of Ppargc1a were further reduced to 25% by TFA+MSG treatment. MSG exacerbates TFA-induced NAFLD.

Developmental Comparison of Ceramide in Wild-Type and Cln3 Δex7/8 Mouse Brains and Sera.

CLN3 disease is a neurodevelopmental disease leading to early visual failure, motor decline, and death. CLN3 pathogenesis has been linked to dysregulation of ceramide, a key intracellular messenger impacting various biological functions. Ceramide is upregulated in brains of CLN3 patients and activates apoptosis. Ceramide levels over the lifespan of WT and Cln3 Δex7/8 mice were measured using the DGK assay. Ceramide subspecies were determined by LC-MS. Ceramide synthesis enzymes and pre- and post-synaptic mRNA expression was measured in Cln3 Δex7/8 and normal mouse brains. Neuronal cell death was established by PARP cleavage and Caspases 3/6/9 and cytochrome C mRNA expression in Cln3 Δex7/8 and normal mouse brains. In WT mouse, a ceramide peak was noted at 3 weeks of age. The absence of this peak in Cln3 Δex7/8 mice might be related to early disease pathogenesis. Increase of ceramide in Cln3 Δex7/8 mouse brain at 24 weeks of age precedes neuronal apoptosis. The correlation between serum and brain ceramide in WT mice, and dysregulation of ceramide in serum and brain of Cln3 Δex7/8 mice, and the significant increase in ceramide in Cln3 Δex7/8 mouse brains and sera argue for use of easily accessible serum ceramide levels to track response to novel therapies in human CLN3 disease.

Zidovudine and interferon-alpha treatment induces a high response rate and reduces HTLV-1 proviral load and VEGF plasma levels in patients with adult T-cell leukemia from North East Iran.

Human T-cell lymphotropic virus type I (HTLV-I) associated adult T-cell leukemia/lymphoma (ATLL) is endemic in southern Japan, the Caribbean, intertropical Africa, and Brazil. Recently north east Iran, particularly the region of Mashhad, has been recognized as a new endemic region. ATLL is an aggressive T-cell lymphoproliferative disorder. Patients with ATLL have high plasma levels of VEGF that induce angiogenesis. Prognosis of ATLL remains poor because of immunosuppression and intrinsic resistance to chemotherapy. Important advances in the treatment of ATLL were reported with the combination of zidovudine (AZT) and interferon-alpha. We investigated the effect of AZT/IFN treatment on vascular endothelium growth factor (VEGF) plasma levels and HTLV-I proviral load in ATLL patients from the region of Mashhad. We confirmed that AZT/IFN treatment induces a high response rate and prolonged survival with minimal side effects. We also confirmed that VEGF plasma levels and HTLV-I proviral load are higher in ATLL patients than in asymptomatic carriers. We finally showed that AZT/IFN treatment reduced both HTLV-I proviral load and importantly VEGF plasma levels, suggesting a potential antiangiogenic effect of this therapy. These results provide further evidence for the efficacy and the mechanism of action of AZT/IFN therapy for ATLL in a developing country.

Candidate Genes for Inherited Autism Susceptibility in the Lebanese Population.

Autism spectrum disorder (ASD) is characterized by ritualistic-repetitive behaviors and impaired verbal/non-verbal communication. Many ASD susceptibility genes implicated in neuronal pathways/brain development have been identified. The Lebanese population is ideal for uncovering recessive genes because of shared ancestry and a high rate of consanguineous marriages. Aims here are to analyze for published ASD genes and uncover novel inherited ASD susceptibility genes specific to the Lebanese. We recruited 36 ASD families (ASD: 37, unaffected parents: 36, unaffected siblings: 33) and 100 unaffected Lebanese controls. Cytogenetics 2.7 M Microarrays/CytoScan™ HD arrays allowed mapping of homozygous regions of the genome. The CNTNAP2 gene was screened by Sanger sequencing. Homozygosity mapping uncovered DPP4, TRHR, and MLF1 as novel candidate susceptibility genes for ASD in the Lebanese. Sequencing of hot spot exons in CNTNAP2 led to discovery of a 5 bp insertion in 23/37 ASD patients. This mutation was present in unaffected family members and unaffected Lebanese controls. Although a slight increase in number was observed in ASD patients and family members compared to controls, there were no significant differences in allele frequencies between affecteds and controls (C/TTCTG: γ2 value = 0.014; p = 0.904). The CNTNAP2 polymorphism identified in this population, hence, is not linked to the ASD phenotype.

RYR2, PTDSS1 and AREG genes are implicated in a Lebanese population-based study of copy number variation in autism.

Autism Spectrum Disorders (ASDs) are a group of neurodevelopmental disorders characterized by ritualistic-repetitive behaviors and impaired verbal and non-verbal communication. Objectives were to determine the contribution of genetic variation to ASDs in the Lebanese. Affymetrix Cytogenetics Whole-Genome 2.7 M and CytoScan(™) HD Arrays were used to detect CNVs in 41 Lebanese autistic children and 35 non-autistic, developmentally delayed and intellectually disabled patients. 33 normal participants were used as controls. 16 de novo CNVs and 57 inherited CNVs, including recognized pathogenic 16p11.2 duplications and 2p16.3 deletions were identified. A duplication at 1q43 classified as likely pathogenic encompasses RYR2 as a potential ASD candidate gene. This previously identified CNV has been classified as both pathogenic, and, of uncertain significance. A duplication of unknown significance at 10q11.22, proposed as a modulator for phenotypic disease expression in Rett syndrome, was also identified. The novel potential autism susceptibility genes PTDSS1 and AREG were uncovered and warrant further genetic and functional analyses. Previously described and novel genetic targets in ASD were identified in Lebanese families with autism. These findings may lead to improved diagnosis of ASDs and informed genetic counseling, and may also lead to untapped therapeutic targets applicable to Lebanese and non-Lebanese patients.

Gene expression profiling of breast cancer in Lebanese women.

Breast cancer is commonest cancer in women worldwide. Elucidation of underlying biology and molecular pathways is necessary for improving therapeutic options and clinical outcomes. Molecular alterations in breast cancer are complex and involve cross-talk between multiple signaling pathways. The aim of this study is to extract a unique mRNA fingerprint of breast cancer in Lebanese women using microarray technologies. Gene-expression profiles of 94 fresh breast tissue samples (84 cancerous/10 non-tumor adjacent samples) were analyzed using GeneChip Human Genome U133 Plus 2.0 arrays. Quantitative real-time PCR was employed to validate candidate genes. Differentially expressed genes between breast cancer and non-tumor tissues were screened. Significant differences in gene expression were established for COL11A1/COL10A1/MMP1/COL6A6/DLK1/S100P/CXCL11/SOX11/LEP/ADIPOQ/OXTR/FOSL1/ACSBG1 and C21orf37. Pathways/diseases representing these genes were retrieved and linked using PANTHER®/Pathway Studio®. Many of the deregulated genes are associated with extracellular matrix, inflammation, angiogenesis, metastasis, differentiation, cell proliferation and tumorigenesis. Characteristics of breast cancers in Lebanese were compared to those of women from Western populations to explain why breast cancer is more aggressive and presents a decade earlier in Lebanese victims. Delineating molecular mechanisms of breast cancer in Lebanese women led to key genes which could serve as potential biomarkers and/or novel drug targets for breast cancer.

Association between CLN3 (Neuronal Ceroid Lipofuscinosis, CLN3 Type) Gene Expression and Clinical Characteristics of Breast Cancer Patients.

Breast cancer is the most common cancer in women worldwide. Elucidation of underlying biology and molecular pathways is necessary for improving therapeutic options and clinical outcomes. CLN3 protein (CLN3p), deficient in neurodegenerative CLN3 disease is anti-apoptotic, and defects in the CLN3 gene cause accelerated apoptosis of neurons in CLN3 disease and up-regulation of ceramide. Dysregulated apoptotic pathways are often implicated in the development of the oncogenic phenotype. Predictably, CLN3 mRNA expression and CLN3 protein were up-regulated in a number of human and murine breast cancer-cell lines. Here, we determine CLN3 expression in non-tumor vs. tumor samples from fresh and formalin-fixed/paraffin-embedded (FFPE) breast tissue and analyze the association between CLN3 overexpression and different clinicopathological characteristics of breast cancer patients. Additionally, gene expression of 28 enzymes involved in sphingolipid metabolism was determined. CLN3 mRNA is overexpressed in tumor vs. non-tumor breast tissue from FFPE and fresh samples, as well as in mouse MCF7 breast cancer compared to MCF10A normal cells. Of the clinicopathological characteristics of tumor grade, age, menopause status, estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), only absence of HER2 expression correlated with CLN3 overexpression. Sphingolipid genes for ceramide synthases 2 and 6 (CerS2; CerS6), delta(4)-desaturase sphingolipid 2 (DEGS2), and acidic sphingomyelinase (SMPD1) displayed higher expression levels in breast cancer vs. control tissue, whereas ceramide galactosyltransferase (UGT8) was underexpressed in breast cancer samples. CLN3 may be a novel molecular target for cancer drug discovery with the goal of modulation of ceramide pathways.

Prediabetic changes in gene expression induced by aspartame and monosodium glutamate in Trans fat-fed C57Bl/6 J mice.

BACKGROUND: The human diet has altered markedly during the past four decades, with the introduction of Trans hydrogenated fat, which extended the shelf-life of dietary oils and promoted a dramatic increase in elaidic acid (Trans-18.1) consumption. Food additives such as monosodium glutamate (MSG) and aspartame (ASP) were introduced to increase food palatability and reduce caloric intake. Nutrigenomics studies in small-animal models are an established platform for analyzing the interactions between various macro- and micronutrients. We therefore investigated the effects of changes in hepatic and adipose tissue gene expression induced by the food additives ASP, MSG or a combination of both additives in C57Bl/6 J mice fed a Trans fat-enriched diet. METHODS: Hepatic and adipose tissue gene expression profiles, together with body characteristics, glucose parameters, serum hormone and lipid profiles were examined in C57Bl/6 J mice consuming one of the following four dietary regimens, commencing in utero via the mother's diet: [A] Trans fat (TFA) diet; [B] MSG + TFA diet; [C] ASP + TFA diet; [D] ASP + MSG + TFA diet. RESULTS: Whilst dietary MSG significantly increased hepatic triglyceride and serum leptin levels in TFA-fed mice, the combination of ASP + MSG promoted the highest increase in visceral adipose tissue deposition, serum free fatty acids, fasting blood glucose, HOMA-IR, total cholesterol and TNFα levels. Microarray analysis of significant differentially expressed genes (DEGs) showed a reduction in hepatic and adipose tissue PPARGC1a expression concomitant with changes in PPARGC1a-related functional networks including PPARα, δ and γ. We identified 73 DEGs common to both adipose and liver which were upregulated by ASP + MSG in Trans fat-fed mice; and an additional 51 common DEGs which were downregulated. CONCLUSION: The combination of ASP and MSG may significantly alter adiposity, glucose homeostasis, hepatic and adipose tissue gene expression in TFA-fed C57Bl/6 J mice.

Nutrigenomics of hepatic steatosis in a feline model: effect of monosodium glutamate, fructose, and Trans-fat feeding.

Nonalcoholic fatty liver disease begins with a relatively benign hepatic steatosis, often associated with increased adiposity, but may progress to a more severe nonalcoholic steatohepatitis with inflammation. A subset of these patients develops progressive fibrosis and ultimately cirrhosis. Various dietary components have been shown to contribute to the development of liver disease, including fat, sugars, and neonatal treatment with high doses of monosodium glutamate (MSG). However, rodent models of progressive disease have been disappointing, and alternative animal models of diet-induced liver disease would be desirable, particularly if they contribute to our knowledge of changes in gene expression as a result of dietary manipulation. The domestic cat has previously been shown to be an appropriate model for examining metabolic changes-associated human diseases such as diabetes. Our aim was therefore to compare changes in hepatic gene expression induced by dietary MSG, with that of a diet containing Trans-fat and high fructose corn syrup (HFCS), using a feline model. MSG treatment increased adiposity and promoted hepatic steatosis compared to control (P < 0.05). Exposure to Trans-fat and HFCS promoted hepatic fibrosis and markers of liver dysfunction. Affymetrix microarray analysis of hepatic gene expression showed that dietary MSG promoted the expression of genes involved in cholesterol and steroid metabolism. Conversely, Trans-fat and HFCS feeding promoted the expression of genes involved in lipolysis, glycolysis, liver damage/regeneration, and fibrosis. Our feline model examining gene-diet interactions (nutrigenomics) demonstrates how dietary MSG, Trans-fat, and HFCS may contribute to the development of hepatic steatosis.

Interactive effects of neonatal exposure to monosodium glutamate and aspartame on glucose homeostasis.

BACKGROUND: Recent evidence suggests that the effects of certain food additives may be synergistic or additive. Aspartame (ASP) and Monosodium Glutamate (MSG) are ubiquitous food additives with a common moiety: both contain acidic amino acids which can act as neurotransmitters, interacting with NMDA receptors concentrated in areas of the Central Nervous System regulating energy expenditure and conservation. MSG has been shown to promote a neuroendocrine dysfunction when large quantities are administered to mammals during the neonatal period. ASP is a low-calorie dipeptide sweetener found in a wide variety of diet beverages and foods. However, recent reports suggest that ASP may promote weight gain and hyperglycemia in a zebrafish nutritional model. METHODS: We investigated the effects of ASP, MSG or a combination of both on glucose and insulin homeostasis, weight change and adiposity, in C57BL/6 J mice chronically exposed to these food additives commencing in-utero, compared to an additive-free diet. Pearson correlation analysis was used to investigate the associations between body characteristics and variables in glucose and insulin homeostasis. RESULTS: ASP alone (50 mg/Kgbw/day) caused an increase in fasting blood glucose of 1.6-fold, together with reduced insulin sensitivity during an Insulin Tolerance Test (ITT) P < 0.05. Conversely MSG alone decreased blood triglyceride and total cholesterol (T-CHOL) levels. The combination of MSG (120 mg/Kgbw/day) and ASP elevated body weight, and caused a further increase in fasting blood glucose of 2.3-fold compared to Controls (prediabetic levels); together with evidence of insulin resistance during the ITT (P < 0.05). T-CHOL levels were reduced in both ASP-containing diets in both genders. Further analysis showed a strong correlation between body weight at 6 weeks, and body weight and fasting blood glucose levels at 17 weeks, suggesting that early body weight may be a predictor of glucose homeostasis in later life. CONCLUSIONS: Aspartame exposure may promote hyperglycemia and insulin intolerance. MSG may interact with aspartame to further impair glucose homeostasis. This is the first study to ascertain the hyperglycemic effects of chronic exposure to a combination of these commonly consumed food additives; however these observations are limited to a C57BL/6 J mouse model. Caution should be applied in extrapolating these findings to other species.

Gender dimorphism in aspartame-induced impairment of spatial cognition and insulin sensitivity.

Previous studies have linked aspartame consumption to impaired retention of learned behavior in rodents. Prenatal exposure to aspartame has also been shown to impair odor-associative learning in guinea pigs; and recently, aspartame-fed hyperlipidemic zebrafish exhibited weight gain, hyperglycemia and acute swimming defects. We therefore investigated the effects of chronic lifetime exposure to aspartame, commencing in utero, on changes in blood glucose parameters, spatial learning and memory in C57BL/6J mice. Morris Water Maze (MWM) testing was used to assess learning and memory, and a random-fed insulin tolerance test was performed to assess glucose homeostasis. Pearson correlation analysis was used to investigate the associations between body characteristics and MWM performance outcome variables. At 17 weeks of age, male aspartame-fed mice exhibited weight gain, elevated fasting glucose levels and decreased insulin sensitivity compared to controls (P<0.05). Females were less affected, but had significantly raised fasting glucose levels. During spatial learning trials in the MWM (acquisition training), the escape latencies of male aspartame-fed mice were consistently higher than controls, indicative of learning impairment. Thigmotactic behavior and time spent floating directionless was increased in aspartame mice, who also spent less time searching in the target quadrant of the maze (P<0.05). Spatial learning of female aspartame-fed mice was not significantly different from controls. Reference memory during a probe test was affected in both genders, with the aspartame-fed mice spending significantly less time searching for the former location of the platform. Interestingly, the extent of visceral fat deposition correlated positively with non-spatial search strategies such as floating and thigmotaxis, and negatively with time spent in the target quadrant and swimming across the location of the escape platform. These data suggest that lifetime exposure to aspartame, commencing in utero, may affect spatial cognition and glucose homeostasis in C57BL/6J mice, particularly in males.