Gursev S. Dhaunsi

Background. The pathogenesis of classical galactosemia, a rare metabolic disorder associated with developmental complications in neonates and children due to inherited deficiency of galactose-1-phosphate (Gal-1-P) uridylyltransferase (GALT), is known to be mediated by elevated Gal-1-P levels and involves a cascade of cytokines, reactive oxygen species (ROS) and growth factors. Objectives. To examine ex vivo the effect of Gal-1-P on the mitogenic activity of different growth factors, particularly insulin-like growth factor-1 (IGF-1), known to regulate growth and development from the fetal stage to adulthood. Material and methods. Fibroblasts derived from the foreskin of 3-8-day-old healthy neonates were cultured for 1-14 days with 0-20 mM galactose or 0-10 mM Gal-1-P and then stimulated with 5% fetal bovine serum (FBS) or 50 ng/mL of platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) or IGF-1 for 24 h. DNA synthesis was measured and protein expression of PDGFR, FGFR and IGF-1R was assessed with western blotting. Results. Supra-physiological concentrations of galactose significantly decreased FBS- and IGF-1-induced BrdU incorporation. The presence of Gal-1-P (5-10 mM) in culture medium for 7-14 days significantly (p < 0.01) decreased IGF-1-, PDGF- and FBS-stimulated DNA synthesis. While treatment with Gal-1-P selectively and significantly (p <0.01) reduced the protein expression of IGF-1 receptor, galactose treatment did not have any marked effect on examined growth factor receptors. Conclusions. This study demonstrates that Gal-1-P impairs IGF-1 activity through IGF-1-receptor impairment, thereby providing a new insight into the molecular mechanisms of galactosemia pathogenesis.

The incidence of type 1 diabetes mellitus (T1DM) in Kuwait is amongst the highest in the world. Vitamin D is considered to be involved in immune modulation and its deficiency contribute to autoimmune destruction of insulin producing beta cells in T1DM patients. Vitamin D has been shown to exert its effects via a nuclear vitamin D receptor (VDR) and therefore, VDR gene may be considered a candidate for T1DM susceptibility. The genotypes of four VDR gene polymorphisms were determined in 253 Kuwaiti Arab T1DM patients and 214 healthy controls by PCR-RFLP analysis. Serum concentrations of three autoantibodies i.e. ICA (Islet cell autoantibody), GADA (Glutamic acid decarboxylase) and INS (Insulin autoantibody) were determined by radio-immunoassays. Statistically significant differences were detected between the genotypes of two VDR gene polymorphisms (FokI, C > T, rs10735810 and TaqI, C > T, rs731236) between T1DM patients and controls (P < 0.0001). In both, the frequency of variant alleles was considerably high in T1DM than in the controls. In contrast, the VDR gene ApaI (G > T, rs7975232) and BsmI (A > G, rs1544410) polymorphisms did not show association with T1DM. The homozygous variant genotypes of FokI, ApaI and TaqI polymorphisms show significant differences between various age-of-onset subgroups while no such association was detected in the case of BsmI polymorphism. Significant differences were also noted between heterozygous genotypes of all four polymorphisms especially between 4-6y and > 6y age-of-onset subgroups of T1DM patients. Three autoantibodies, ICA (Islet cell), GADA (glutamate decarboxylase) and INS (insulin) were positively associated to, varying degrees, with T1DM in Kuwaiti Arabs harboring different VDR gene polymorphism genotypes. Our results demonstrate a significant effect of two VDR gene polymorphisms (FokI and TaqI) and three autoantibodies on genetic susceptibility of T1DM in Kuwaiti Arabs along with other factors.

Molecular mechanisms of the beneficial effects of angiotensin-(1-7), Ang-(1-7), in diabetes-related complications, including erectile dysfunction, remain unclear. We examined the effect of diabetes and/or Ang-(1-7) treatment on vascular reactivity and cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) in corpus cavernosum. Male Wistar rats were grouped as (1) control, (2) diabetic (streptozotocin, STZ, treated), (3) control + Ang-(1-7), and (4) diabetic + Ang-(1-7). Following 3 weeks of Ang-(1-7) treatment subsequent to induction of diabetes, rats were sacrificed. Penile cavernosal tissue was isolated to measure vascular reactivity, PDE gene expression and activity, and levels of p38MAP kinase, nitrites, and cGMP. Carbachol-induced vasorelaxant response after preincubation of corpus cavernosum with PE was significantly attenuated in diabetic rats, and Ang-(1-7) markedly corrected the diabetes-induced impairment. Gene expression and activity of PDE and p38MAP kinase were significantly increased in cavernosal tissue of diabetic rats, and Ang-(1-7) markedly attenuated STZ-induced effects. Ang-(1-7) significantly increased the levels of nitrite and cGMP in cavernosal tissue of control and diabetic rats. Cavernosal tissue of diabetic rats had significantly reduced cGMP levels and Ang-(1-7) markedly prevented the STZ-induced cGMP depletion. This study demonstrates that attenuation of diabetes-induced PDE activity might be one of the key mechanisms in the beneficial effects of Ang-(1-7).

Type 1 diabetes mellitus (T1DM) is highly prevalent in Kuwait with incidence of around 40.1/100,000 individuals. Evidence indicate that vitamin D plays an important role in modulating the immune system and could thus impact the onset and high prevalence of T1DM. We report serum vitamin D levels in Kuwaiti children with T1DM and non-diabetic controls to explore its relationship with prevalence and onset of the disease. This study included 216 Kuwaiti Arab children with T1DM. The diagnosis of T1DM was based on the ISPAD criteria. The control subjects (204 Kuwaitis) were age and gender matched, healthy, non-diabetic, and had no close relative with T1DM. Vitamin D levels were determined in serum using an enzyme immunoassay (EIA) method. The age of onset of T1DM was 6y in 52 % patients. In T1DM patient group, 84 % subjects were found to be deficient in serum vitamin D level compared to 77 % of the controls (p = 0.046). Collectively, the deficient and insufficient vitamin D status was detected in 99 % of the T1DM patients compared to 92 % of the controls (p = 0.027). The mean serum vitamin D levels were found to be significantly different in early onset cases (age <4y) compared to the late onset sub-group (p = 0.001). A significant correlation was found between some elements of socioeconomic status, SES (i.e. parent’s profession and family’s income) and lower vitamin D levels in Kuwaiti T1DM children. There was no significant difference between mean serum vitamin D levels during winter and summer months in the T1DM patients. The proportion of cases with a deficient vitamin D status was significantly high in Kuwaiti T1DM children compared to the controls. The serum vitamin D levels were found to be significantly different in early onset and late onset T1DM patients. Therefore, serum vitamin D status can be considered an important contributor in high prevalence of T1DM in Kuwaiti children.

Phytanic acid (PA) has been implicated in development of cancer and its defective metabolism is known to cause life-threatening conditions, such as Refsum disease, in children. To explore molecular mechanisms of phytanic acid-induced cellular pathology, we investigated its effect on NADPH oxidase (NOX) and epidermal growth factor receptor (EGFR) in rat aortic smooth muscle cells (RASMC). Smooth muscle cells were isolated from rat aortae using enzymic digestion with collagenase and elastase. Cultured RASMC were treated with varying concentrations (0.5-10 μg/ml) of phytanic acid in the presence/absence of fetal bovine serum (FBS) and/or EGFR inhibitor, AG1478. Following treatment with experimental agents, NOX activity was assayed in RASMC cultures by luminescence method. Gene expression of NOX-1 and p47phox was assessed using RT-PCR. NOX-1, p47phox and, total EGFR protein and its phosphorylated form were measured by Western blotting. Treatment of RASMC with supraphysiological concentrations (>2.5 μg/ml) of PA significantly (p < 0.01) increased the NOX activity. PA also significantly increased gene/protein expression of NOX-1 and p47phox whereas p22phox and p67phox remained unaffected. Interestingly, PA (2.5-10 μg/ml) markedly (2–3 folds) increased the total and phosphorylated EGFR. Treatment of cells with EGFR inhibitor, AG1478, significantly blocked the PA-induced enhancement of NOX activity. Our findings that PA transactivates EGFR and induces NOX activity in vascular smooth muscle cells provide new insights into molecular mechanisms of PA’s role in cancer and Refsum disease.

Background: Galactosemia is a severe metabolic disorder known to cause hepatosplenomegaly, jaundice and cataracts in neonates, and many patients develop later complications such as mental retardation, disorders of motor function or speech, and hypergonadotrophic hypogonadism. The pathogenetic mechanisms of classical galactosemia are unclear; however, nitric oxide (NO) has been suggested to play a role. Objectives: Insulin-like growth factor-1 (IGF-1) is important for the growth and development of children, and the aim of this study was to examine the association of NO production with IGF-1 gene expression under galactosemic conditions. Methods: Serum levels of IGF-1 and nitrite were measured in 15 galactosemia patients and 15 age- and gender-matched healthy controls. Fibroblast cultures established from postcircumcision foreskin of 3- to 8-day-old healthy neonates were treated for 72 h with 0-10 mM of galactose or 0-5 mM of galactose-1-phosphate (Gal-1-P) in the presence or absence of NO synthase inhibitor (L-NAME), and inducible NO synthase (iNOS) protein was measured using Western blot analysis. RT-PCR was performed to assess the IGF-1 gene expression. Results: Galactosemia patients were observed to have significantly (p < 0.01) elevated serum nitrites and markedly decreased levels (p < 0.01) of serum IGF-1 as compared to healthy controls. The cotreatment of neonate skin fibroblast cultures with galactose and Gal-1-P significantly (p < 0.01) increased cellular levels of NO and iNOS protein expression, and decreased (p < 0.01) IGF-1 mRNA levels. Treatment with L-NAME, a NOS inhibitor, significantly (p < 0.05) alleviated a galactose/Gal-1-P-induced decrease in IGF-1 mRNA levels. Conclusion: These results suggest that NO mediates the downregulation of IGF-1 by Gal-1-P/galactose, thereby providing a new molecular mechanism and possible therapeutic insight for galactosemia-related complications.

BACKGROUND: Congenital disorders of glycosylation (CDG) patients share a basic feature of protein hypoglycosylation. Activity of growth factors and their receptors, glycoproteins playing a pivotal role during child development, remains unexplored in CDG patients. METHODS: Peripheral blood lymphocytes (PBL) isolated from 9 CDG patients and 12 healthy controls were cultured in the presence of fetal bovine serum (FBS), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1), and BrdU incorporation was measured. Levels of plasma IGF-1 and PBL IGF-1 receptor (IGF-1R) and its glycosylation were detected using immunoassay and western blot. RESULTS: CDG patients showed significantly less (P < 0.01) serum-induced 5'-Bromo-2'-deoxyuridine (BrdU) incorporation in PBL than in controls. PDGF-/FGF-stimulated BrdU incorporation showed no difference in patients and controls, whereas IGF-1-induced DNA synthesis was significantly (P < 0.01) less in patients. Plasma IGF-1 levels and PBL IGF-1 receptor protein were significantly (P < 0.01) reduced in patients as compared to controls. IGF-1 receptor in PBL of all CDG patients had significantly (P < 0.01) reduced carbohydrate content when compared with control. CONCLUSIONS: These results show selective impairment of IGF-1-induced DNA synthesis in lymphocytes of CDG patients through decreased gene expression and hypoglycosylation of the IGF-1 receptor.

Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

Objectives: Insulin-like growth factor-1 (IGF-1) regulates several biological functions, and low plasma levels of IGF-1 are known to contribute towards the pathogenesis of rheumatoid arthritis (RA). In view of the biological significance of IGF-1, we investigated the association of RA with the polymorphism of a 192-bp allele which is cytosine–adenosine repeat located 1 kb upstream from the IGF-1 gene transcription site and is known to regulate serum IGF-1 levels. Methods: Blood samples were collected from 52 healthy controls (HC) and 68 RA patients to measure the levels of IGF-1 and to isolate genomic DNA. Polymorphism of the IGF-1 gene was examined using polymerase chain reaction (PCR). Disease severity, duration, and activity were recorded for all RA patients. Results: We observed that 97% of all the subjects who participated in this study showed the presence of a 192-bp allele of the IGF-1 gene. All healthy controls exhibited the presence of 192-bp wild-type allele. All non-carriers of the 192-bp allele were Arabs and had RA. Gender correlated significantly with allele frequencies as 14% of the male and only 2% of the female RA patients were non-carriers of 192-bp allele. Plasma IGF-1 levels were significantly lower (p < 0.01) in RA patients compared to HC, and all RA patients who were non-carriers of the 192-bp allele had a significantly high disease activity score. No correlation was found between the duration of RA and the presence or absence of this allele. Conclusions: This study suggests a possible association of the IGF-1 gene polymorphism with developing RA, particularly in males as non-carriers of the 192-bp allele.