Differential glycosylation of tissue non-specific alkaline phosphatase in mesenchymal stromal cells differentiated into either an osteoblastic or adipocytic phenotype.

It has long been known that tissue non-specific alkaline phosphatase (TNAP) is essential for the correct formation of bone, as altered expression or function of this enzyme results in hypophosphatasia, a disease characterised by compromised bone structure, density and strength. However, recent evidence strongly suggests that the enzyme also has a role in lipid accrual and adipogenesis, a function that seems far removed from bone formation. Given that mesenchymal stromal cells (MSCs) are progenitors of both osteoblasts and adipocytes, the question arises of how TNAP is regulated to potentially have a different function when MSCs undergo either osteogenesis or adipogenesis. As the primary protein sequence is unchanged for the enzyme during both types of differentiation, any differences in function must be attributed to post-translational modification and/or localisation. We therefore examined the location of TNAP in bone- or adipose-derived MSCs differentiated into an adipocytic phenotype and compared the glycosylation state of the enzyme in MSCs differentiated into either osteoblasts or adipocytes. TNAP was found to co-locate with perilipin around lipid droplets in MSCs from bone, subcutaneous- and visceral adipose tissue during adipocytic differentiation. Treatment of TNAP with wheat germ lectin followed by electrophoresis showed minor differences in glycosylation between the phosphatase isolated from cells from these tissues, whereas electrophoresis after neuraminidase digestion highlighted differential glycosylation between cell types and during adipogenesis and osteoblastogenesis. This infers that post-translational modification of TNAP is altered during differentiation and is dependent on the eventual phenotype of the cells.

The association of vitamin D binding protein levels and genotypes with type 1 diabetes in the black South African population.

BACKGROUND: Vitamin D deficiency and the vitamin D pathway have previously been associated with type 1 diabetes (T1D). The majority of vitamin D is transported through the blood bound to the vitamin D binding protein (VDBP). Two polymorphisms in the VDBP gene (rs4588 and rs7041) result in different VDBP variants and have been associated with T1D, however the results are not consistent. The association of VDBP levels and its polymorphisms with T1D have not been investigated in the black South African population. Therefore, this study aimed to determine whether rs4588, rs7041 or serum VDBP levels were associated with T1D in this population. METHODS: Participants with type 1 diabetes and controls were recruited from the greater Johannesburg area, South Africa. Participants were genotyped for rs4588 and rs7041 using PCR-RFLP and serum VDBP levels were determined by ELISA. RESULTS: There was no difference in VDBP allelic or genotypic frequencies between participants with T1D and controls (rs4588 C allele frequency 0.92 vs. 0.94; p = 0.390 and rs7041 T allele frequency 0.95 vs. 0.95; p = 0.890). In univariate analysis, the rs4588 CC genotype was associated with increased serum VDBP levels, however, this association was lost with multivariate analysis. The VDBP genotypes were not associated with any other study variables. In logistic regression analysis, higher VBDP levels were associated with T1D (OR: (95% CI): 6.58 (1.45-29.9); p = 0.015), and within a linear regression analysis, T1D disease status was found to be associated with 0.044 mg/ml higher VDBP levels (p = 0.028). CONCLUSIONS: These data suggest that serum VDBP levels are positively associated with the presence of T1D in the African population. Whether VDBP lies in the causal pathway or its elevation is an effect of T1D is uncertain and requires further investigation.

A new perspective on the function of Tissue Non-Specific Alkaline Phosphatase: from bone mineralization to intra-cellular lipid accumulation.

Tissue-nonspecific alkaline phosphatase (TNAP) is one of four isozymes, which include germ cell, placental and intestinal alkaline phosphatases. The TNAP isozyme has 3 isoforms (liver, bone and kidney) which differ by tissue expression and glycosylation pattern. Despite a long history of investigation, the exact function of TNAP in many tissues is largely unknown. Only the bone isoform has been well characterised during mineralization where the enzyme hydrolyses pyrophosphate to inorganic phosphate, which combines with calcium to form hydroxyapatite crystals deposited as new bone. The inorganic phosphate also increases gene expression of proteins that support tissue mineralization. Recent studies have shown that TNAP is expressed in preadipocytes from several species, and that inhibition of TNAP activity causes attenuation of intracellular lipid accumulation in these and other lipid-storing cells. The mechanism by which TNAP stimulates lipid accumulation is not known; however, proteins that are important for controlling phosphate levels in bone are also expressed in adipocytes. This review examines the evidence that inorganic phosphate generated by TNAP promotes transcription that enhances the expression of the regulators of lipid storage and consequently, that TNAP has a major function of lipid metabolism.

Zinc transporter 8 (ZnT8) autoantibody prevalence in black South African participants with type 1 diabetes.

BACKGROUND: Autoantibodies to ß-cell specific antigens are markers of type 1 diabetes. The most recently identified autoantibodies are targeted to the zinc transporter 8 (ZnT8) protein located in the membrane of ß-cell insulin secretory granules. The prevalence of ZnT8 autoantibodies in newly diagnosed participants with type 1 diabetes has been found to range from 33 to 80 %. Due to the lack of data on the immunological aetiology of type 1 diabetes in African populations, this study aimed to determine the prevalence of ZnT8 autoantibodies in black South Africans with type 1 diabetes and whether ZnT8 autoantibody positivity was associated with age at diagnosis and disease duration. METHODS: Participants with type 1 diabetes and controls were recruited from the greater Johannesburg area, South Africa. Positivity for ZnT8, GAD65 and IA2 autoantibodies was determined by ELISA. RESULTS: Participants with type 1 diabetes (n = 183) and controls (n = 49) were matched for age (29.1 ± 9.53 vs. 27.3 ± 7.29, respectively; p = 0.248). The mean age at diagnosis for participants with type 1 diabetes was 20.8 ± 8.46 years. The prevalence of ZnT8 autoantibody positivity was 17.5 % (32 of 183) in participants with type 1 diabetes with a median disease duration of 7.00 [2.00; 11.0] years. ZnT8 autoantibody prevalence in newly diagnosed participants (< 1 year duration) was 27.3 % (6 of 22). Logistic regression analysis found an association between ZnT8 autoantibody positivity and shorter disease duration (OR: 0.9 (0.81-1.00); p = 0.042). In addition, ZnT8 autoantibody positivity was significantly associated with an increased chance of being GAD65 (OR: 3.37 (1.10-10.3)) and IA2 (OR: 8.63 (2.82-26.4)) autoantibody positive. Multiple regression analysis found no association between ZnT8 autoantibody positivity and age at diagnosis. However, the presence of ≥ 2 autoantibodies was associated with a younger age at diagnosis of type 1 diabetes when compared to participants with ≤ 1 autoantibody (B = -5.270; p = 0.002). CONCLUSIONS: The presence of ZnT8 autoantibodies was not related to a younger age at diagnosis in black South African patients with type 1 diabetes. However, the greater the numbers of autoantibodies present in an individual the earlier the age at diagnosis. ZnT8 autoantibodies decline with disease duration in the black South African population.

A Polymorphism in the Gene Encoding the Insulin Receptor Binding Protein ENPP-1 Is Associated with Decreased Glomerular Filtration Rate in an Under-Investigated Indigenous African Population.

INTRODUCTION: The C allele of the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP-1) rs1044498 polymorphism has previously been associated with increased binding of ENPP-1 to the insulin receptor (IR), resulting in decreased IR signalling and enhanced insulin resistance. It has also been associated with reduced kidney function in participants with diabetes of predominantly European and Asian descent. The association of this polymorphism with kidney disease in healthy Black South African participants has yet to be ascertained. OBJECTIVE: This study, therefore, aimed to determine whether the K121Q polymorphism is associated with estimated glomerular filtration rate (eGFR) in a Black South African cohort. METHODS: Black South African participants (n = 348) from an existing cohort with known eGFR levels were genotyped for the K121Q polymorphism using PCR-RFLP and assessed for any statistical association between genotype and kidney function. RESULTS: Individuals with the A allele had significantly lower eGFR levels than individuals with the CC genotype (86.52 ± 18.95 vs. 93.29 ± 23.55 mL/min; p = 0.022). The association of the A allele with lower eGFR levels remained after controlling for sex, blood pressure, insulin resistance, age, smoking, thyroid-stimulating hormone, insulin-like growth factor-1, and BMI (R2 = 0.030, p < 0.001). CONCLUSION: The rs1044498 A allele was significantly associated with lower eGFR levels in a cohort of apparently healthy Black South Africans, through an unknown mechanism that was independent of insulin resistance. It is possible that the rs1044498 polymorphism affects kidney function by altering the role of ENPP-1 in endothelial wound healing, podocyte signalling, or oxidative stress. Thus, the presence of this polymorphism may predispose individuals to a greater risk of CKD even in the absence of diabetes.

Tissue non-specific alkaline phosphatase mediates the accumulation of cholesterol esters in the murine Y1 adrenal cortex cell line.

BACKGROUND: Cholesterol esters (CEs) accumulate in the cells of the adrenal cortex and are used for the synthesis of steroid hormones. The full molecular pathways involved in mediating the accumulation of CEs within the adrenal cortex are yet to be elucidated. Tissue non-specific alkaline phosphatase (TNAP) is needed for intracellular lipid accumulation of triglycerides in adipocytes and is also expressed in the cortical cells of the adrenal gland. Therefore we aimed to determine if TNAP is needed for the accumulation of CEs within the murine Y1 adrenal cortex cell line. METHODS: Y1 cells were induced to accumulate lipids. Lipid accumulation and TNAP activity and expression were determined throughout intracellular lipid accumulation. The location of TNAP within the cell was determined through immunohistochemical analysis. Lipid accumulation in the cells was associated with a rise in TNAP activity and TNAP was localised to lipid droplets within the Y1 cells. Inhibition of TNAP with a specific inhibitor (levamisole) resulted in the cessation of CE accumulation. DISCUSSION AND CONCLUSIONS: These data demonstrate that TNAP plays a role in the control of lipid accumulation in this adrenal cortex cell line. Therefore, in both triglyceride and CE storing cell types TNAP would seem to be essential for intra-cellular lipid storage.

The Use of 3T3-L1 Murine Preadipocytes as a Model of Adipogenesis.

The 3T3-L1 murine preadipocyte cell line is a commonly used tool for analysis of the subcellular pathways involved in preadipocytic cell differentiation (a process also commonly known as adipogenesis). The major characteristic of adipogenesis is the intracellular accumulation of membrane-bound lipid droplets. Here, we describe methods used for the culture and transformation of these preadipocytes into mature adipocytes and quantification of intracellular lipid accumulation using the lipid specific dye, Oil Red O.

Suppression of Caco-2 and HEK-293 cell proliferation by Kigelia africana, Mimusops zeyheri and Ximenia caffra seed oils.

Animal-derived oils and purified fatty acids, but not indigenous fruit-tree-derived seed oils, have been used to study cell growth and differentiation. In this study, we determined the effects of the Kigelia africana, the Mimusops zeyheri and the Ximenia caffra seed-oil on cell proliferation in culture. Human colon adenocarcinoma (Caco-2) and human embryonic kidney (HEK-293) cells were maintained and treated with various concentrations (0, 20, 40, 80, 100 and 120 mg/l) of K. africana, M. zehyeri and X. caffra seed oil. The trypan blue dye exclusion method was used to determine cell growth 48-hours after oil treatment. All three tree seed oils suppressed both Caco-2 and HEK-293 cell growth in a dose-dependent manner. Importantly, the tree seed oils did not cause increased cell death as the number of dead cells remained unchanged under control and oil-treated conditions. K. africana oil significantly suppressed Caco-2 cell growth compared to HEK-293 cell growth at all oil concentrations, whereas M. zeyheri and X. caffra seed oils significantly suppressed HEK-293 and Caco-2 cell growth, only at a concentration of 80 mg/l. The suppression of Caco-2 and HEK-293 cell proliferation by K. africana, M. zeyheri and X. caffra seed oils suggest a potential antiproliferative effect of these tree seed oils on the two cell lines.

Limited neutralizing antibody specificities drive neutralization escape in early HIV-1 subtype C infection.

We previously showed that HIV-1 subtype C viruses elicit potent but highly type-specific neutralizing antibodies (nAb) within the first year of infection. In order to determine the specificity and evolution of these autologous nAbs, we examined neutralization escape in four individuals whose responses against the earliest envelope differed in magnitude and potency. Neutralization escape occurred in all participants, with later viruses showing decreased sensitivity to contemporaneous sera, although they retained sensitivity to new nAb responses. Early nAb responses were very restricted, occurring sequentially and targeting only two regions of the envelope. In V1V2, limited amino acid changes often involving indels or glycans, mediated partial or complete escape, with nAbs targeting the V1V2 region directly in 2 cases. The alpha-2 helix of C3 was also a nAb target, with neutralization escape associated with changes to positively charged residues. In one individual, relatively high titers of anti-C3 nAbs were required to drive genetic escape, taking up to 7 weeks for the resistant variant to predominate. Thereafter titers waned but were still measurable. Development of this single anti-C3 nAb specificity was associated with a 7-fold drop in HIV-1 viral load and a 4-fold rebound as the escape mutation emerged. Overall, our data suggest the development of a very limited number of neutralizing antibody specificities during the early stages of HIV-1 subtype C infection, with temporal fluctuations in specificities as escape occurs. While the mechanism of neutralization escape appears to vary between individuals, the involvement of limited regions suggests there might be common vulnerabilities in the HIV-1 subtype C transmitted envelope.

Silencing of HIV-1 subtype C primary isolates by expressed small hairpin RNAs targeted to gag.

Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.