Neuro- and immunotoxic effects of fumonisin B1 in cells

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other agricultural products. Fusarium species can also be found in moisture-damaged buildings, and therefore there may also be human exposure to Fusarium mycotoxins, including FB1. FB1 affects the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase. It is neuro-, hepato- and nephrotoxic, and it is classified as possibly carcinogenic to humans. This study aimed to clarify the mechanisms behind FB1-induced neuro- and immunotoxicity. Four neural and glial cell lines of human, rat and mouse origin were exposed to graded doses of FB1 and the effects on the production of reactive oxygen species, lipid peroxidation, intracellular glutathione levels, cell viability and apoptosis were investigated. Furthermore, the effects of FB1, alone or together with lipopolysaccharide (LPS), on the mRNA and protein expression levels of different cytokines and chemokines were studied in human dendritic cells (DC). FB1 induced oxidative stress and cell death in all cell lines studied. Generally, the effects were only seen after prolonged exposure at 10 and 100 µM of FB1. Signs of apoptosis were also seen in all four cell lines. The sensitivities of the cell lines used in this study towards FB1 may be classified as human U-118MG glioblastoma > mouse GT1-7 hypothalamic > rat C6 glioblastoma > human SH-SY5Y neuroblastoma cells. When comparing cell lines of human origin, it can be concluded that glial cells seem to be more sensitive towards FB1 toxicity than those of neural origin. After exposure to FB1, significantly increased levels of the cytokine interferon-γ (IFNγ) were detected in human DC. This observation was further confirmed by FB1-induced levels of the chemokine CXCL9, which is known to be regulated by IFNγ. During co-exposure of DC to both LPS and FB1, significant inhibitions of the LPS-induced levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β, and their regulatory chemokines CCL3 and CCL5 were observed. FB1 can thus affect immune responses in DC, and therefore, it is rather likely that it also affects other types of cells participating in the immune defence system. When evaluating the toxicity potential of FB1, it is important to consider the effects on different cell types and cell-cell interactions. The results of this study represent new information, especially about the mechanisms behind FB1-induced oxidative stress, apoptosis and immunotoxicity, as well as the varying sensitivities of different cell types towards FB1.

Effects of dairy proteins and calcium on diet-induced obesity in mice

Diet high in dairy products is inversely associated with body mass index, risk of metabolic syndrome and prevalence of type 2 diabetes in several populations. Also a number of intervention studies support the role of increased dairy intake in the prevention and treatment of obesity. Dairy calcium has been suggested to account for the effect of dairy on body weight, but it has been repeatedly shown that the effect of dairy is superior to the effect of supplemental calcium. Dairy proteins are postulated to either enhance the effect of calcium or have an independent effect on body weight, but studies in the area are scarce. The aim of this study was to evaluate the potential of dairy proteins and calcium in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. The effect of dairy proteins and calcium on the liver and adipose tissue was also investigated in order to characterise the potential mechanisms explaining the reduction of risk for metabolic syndrome and type 2 diabetes. A high-calcium diet (1.8%) in combination with dietary whey protein inhibited body weight and fat gain and accelerated body weight and fat loss in high-fat-fed C57Bl/6J mice during long-term studies of 14 to 21 weeks. α-lactalbumin, one of the major whey proteins, was the most effective whey protein fraction showing significantly accelerated weight and fat loss during energy restriction and reduced the amount of visceral fat gain during ad libitum feeding after weight loss. The microarray data suggest sensitisation of insulin signalling in the adipose tissue as a result of a calcium-rich whey protein diet. Lipidomic analysis revealed that weight loss on whey protein-based high-calcium diet was characterised by significant decreases in diabetogenic diacylglycerols and lipotoxic ceramide species. The calcium supplementation led to a small, but statistically significant decrease in fat absorption independent of the protein source of the diet. This augments, but does not fully explain the effects of the studied diets on body weight. A whey protein-containing high-calcium diet had a protective effect against a high-fat diet-induced decline of β3 adrenergic receptor expression in adipose tissue. In addition, a high-calcium diet with whey protein increased the adipose tissue leptin expression which is decreased in this obesity-prone mouse strain. These changes are likely to contribute to the inhibition of weight gain. The potential sensitisation of insulin signalling in adipose tissue together with the less lipotoxic and diabetogenic hepatic lipid profile suggest a novel mechanistic link to explain why increased dairy intake is associated with a lower prevalence of metabolic syndrome and type 2 diabetes in epidemiological studies. Taken together, the intake of a high-calcium diet with dairy proteins has a body weight lowering effect in high-fat-fed C57Bl/6J mice. High-calcium diets containing whey protein prevent weight gain and enhance weight loss, α-lactalbumin being the most effective whey protein fraction. Whey proteins and calcium have also beneficial effects on hepatic lipid profile and adipose tissue gene expression, which suggest a novel mechanistic link to explain the epidemiological findings on dairy intake and metabolic syndrome. The clinical relevance of these findings and the precise mechanisms of action remain an intriguing field of future research.

Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.