Aksel Kornerup Hansen

Consumption of isoflavones may prevent adiposity, hepatic steatosis, and dyslipidaemia. However, studies in the area are few and primarily with genistein. This study investigated the effects of formononetin and its synthetic analogue, 2-heptyl-formononetin (C7F), on lipid and cholesterol metabolism in C57BL/6J mice. The mice were fed a cholesterol-enriched diet for five weeks to induce hypercholesterolemia and were then fed either the cholesterol-enriched diet or the cholesterol-enriched diet-supplemented formononetin or C7F for three weeks. Body weight and composition, glucose homeostasis, and plasma lipids were compared. In another experiment, mice were fed the above diets for five weeks, and hepatic triglyceride accumulation and gene expression and histology of adipose tissue and liver were examined. Supplementation with C7F increased plasma HDL-cholesterol thereby increasing the plasma level of total cholesterol. Supplementation with formononetin did not affect plasma cholesterol but increased plasma triglycerides levels. Supplementation with formononetin and C7F induced hepatic steatosis. However, formononetin decreased markers of inflammation and liver injury. The development of hepatic steatosis was associated with deregulated expression of hepatic genes involved in lipid and lipoprotein metabolism. In conclusion, supplementation with formononetin and C7F to a cholesterol-enriched diet adversely affected lipid and lipoprotein metabolism in C57BL/6J mice.

For oral glucose tolerance test (OGTT) in mice, multiple blood samples need to be taken within a few hours from conscious mice. Today, a number of essential parameters may be analysed on very small amounts of plasma, thus reducing the number of animals to be used. It is, however, crucial to obtain high-quality plasma or serum in order to avoid increased data variation and thereby increased group sizes. The aim of this study was to find the most valid and reproducible method for withdrawal of blood samples when performing OGTT. Four methods, i.e. amputation of the tail tip, lateral tail incision, puncture of the tail tip and periorbital puncture, were selected for testing at 21°C and 30°C after a pilot study. For each method, four blood samples were drawn from C57BL/6 mice at 30 min intervals. The presence of clots was registered, haemolysis was monitored spectrophotometrically at 430 nm, and it was noted whether it was possible to achieve 30–50 μL blood. Furthermore, a small amount of extra blood was sampled before and after the four samplings for testing of whether the sampling induced a blood glucose change over the 90 min test period. All methods resulted in acceptable amounts of plasma. Clots were observed in a sparse number of samples with no significant differences between the methods. Periorbital puncture did not lead to any haemolysed samples at all, and lateral tail incision resulted in only a few haemolysed samples, while puncture or amputation of the tail tip induced haemolysis in a significant number of samples. All methods, except for puncture of the tail tip, influenced blood glucose. Periorbital puncture resulted in a dramatic increase in blood glucose of up to 3.5 mmol/L indicating that it is stressful. Although lateral tail incision also had some impact on blood glucose, it seems to be the method of choice for OGTT, as it is likely to produce a clot-free non-haemolysed sample, while periorbital sampling, although producing a high quality of sample, induces such a dramatic change in blood glucose that it should not be applied for OGTT in mice.

It is well established that gluten‐free diet reduces the incidence of type 1 diabetes mellitus (T1D) in non‐obese diabetic (NOD) mice, though the mechanism is not known. However, regulatory T cells (Treg) are likely to play an important role. Also, it is known that dietary gluten induces an intestinal increase in the bacterium Lactococcus garvieae, but the importance of this phenomenon for T1D development is doubtful. Our hypothesis is that gluten is responsible for mediating its effect on T1D through the influence on Treg development independent of gluten‐induced Lactococci. Four groups of female NOD and BALB/c mice of 3 week old were fed either a gluten‐free diet or a standard diet. Lactococcus garvieae or saline water was administered per oral to one of each dietary group. Spleen and Peyer′s patches were sampled from BALB/c mice for flow cytometric monitoring of IL‐10 and Treg. NOD mice were diagnosed diabetic with blood glucose level >12 mmol/l. Dietary gluten significantly decreased the occurrence of Tregs by 10–15% (P < 0.05) in mice compared with those fed a standard diet. These results and the diabetes incidence were independent of the gluten‐induced bacterial factor Lactococci. The prevalence of Treg was 5‐ to 10‐fold more abundant in the Peyer′s patches than in the spleen (P < 0.001). In conclusion, dietary gluten has a significant negative quantitative impact on the generation of Treg in mice, independent of gluten‐induced Lactococcus garvieae, and Treg are far more abundant in Peyer′s patches than in the spleen.

Sulphatide is a newly described autoantigen in insulin-dependent diabetes mellitus; it is present in the islets of Langerhans, and anti-sulphatide antibodies are found in diabetic patients and in spontaneously diabetic BB rats. The aim of the study was to treat neonatal BB rats with sulphatide in order to induce tolerance and thereby possibly influence later diabetes development. One hundred and twelve newborn BB rats, divided into three groups, were treated once daily during the first 6 days of life with intrathymic injections of sulphatide, galactosyl-ceramide (which is similar to sulphatide but without sulphate) or phosphate buffer alone. Although the results showed no difference in diabetes incidence among the three groups, there was a delayed onset of diabetes in the sulphatide-treated group, which developed diabetes on average at 77 +/- 1 days of age, compared to 70 +/- 2 days (p < 0.02) for the galactosyl-ceramide-treated group and 70 +/- 1 days (p < 0.01) for the buffer-treated group. The degree of insulitis and the size of islets of Langerhans were studied histologically for the diabetic animals in all three groups; there were no significant differences although the sulphatide-treated group tended to have a more normal histology. The blood glucose levels for the diabetic BB rats were similar in all three groups. Thus, neonatal treatment with the diabetic autoantigen sulphatide at the chosen dosage does not influence the incidence of diabetes, but delays the onset of disease.

The off-spring in a barrier maintained colony of spontaneously type 1 diabetic BB/Wor/Mol-BB rats was treated with a daily injection of either saline, forskolin, arginine, glucose or both glucose and arginine for the first six days after birth. The incidence was reduced from 88% to 72% by the neonatal stimulation with arginine and glucose in combination, which also delayed the onset time from 76.0 ± 2.2 days to 88.1 ± 2.3 days. No such effect was observed after stimulation with either one of the compunds. Neonatal stimulation with forskolin also delayed the onset to 87.9 ± 3.8 days, however without reducing the incidence. A higher incidence of diabetes was observed in the barrier-protected rats taking part in this study than in an earlier study using BB rats of the same origin, but kept under conventional conditions.