Troels Bock

Gluten‐free diet prevents diabetes in NOD mice

Epidemiological as well as animal studies have shown that environmental factors such as nutrition contribute to the development of diabetes. In this study we investigated whether the early introduction of a gluten‐free diet can influence the onset and/or incidence of diabetes, as well as insulitis and the number of gut mucosal lymphocytes, in non‐obese diabetic (NOD) mice.

Unbiased estimation of total β-cell number and mean β-cell volume in rodent pancreas

We describe a method for unbiased assumption‐free estimation of the total number of β‐cells and the mean β‐cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of β‐cells was 1.06±0.07±106 (mean±SEM) per pancreas with a mean β‐cell volume of 1280±17 μm3, while in rats the total β‐cell number was 2.76±0.42±106 per pancreas with a mean β‐cell volume of 1170±65 μm3. Furthermore, the results showed that in both species the biological variability in the total β‐cell volume is due to differences in the number of β‐cells rather than variability of the mean β‐cell volume. The method can be used to give a precise description of number and volume of β‐cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyperlhypoplasia to a given induced or spontaneous change in the total β‐cell mass.

Intermittent feeding and fasting reduces diabetes incidence in BB rats.

Food intake may be one of several factors which influence the risk of development of insulin dependent diabetes mellitus, but the influence of the pattern of food supply has not been studied previously. The aim of the present study was to investigate the effect of intermittent feeding and fasting upon diabetes in BB rats. This study included three groups. Group 1 served as control and included 77 animals, 79% became diabetic. In groups 2 and 3, after weaning, food but not water was withdrawn from the animals: 24 h twice a week in group 2; 24 h every second day in group 3. Group 2 included 40 BB rats, 50% (p < 0.004) became diabetic. Group 3 included 44 BB rats, 52% (p < 0.01) became diabetic. No differences were seen between sexes. Degree of insulitis was not influenced by changed food supply. Regarding blood glucose, no influence was seen among diabetic animals, among non-diabetic animals changed food supply reduced blood glucose values obtained at the end of the study. Intermittent feeding and fasting tended to reduce mean age at the time of diagnosis of diabetes, significance was reached only in female animals from group 3 compared to group 1. Body weight was obtained weekly. Intermittent feeding and fasting caused a reduced weight gain in group 2 as well as in group 3 compared to control animals; however, most pronounced in group 3 and also more pronounced among males compared to females. For pre-diabetic and non-diabetic animals comparable influence on body weight was seen. The main conclusion in the study is that intermittent feeding and fasting reduced diabetes incidence in BB rats.

Perinatal Determinants Among Children Who Later Develop IDDM

OBJECTIVE The aim of this study was to investigate whether children who develop insulin-dependent diabetes mellitus (IDDM) differ in some aspects from a matched control group at the time of birth. RESEARCH DESIGN AND METHODS We studied all children who were born in Denmark during the period 1973–1977 and admitted to a Danish hospital with a diagnosis of IDDM during 1978–1989. The study was conducted by combining two nationwide registries, The National Patient Registry and The Birth Registry. RESULTS The criteria were fulfilled by 837 children. Data regarding the age of the parents, the number of previous pregnancies of the mother, the month of birth, and the birth weight and length of the children who developed IDDM were compared with the data of an age- and sex-matched control group of 837 children without IDDM. We did not detect any significant differences between the two groups with respect to the parameters studied. CONCLUSIONS No differences in perinatal determinants could be demonstrated among Danish children who develop IDDM compared with children without diabetes.

The first fatal case of Vibrio vulnificus infection in Denmark.

Vibrio vulnificus can cause severe infections in humans and persons with preexisting liver disorders are especially at risk. In this paper we report what is to our knowledge the first fatal case of V. vulnificus infection in Denmark. The patient was a 68‐year‐old man with a history of chronic lymphatic leukemia and hepatic cirrhosis. Physicians should be aware of the clinical manifestations of this disease and should be especially attentive to patients at risk of acquiring the infection if there has been possible exposure to V. vulnificus by contact with seawater or contaminated material such as eels.

Reduction of diabetes incidence in NOD mice by neonatal glucose treatment

The aim of this study was to investigate whether neonatal glucose treatment influences the incidence of diabetes in NOD mice. Thirty‐nine NOD mice (19 males, 20 females) were treated with 8 g glucose/ kg BW/day administered by subcutaneous injections twice a day for the first six days of life. Thirty‐six untreated NOD mice (20 males, 16 females) served as a control group. In the glucose‐treated group, 33% became diabetic compared with 58% in the control group (X2= 5.3, p = 0.021). Among the glucose‐treated males, 16% became diabetic compared with 50% of the untreated males (X2= 5.5, p = 0.019), whereas 50% of the glucose‐treated females became diabetic compared with 69% of the untreated females (X2= 1.1, NS). We conclude that neonatal glucose treatment can reduce the diabetes incidence in NOD mice. These results could have implications for the prevention of type 1 diabetes mellitus in humans.

HIGH JUVENILE BODY WEIGHT AND LOW INSULIN LEVELS AS MARKERS PRECEDING EARLY DIABETES IN THE BB RAT

Diabetes incidence in BB rats is 60-80% and our aim was to investigate whether it is possible to characterize those rats destined to develop diabetes. While the genetic background as well as the environmental factors affecting BB-rat littermates are very similar, body weight reflects some existing variance. The study involved 151 BB rats, and the body weight of each animal was measured daily from birth. Thirty-four animals became diabetic before 100 days of age, and their body weight showed a 5-10% increase compared to the non-diabetic animals for each day of life from day 1 to day 45 (p values 0.0001 to 0.05). This increased body weight in individuals destined for diabetes was seen in both sexes. When investigating whether juvenile body weight has any predictive value, we found that the incidence of diabetes at 100 days of age increased from 22.5% to 46.7% (p < 0.01) when the heaviest animals in each litter were selected. Insulin content in pancreas was examined at day 10 and 20, and was found to be significantly reduced in the BB rats with highest body weight compared with rest of the litter (p = 0.02 and p = 0.0005, respectively). The insulin concentration in peripheral blood was significantly reduced in the BB rats with highest body weight at 20 days of age (p = 0.002). When early and late diabetic BB rats were compared at time of diagnosis regarding blood glucose, degree of insulitis and number of small and large islets, no significant differences were found between the groups.

TOTAL NUMBER AND SIZE DISTRIBUTION OF MOTOR NEURONS IN THE SPINAL CORD OF NORMAL AND EMC-VIRUS INFECTED MICE : A STEREOLOGICAL STUDY

The encephalomyocarditis virus of the diabetogenic M‐strain (EMC‐M) is known to cause diabetes in mice. The EMC‐M virus has also been shown to cause paresis in some of the infected animals. The clinical features include an acute ascending predominantly motor paralysis, developing within days. This resembles acute idiopathic polyneuritis. The alpha motor neurons would be a possible target for the virus, so two parameters, the total number and the size distribution of motor neurons, were therefore selected for further investigation in 6 mice with neurological involvement and compared with 6 control mice. The optical fractionator method was applied for estimating the total number of motor neurons and the 3D size distribution was estimated using the rotator method in a vertical design. No difference was found in the total number of motor neurons and the size distributions were similar in the 2 groups. This design can be used as a model for the estimation of the total number of motor neurons and their size distribution in other experimental animal models.

Neonatal Treatment with Beta-cell Stimulatory Agents Reduces the Incidence of Diabetes in BB Rats

The aim of the study was to investigate whether various beta-cell stimulatory drugs, given neonatally, influence the incidence of diabetes in BB rats. Newborn BB rats were treated twice daily for 6 days and diabetes development was observed during the following 200-day study period. Compared to a diabetes incidence of 63.8% in 163 control BB rats which received saline or were untreated, the percentage of experimental BB rats that developed diabetes was as follows in the different subgroups: arginineglucose: 47% (n= 73, p < 0.02); glucagon: 37% (n = 93, p < 0.0001); tolbutamide-glucose: 36% (n = 58, p < 0.0005); and theophylline-glucose: 39% (n = 41, p < 0.005). A long-term arginine-glucose treatment was not superior to the shorter neonatal treatment. Histological examination revealed a higher degree of insulitis in diabetic than in non-diabetic animals but no difference according to the kind of treatment was observed. Finally, we found that the diabetes incidence in BB rats was higher in the first litter compared to subsequent litters (p = 0.04). Thus, neonatal treatment with various beta-cell stimulatory agents reduces diabetes incidence in BB rats. The theory behind the study, that the treatment accelerates beta-cell maturation leading to increased immunological tolerance towards beta cells, is discussed.

Beta-cell expression of 65-kDa heat-shock protein mRNA is function- and age-dependent

This study examined the expression of mRNA coding for the 65‐kDa heat‐shock protein (HSP) in rat islet cells of different functional states and different ages. In addition, beta cells and non‐beta cells purified by fluorescence‐activated cell sorting were studied. Total RNA from islet cells and insulin‐producing RINm5F cells was isolated and analyzed by Northern blotting using a cDNA probe coding for the human homologue to the mycobacterial 65‐kDa HSP, after which blots were quantified by densitometric scanning. Isolated beta cells were found to express 65‐kDa HSP mRNA. The expression was increased in Lewis islet cells exposed to heat shock or high glucose concentration, four‐and three‐fold, respectively (p<0.01). In isolated beta cells cultured at high glucose concentration a doubling in the content of 65‐kDa HSP mRNA was seen compared with islets cultured at low glucose concentration (p<0.05). In islets from Lewis rats fasted for 24 h, the content of 65‐kDa HSP mRNA was 42% lower than in islets isolated from normally fed Lewis rats (p<0.01). Both in BB rats and Wistar Furth rats the content of 65‐kDa HSP mRNA was found to be higher in the 30‐ and the 60‐day‐old rats compared with the neonatal animals (p<0.01). The expression of 65‐kDa HSP mRNA was increased in RINm5F cells following heat shock, while no induction was seen after stimulation with glucose, TPA or IBMX. It is concluded that the 65‐kDa heat‐shock protein belongs to the family of inducible functional antigens in beta cells, which strengthens the interest in 65‐kDa HSP as an antigen possibly involved in the initiation of autoimmune beta‐cell destruction.