Novel genes on rat chromosome 10 are linked to body fat mass, preadipocyte number and adipocyte size.

BACKGROUND: The genetic architecture of obesity is multifactorial. We have previously identified a quantitative trait locus (QTL) on rat chromosome 10 in a F2 cross of Wistar Ottawa Karlsburg (WOKW) and Dark Agouti (DA) rats responsible for obesity-related traits. The QTL was confirmed in congenic DA.WOKW10 rats. To pinpoint the region carrying causal genes, we established two new subcongenic lines, L1 and L2, with smaller refined segments of chromosome 10 to identify novel candidate genes. METHODS: All lines were extensively characterized under different diet conditions. We employed transcriptome analysis in visceral adipose tissue (VAT) by RNA-Seq technology to identify potential underlying genes in the segregating regions. Three candidate genes were measured in human paired samples of VAT and subcutaneous (SC) AT (SAT) (N=304) individuals with a wide range of body weight and glucose homeostasis parameters. RESULTS: DA.WOKW and L1 subcongenic lines were protected against body fat gain under high-fat diet (HFD), whereas L2 and DA had significantly more body fat after high-fat feeding. Interestingly, adipocyte size distribution in SAT and epigonadal AT of L1 subcongenic rats did not undergo typical ballooning under HFD and the number of preadipocytes in AT was significantly elevated in L2 compared with L1 and parental rats. Transcriptome analysis identified three candidate genes in VAT on rat chromosome 10. In humans, these candidate genes were differentially expressed between SAT and VAT. Moreover, HID1 mRNA significantly correlates with parameters of obesity and glucose metabolism. CONCLUSIONS: Our data suggest novel candidate genes for obesity that map on rat chromosome 10 in an interval 102.2-104.7 Mb and are strongly associated with body fat mass regulation, preadipocyte number and adipocyte size in rats. Among those genes, AT head involution defective (HID1) mRNA expression may be relevant for human fat distribution and glucose homeostasis.

Combining QTL data for HDL cholesterol levels from two different species leads to smaller confidence intervals.

Quantitative trait locus (QTL) analysis detects regions of a genome that are linked to a complex trait. Once a QTL is detected, the region is narrowed by positional cloning in the hope of determining the underlying candidate gene-methods used include creating congenic strains, comparative genomics and gene expression analysis. Combined cross analysis may also be used for species such as the mouse, if the QTL is detected in multiple crosses. This process involves the recoding of QTL data on a per-chromosome basis, with the genotype recoded on the basis of high- and low-allele status. The data are then combined and analyzed; a successful analysis results in a narrowed and more significant QTL. Using parallel methods, we show that it is possible to narrow a QTL by combining data from two different species, the rat and the mouse. We combined standardized high-density lipoprotein phenotype values and genotype data for the rat and mouse using information from one rat cross and two mouse crosses. We successfully combined data within homologous regions from rat Chr 6 onto mouse Chr 12, and from rat Chr 10 onto mouse Chr 11. The combinations and analyses resulted in QTL with smaller confidence intervals and increased logarithm of the odds ratio scores. The numbers of candidate genes encompassed by the QTL on mouse Chr 11 and 12 were reduced from 1343 to 761 genes and from 613 to 304 genes, respectively. This is the first time that QTL data from different species were successfully combined; this method promises to be a useful tool for narrowing QTL intervals.

Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.

Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been suggested to participate in chronic disorders, such as diabetes and its complications. In contrast to the short and transient activation of NF-kappaB in vitro, we observed a long-lasting sustained activation of NF-kappaB in the absence of decreased IkappaBalpha in mononuclear cells from patients with type 1 diabetes. This was associated with increased transcription of NF-kappaBp65. A comparable increase in NF-kappaBp65 antigen and mRNA was also observed in vascular endothelial cells of diabetic rats. As a mechanism, we propose that binding of ligands such as advanced glycosylation end products (AGEs), members of the S100 family, or amyloid-beta peptide (Abeta) to the transmembrane receptor for AGE (RAGE) results in protein synthesis-dependent sustained activation of NF-kappaB both in vitro and in vivo. Infusion of AGE-albumin into mice bearing a beta-globin reporter transgene under control of NF-kappaB also resulted in prolonged expression of the reporter transgene. In vitro studies showed that RAGE-expressing cells induced sustained translocation of NF-kappaB (p50/p65) from the cytoplasm into the nucleus for >1 week. Sustained NF-kappaB activation by ligands of RAGE was mediated by initial degradation of IkappaB proteins followed by new synthesis of NF-kappaBp65 mRNA and protein in the presence of newly synthesized IkappaBalpha and IkappaBbeta. These data demonstrate that ligands of RAGE can induce sustained activation of NF-kappaB as a result of increased levels of de novo synthesized NF-kappaBp65 overriding endogenous negative feedback mechanisms and thus might contribute to the persistent NF-kappaB activation observed in hyperglycemia and possibly other chronic diseases.

Toxic effects of cyclosporine on the endocrine pancreas of Wistar rats.

The widely used immunosuppressive drug cyclosporine exerts toxic effects on various parenchymal organs including the liver and kidney. This study was performed with the aim of testing whether cyclosporine also affects the endocrine pancreas. Daily cyclosporine doses of 50 mg/kg body weight over 3 weeks in rats enhanced the serum bilirubin and creatinine concentrations, led to light-microscopic destruction in the liver and kidneys, and resulted in the development of an impaired glucose tolerance--and, later on, of hyperglycemia. The pancreatic insulin content decreased to 33% of values observed in vehicle-treated controls, which can be ascribed to a 50% decrease of beta-cell volume and a slightly smaller reduction of islet insulin content. The reduction of the cyclosporine dose to 15 mg/kg body weight daily, which also reduced the popliteal lymph node weight gain after allogeneic stimulation, was not accompanied by serochemical or morphological alterations of livers or kidneys in the rats when treated for 3 weeks. However, the animals had already developed an impaired glucose tolerance, accompanied by a decrease in pancreatic insulin content (to 50% that of controls), a decrease of islet insulin content (to 70%) and a reduced pancreatic beta cell volume (to 72%). The findings let us conclude that pancreatic beta cells are sensitive to toxic effects of cyclosporine in vivo. We suggest that the measurement of glucose tolerance, as a sensitive parameter of a toxic cyclosporine action, should be included in the monitoring of grafted patients under cyclosporine treatment.

Effect of syngeneic islet antigen administration on complement-dependent antibody-mediated cytotoxicity to islet cells and diabetes onset in diabetes-prone BB/OK rats.

Diabetes-prone BB/OK rats aged 30 to 35 days were subjected to three sequential intrasplenic injections of unfractionated homogenate prepared from Langerhans' islets of newborn syngeneic BB/OK rats. Syngeneic islet antigen administration resulted in increased complement-dependent antibody-mediated cytotoxicity (C'AMC) to rat pancreatic islet cells in serum, compared to buffer-treated control animals as detected by 51Cr-release assay. However, the increase of anti-islet-cell cytotoxicity neither impaired glucose tolerance nor affected the incidence of diabetes and the age at manifestation. In contrast to BB/OK rats developing diabetes, animals remaining long-term normoglycaemic did not show an enhancement of cytotoxicity to islet cells within twelve days after the first islet antigen injection as revealed retrospectively. In conclusion, humoral mediated anti-islet-cell cytotoxicity is not decisively involved in pancreatic beta-cell destruction and promotion of diabetes development in BB/OK rats, but animals becoming diabetic seem to be characterized by a stronger immunological reactivity upon syngeneic islet antigen challenge as indicated by an increase of anti-islet C'AMC compared to long-term normoglycaemic rats.

Scanning electron microscopic examinations on retarded bone defect healing in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats.

To date, no detailed knowledge from animal experiments is available on the kind and extent of osseous and mineral metabolic disorders in genetically determined, insulin-dependent Type I diabetes. The purpose of this study was to examine the influence of the diabetic metabolic state in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats on bone defect healing. Eighty spontaneously-diabetic BB/OK rats with a blood-glucose value of 391 +/- 106 mg% (mean +/- SD) at the time of manifestation were used in the study. Based on blood-glucose values at the time of surgery (mg%), postoperative blood-glucose course (mg%) and postoperative insulin requirements (IU/kg), the animals were divided into groups with well-compensated (n = 40, 170 +/- 101 mg%; 221 +/- 120 mg%; 2.1 +/- 1 IU/kg) or poorly compensated (n = 40; 371 +/- 158 mg%; 357 +/- 83 mg%; 5.2 +/- 1.4 IU/kg) metabolic state. Forty LEW.1A rats served as the normoglycemic controls (95 +/- 18 mg%). Using a 1-mm-diameter Kirschner wire, a hole of femoral bone ca. 1 cm proximal to the knee joint space was centrally drilled. Ten animals from each group were killed on postoperative days 7, 14, 24, and 42, and specimens were taken for analysis. Using SEM to measure regions of new bone semiautomatically and quantitatively, also determining the number, area, and circumference of regions not yet filled with new bone. Up to postoperative day 14, very significant differences (p < 0.0001) for all investigated characteristics were found between the spontaneously-diabetic BB/OK rats and the control animals--in favor of the controls--and up to postoperative day 24 within the group of spontaneously-diabetic BB/OK rats, where the well-compensated animals had significantly better results in terms of number and area of regions of bone not yet filled with new bone formations. Forty-two days postoperatively, SEM observations showed no differences between examination groups. The process of bone defect healing in spontaneously-diabetic rats was disturbed only in the early phase and exhibited retardation in its progression. After 42 days, bone defect healing was complete, regardless of the diabetic metabolic state; no differences were detected with the SEM between examination groups at this time point.

Diabetes and hypertension in rodent models.

As shown by ourselves and others, animals models closely resembling human complex diseases like IDDM in BB/OK and hypertension in SHR/Mol rats can be used to dissect a complex disease into discrete genetic factors as has been done for hypertension in (BB/OK x SHR/Mol) cross hybrids. Discrete genetic factors, so-called QTLs, were detected on chromosomes 1, 10, 18, 20, and X. To gain additional information about the physiologic effect of the mapped blood pressure QTLs, genetically defined regions of the SHR rat were transferred onto the genetic background of diabetes-prone BB/OK rats. Four new congenic BB.SHR rats named BB.Sa, BB.Bp2, BB.1K, and BB.Xs were generated and characterized telemetrically for blood pressure, heart rate, and motor activity. The data demonstrate clearly that each single blood pressure QTL of the SHR rat causes a significant increase of the systolic blood pressure and has a different influence on diastolic blood pressure, heart rate, and motor activity. The effects were modified differently by the diabetic state in BB.Sa, BB.Bp2, and BB.Xs rats carrying all diabetogenic genes of the BB/OK rats. The results demonstrate that these newly established congenic strains are a unique tool to study the physiological control of blood pressure by a single blood pressure QTL on the one hand and their interaction with hyperglycemia on the other. It is well within the bounds of possibility that diabetic congenics reflect the diabetic hypertension seen in diabetic patients. Because of the synteny conservation in gene order between different mammals, genes of the appropriate human region could therefore be candidate genes for hypertension in diabetics. Furthermore, these congenic strains can also be used to study interactions between a blood pressure QTL and various selected environmental conditions. In this way, one could learn which QTL can be influenced by environmental factors and to what extent. Another point is the study of gene interactions. Because congenics are genetically identical except for the defined transferred region, congenics can be crossed to investigate the interaction between two or three blood pressure QTLs selected by the investigator and not by nature. These QTL combinations can be studied in the nondiabetic as well as diabetic state. Although the advantage of congenic strains has been shown, the transferred chromosomal regions are too large to pinpoint the gene responsible for the phenotypic change. Therefore, regions on each chromosome must be systematically whittled down, which can be done by crossing the congenics with BB/OK rats and intercrossing their progeny to generate recombinants. These can then be used for the creation of new congenic lines carrying a much smaller region of the SHR/Mol rat. This has been started for the region on chromosome 1 spanning a 16-cM region from the Sa to the Igf2 gene. BB.Sa rats were therefore backcrossed onto BB/OK rats and the resulting progeny were intercrossed. The aim will be to create at least three new congenic BB.Sa rat strains homozygous for the SHR alleles of Sa, Lsn, or Igf2 genes. However, new problems will emerge with these new congenics. To genetically define small regions requires more dense polymorphic markers than are currently available. Dense polymorphic markers will also be necessary to split the other regions on chromosomes 10, 18, 20, and X. We expect that in the near future it will be possible using this approach to define small regions of < 0.5 cM. The recent progress in gene mapping in the rat gives hope that the use of such congenic lines will allow the identification and recovery of the blood pressure genes in the near future.

Features of the metabolic syndrome in the spontaneously hypertriglyceridemic Wistar Ottawa Karlsburg W (RT1u Haplotype) rat.

The aim of this study was to characterize the Wistar Ottawa Karlsburg W ([WOKW] RT1u haplotype) rat in a cross-sectional study (up to 14 weeks of age) for traits with pathophysiological relevance to the metabolic syndrome in comparison to the Dark Agouti (DA) rat, to determine the age at which the WOKW rat begins to manifest the characteristics of the metabolic syndrome. The findings indicate that the WOKW rat is dyslipidemic (high serum triglycerides and low high-density lipoprotein [HDL] cholesterol), hyperinsulinemic, and obese. The interval between 8 and 10 weeks appears to be the crucial age after which the most dramatic changes were observed in the measured phenotypic traits in the WOKW rat, as well as the most expressive differences between the WOKW and DA strains. Considering the phenotypic differences between WOKW and DA rats, the DA rat provides an appropriate control strain for crossing studies with the WOKW rat, which might contribute to the explanation of the genetic basis for traits of the metabolic syndrome in this model.

Congenic BB.SHR rat provides evidence for effects of a chromosome 4 segment (D4Mit6-Npy approximately 1 cm) on total serum and lipoprotein lipid concentration and composition after feeding a high-fat, high-cholesterol diet.

Congenic BB.SHR (previously referred to as BB.LL) rats were generated by transferring the segment of chromosome 4 flanked by the D4Mit6 and Spr loci from the spontaneously hypertensive rat (SHR/Mol) onto the genetic background of the diabetes-prone BB/OK rat. In this study, the influence of the above-mentioned region of chromosome 4 on triglyceride, cholesterol, and phospholipid phenotypes after a high-fat, high-cholesterol diet was examined by comparison of BB.SHR congenic rats with BB/OK rats. BB/OK and BB.SHR had comparable concentrations of basal and postdietary serum insulin, as well as of basal total serum triglycerides and had an identical body weight and food intake at the beginning of the test period. However, after 4 weeks on the test diet, BB.SHR rats were significantly heavier than BB/OK rats and had significantly higher food intake and lower total serum triglyceride concentrations. The basal serum leptin level was significantly lower, but postdietary serum leptin concentration did not show a significant difference between the 2 strains. Furthermore, significantly higher basal total serum cholesterol and phospholipid levels were observed in BB.SHR rats, but this difference disappeared after feeding the high-fat, high-cholesterol diet. Postdietary high-density lipoprotein (HDL)(2) cholesterol and phospholipid levels were significantly elevated in BB.SHR rats when compared with BB/OK rats. The 2 strains also differed slightly, but significantly, with respect to the other HDL phospholipid concentrations. In addition to previously described differences between BB/OK and BB.SHR rats, the results of this study clearly show the impact of genes, lying within the transferred segment, on serum lipid phenotypes after high-fat, high-cholesterol diet.

Islet neuronal abnormalities associated with impaired insulin secretion in type 2 diabetes in the Chinese hamster.

This study examined the relationship between islet neurohormonal characteristics and the defective glucose-stimulated insulin secretion in genetic type 2 diabetic Chinese hamsters. Two different sublines were studied: diabetes-prone CHIG hamsters and control CHIA hamsters. The CHIG hamsters were divided into three subgroups, depending on severity of hyperglycemia. Compared to normoglycemic CHIG hamsters and control CHIA hamsters, severely hyperglycemic CHIG hamsters (glucose > 15 mmol/l) showed marked glucose intolerance during i.p. glucose tolerance test and 75% impairment of glucose-stimulated insulin secretion from isolated islets. Mildly hyperglycemic CHIG animals (glucose 7.2-15 mmol/l) showed only moderate glucose intolerance and a 60% impairment of glucose-stimulated insulin secretion from the islets. Immunostaining for neuropeptide Y and tyrosine hydroxylase (markers for adrenergic nerves) and for vasoactive intestinal peptide (marker for cholinergic nerves) revealed significant reduction in immunostaining of islets in the severely but not in the mildly hyperglycemic animals, compared to control CHIA hamsters. The study therefore provides evidence that in this model of type 2 diabetes in Chinese hamsters, severe hyperglycemia is accompanied not only by marked glucose intolerance and islet dysfunction but also by reduced islet innervation. This suggests that islet neuronal alterations may contribute to islet dysfunction in severe but not in mild diabetes.

Relationship between the histopathology of the endocrine-exocrine pancreas parenchyma and beta-cell function in the Chinese hamster CHIG/Han subline.

To investigate pancreatic histopathology in relation to islet function in type 2 diabetes, pancreatic tissue was examined at disease onset and after death in the genetically diabetic Chinese hamster CHIG/Han subline. The pancreatic islets displayed vacuolation, intraislet fibrosis, variable stages of degranulation, and beta-cell necrosis, but no insulitis. These lesions were associated with changes in immunostaining of major islet peptides, impaired glucose-stimulated (10 mM) insulin secretion, reduced islet insulin content, and the severity of hyperglycemia. The exocrine pancreas was characterized by peri- and intrapancreatic fat and mononuclear cell infiltration. Biopsy of the pancreas had a marked effect on plasma glucose such that at 2 weeks after excision, in 9 and 18% of the severely hyperglycemic hamsters, plasma glucose levels decreased to <7.2 and 15 mM, respectively, and 45% of the mildly hyperglycemic hamsters became transiently normoglycemic (<7.2 mM). The results showed that insulitis is not involved in beta-cell failure of diabetic CHIG/Han hamsters. Vacuolation of islets was the most prominent lesion associated with a functional islet abnormality and development of hyperglycemia. Attenuation of hyperglycemia after biopsy suggests that the pancreas, in type 2 diabetes, maintains the ability to respond to impairment with amelioration of the diabetic state.

Metabolic features of newly established congenic diabetes-prone BB.SHR rat strains.

The well-known association of hypertension and diabetes mellitus and the lack of suitable animal models to study diabetic hypertension prompted us to transfer 4 chromosomal regions with quantitative trait loci (QTLs) for blood pressure of the spontaneously hypertensive SHR rat onto the genetic background of the diabetes-prone and normotensive BB/OK rat. Four congenic strains developed are named as BB. Sa (Chr.1), BB.Bp2 (Chr.18), BB.1K (Chr.20) and BB.Xs (Chr.X). Because the systolic blood pressure is significantly elevated in all congenics, renal related traits were investigated in serum and urine. Comparing BB/OK and their congenic derivatives, significant differences were found in all serum and in 7 out of 8 urine constituents studied. Most significant differences were found between BB/OK and BB.Bp2 rats. Significant differences were also found between the different congenic strains indicating that each congenic strain has its own phenotype and that each chromosomal region contains most probably further QTLs for some of the traits studied.

Functional alterations in the rat kidney induced either by diabetes or high protein diet.

The aim of this study was to compare the effect of long-term diabetes with that of a long-term high protein diet in vivo on the kidney function and in vitro on cellular parameters of isolated glomeruli of BB rats. Four groups of rats were investigated: Group 1 = normoglycaemic (N) rats > 250 days old; group 2 = age-matched diabetic (D) rats with a diabetes duration of more than 150 days; group 3 = BB rats which were fed a protein diet of 8% (low protein = LP) and group 4 = rats which received a high protein (HP) diet (32%) for more than 80 weeks. From 24-h-urine samples albumin, urea, creatinine and electrolyte excretion were estimated. At the end of the study the total kidney weight was determined and glomeruli were isolated for in vitro experiments. Hyperglycaemic and HP fed rats were characterised by a significantly increased excretion of albumin, urea and different electrolytes compared to normoglycaemic or LP fed animals. Creatinine excretion was neither affected by hyperglycaemia nor HP diet. HP and D caused a significant increase in total kidney weight when compared to the LP and N group, respectively (N: 1.79 +/- 0.08; D: 2.33 +/- 0.09; LP: 2.26 +/- 0.18; HP: 3.28 +/- 0.46 g; p < 0.05). The increased kidney weight of diabetic and HP rats correlated well with a significant enhanced DNA content of isolated glomeruli (N: 3.41 +/- 0.15; D: 4.45 +/- 0.19; LP: 4.18 +/- 0.35; HP: 6.40 +/- 0.62 micrograms/1000 glomeruli; p < 0.02). In addition, glomeruli obtained from either D or HP fed BB rats incorporated significantly more 3H-thymidine into their DNA indicating an elevated rate of DNA synthesis. The results demonstrate that HP diet caused markedly altered kidney function and induced cellular changes of glomeruli which are interpreted as enhanced proliferative processes. These alterations are comparable to those associated with diabetes mellitus. An unbalanced high protein diet represents a considerable risk factor for the development of functional and structural impairments of the kidney and should absolutely be avoided in patients suffering from diseases which are known to be associated with kidney alterations.

Circadian variations in blood pressure and heart rate in diabetes prone and resistant rat strains compared with spontaneously hypertensive rats.

Circadian blood pressure (BP), heart rate (HR) and motor activity (MA) of nondiabetic (nd) and spontaneously diabetic (d) BB/OK rats were compared with that of spontaneously hypertensive rats (SHR). In addition a diabetes-resistant and non-hypertensive rat strain (LEW.1W) was monitored for the same parameters. Systolic and diastolic BP (SBP, DBP), HR and MA were measured telemetrically. In d BB rats, the 24 h mean value of SBP (132 +/- 0.15 mm-Hg) was significantly increased compared to nd BB rats (125 +/- 0.18 mmHg). No differences were found in DBP between d and nd BB rats (93 +/- 0.13 v.s. 94 +/- 0.15 mmHg). Both, d and nd BB rats were significantly different in SBP and DBP to that of SHR (155 +/- 0.19 and 110 +/- mmHg). Nondiabetic BB rats did not significantly differ from LEW.1W rats in SBP and DBP (125 vs. 123 mmHg and 94 vs. 94 mmHg). The heart rate was lowest in diabetic BB rats compared with all other strains. Compared to nd BB the diabetic rats had an altered daily rhythm in BP. The results demonstrate that the diabetic BB rats develop circadian variations in BP and HR similar to those observed in hypertensive rats.

Insulin treatment improves islet function in type 2 diabetic Chinese hamsters.

To study whether normalization of hyperglycemia improves islet function in long-standing type 2 diabetes, hyperglycemic CHIG/Han subline of the genetic type 2 diabetic Chinese hamster (>15 mmol/l: n=23) were either treated with insulin implants (liberating 1 U/day) or vehicle for two weeks. Islets were isolated and incubated for 3 h in the presence of 10 mmol/l glucose with or without 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). Specimens were also taken for immunocytochemical analysis of insulin cells. Glucose-stimulated insulin secretion was reduced by 83% in the vehicle-treated diabetic hamsters compared to non-diabetic controls (p<0.001). This impairment was not improved by the two-week insulin treatment. IBMX potentiated glucose-stimulated insulin secretion; this effect was markedly reduced in vehicle-treated diabetics compared to controls (p<0.001). In fact, the linear relation between IBMX-potentiated and glucose-stimulated insulin secretion in controls was absent in islets from diabetic animals. The two week insulin treatment normalized this relation, although still the total insulin secretory response to IBMX and glucose was lower than in controls. Furthermore, the islet insulin content was significantly increased by the 2 week normalization of glucose and, finally, the severe degranulation and lowering of insulin staining in islet beta cells in diabetic animals were markedly improved by insulin treatment. The results suggest that two-weeks of normalization of glycemia in long-standing type 2 diabetes in non-obese Chinese hamster improves beta cell signaling induced by the cyclic AMP pathway in conjunction with improved islet insulin content and beta cell morphology.

Impaired beta-cell function in the Chinese hamster CHIG/Han subline.

Plasma glucose and insulin levels were measured in the genetically diabetic CHIG/Han and the diabetes-resistant CHIA/Han subline of the Chinese hamster. At 31 +/- 8 wk of age, the CHIG hamsters were grouped into nondiabetic, mildly and severely diabetic, according to their levels of glycemia. Hyperinsulinemia, occurring in nondiabetic and mildly diabetic CHIG hamsters, was attenuated in severely diabetic animals. Light microscopy and immunohistochemistry revealed initial beta-cell hyperplasia, followed by extensive degranulation and loss of immunoreactive insulin in islets of severely diabetic animals. Staining intensity of glucagon-immunoreactive cells was unchanged in nondiabetic and mildly diabetic animals, but was increased in islets from the severely diabetic hamsters. A static incubation system was used to examine the insulin response of pancreatic islets isolated from the diabetic and nondiabetic CHIG hamsters, and the diabetes-resistant CHIA subline. Compared with the nondiabetic CHIG hamsters, islets from mildly and severely diabetic animals displayed increased basal insulin release at 1.5 mmol/l and a deficient response at 10 mmol/l glucose which was associated with 61 and 77% decreases (p < 0.01 and p < 0.001) in the islet insulin content. The addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced glucose-stimulated insulin release from islets of nondiabetic and mildly diabetic CHIG hamsters, although the response elicited was lower than from CHIA islets. However, IBMX failed to significantly increase the glucose-stimulated insulin response of islets from severely diabetic hamsters. A negative correlation (r = -0.73, p < 0.001, n = 48) was found between islet insulin content and plasma glucose levels. The data suggest that the reduced secretory capacity represents an early islet beta-cell dysfunction, and the decrease in the insulin content contributes to the islet abnormalities in the diabetes-susceptible CHIG hamsters.

Quantitative trait loci for blood glucose confirm diabetes predisposing and protective genes, Iddm4 and Iddm5r, in the spontaneously diabetic BB/OK rat.

Several crossing studies using diabetic BB/OK and diabetes-resistant rat strains have clearly shown that the MHC class-II-genes of the RT1u haplotype (Iddm1) and the lymphopenia (Iddm2) are essential but not sufficient for type 1 diabetes development. The search for additional diabetogenic genes revealed predisposing non-MHC genes, Iddm3 and Iddm4, and a diabetes protective gene, Iddm5r, cosegregating with diabetes in the BB/OK rat subline. These findings were based on cosegregation studies comparing allele frequencies between diabetic and non-diabetic cross hybrids. Since, type 1 diabetes is characterised by hyperglycaemia we analysed 22 diabetic and 43 non-diabetic [(BB x SHR)FI x BB] backcross hybrids (28M:37F) which were already homozygous for Iddml and Iddm2 to search for quantitative trait loci (QTLs) affecting blood glucose in BB/OK rats. The QTL analysis using 117 microsatellite markers located on 19 autosomal chromosomes and the X chromosome, revealed suggestive linkage for blood glucose at the same position for diabetics (lod score 3.1) and non-diabetics at an age of 16 weeks at locus D6Mgh2 on chromosome 6 (lod score 1.9). In contrast, the peak for nondiabetics at an age of 28 weeks (lod score 3.1) was located in the region on chromosome 1 flanked by D1Mgh12 and D1Mit14, whereas the peak for diabetics (lod score 1.9) was found between Sa and Igf2. The distance between two peaks is ca. 50 cM. These findings are consistent with previously described results and provide strong evidence on the relevance of the described region for the development of diabetes not only in the rat, but, regarding the chromosomal homology also in human.

Crosses between diabetic BB/OK and wild rats confirm that a third gene is essential for diabetes development.

Several crossing studies with diabetic BB rats have shown that in addition to the lymphopenia (Iddm1) and the MHC class II genes of the RT1U haplotype (Iddm2) there are further non-MHC genes essential for diabetes development. Because diabetes-resistant inbred rat strains may be homozygous for one of the diabetogenic non-MHC genes, masking the expression of diabetogenic genes and leading to an underestimation of the number of diabetogenic genes, we crossed wild and diabetic BB/OK rats. The F1 hybrids were backcrossed onto diabetic female (BC1W-F, n=97) and male BB/OK rats (BC1W-M, n=98) transferred to a specified-pathogen-free environment and studied for the frequency and age at onset of diabetes up to an age of 30 weeks. Comparing the results of these BC1 W hybrids with similarly derived hybrids using diabetes-resistant DA rats (BC1DA-F, n=113; BC1DA-M, n=216), the diabetes frequency in total was comparable indicating the action of three recessive genes. The percentage of diabetics in Iddm1 and Iddm2 homozygotes confirmed the existence of the third gene, Iddm3, but there were some sex differences; significantly more male than female BC1W-F and significantly more BC1DA-M than BC1DA-F males were diabetic. Regarding the age at onset, the BC1W-F hybrids manifested not only significantly earlier, but also more uniformly than BC1DA-F and BC1-M hybrids.

Characterization of monoclonal islet cell reactive autoantibodies from the diabetic biobreeding (BB/OK) rat.

Two fusion experiments using the heteromyeloma cell line CB-F7 and splenocytes from two diabetes-prone BB (BioBreeding) rats at the onset of diabetes resulted in 128 islet cell reactive autoantibodies primarily detected with permeabilized insulin-producing rat insulinoma cells (RIN) by a cellular enzyme-linked immunosorbent assay. Seventy-nine (62%) of 128 RIN cell reactive supernatants exhibited a cross-reactivity with rat splenic lymphocytes. Six stable hybridomas secreting monoclonal ICSA (islet cell surface antibodies) were established, but only one monoclonal antibody, R4B10, showed preferential beta-cell binding. Six monoclonal antibodies showed a dual reactivity as ICA (islet cell cytoplasmic antibodies) detected by immunostaining of pancreatic islet cryosections and as ICSA on the surface of viable islet cells, whereas two reacted only with an ICA-like pattern. One monoclonal ICSA was specifically displaced from the RIN cell surface by sera of type 1 diabetic patients.

Antibody response to sheep red blood cells in four substrains of BB rats, some other strains and interstrain F2 hybrids.

Primary and secondary antibody responses to SRBC were assayed in three substrains of dp BB rats, one strain of ndp BB rats, in their parental WOK rats, in highly inbred LEW rats, in two RT1 congenic strains LEW.1A and LEW.1W, and in the recombinant LEW.1WR2 strain. Furthermore, interstrain F1 and F2 hybrids of two dp BB substrains with congenic animals were tested. A significant lowering of the primary antibody response was found in two dp BB substrains and in the recombinant LEW.1WR2 strain. A transient lowering was shown in ndp BB rats. The third dp BB substrain responded well. The observed differences among three dp BB substrains only indicate that the level of antibody response to SRBC cannot be taken as a further marker, besides the RT1uv haplotype, for spontaneous development of the IDDM in BB rats. The observed decrease in the primary antibody response in the RT1 recombinant LEW.1WR2 rats and in one group of interstrain F2 hybrids typing as homozygous RT1uv may have another meaning. They could indicate some immunoregulatory effects of the RT1 haplotype on the supposedly multigenic response to SRBC in rats. No significant differences were found in the secondary antibody responses to SRBC.