The newly inbred cohen diabetic rat: a nonobese normolipidemic genetic model of diet-induced type 2 diabetes expressing sex differences.

The newly inbred Cohen diabetic rat is an exceptional experimental model of diet-induced type 2 diabetes mellitus that is the result of secondary inbreeding nearly 30 years after it originally had been established. Animals from the original colony were selectively inbred by stringent criteria for 10 additional generations, bringing overall inbreeding to >50 generations. The metabolic phenotypes of the resulting contrasting strains, designated as the Cohen diabetic-sensitive (CDs) and -resistant (CDr) rats, were characterized. The phenotype of the CDs strain that was fed a regular diet consisted of fasting normoglycemia, normal glucose tolerance to intraperitoneal glucose loading, normal fasting insulin levels, and a normal insulin response to glucose loading. In contrast, CDs rats that were fed a custom-prepared high-sucrose low-copper diabetogenic diet became overtly diabetic: fasting glucose levels were normal or elevated, and the blood glucose insulin response to glucose loading was markedly abnormal. CDr rats that were fed a regular or diabetogenic diet did not develop diabetes and maintained normal glucose tolerance and insulin secretion. A striking sex difference was observed in CDs rats that were fed a diabetogenic diet: males had a lower growth rate and a more severe glucose intolerance pattern than females. Gonadectomy shortly after weaning did not prevent the development of the diabetic phenotype in its early phase in either sex but markedly attenuated its expression in males at a later phase, abolishing the sex differences. Alternate-day feeding, as opposed to daily feeding, also attenuated the metabolic phenotype in males. The development of the diabetic phenotype in CDs rats that were fed a diabetogenic diet was not accompanied by obesity or hyperlipidemia. The genetic profile of the strains was established using 550 microsatellite markers evenly distributed throughout the rat genome. The rate of homozygosity within strain was > or = 96%. The rate of polymorphism between the contrasting strains was 43%. We conclude that the metabolic phenotypes of the rebred colony of CDs and CDr rats and their genetic makeup render the Cohen diabetic rat a useful experimental model that is highly suitable for studying the interaction between nutritional-metabolic environmental factors and genetic susceptibility (sensitivity and resistance) for the development of type 2 diabetes. The model is also distinctively useful for investigating the effect of sex on the expression of the diabetic phenotype.

Sublines of the Burkitt line Daudi, selected on the basis of reactivity with soybean agglutinin, differ in tumorigenicity in athymic mice.

Two subpopulations were isolated on the basis of soybean agglutinin (SBA) binding, from the human Burkitt lymphoma line Daudi. The low- and high-binder sublines maintained this characteristic in continuous passages. Their surface marker profiles, antibodies, scanning electron microscope (SEM), cytochemical reactions and binding of other lectins (concanavalin A and wheatgerm agglutinin) were not different. They differed, however, in growth potential in athymic mice. The low-binder subline had lower frequency of takes, tumor weight and volume, and did not metastasize as compared to the high-binder subline. However, the reaction with F-SBA of all the tumor cells examined was strong (greater than 70%), indicating in vivo selection and tumor development of high binder cells.

Interaction of soybean agglutinin with human leukemia-lymphoma lines at various stages of differentiation.

Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for reactivity with soybean agglutinin (SBA). The binding and redistribution pattern of soybean surface receptors was determined with fluorescein-isothiocyanate conjugated SBA (F-SBA) by ultraviolet microscopy, and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between SBA labelling--distribution and the stage of lymphoid cell differentiation. The SBA labelling on the membrane of null lines was undetectable by U.V. microscopy and flow cytometry. A gradual increase in SBA labelling correlating with the stage of differentiation was observed on cell lines of both B and T origin. However the maximal fluorescence intensity of the T lines was lower than the B lines. The redistribution pattern of SBA on the membrane of T lines was rings and mild patches, whereas that on the B lines was moderate to large patches. The reactivity of the lymphoid lines with SBA was not affected by growth conditions. The binding of SBA to normal lymphoblastoid lines was generally low and the fluorescence intensity weak. The reactivity of these lines with SBA was not associated with their origin or "age". It is suggested that the differences in the reactivity of SBA with human hematopoietic lines at various stages of maturation may be of value in future understanding the differences in structure and function of the surface membrane between normal and malignant cells, and the relation to normal and abnormal cell differentiation.

Adaptation to hypoxia in the diabetic rat kidney.

Hypoxia of the kidney in diabetes could predispose it to develop acute and chronic renal failure. To examine the relationship between renal hypoxia and renal failure, we measured hypoxia (as a pimonidazole adducts), hypoxia-inducible factors (HIFs), and a hypoxia target gene heme oxygenase-1. The studies were performed in rats with streptozotocin (STZ)-induced diabetes, Cohen diabetes sensitive rats, and during short-term artificial hyperglycemia in rats induced by intravenous glucose and octreotide. STZ-treated rats received insulin, the superoxide dismutase mimetic tempol, or contrast medium. Radiocontrast media causes hypoxia and HIF induction. Hypoxia, HIFs, and heme oxygenase were undetectable in controls, but transiently activated in STZ-treated and the Cohen diabetes sensitive rats. Different patterns of HIFs and pimonidazole were observed between the three models. Insulin abolished pimonidazole and HIF induction, whereas tempol lead to increased HIFs and heme oxygenase induction at similar levels of pimonidazole. When compared with control rats, STZ-treated rats exhibited more intense and protracted renal pimonidazole, with augmented hypoxia inducible factor production and reduced GFR following contrast media. Our data suggest that both regional hypoxia and hypoxia adaptation transiently occur in early stages of experimental diabetes, largely dependent on hyperglycemia or after contrast media. Tempol may augment the HIF response in diabetes.

Opioid peptides mediate sympathetic inhibition in response to baroreceptor activation in a distinct genetic strain of rabbit.

1. Two strains of rabbits have been bred with marked differences in their cardiac baroreflex sensitivity (BRS). The difference in cardiac BRS was attenuated by naloxone. We compared the sympathetic responses to a pressor stimulus in these two strains, by measuring the changes in plasma catecholamines in the presence and absence of naloxone. 2. Cardiac BRS was assessed in eight rabbits of each group by the steady-state method. Two weeks later, both ear arteries and one ear vein were cannulated. Mean arterial pressure (MAP) and heart rate (HR) were recorded from one artery and blood samples (5 mL) for plasma catecholamines (CA) taken before, and during the peak of the pressor response to intravenous phenylephrine (PE, 20 micrograms/kg) from the other. The experiment was repeated 2-3 weeks later in rabbits with high BRS (Group I) after injection of naloxone 0.1 mg/kg, i.v. 3. Resting MAP and HR did not differ in the two groups. The mean gains of the cardiac baroreflex were 23.3 +/- 2.2 ms/mmHg in Group I and 6.3 +/- 1.1 ms/mmHg in Group II. After PE, MAP rose by 54.5 +/- 1.8 mmHg in Group II and 40.3 +/- 3.6 mmHg in Group I (P less than 0.02). The pressor response was associated with a 31% reduction in plasma noradrenaline (NA) in Group I and a 34% increase in Group II. The reduction in NA was significantly correlated with the degree of bradycardia in Group I (r = 0.72, P less than 0.05) and with BRS in both groups (r = 0.78, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Contrasting influences of central and peripheral opioids on cardiac baroreflex sensitivity in rabbits.

We studied the effects of naloxone [0.1 mg/kg intravenously (i.v.) or 7.5 micrograms/kg intracisternally (i.c.)], naloxone methyl iodide (NMI, 0.2 mg/kg, i.v. or 15 micrograms/kg i.c.) and morphine (2 mg/kg i.v.) on the cardiac baroreflex elicited in conscious rabbits especially bred for high and low baroreflex sensitivity (BRS) (group I, BRS > 5.5 beats/min/mm Hg and group II BRS < 4 beats/min/mm Hg, respectively). Full sigmoid barocurves were produced in 37 rabbits by i.v. injection of phenylephrine (1-15 micrograms/kg) and nitroglycerin (1-20 micrograms/kg) after pretreatment with saline or one of the above drugs. In group I, both naloxone i.v. and i.c. and NMI i.c. significantly reduced BRS and decreased the degree of bradycardia in response to a pressor stimulus; neither morphine nor NMI i.v. had any effect. In group II, naloxone i.v. and i.c. and NMI i.c. had no effect on BRS, but both morphine and NMI i.v. significantly increased BRS. An even greater increase was achieved by a combination of these drugs, which also increased specifically the degree of tachycardia in response to a decrease in blood pressure (BP). The results suggest that baroreceptor activation in group I released in the brainstem an opioid peptide that acts to increase BRS. In contrast, group II rabbits responded by peripheral opioid activation, which results in a decrease in BRS, possibly by inhibiting norepinephrine (NE) release from cardiac neurons. The predominance of peripheral or central opioid involvement in BRS modulation appears to be due to genetic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Predictability of heterozygosity scores and polymorphism information content values for rat genetic markers.

Construction of a genetic linkage map of the laboratory rat, Rattus norvegicus, establishes the rat as a genetic model. Allele sizes were reported for 432 simple sequence length polymorphisms (SSLPs) genotyped in 12 different substrains belonging to nine different inbred strains of rats. However, these nine strains represent only a fraction of the more than 140 inbred strains available. If allele sizes are not known, alternative indices of markers' polymorphism content can be used, such as heterozygosity (H) and polymorphism information content (PIC). Here, we have determined heterozygosity scores and PIC values for all markers of the rat genetic linkage map, and we evaluate the predictability of the heterozygosity and the PIC values. Correlation analysis between the nine inbred strains reported for the rat map and ten "test" strains yielded r = 0.42 and r = 0.44 for heterozygosity and PIC values, respectively. While the correlation of the indices between the two groups of animals is low, these indices do provide a means of predicting whether a genetic marker will be informative in strains where allele sizes are not known.

Topical nitrates potentiate the effect of botulinum toxin in the treatment of patients with refractory anal fissure.

BACKGROUND: Anal fissure is perpetuated by high sphincter pressures and secondary local ischaemia. Pharmacological approaches include topical nitrates and botulinum toxin (BT) which act to reduce anal pressure. BT lowers anal pressure by preventing acetylcholine release from nerve terminals while topical nitrates act by donating nitric oxide (NO). The aims of the present study were to compare the therapeutic effect and lowering action on internal anal sphincter pressure of BT injection and local application of isosorbide dinitrate (ID) compared with BT given alone, in patients with chronic anal fissure (CAF) refractory to treatment with ID. METHODS: Thirty consecutive patients with CAF who did not respond to previous topical ID treatments were randomly assigned to receive one of the following treatments: group A, injection of BT (20 U into the internal anal sphincter) and subsequent daily applications of ID (2.5 mg three times daily for three months); and group B, BT injection only (20 U). If at the end of six weeks following BT injection no improvement was seen in group B, ID was added. A series of anal pressure measurements, including resting basal pressure and resting pressure following topical ID (1.25, 2.5, and 3.75 mg), was carried out both before and two weeks after 20 U of BT injection into the internal anal sphincter. At the end of the trial, patients were followed up for an average period of 10 months. FINDINGS: At six weeks the fissure healing rate was significantly higher in group A patients (10/15 (66%)) compared with group B (3/15 (20%)) (p=0.025). At eight and 12 weeks, no significant differences were seen: 11/15 (73%) v 11/15 (73%) and 9/15 (60%) v 10/15 (66%), group A v group B, respectively. Maximum anal resting pressure (MARP) was significantly lower two weeks after BT injection than baseline MARP (90 (4) v 110 (5) mm Hg; p<0.001). A significantly greater reduction in MARP following local application of ID was achieved after BT injection compared with that achieved before BT injection (p=0.037) INTERPRETATION: (1) Combined BT injection and local application of ID in patients with CAF who failed previous treatment with ID was more effective than BT alone. This treatment modality appears to be safe and promising. (2) ID application induced a greater reduction in MARP following BT injection compared with ID application before BT injection. The improved potency of ID on MARP after BT injection suggests a primary cholinergic tonus dominance in some patients and not, as previously claimed, anal sphincter insensitivity to nitrates.

Treatment of experimental colitis by oral tolerance induction: a central role for suppressor lymphocytes.

OBJECTIVE: Inflammatory bowel diseases (IBD) are immune-mediated disorders wherein an imbalance between proinflammatory (Th1) and antiinflammatory (Th2) cytokines is thought to play a role in the pathogenesis. The aim of this study was to test whether induction of oral tolerance to proteins extracted from inflammatory colon alleviates experimental colitis, and whether oral tolerization mediated by suppressor cells can induce immune tolerance. METHODS: Colitis was induced in rats by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Rats received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Splenocytes harvested from tolerized and control rats were transplanted into irradiated naive rats. RESULTS: Feeding of colitis-extracted proteins ameliorated colonic inflammation, as shown by reduction of colonic ulcerations, as well as decreased diarrhea, intestine and peritoneal adhesions, wall thickness, and edema. A marked reduction of the fraction of injured colonic area and colon weight, and decrease in colon weight, were observed in tolerized rats versus controls. Histological parameters for colitis were markedly improved in tolerized animals that showed significant reduction in inflammatory response and mucosal ulcerations. Tolerized rats developed an increase in TGFbeta1 and a decrease in IFNgamma serum levels. TNBS-induced colitis was significantly attenuated in naive recipients of splenocytes from tolerized rats, compared with rats that received splenocytes from control donors. CONCLUSIONS: Induction of oral tolerance to colitis-extracted proteins downregulates the anticolon immune response, thereby ameliorating experimental colitis. Suppressor lymphocytes mediate the tolerance by induction of a shift from a proinflammatory to an antiinflammatory immune response.

Mast cells involvement in the inflammation and fibrosis development of the TNBS-induced rat model of colitis.

BACKGROUND: Mast cells have been implicated in chronic inflammatory conditions resulting in fibrosis, such as Crohn disease. However, a link between inflammation, fibrosis and mast cells has not been demonstrated in human or animal intestinal diseases. This work was undertaken to analyze whether mast cells play a role in inflammation and fibrosis in the TNBS-induced rat colitis. METHODS: Rats were rectally instilled 2,4,6,-trinitrobenzene sulfonic acid in ethanol, and immediately or 4 days later injected daily i.p. with nedocromil sodium, a mast cell stabilizer, compound 48/80, a mast cell activator, or saline. Rats were sacrificed 5 days post-TNBS, or on day 21. Intestinal inflammation and fibrosis were assessed by gross and histopathological evaluation. Colonic mast cell numbers (toluidine blue) and collagen (type I mRNA expression) were evaluated. Mast cell sonicate was added to rat colon fibroblasts. Fibroblast proliferation (3H-thymidine), collagen synthesis (3H-proline) and contractile activity (tridimensional collagen lattice contraction) were then assessed. RESULTS: Nedocromil reduced inflammation and fibrosis possibly by decreasing mast cell numbers and activation and consequent collagen production. Compound 48/80 slightly enhanced the severity of the disease by activating mast cells. Mast cells increased fibroblast proliferation, collagen production and contractile activity. CONCLUSIONS: Mast cells are involved in the gastrointestinal tract inflammation and fibrosis of the TNBS-colitis rats.

Time course of nitric oxide synthase generation after gluten exposure in the rectal mucosa of gluten-sensitive patients.

BACKGROUND: Nitric oxide is thought to play an important role in modulating chronic inflammatory responses as well as in immune-mediated inflammation. We reproduced a gluten-mediated mucosal response in the rectum of celiac and control subjects in order to determine the role of inducible and constitutive nitric oxide synthases in the pathogenesis of this process. MATERIAL: Nine patients with confirmed celiac disease and five healthy controls underwent a long-term rectal gluten challenge (48 h) after an enema of 6 g of crude gluten, and constitutive and inducible nitric oxide synthase activity were determined in rectal biopsies. The histological localization of inducible nitric oxide synthase was determined by immunohistochemistry. RESULTS: Activity of both isoforms of nitric oxide synthase in control subjects did not change significantly after gluten instillation. In celiac patients, constitutive nitric oxide synthase on rectal mucosa also showed no significant changes after challenge with gluten. Inducible nitric oxide synthase isoform exhibited a modest increase 4 h after gluten instillation in celiac patients (mean increase 35% compared with baseline levels) but, 8 h after challenge, generation of iNO synthase was significantly higher: 54% more than pre-challenge production (P < 0.05) and higher than control values (P < 0.05). Inducible nitric oxide synthase staining was mostly localized in mononuclear cells of the epithelium and the lamina propria. After gluten instillation, the enhanced staining was mainly localized in subepithelial areas of the lamina propria. CONCLUSION: Our data suggest a role for nitric oxide, generated by inducible nitric oxide synthase, in the process of rectal mucosa injury by local gluten instillation in sensitized patients. We could not, however, determine if the role of nitric oxide in the ensuing injury of this gluten-induced immune inflammation model is a protective one, or merely a by-product generated by the activation of the inflammatory cells.

Identification of quantitative trait loci for non-insulin-dependent diabetes mellitus that interact with body weight in the Otsuka Long-Evans Tokushima Fatty rat.

Non-insulin-dependent diabetes mellitus (NIDDM) is a prototypical multifactorial disease. Genetic predisposition and obesity are major risk factors for NIDDM development and the interactions between these factors are likely to be important in the etiology of this disease. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is one of the best animal models of NIDDM, since the OLETF rat develops NIDDM with mild obesity that is very similar to human NIDDM. Therefore, the OLETF rat is a powerful model for investigating the interaction between genetic susceptibility to NIDDM and obesity. In this study, our goal was to clarify the relationship between an individual NIDDM susceptibility locus and obesity in the OLETF using a molecular genetics approach. We identified four novel quantitative trait loci (QTLs) that contribute to the susceptibility to NIDDM, none of which shows significant linkage with body weight. However, Nidd1/of on chromosome 7 and Nidd2/of on chromosome 14 have an interaction with body weight. In contrast, one locus was mapped to chromosome 10 for body weight, but not to fasting or postprandial glucose levels. These data illustrate that NIDDM and body weight are under separate genetic control in the OLETF yet interact to yield the final disease phenotype in the two Nidd/of loci. In addition, body weight could be used in place of body mass index as an indicator of obesity in our experimental system of genetic study. This study will facilitate the understanding of the complex interaction between genetic susceptibility to NIDDM and obesity.

A reference cross DNA panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms.

The ultimate informativeness of the zebrafish mutations described in this issue will rest in part on the ability to clone these genes. However, the genetic infrastructure required for the positional cloning in zebrafish is still in its infancy. Here we report a reference cross panel of DNA, consisting of 520 F2 progeny (1040 meioses) that has been anchored to a zebrafish genetic linkage map by 102 simple sequence length polymorphisms. This reference cross DNA provides: (1) a panel of DNA from the cross that was used to construct the genetic linkage map, upon which polymorphic gene(s) and genetic markers can be mapped; (2) a fine order mapping tool, with a maximum resolution of 0.1 cM; and (3) a foundation for the development of a physical map (an ordered array of clones each containing a known portion of the genome). This reference cross DNA will serve as a resource enabling investigators to relate genes or genetic markers directly to a single genetic linkage map and avoid the problem of integrating different maps with different genetic markers, as must be currently done when using randomly amplified polymorphic DNA markers, or as has occurred with human genetic linkage maps.