Inheritance of hydronephrosis in the inbred mouse strain DDD

The mode of inheritance of hydronephrosis was investigated by crossing inbred DDD mice having a high incidence of hydronephrosis and C57BL/6 mice having normal kidneys. In the males, incidences of hydronephrosis in F1 animals were intermediate between the two parental strains at a rate of 32.6% in (DDD x C57BL/6)F1 and 23.4% in reciprocal F1. The same tendency was observed in F2 male animals. In BCF1 males, the number of affected mice was higher in (C57BL/6 x DDD) F1 x DDD (72.4%) than in (DDD x C57BL/6)F1 x C57BL/6 (11.1%). A few affected mice were found among the females of hybrids F1, F2 and BCF1. These results suggested that hydronephrosis in the DDD strain of mice was controlled by polygenes, and that male hormones may have some effect on the occurrence of hydronephrosis.     

Influence of testosterone on hydronephrosis in the inbred mouse strain DDD

Hereditary hydronephrosis was detected in all of the male mice of DDD inbred strain maintained at the National Institute of Animal Health Japan, but in only a few of the females. From the standpoint that male hormones are related to the development of hydronephrosis in this strain, the incidence and severity of the disease were investigated in gonadectomized mice treated with testosterone. In the males, the incidence of hydronephrosis was 50% (7/14) in the control (gonadectomized) group, and 73.3% (11/15), 100% (13/13) and 100% (12/12) in the 0.1 mg, 3.3 mg and 10 mg treatment groups respectively. The same tendency was observed in the female animals, though the incidence in each group was not so high. In both sexes the degree of severity increased in proportion to the dose of testosterone administrated. Blood testosterone levels were higher in intact DDD mice than in C57BL/6 mice, which had normal kidneys in both sexes. These results suggest that male hormones play a significant role in the development of hydronephrosis.     

Incomplete protection mechanism against vesico-ureteral reflux and hydronephrosis in the inbred mouse strain DDD

We have previously reported the occurrence and inheritance mode of hydronephrosis in the inbred mouse strain DDD. The present investigation examined the possibility that hydronephrosis may be caused by a vesico-ureteral reflux (VUR). Bladder pressure was measured under anaesthesia in 3 strains of mice (DDD, ddY and C57BL/6). VUR was demonstrated by lower bladder pressure only in the DDD strain. Loading pressure into the renal pelvis (LPP) was significantly higher in DDD than in C57BL/6 (P less than 0.001). Correlations between LPP and severity of hydronephrosis were significantly positive in DDD and ddY, indicating that mice showing higher LPPs had the severest disease. Scanning electron micrography revealed that the ureteral orifice of DDD (100-300 microns) was much larger than in C57BL/6 (30 microns), and thus offered scant protection against VUR in DDD. These results suggest that DDD hydronephrosis caused by VUR is related primarily to the absence of an adequate protection mechanism in the ureteral orifice.     

Substance P, neurokinin A, vasoactive intestinal polypeptide and gastrin releasing peptide in the intestine and pancreas of spontaneously obese-diabetic mice

The concentrations and contents of immunoreactive substance P (SP), neurokinin A (NKA), vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) were measured in acid-ethanol extracts of intestine (duodenum-jejunum-ileum) and pancreas of C57BL/KsJ diabetes-obese (db/db) mice, Aston obese-hyperglycaemic (ob/ob) mice, and their respective lean controls. The intestinal concentration of GRP and pancreatic concentrations of VIP and GRP were 36-57% lower in lean Aston mice than lean C57BL/KsJ mice, indicating the influence of genetic background in control mice. Intestinal concentrations of SP and NKA were reduced by 19-33% in the db/db and ob/ob mutants compared with their lean controls, but the intestinal contents of these peptides were normal or greater than normal due to intestinal hypertrophy of the mutant mice. The intestinal VIP concentration was not altered, but the content was increased by 87% and 25% respectively in db/db and ob/ob mice, whereas the intestinal GRP concentration was reduced by 51% in ob/ob mice. Pancreatic concentrations and contents of NKA, VIP and GRP were similar in lean and db/db C57BL/KsJ mice. However, pancreatic concentrations and contents of VIP and GRP were reduced by 51-55% in ob/ob mice compared with their lean controls. The sensitivity of the present assay did not permit accurate determination of the low pancreatic concentrations of SP. The results suggest that the spontaneous ob/ob and db/db syndromes of obesity and diabetes in mice are associated with reduced intestinal concentrations of SP and NKA. The ob/ob mouse also exhibited reductions of intestinal GRP and pancreatic GRP and VIP concentrations. These changes in regulatory peptides may relate to abnormalities of intestinal and possibly pancreatic function in obese and diabetic mutant mice.     

Quantitation of glycogen synthase and phosphorylase protein in mouse liver: correlation between enzymatic protein and enzyme activity

Phosphorylase and glycogen synthase protein were measured in normal and genetically diabetic (C57BL/KsJ db/db) mice liver extracts using rocket immunoelectrophoresis, and these data correlated with measurements of total phosphorylase and total glycogen synthase activities, respectively. Phosphorylase protein in 5-week-old normal mice was about 5 micrograms/mg protein and reached 8 micrograms/mg protein by 9 weeks. In comparison, the diabetic mice had elevated levels of phosphorylase protein (11-13 micrograms/mg protein) which correlated with an increased total phosphorylase activity compared to normals. The correlation coefficient for the phosphorylase activity vs protein plot was highly significant (r = 0.73, P less than 0.001). The molar concentration of phosphorylase subunit in normal mouse liver was calculated to be 11 microM and up to 23 microM in the diabetic mice. The liver concentration of glycogen synthase was relatively constant in normal mice at 400 ng/mg protein (corresponding to approximately 1.4 microM) but varied from 230 to 441 ng/mg protein (0.9 to 1.8 microM) in diabetic mice. There was little correlation between glycogen synthase activity and enzymatic protein (r = 0.15). These results indicate (1) that phosphorylase is present at concentrations approximately 10 times that of glycogen synthase, and (2) that glycogen synthase activity is relatively more dependent upon factors other than the amount of enzymatic protein.     

Increase in insulin secretion induced by plasma from mice injected with allogeneic lymphocytes

Plasma from BALB/c mice bled 90 minutes after allogeneic lymphocyte injection significantly rises glucose induced insulin secretion. This rise is observed in pancreas either from non-treated or from allogeneized mice. This rise is time and dose-dependent. An 1/40 dilution is enough to bring about a significant increase on insulin secretion. This effect is seen when mice are bled between 60 and 180 minutes after injection with a maximum effect at 90-120 minutes. Plasma from BALB/c mice injected with C57BL/6 J lymphocytes rises insulin secretion from BALB/c, C57BL/6 J, C3h and C57BL/KsJ mice pancreas. Plasma from streptozotocin diabetic BALB/c mice and from genetically diabetic C57BL/KsJ mdb-mdb mice injected with allogeneic lymphocytes stimulates glucose induced insulin secretion but to a lesser extent than plasma from normal non-diabetic mice does.     

Hepatic adrenergic receptors in the genetically diabetic C57 BL/KsJ (db/db) mouse

1. At 30 weeks of age, homozygote diabetic C57 BL KsJ (db/db) mice were grossly obese, lethargic and displayed moderate hair loss relative to heterozygote control C 57 BL KsJ (db/+) mice. 2. In diabetic mice, compared to control, the total body weights, liver weight: body weight ratios, and blood glucose levels were increased 2.3 fold, 20% and 3.1 fold, respectively. 3. Analysis of plasma membranes isolated from control and diabetic mouse liver established that comparable purity levels were achieved since relative specific activities of the plasma membrane markers 5'-nucleotidase and gamma-glutamyltranspeptidase were similar: 10.2 and 11.4 fold with respect to 5'-nucleotidase in control and diabetic states respectively; and 8.0 and 8.3 fold with respect to gamma-glutamyltranspeptidase in control and diabetic states respectively. 4. A select effect of diabetes on gamma-glutamyltranspepetidase, however, was observed. The activity of this enzyme was found to be reduced 16% in diabetic liver compared to control liver. 5. Assessment of [3H]prazosin and [3H]dihydrolalprenolol binding to mouse liver plasma membranes indicated that although there was no difference in beta-adrenergic receptor binding in control and diabetic states, alpha 1-adrenergic receptor binding was found to be reduced 43% in diabetic mouse liver plasma membranes. 6. Scatchard analyses of kinetic studies indicate that the reduction is a reflection of decreases in alpha 1-adrenergic receptor numbers with no change in alpha 1 receptor affinity in the diabetic state: since for diabetic and control liver plasma membranes, Kd values were 3.41 +/- 0.02 nM and 3.40 +/- 0.01 nM respectively; and Bmax were 650.12 +/- 16.44 fmol mg-1 and 380.76 +/- 12.92 fmol mg-1, respectively.     

Depressed progesterone accumulation by the brain and peripheral tissues of diabetic C57BL/KsJ mice: normalization by estrogen therapy

The effects of diabetes on the uptake and incorporation of 3H-progesterone (P) in various brain areas and peripheral tissues were analyzed in C57BL/KsJ mice. Littermate control (+/?) and diabetic (db/db) mice were pulse-treated (60) min with 10 mu Ci of 3H-P at either 8 or 16 weeks of age. Subsequently, the peripheral tissues and brains were dissected, weighed, digested and the amount of incorporated 3H-P assessed. The pituitary, amygdala, septum and hypothalamus were found to concentrate the highest levels of 3H-P of all the brain areas examined. The brain areas of 16-week-old (+/?) mice accumulated higher concentrations of 3H-P than the comparable 8-week-old brain tissues. In addition, all the brain areas of 8-week-old (db/db) mice accumulated equal or more 3H-P than the comparable (+/?) brains. By 16 weeks of age, however, the 3H-P accumulation rate was significantly higher in all brain areas of (+/?), as compared to (db/db), mice. Of the peripheral tissues examined, the ovary, uterus, pancreas, kidney and mesometrial fat pad consistently exhibited the highest rates of 3H-P accumulation in both (+/?) and (db/db) mice. By 16 weeks of age, all peripheral tissues of (+/?) mice exhibited greater accumulation rates of 3H-P than the comparable (db/db) tissues. Following 4 days of estradiol treatment (10 micrograms/day s.c.), the 3H-P accumulation rates in 16-week-old (+/?) and (db/db) brain were essentially equal. Only the hypothalamus, septum and amygdala of the (db/db) mice failed to normalize to (+/?) levels following estradiol treatment. In a similar manner, the pancreas, uterus, ovary and fat of (db/db) mice exhibited 3H-P accumulation rates similar to those of (+/?) mice following estradiol treatment. These data indicate that estradiol therapy effectively normalizes the normal age- and diabetes-related declines in brain and peripheral tissue sensitivity and/or responsitivity to progesterone in C57BL/KsJ mice.     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.     

Genetic background determines the extent of islet amyloid formation in human islet amyloid polypeptide transgenic mice

Genetic background is important in determining susceptibility to metabolic abnormalities such as insulin resistance and beta-cell dysfunction. Islet amyloid is associated with reduced beta-cell mass and function and develops in the majority of our C57BL/6J x DBA/2J (F(1)) male human islet amyloid polypeptide (hIAPP) transgenic mice after 1 yr of increased fat feeding. To determine the relative contribution of each parental strain, C57BL/6J (BL6) and DBA/2J (DBA2), to islet amyloid formation, we studied male hIAPP mice on each background strain (BL6, n = 13; and DBA2 n = 11) and C57BL/6J x DBA/2J F(1) mice (n = 17) on a 9% (wt/wt) fat diet for 1 yr. At the end of 12 mo, islet amyloid deposition was quantified from thioflavin S-stained pancreas sections. The majority of mice in all groups developed islet amyloid (BL6: 91%, F(1): 76%, DBA2: 100%). However, the prevalence (%amyloid-positive islets; BL6: 14 +/- 3%, F(1): 44 +/- 8%, DBA2: 49 +/- 9%, P < 0.05) and severity (%islet area occupied by amyloid; BL6: 0.03 +/- 0.01%, F(1): 9.2 +/- 2.9%, DBA2: 5.7 +/- 2.3%, p < or = 0.01) were significantly lower in BL6 than F(1) and DBA2 mice. Increased islet amyloid severity was negatively correlated with insulin-positive area per islet, in F(1) (r(2) = 0.75, P < 0.001) and DBA2 (r(2) = 0.87, P < 0.001) mice but not BL6 mice (r(2) = 0.07). In summary, the extent of islet amyloid formation in hIAPP transgenic mice is determined by background strain, with mice expressing DBA/2J genes (F(1) and DBA2 mice) being more susceptible to amyloid deposition that replaces beta-cell mass. These findings underscore the importance of genetic and environmental factors in studying metabolic disease.     

Maintenance of Islet Morphology Is Beneficial for Transplantation Outcome in Diabetic Mice

We have previously shown that co-transplantation of islets and Mesenchymal Stem Cells (MSCs) improves islet graft function and revascularisation, which was associated with the maintenance of normal islet morphology. The aim of the current study was to determine whether maintaining islet morphology in the absence of additional islet-helper cells would improve transplantation outcome in diabetic mice. Islets were isolated from C57BL/6 mice. Recipient streptozotocin-diabetic C57BL/6 mice were transplanted with a minimal mass of 150 islets as a single pellet or islets that were either manually dispersed or dispersed within a matrigel plug beneath the kidney capsule. Blood glucose concentrations were monitored for one month. Islet graft morphology and vascularisation were analysed by histology. Islets dispersed either alone or within matrigel plugs maintained near normal morphology, in contrast to pelleted islets, where individual islets fused to form large endocrine aggregates. The vascularisation of manually dispersed islets and islets dispersed within matrigel plugs was increased relative to respective control pelleted islet grafts. After one month 1/6 mice transplanted with pelleted islets cured compared to 5/6 mice transplanted with manually dispersed islets. The curative capacity of islets dispersed in matrigel was also better than that of pelleted islets (5/8 islet-matrigel implanted mice vs. 1/7 mice transplanted with pelleted islets cured by one month). Therefore, this study demonstrates that the maintenance of islet morphology is associated with improved graft function and revascularisation in diabetic mice.     

Modification of fatty acid composition in adipocyte plasma membranes by an oral treatment with a new antidiabetic agent, AS-6, in genetically obese diabetic mice, db/db

Male 12-week-old C57BL/KsJ db/db mice were treated for 1 week with a dietary admixture of an experimental antidiabetic agent, AS-6 (4-O-carboxymethylascochlorin, 0.1%). The fatty acid composition of the adipose tissue and its plasma membranes in the treated mice was compared with that in untreated db/db mice and their lean littermates. The results indicate that, when compared with the lean, the db/db adipose tissue and its plasma membrane are extremely rich in nonessential fatty acids, and AS-6 treatment modifies the fatty acyl composition only in the membranes in which 16:1 and 18:1 increase and C18 decreases.     

Pancreatic lesions of small-obese mice (C57BL/6 J-ob/ob) with hyperglucagonemia

Male Small-obese mice (Small-ob) which derived from a C57BL/6 J-ob/ob mouse colony were examined histopathologically at 13-, 39-, and over 52-week-old. C57BL/6 J-ob mice (?/+: Non-ob, ob/ob: Ob) were studied as controls. In Small-ob mice, plasma glucagon concentration was higher than that of the Ob mice (this difference was highly significant), and serum levels for insulin was within normal limits. Microscopically, hypertrophy and hyperplasia of the islets of Langerhans were found only in the pancreas of Ob mice. The increase in the number of A-cells and the decrease in the number of B-cells were revealed immunohistochemically in the islets of Small-ob mice. These changes were more severe with advance of age. In the aged Small-ob mice, perivascular and periductular cell infiltration were found, but inflammatory change of islet tissue was not confirmed in any animals examined. Diabetic symptoms in Small-ob mice seems to stem from the disparity in insulin/glucagon (I/G) ratio associated with hyperglucagonemia which result from increased number of A-cells of pancreatic islets.     

Influence of the mutation "diabetes" on insulin release and islet morphology in mice of different genetic backgrounds

Mice, 7–8-mo old, of the C57BL/KsJ-db strain and homozygotic for the mutant gene db, exhibited marked hyperglycemia and moderately elevated serum insulin levels. Light and electron microscopy provided evidence of a slightly decreased proportion of β cells in the pancreatic islets, irregular islet architecture with intraislet ducts, and degenerative as well as hypertrophic changes in the individual β cells. As a rule, islets microdissected from these mice did not release insulin in response to glucose, theophylline, iodoacetamide, or chloromercuribenzene-p-sulphonic acid. The absence of secretory responses was not simply due to lack of insulin. Although the islet content of insulin was decreased in C57BL/KsJ-db/db mice, the remaining amount was severalfold larger than that released from stimulated islets of normal controls. Another mutation, db^2J, an allele of db with identical phenotypic expressions in the C57BL/KsJ strain, was studied on the genetic background C57BL/6J. In contrast to the severely diabetic C57BL/KsJ-db/db animals, the C57BL/6J-db^2J/db^2J mice were characterized by highly elevated serum insulin levels and only moderate hyperglycemia. Their endocrine pancreas was enlarged and showed an increased proportion of β cells. Like the islets of normal mice, those of C57BL/6J-db^2J/db^2J mice responded to glucose and chloromercuribenzene-p-sulphonic acid, the glucose-induced responses being potentiated by theophylline or iodoacetamide. C57BL/KsJ-db/db mice should provide a valuable model for studying defects in insulin secretion in relation to diabetes mellitus. Mice of the C57BL/6J strain offer a control material that may help to elucidate the dependence of the insulin secretory defect on the background genome.     

Endocrine pancreatic cells of postnatal "diabetes" (db) mice in cell culture.

Ultrastructural characteristics as well as secretory and biosynthetic behavior of monolayer pancreatic cell cultures established from 4-day-old C57BL/KsJ misty diabetic (m db/m db) mice have been studied in comparison to normal littermate controls. Hypersecretion of glucagon by alpha-cells from BL/Ks misty diabetic mice after 2 days in vitro was found to precede any hyperfunction of the insulin-secreting beta-cells. The increased level of glucagon-release in BL/Ks cell cultures from diabetic mice was accompanied by a greatly enhanced level of incorporation of [3H]tryptophan into glucagon-like molecules whose specific radioactivity was up to 15-fold higher than that observed in cultures from genetic controls. The finding of an alpha-cell dysfunction in cultures established from preweaning diabetic BL/Ks mice suggests that glucagon could play an early role in shaping the events that culminate in the expression of frank diabetes in this inbred strain.     

Origin of hematopoietic cells in a syngenous and semisyngenous focus of heterotopic hematopoiesis]

Heterotopic hemopoiesis foci were produced by the bone marrow of C57BL/6 or (CBA X C57BL)F1 mice grafted under the renal capsule of (CBAT6T6XC57BL)F1 mice, bearing the chromosomal translocation. The cytogenetic analysis of the hemopoietic cells in the foci 20 to 120 days after the transplantation showed that in 40% of the transplants only the recipient's hemopoietic cells proliferated, whereas the rest were mosaic and contained on the average less than 20% of donor's cells both in the syngeneic and in the semisyngeneic systems. These characteristics remained stable for at least 4 months. The data obtained suggest a single inflow of not less than 10 effective hemopoietic stem cells per graft. The clone stability indicated that during the steady-state hemopoiesis the cell exchange between various regions of the hemopoietic system was not great, if any.     

Mast cells accumulate in the renal capsule adjacent to transplanted pancreatic islets in rats

Mast cells are important mediators of normal angiogenesis, and participate in normal would healing, i.e. processes involved in pancreatic islet engraftment. The aim of the study was to evaluate if mast cells are present in islet grafts. For this purpose, male normoglycaemic Wistar-Furth rats were either untreated or syngeneically implanted with 250 islets under the renal capsule. The animals were killed 1 month later, and the kidneys and endogenous pancreas were removed, fixed and embedded in paraffin. The distribution of mast cells was studied in Alcian Blue stained sections. Mast cells were rarely encountered in endogenous islets, but were frequent in the renal capsule adjacent to islet grafts. Mast cells interspersed between graft endocrine cells were as rare as in the endogenous pancreas. We conclude that mast cells may contribute to the engraftment after islet transplantation.     

Changes in glucose-stimulated insulin secretion after long-term treatment of C57BL mice with glibenclamide

An insulin response to increased glucose concentrations could not be found in vivo and in vitro after long-term treatment of C57BL/KsJ and C57BL/6J mice with Glibenclamide. This missing stimulation of insulin secretion was not the result of an exhaustion of the islets or a disturbed (pro)insulin biosynthesis as demonstrated by measurements of insulin content of the islets and by in vitro (pro)insulin biosynthesis experiments. In the presence of glucose (15 mmol/l) theophylline increased the insulin secretion of isolated islets of Glibenclamide-treated mice to values similar to control islets. The insulin response to an i.p. glucose loading was found to be normal in comparison with control mice 1-2 weeks after the Glibenclamide treatment had been finished.     

Mouse (C57BL/KsJ) liver phosphofructokinase. Allosteric kinetics and age-related changes in the genetically diabetic state

The regulatory kinetic properties of phosphofructokinase partially purified from the livers of C57BL/KsJ mice were studied. The fructose 6-phosphate saturation curves were highly pH dependent. At a fixed MgATP concentration (1 mM), allosteric kinetics was observed in the range of pH studied (7.3 to 8.3) and the S0.5 values for fructose 6-phosphate decreased by about 0.2 to 0.3 mM for each 0.1-unit increment in pH. Allosteric effects on the sigmoidal response to fructose 6-phosphate: activation by AMP, NH4+, and glucose 1,6-bisphosphate, inhibition by MgATP2-, and synergistic inhibition between ATP and citrate, were all present at pH 8.0 to 8.2. Comparative kinetic studies with liver phosphofructokinase isolated from both the normal (C57BL/KsJ) and the genetically diabetic (C57BL/KsJ-db) mice of 9 to 10 and 15 to 16 weeks of age showed that the enzyme from the livers of diabetic mice exhibited decreased activity at subsaturating concentrations of fructose 6-phosphate. However, phosphofructokinase isolated from the livers of normal and genetically diabetic mice of 4 to 5 weeks of age showed no difference in kinetic properties. Thus, there appears to be a correlation between the change in properties of liver phosphofructokinase and the expression of hyperglycemia and obesity in the genetically diabetic mice. The decreased activity of liver phosphofructokinase in the older diabetic animals may well be one of the causes of the increased blood glucose levels. The results are also discussed in a general context with regard to the possible role of phosphofructokinase in the regulation of hepatic gluconeogenesis.