Type 1 diabetes in BioBreeding rats is critically linked to an imbalance between Th17 and regulatory T cells and an altered TCR repertoire.

Diabetes-prone BioBreeding (DP-BB) rats spontaneously develop type 1 diabetes mellitus (T1DM) on grounds of their MHC haplotype RT1(u) and a point mutation in the Gimap5 gene. In this study, we report that DP-BB rats exhibit an increasingly severe imbalance, in particular between Th17 and regulatory T (T(reg)) cells, within the first months of age. This can be assigned to an excess in effector T cells because neither the percentage nor the function of the T(reg) cells is compromised. Flow cytometric analysis of Vbeta segment usage and CDR3 spectratyping further suggest that the disturbed repertoire of peripheral T cells may also contribute to the development of T1DM in DP-BB rats. Importantly, expansion of T(reg) cells in vivo by means of a CD28 superagonistic Ab as well as adoptive transfer of T(reg) cells efficiently interferes with the development of T1DM in DP-BB rats, whereas treatment with conventional Th cells does not afford protection. Using a newly generated strain of enhanced GFP transgenic rats, we could further demonstrate that the transferred T(reg) cells persist in the recipient rats for several months and partially correct the imbalance between Th17 and T(reg) cells. Thus, our data support the hypothesis that unchecked effector T cell action and a disturbed T cell repertoire contribute to the development of T1DM in DP-BB rats, which may also have implications for a better understanding of the human disease.

Increase in Bone Mass Before Onset of Type 1 Diabetes Mellitus in Rats.

BACKGROUND/AIM: The typical insulin deficiency in type 1 diabetes mellitus has general effects on metabolism and also affects bone quality. MATERIALS AND METHODS: Two diabetic rat lines (BB/OK; BB.6KWR) and two non-diabetic rat strains (KWR and BB.14+18KWR), as control group, were included in the study. Bone mineral density, bone mineral content and body structure measurements were performed. The measurements took place before the onset of diabetes mellitus Results: A comparison of the groups showed increased bone density values of the diabetic rats in relation to the control groups. A new finding of increased bone density in the diabetic rats occurs. CONCLUSION: Diabetic rats showed no osteoporotic bone metabolism before the onset of clinically relevant type 1 diabetes mellitus, but rather increased bone metabolic activity.

High-fat diet protects BB/OK rats from developing type 1 diabetes.

BACKGROUND: It is well known that lipid metabolism plays an important role in the early stages of type 1 diabetes (T1D). For that reason, we examined factors that influence lipid metabolism of BioBreeding/Ottawa Kalsburg (BB/OK) rats that spontaneously develop an insulin-dependent T1D. METHODS: BB/OK female rats were fed a high-fat diet during pregnancy (Ssniff R-Z + 10% tallow) and their progeny were also given this diet up to an age of 30 weeks (n = 55) or 4 weeks (n = 14) to study gene expression of Pparg, Fasn, Lep, Adipoq, Repin1, Rarres 2, and Glut4 in adipose tissue. Forty-two BB/OK rats fed the normal diet (Ssniff R-Z) during pregnancy and the observation period served as controls. RESULTS: The high-fat diet significantly decreased diabetes frequency in BB/OK rats when compared with control rats (71 versus 95%, p = 0.002). Although this difference was also reflected in the male rats (68 versus 100%, p = 0.003), no significant variation was observed in female rats (73 versus 90%, p = 0.23). The high-fat diet resulted in significantly reduced mRNA expression of examined genes in subcutaneous adipose tissue, but not in visceral adipose tissue, except for Fasn and Repin1 expression. CONCLUSIONS: A high-fat diet seems to protect BB/OK rats from T1D in a sex-specific manner. The data suggest that a high-fat diet might influence fat accumulation and/or fat metabolism and prevent T1D development in male rats, which is supported by changes in adipose tissue gene expression.

Repin1 maybe involved in the regulation of cell size and glucose transport in adipocytes.

Replication initiator 1 (Repin1) is highly expressed in liver and adipose tissue and has been suggested as candidate gene for obesity and its related metabolic disorders in congenic and subcongenic rat strains. The cellular localization and function of Repin1 has remained elusive since its discovery in 1990. To characterize the role of Repin1 in adipocyte biology, we used siRNA knockdown technology to reduce the expression of Repin1 by electroporation of semiconfluent 3T3-L1 preadipocytes. Glucose transport, palmitate uptake as well as triglyceride content were measured. In paired samples of human visceral and subcutaneous adipose tissue, we investigated whether Repin1 mRNA expression is related to measures of fat accumulation and adipocyte size. We demonstrate that Repin1 increases during adipogenesis. RNA interference based Repin1 downregulation in mature adipocytes significantly reduces adipocyte size and causes reduced basal, but enhanced insulin stimulated glucose uptake into 3T3-L1 cells. Additionally, knockdown of Repin1 resulted in reduced palmitate uptake and significantly changed the mRNA expression of genes involved lipid droplet formation, adipogenesis, glucose and fatty acid transport. Furthermore, we found significant correlations between Repin1 mRNA expression in human paired visceral and subcutaneous adipose tissue and total body fat mass as well as adipocyte size. We have identified a potential role for Repin1 in the regulation of adipocyte size and expression of glucose transporters GLUT1 and GLUT4 in adipocytes.

Determination of glutamic acid decarboxylase (GAD65) in pancreatic islets and its in vitro and in vivo degradation kinetics in serum using a highly sensitive enzyme immunoassay.

Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37 degrees C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65.

Involvement of cocaine-amphetamine regulated transcript in the differential feeding responses to nociceptin/orphanin FQ in dark agouti and Wistar Ottawa Karlsburg W rats.

Wistar Ottawa Karlsburg W (WOKW) rats and their controls, dark agouti (DA), present different features: in particular, DA rats are lean, while the WOKW are obese and present symptoms of hypertension, dyslipidemia, hyperinsulinemia, and impaired glucose tolerance. The present study tested the hypothesis that these two strains would demonstrate different sensitivity to nociceptin/orphanin FQ (N/OFQ). N/OFQ was injected into the lateral brain ventricle (LBV) of sated DA and WOKW rats, and corticosterone levels in both strains were measured after LBV injection of N/OFQ. LBV N/OFQ injections dose-dependently produced a significant increase in food intake (4 h) in DA rats, but not in WOKW. However, corticosterone levels were increased by N/OFQ to a greater degree in WOKW than in DA rats. Gene sequencing and gene expression of ORL1 receptor and cocaine-amphetamine regulated transcript (Cart) peptide were evaluated to study the difference in N/OFQ-induced feeding behavior in the two strains. WOKW rats had a different amino acid sequence of Cart peptide and a significantly higher expression of Cart in the hypothalamus. The present data show that DA and WOKW rats demonstrate different sensitivity to N/OFQ, and suggest that Cart peptide might be the underlying mechanism of this difference.

Is there an autoimmune process in bone? Gene expression studies in diabetic and nondiabetic BB rats as well as BB rat-related and -unrelated rat strains.

It is well known that type 1 diabetes is associated with a decrease in bone mass and delayed healing of fractures in human and in animal models of type 1 diabetes. Using well- and poorly compensated diabetic BB/O(ttawa) K(arlsburg) rats spontaneously developing insulin-dependent type 1 diabetes, it was recently shown that, in contrast to all other tissues studied, bone is most influenced by metabolic state and seems to be regulated in a manner different from other organs. Therefore, we studied the expression of additional genes (Bmp-1, Bmp-4, Vegf, Bglap, Il-1b, Infg, Tnfa, Calca, Sp1, Yy1) in bone of nondiabetic BB rats compared with newly diagnosed and well- and poorly compensated diabetic rats as well as two nondiabetes-prone congenic BB.SHR rats, BB rat-related (WOKW) and -unrelated rat strains (F344). Six males of each group were euthanized, the tibial bone was removed, and total RNA was extracted, transcribed in complementary DNA, and used for real-time PCR. In a comparison of nondiabetic with diabetic groups, the relative gene expression was reduced by >80% in newly diagnosed and in well-compensated diabetic BB/OK rats. The gene expression in poorly compensated rats increased significantly in 7 of 10 genes and was comparable with those of nondiabetic BB/OK rats. In a comparison of gene expression between diabetes-prone BB/OK and nondiabetes-prone BB.1K, BB.4S, WOKW, and F344 rats, there were no significant differences between newly diagnosed and well-compensated BB/OK diabetic rats and nondiabetic BB.1K, BB.4S, WOKW, and F344 rats. On the basis of these findings, we concluded that spontaneous diabetes influences bone gene expression in BB/OK rats, which may be attributed to the genetically determined autoimmune process not only affecting pancreatic beta-cells but also bone formation and resorption.

Potential involvement of nicotinamide N-methyltransferase in the pathogenesis of metabolic syndrome.

BACKGROUND: Metabolic syndrome is a complex disorder characterized by the presence of insulin resistance (IR), type 2 diabetes mellitus (T2DM), impaired glucose tolerance (IGT), or impaired fasting glucose (IFG), plus at least two of the following conditions--hypertension, hyperlipidemia, obesity, and microalbuminuria. Metabolic syndrome exposes patients to a greater risk of developing cardiovascular disease (CVD) and is often associated with elevated levels of homocysteine (Hcy). In the current work, we analyzed the expression of nicotinamide N-methyltransferase (NNMT). Because NNMT is involved in Hcy metabolism and participates in the regulation of the cellular and plasma levels of this compound, we explored the role played by the enzyme in metabolic syndrome. METHODS: Real-time PCR, immunohistochemistry, western blot analysis, and catalytic activity assay were performed to evaluate NNMT expression levels in adipose tissue from 10 Wistar Ottawa Karlsburg W (WOKW) rats, which are an animal model for metabolic syndrome, and from 10 Dark Agouti (DA) rats as the disease-resistant control strain. RESULTS: NNMT mRNA, protein, and activity levels were significantly higher in adipose tissue obtained from WOKW rats compared with those observed in adipose tissue of DA rats. CONCLUSION: Data reported in this study represent the first evidence supporting the hypothesis that NNMT could play an important role in the pathogenesis of metabolic syndrome and could have a potential for the development of a targeted therapy.

Phenotypic and genetic analyses of subcongenic BB.SHR rat lines shorten the region on chromosome 4 bearing gene(s) for underlying facets of metabolic syndrome.

Congenic BB.SHR (D4Got41-Npy-Tacr1; BB.4S) rats develop an incomplete metabolic syndrome with obesity, hyperleptinemia, and dyslipidemia compared with their progenitor strain, the diabetes-prone BB/OK rat. To narrow down the underlying gene(s), two subcongenic BB.SHR rat lines, briefly termed BB.4Sa and BB.4Sb, were generated. Male BB.4S (n = 20), BB.4Sa (n = 24), and BB.4Sb (n = 26) were longitudinally characterized for facets of the metabolic syndrome and analyzed for expression of genes located in the region of interest in liver and blood. Body weight gain was comparable, serum triglycerides and leptin were significantly increased, and total cholesterol and HDL-cholesterol ratio were decreased in BB.4S compared with both subcongenics. Serum insulin was significantly higher in BB.4S and BB.4Sa than in BB.4Sb. The adiposity index showed a graduated decrease from BB.6S to BB.4Sb. Obvious differences in relative expression were found in 6 of 10 genes in liver and in 2 of 9 genes in blood. Only one gene, the eukaryotic translation initiation factor 2alpha kinase 3 (Eif2ak3 also called Perk or Pek), was significantly less expressed in liver and in blood of both subcongenic BB.4Sa and BB.4Sb compared with their "parental" BB.4S rats. Based on the phenotype and genotype in BB.4S and its subcongenic derivatives, the most important region on chromosome 4 can be said to lie between D4Got72 and Tacr1. Eif2ak3 is mapped in this region. Considering the function of Eif2ak3, it may be a candidate gene for the development of glucose intolerance found in both subcongenics but not in BB.4S. Allelic variants between BB/OK and SHR could influence Eif2ak3 function, possibly leading not only to glucose intolerance but also to the disturbances in hepatic and renal function found in human Wolcott-Rallison syndrome.

Insulin receptor substrate-4 is expressed in muscle tissue without acting as a substrate for the insulin receptor.

Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.

Histomorphometric evaluation of the influence of the diabetic metabolic state on bone defect healing depending on the defect size in spontaneously diabetic BB/OK rats.

Insulin-dependent type 1 diabetes mellitus (IDDM) has been shown to alter the properties of bone and impair bone repair in both humans and animals. The objective of this study was the detailed histomorphometric evaluation of the influence of the diabetic metabolic state on bone formation and remodeling during bone defect healing depending on the defect size in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats, a rat strain that represents a close homology to IDDM in man. Based on blood-glucose values at the time of surgery, postoperative blood-glucose course, and postoperative insulin requirements, 80 spontaneously diabetic BB/OK rats were divided into groups with well-compensated or poorly compensated metabolic state. Forty LEW.1A rats served as normoglycemic controls. Using a Kirschner wire, bone defects of different sizes were created proximal to the knee joint space in both femora. Ten animals from each group were killed on postoperative days 7, 14, 24, and 42, and specimens were processed undecalcified for quantitative bone histomorphometry. In terms of bone histomorphometry, our study did not show any differences in bone defect healing between the groups where the defect size was 0.4 mm. Larger bone defects (0.8 mm) only showed significant differences in the structural calculations after the 24th postoperative day exclusively in poorly compensated diabetic rats compared to well-compensated diabetic and control rats (P < 0.05 or P < 0.01). In bone defect sizes more than 1.2 mm, severe mineralization disorders occurred within the first 14 days exclusively in rats with poorly compensated diabetic metabolic state with a highly significant (P < 0.001) or significant (P < 0.01) decrease of all fluorochrome-based parameters of mineralization, apposition, formation, and timing of mineralization in comparison to spontaneously diabetic rats with well-compensated diabetic metabolic state and control rats. These results demonstrate that the bone repair of minor bone defects (0.4 mm) is independent of the diabetic metabolic state in spontaneously diabetic BB/OK rats. In larger bone defects (more than 0.8 mm), the bone defect healing in spontaneously diabetic BB/OK rats is impaired exclusively in poorly compensated diabetic metabolic states. This study suggests that strictly controlled insulin treatment resulting in a well-compensated diabetic metabolic state will ameliorate the impaired histomorphometric parameters of IDDM bone defect healing.

Long-term arterial pressure in spontaneously hypertensive rats is set by the kidney.

OBJECTIVES: We investigated whether arterial pressure in spontaneously hypertensive rats (SHR) can be normalized by a kidney graft from normotensive histocompatible donors. In addition, the effect of differential genetic predisposition to hypertension of recipients of an SHR kidney on the development of post-transplantation hypertension was studied. METHODS: SHR were transplanted with a kidney from congenic rats (BB.1K) homozygous for a 2 cM segment of SHR chromosome 20, including the major histocompatibility complex class Ia and class II genes. BB.1K and F1 hybrids (F1H, SHR x Wistar-Kyoto rats) were transplanted with an SHR kidney and the development of renal post-transplantation hypertension was monitored. RESULTS: Thirty days after renal transplantation, mean arterial pressure (MAP) was 116 +/- 4 mmHg in SHR with a BB.1K kidney (n = 8) versus 168 +/- 2 mmHg in sham-operated SHR (n = 10); P < 0.001. Cumulative renal sodium balance (mmol/100 g body weight) over 21 days after bilateral nephrectomy was 6.8 +/- 0.6 in SHR with a BB.1K kidney versus 10.8 +/- 1.6 in sham-operated SHR (P < 0.05). Within 60 days of transplantation, MAP increased in BB.1K and in F1H transplanted with an SHR kidney (n = 7 per group) by 38 +/- 5 mmHg and 43 +/- 8 mmHg, respectively. CONCLUSIONS: In SHR, arterial pressure can be normalized by a kidney graft from normotensive donors. The genetic predisposition of the recipients to hypertension does not modify the rate and the extent of the arterial pressure rise induced by an SHR kidney graft.

Cytokine sensitivity of Langerhans' islets of diabetes-prone BB/OK rats under hypoglycemic conditions.

Islets of Langerhans isolated from diabetes-prone BB/OK rats were exposed to interleukin-1beta (IL-1beta) or to a combination of tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) under hypoglycemia at glucose concentrations of 2.2 and 3.2 mmol/l or in the presence of stimulatory conditions at 6.0 and 11 mmol/l glucose. For estimating cytokine effects the islets were functionally assayed by measurement of glucose stimulated insulin secretion. Pancreatic islets exposed for 24 h to IL-1beta at a glucose concentration of 6.0 mmol/l exhibited a reduced insulin secretion following a 48h recovery period compared to islets which were cytokine treated at 2.2 or 3.2mmol/l glucose, respectively. Islets pre-exposed for 24h to TNF-alpha plus IFN-gamma at 2.2, 3.2 or 6.0 mmol/l glucose displayed no alterations of insulin secretion following a 48 h regeneration. A temporary (3 h) influence of IL-1beta under hyperglycemic conditions at 11 mmol/l glucose caused a reduction of the subsequent insulin secretion of Langerhans' islets prior incubated for 24 h at 6.0 mmol/l glucose without cytokines, but not of islets precultured at 2.2 mmol/l glucose. In contrast, a 3 h treatment with TNF-alpha plus IFN-gamma at 11 mmol/l glucose did not affect insulin secretion of islets prior held at 6.0 mmol/l glucose, whereas a transient exposure for 6h to IL-1beta as well as TNF-alpha plus IFN-gamma under similar conditions diminished insulin secretion of islets preincubated at 2.2 or 6.0 mmol/l glucose. In conclusion, hypoglycemia reduces the sensitivity of BB/OK rat islets to IL-1beta, whereas a slight elevation of glucose concentration to 6.0 mmol/l increases again their vulnerability. TNF-alpha plus IFN-gamma at concentrations capable to decrease insulin secretion of islets during hyperglycemia do not affect the insulin output in a range between 2.2 and 6.0 mmol/l glucose. During glucose stimulation at 11 mmol/l islets' insulin secretory machinery is protected from IL-1beta as well as TNF-alpha plus IFN-gamma for 3 h by a preceding 24 h hypoglycemia, but its vulnerability is restored within additional 3 h.

Endocrine pancreas histology of congenic BB-rat strains with reduced diabetes incidence after genetic manipulation on chromosomes 4, 6 and X.

Congenic BB.SHR rat strains were established by crossing of spontaneously diabetic BB/OK rats and diabetes-resistant SHR rats. Chromosomal regions on which the genes Iddm 4 (BB.6s), Iddm6 (BB.Xs) and Iddm 2 (BB.LL) are located were exchanged. As a result of genetic manipulation diabetes incidence was markedly reduced from 80% in BB/OK to 50% in BB.SHR (Chr. X), to 14% in BB.SHR (Chr. 6) and to 0% in BB.LL rats. Pancreata of these newly generated BB.SHR rats were investigated histologically. In newly diagnosed diabetic rats of congenic strains pancreatic insulin content (BB.6s: p < 0.05; BB.Xs p < 0.01) and relative volume of insulin-positive cells (BB.Xs: p < 0.001) were significantly higher than in BB/OK rats. The degree of insulitis was not different in 90-day-old and newly diagnosed diabetic animals. Surprisingly, in 30-day-old rats we observed an increase of the degree of insulitis with decreasing diabetes incidence. We suppose that by an earlier occurrence of the immunological beta-cell destruction, a part of the animals is able to develop a secondary diabetes resistance. The exchange of the BB-lymphopenia gene by that of SHR-rats prevented the development of hyperglycaemia without altering the auto-reactive immune response, which could be observed in all animals investigated.

Prophylactic insulin treatment of diabetes-prone BB/OK rats by application of a sustained release insulin implant.

Normoglycemic diabetes-prone BB/OK rats aged 33, 45 or 75 days were subjected to prophylactic insulin treatment by means of a single subcutaneous application of a sustained release insulin implant. The single application of a sustained release insulin implant decreased the incidence of diabetes or delayed the onset of the disease in BB/OK rats of all treatment groups. Prophylactic insulin administration caused a transient hypoglycemic period accompanied by an inhibition of glucose stimulated insulin secretion and a decrease of the insulin content of Langerhans' islets as detectable in vitro. Compared to islets of normoglycemic controls pancreatic islets isolated from hypoglycemic BB/OK rats within 7-21 days after the insulin application at 45 days of age displayed a decreased susceptibility of the cells to complement-dependent cytotoxicity of the monoclonal islet cell surface antibody (ICSA) K14D10 but not to the cytotoxic effect of the ICSA M3aG8. The appearance of complement-dependent antibody-mediated cytotoxicity to islet cells and pancreatic exocrine cells in serum regarded as a sign of immune dysregulation in BB/OK rats seems not to be affected by insulin prophylaxis and was detectable during hypoglycemia as well as in the subsequent normoglycemic state. In conclusion, BB/OK rats of different age can be protected from diabetes by a single application of a sustained release insulin implant. Insulin and/or hypoglycemia seem to influence the expression of cell surface antigens, thus render the islets of Langerhans less vulnerable to immune cytolysis, whereas the appearance of humoral immunological abnormalites is not affected.

Stress response of pancreatic islets from diabetes prone BB rats of different age.

Protective and/or repair mechanisms are thought to be activated in pancreatic beta cells in response to injury during insulitis. Manifestation of type-1 diabetes may depend on an imbalance between beta cell damage and repair. To prove this hypothesis, the ability of collagenase-isolated islets to respond to heat stress depending on the age of BB rats was investigated. The islets were exposed either to 44 degrees C (HS) or 37 degrees C (control) for 30 min and then kept at 37 degrees C for 5 h. Immediately and 5 h after heat shock, insulin secretion in response to 20 mmol/l glucose and total protein synthesis of heat-exposed islets were significantly diminished as compared with controls. The islet proteins were separated by SDS-PAGE followed by immunoblotting. Islets from BB rats at an age of 6-90 days responded to heat shock with the expression of major heat shock protein 70 (HSP 70). Islets from 3-day old rats, however, did not respond with induction of HSP 70. In contrast we could detect inducible HSP 70 in islets from 3-day old diabetes-resistant LEW rats. In islets from 90-day old BB rats we observed a decreased amount of HSP 70 compared with islets from 9-, 12-, 30- and 60-day old animals. There was also a higher extent of HSP 70 to observe in islets from 90-day old LEW rats as compared with 90-day old BB rats. Differences in HSP 70 expression between islets of 3-day old BB and LEW rats and other age groups of BB rats might represent distinct stages of maturation of islets whereas diminished expression of HSP 70 in islets of 90-day old BB rats at the age of high probability of developing diabetes might result from reduced ability to induce protective mechanisms.

High-fat diet-induced met-hemoglobin formation in rats prone (WOKW) or resistant (DA) to the metabolic syndrome: effect of CoQ10 supplementation.

The aim of this study was to evaluate the effects of a high-fat diet (HFD) on oxidative indexes in WistarOttawaKarlsburg W (WOKW) rats used as a model of metabolic syndrome in comparison with Dark Agouti (DA) rats used as a control strain. This syndrome is defined by the occurrence of two or more risk factors including obesity, hypertension, dyslipidemia, and insulin resistance. Forty rats were used in the study and the effect of HFD was evaluated in terms of body weight and both hemoglobin and CoQ oxidative status. Moreover, 16 rats (8 of each strain) were supplemented with 3 mg/100 g b.w. of CoQ10 for 1 month in view of its beneficial properties in cardiovascular disease due to its antioxidant activity in the lipid environment. HFD promoted an increase in body weight, in particular in WOKW males, and in the methemoglobin (met-Hb) index in both strains. Moreover, HFD promoted endogenous CoQ10 oxidation. CoQ10 supplementation was able to efficiently counteract the HFD pro-oxidant effects, preventing met-Hb formation and CoQ oxidation.

Liver-restricted Repin1 deficiency improves whole-body insulin sensitivity, alters lipid metabolism, and causes secondary changes in adipose tissue in mice.

Replication initiator 1 (Repin1) is a zinc finger protein highly expressed in liver and adipose tissue and maps within a quantitative trait locus (QTL) for body weight and triglyceride (TG) levels in the rat. The QTL has further been supported as a susceptibility locus for dyslipidemia and related metabolic disorders in congenic and subcongenic rat strains. Here, we elucidated the role of Repin1 in lipid metabolism in vivo. We generated a liver-specific Repin1 knockout mouse (LRep1(-/-)) and systematically characterized the consequences of Repin1 deficiency in the liver on body weight, glucose and lipid metabolism, liver lipid patterns, and protein/mRNA expression. Hyperinsulinemic-euglycemic clamp studies revealed significantly improved whole-body insulin sensitivity in LRep1(-/-) mice, which may be due to significantly lower TG content in the liver. Repin1 deficiency causes significant changes in potential downstream target molecules including Cd36, Pparγ, Glut2 protein, Akt phosphorylation, and lipocalin2, Vamp4, and Snap23 mRNA expression. Mice with hepatic deletion of Repin1 display secondary changes in adipose tissue function, which may be mediated by altered hepatic expression of lipocalin2 or chemerin. Our findings indicate that Repin1 plays a role in insulin sensitivity and lipid metabolism by regulating key genes of glucose and lipid metabolism.

Gene expression profile in bone of diabetes-prone BB/OK rats fed a high-fat diet.

A high-fat diet (HFD) has been recognized as a risk factor for diseases such as dyslipidemia, atherosclerosis, obesity, and osteoporosis. However, studies analyzing gene expression after HFD in bone are rare. That prompted us to analyze the expression of selected genes in bone of 4-week-old diabetes-prone B(io)B(reeding) rats. Two breeding pairs were fed a HFD (+10 % tallow) or were fed a normal diet (ND; Ssniff R-Z) before mating and afterward during pregnancy. After the birth of progeny, parents continued to be given HFD or ND until the progeny was weaned (3 weeks). Thereafter, offspring were weaned and were fed the same food as their parents up to an age of 4 weeks. Body weight was measured at an age of 4 weeks, and subsequently 13 HFD rats and 13 ND rats were killed and the tibial bone was harvested to analyze the expression of 53 genes in bone. All rats fed HFD were significantly heavier than rats fed ND after 3 and 4 weeks. The diet also influenced the expression of genes in bone. There were significant differences in 20 out of 53 genes studied between rats fed HFD compared with rats fed ND. Four out of 20 had a lower and 17 out of 20 genes a higher expression in HFD rats, but differences in gene expression showed obvious differences between males and females. There were only two genes that were similarly different between males and females: Bmp4 and Atf4. Two genes, Foxg1 and Npy, were inversely expressed in males and females. It seems that the gene expression is differently regulated by diet during pregnancy and later in life between males and females. Nevertheless, it cannot be excluded that HFD also acts as an epigenetic factor in the development of offspring in utero.

Light rhythm and diet differently influence facets of the metabolic syndrome in WOKW rats.

It has previously been shown that high-calorie diet alters the function of the mammalian circadian clock and that obesity has an influence on circadian organization of hormone secretion. That prompted us to test whether inbred Wistar Ottawa Karlsburg W (RT1(u)) (WOKW) rats developing facets of the metabolic syndrome show changes in their metabolic profiles under different feeding conditions (high-fat, high-sugar versus control diet) and under two different 12 h:12 h light-dark (LD) cycles. At the age of four weeks, these rats were divided into four groups. Groups 1 and 2 were kept under initial LD cycle (lights on at 05:00 h). Group 1 was fed with a normal rat diet while group 2 received a high-fat, high-sugar diet from 10 up to the age of 21 weeks. Groups 3 and 4 were kept under a shifted LD cycle (lights on at 11:00 h). Group 3 was given a normal diet while group 4 received a high-fat, high-sugar diet from an age like groups 1 and 2. Several metabolic traits were studied during the observation period of 21 weeks. The blood samples were obtained 2 h before lights off. Body weight gain (P < 0.001), leptin (P < 0.001), triglycerides (P < 0.001) and cholesterol (P < 0.05) were significantly reduced in group 4 versus group 2, but comparable between control groups (1 versus 3). The insulin concentrations were reduced in groups 3 and 4 versus groups 1 and 2 without effect of diet. In conclusion, the results provide evidence that light conditions influence diet induced changes in phenotypic traits like body weight gain, lipids as well as hormone levels (insulin and leptin) in WOKW rats.