Karsten Wassermann

GLP-1 derivative liraglutide in rats with β-cell deficiencies: influence of metabolic state on β-cell mass dynamics

Liraglutide is a long‐acting GLP‐1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and β‐cell mass in rat models of β‐cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats. When liraglutide was dosed s.c. at 150 μg kg−1 b.i.d. for 6 weeks in ZDF rats 6–8 weeks of age at study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mM lower compared to vehicle (P<0.0002), and plasma insulin was 2–3‐fold higher during a normal 24‐h feeding period (P<0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect observed on 24‐h glucose profiles was mediated by a reduction in food intake, which persisted throughout the study and averaged 16% (P<0.02). Histological analyses revealed that β‐cell mass and proliferation were significantly lower in prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle‐treated animals that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide‐treated animals were no longer completely normoglycemic and the β‐cell mass was significantly increased compared to overtly diabetic vehicle‐treated animals, while β‐cell proliferation was unaffected. In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again, part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on β‐cell mass was observed in these virtually normoglycemic animals. In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of β‐cell deficiencies, and the in vivo effect of liraglutide on β‐cell mass may in part depend on the metabolic state of the animals.

Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice.

In the mouse, ovariectomy (OVX) leads to significant reductions in cancellous bone volume while estrogen (17β‐estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2‐dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) α and β expression of primary culture MSCs isolated from OVX and sham‐operated mice. MSCs, treated in vitro with 10−7 M E2, displayed a significant increase in ERα mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERβ mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up‐regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF‐β1, BMP‐2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERα was the predominant form expressed in MSCs obtained from both OVX and sham‐operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERα expression, osteogenic gene expression and, inhibits apoptosis and ERβ expression in MSCs obtained from OVX and sham‐operated mice. Co‐expression of ERα, but not ERβ, and osteogenic differentiation markers might indicate that ERα function as an activator and ERβ function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERα activation. J. Cell. Biochem. Suppl. 36: 144–155, 2001.

The dual PPARα/γ agonist, ragaglitazar, improves insulin sensitivity and metabolic profile equally with pioglitazone in diabetic and dietary obese ZDF rats

1 In 6‐ and 10‐week‐old obesity‐prone (fa/fa) Zucker diabetic fatty (ZDF) rats, effects of prevention and intervention therapies, respectively, were compared between PPARα/γ agonist, ragaglitazar (RAGA) and separate PPARγ and α agonists, pioglitazone (PIO) and bezafibrate (BF). 2 In a separate study, lean (+/+) ZDF rats fed highly palatable chow to induce dietary obesity and insulin resistance were treated similarly. To test insulin‐secretory capacity, all animals underwent a hyperglycaemic clamp. 3 Insulin sensitivity was improved equally by RAGA and PIO in fa/fa rats subjected to both prevention and intervention treatments (e.g., prevention HOMA‐IR: −71 and −72%, respectively), as was hyperglycaemia (both −68%). BF had no effect on either parameter in any study. Plasma lipids were markedly reduced (by 48–77%) by RAGA in all studies, equivalent to PIO, but to a greater extent than BF. 4 RAGA improved β‐cell function (HOMA‐β) more than three‐fold with prevention and intervention therapies, whereas PIO showed improvement only in intervention therapy. Consistent with improved insulin sensitivity, glucose infusion rate during the clamp was 60% higher in RAGA‐treated animals subjected to prevention therapy, but there was little additional insulin‐secretory response, suggesting that insulin secretion was already maximal. 5 Thus, RAGA and PIO equally improve metabolic profile in ZDF rats, particularly when administered early in the course of diabetes. They also improve β‐cell function, although this is better demonstrated through indices incorporating fasting insulin and glucose concentrations than through the hyperglycaemic clamp technique in this model.

Biomonitoring of genotoxic exposure among stainless steel welders

A biosurvey in the Danish metal industry measured the genotoxic exposure from stainless steel welding. The study comprised measurements of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), unscheduled DNA synthesis (UDS) in peripheral lymphocytes and serum immunoglobulin G. Environmental monitoring of welding fumes and selected metal oxides, biomonitoring of chromium and nickel in serum and urine and mutagenic activity in urine, and evaluation of semen quality were also done. Manual metal arc (MMA) welding and tungsten inert gas (TIG) welding were the dominant welding processes. A higher frequency of chromosomal aberrations, classified as translocations, double minutes, exchanges and rings, was observed in stainless steel welders than in non-welders. SCE was lower in welders working with both MMA and TIG welding than in reference persons. N-Acetoxy-N-acetylaminofluorene (NA-AAF)-induced UDS was lower in 23 never-smoking welders than in 19 unexposed never-smokers. Smoking was a confounding factor resulting in significantly higher CA, SCE, NA-AAF binding to DNA and mutagenic activity in urine. Age was also a confounder: CA, SCE, NA-AAF binding to DNA and UDS increased significantly with age. No significant correlation between SCE and CA or between CA and UDS was found. UDS decreased significantly with increasing lymphocyte count and a higher lymphocyte count was seen in MMA welders than in reference persons and in smokers than in non-smokers. Differences in the composition among lymphocytes in exposed persons compared with non-exposed are suggested. MMA welding gave the highest exposure to chromium, an increased number of chromosomal aberrations and a decrease in SCE when compared with TIG welding. Consequently improvements in the occupational practice of stainless steel welding with MMA is recommended.

Selective Estrogen Receptor Modulator Effects in the Rat Brain

The effects in the brain of selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene have not yet been fully elucidated. Based upon the hypothesis that serotonin (5-HT)-steroid hormone interactions are important in mood regulation, we have compared six SERMs (tamoxifen, raloxifene, levormeloxifene, NNC 45-0781, NNC 45-0320, NNC 45-1506) with 17β-estradiol (E2) in terms of their ability to regulate mRNA levels of estrogen receptor (ER)α, ERβ, 5-HT1A receptor, and 5-HT reuptake transporter (SERT) in the midbrain, amygdala, and hypothalamus of ovariectomized (OVX) rats. Female rats (n = 6/group, 8 groups total) were OVX and allowed to recover for 2 weeks. During the third post-OVX week, rats were injected subcutaneously with E2 (0.1 mg/kg) or one of the SERMs (5 mg/kg) once per day for 7 days. Twenty-four hours after the last injection, tissue was collected for the determination of mRNA levels by ribonuclease protection assay (RPA). E2 treatment significantly decreased mRNA levels for ERα, ERβ, and SERT in midbrain and ERα in hypothalamus. Tamoxifen increased ERβ mRNA levels in hypothalamus, while raloxifene increased ERβ mRNA levels in amygdala. NNC 45-0320 decreased ERα mRNA in hypothalamus and decreased ERβ mRNA in amygdala. These results suggest that while SERMs are not full estrogen receptor agonists in the brain, the agonist/antagonist profiles for individual SERMs may differ among brain areas. This raises the possibility of developing new SERMs for selective functions in specific brain areas.

Cellular Resistance to Cancer Chemotherapy

Publisher Summary One of the most challenging aspects of cancer chemotherapy is the problem of resistance to clinical drugs. The reasons for clinical resistance may include pharmacokinetic or cell kinetic factors. It is generally accepted that cellular drug resistance is one of the major reasons that treatment fails. The mechanisms of resistance depend on several factors and circumstances that have given rise to the following classifications: (1) natural ( de novo or intrinsic) versus acquired resistance, (2) experimental versus clinical resistance, (3) resistance developed in rodent versus in human cell lines, (4) in vitro versus in vivo resistance and ( 5 ) low versus high degree of resistance. In addition, the dose schedule may affect resistance. Basically, cellular resistance depends on the biological possibilities which are available for a mammalian cell to escape cellular injury from a cytotoxic drug. Consequently, the mechanisms of resistance found in one cellular system may also occur in another. Considering drug resistance from this point of view, all mechanisms described may be relevant. The chapter begins with a description of different mechanisms of resistance, even though they may have been documented in only one of the classes of resistance, and focuses on cellular resistance to cancer chemotherapy.

Dissociation of antihyperglycaemic and adverse effects of partial perioxisome proliferator-activated receptor (PPAR-γ) agonist balaglitazone

Balaglitazone is a novel thiazolidinedione in clinical development for the treatment of type 2 diabetes. Common side effects associated with PPARgamma receptor agonists are weight gain, oedema and adipogenesis. Balaglitazone is a selective partial PPARgamma agonist and it has been speculated that such compounds have a more favourable safety margin than full agonists. We have compared impact of equi-efficacious antihyperglycaemic doses of balaglitazone with full PPARgamma agonist rosiglitazone on body fluid accumulation, cardiac enlargement, and adipogenesis. Equi-efficacious antihyperglycaemic doses (ED(90)) of balaglitazone (3 mg/kg/day) and rosiglitazone (6 mg/kg/day) were determined in male diabetic db/db mice. In adult male rats treated for up to 42 days, feeding, drinking, anthropometry, and plasma volumes were measured. Total plasma volume was measured with dye dilution technique. Compared to vehicle, rosiglitazone consistently increased food intake throughout the 42 day treatment period. In contrast, balaglitazone increased food intake in the last week of the experiment. However, both rosiglitazone and balaglitazone increased water intake. After 42 days, rosiglitazone treated rats displayed significantly elevated adiposity. Rosiglitazone increased total blood and plasma volumes throughout the treatment. Twenty-one days of balaglitazone treatment had no significant impact on blood or plasma volumes, whilst 42 days of balaglitazone increased plasma volume but to a significantly lesser extent than seen for rosiglitazone (vehicle: 46.1+/-1.5; balaglitazone: 50.8+/-1.21; rosiglitazone: 54.6+/-1.6 ml/kg). Heart weight was significantly elevated only in rosiglitazone treated animals. At doses inducing comparable antihyperglycaemic control, the full PPARgamma agonist, rosiglitazone, induces more pronounced body fluid retention and heart enlargement than seen for the partial PPARgamma agonist, balaglitazone. Thus, partial agonists may pose safer alternative to current anti-diabetic therapy with full PPARgamma agonist.

Synthesis and pharmacology of a novel pyrrolo[2,1,5-cd] indolizine (NNC 45-0095), a high affinity non-steroidal agonist for the estrogen receptor.

1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties. NNC 45-0095 binds with high affinity to the estrogen receptor (IC50=9.5 nM) and exhibits full protection of bone loss in the ovariectomized mouse model for post-menopausal osteoporosis.

Design and synthesis of novel PPARα/γ/δ triple activators using a known PPARα/γ dual activator as structural template

Abstract Using a known dual PPARα/γ activator (5) as a structural template, SAR evaluations led to the identification of triple PPARα/γ/δ activators (18–20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARα/γ/δ activation.

Synthesis and estrogen receptor binding affinities of novel pyrrolo[2,1,5-cd]indolizine derivatives

A series of pyrrolo[2,1,5-cd]indolizine derivatives has been synthesized and evaluated as ligands for the estrogen receptor. Properly substituted mono- and di-hydroxy derivatives showed binding in the low nanomolar range in accordance with their structural resemblance to estrogen.