The insulin receptor (INSR) and the insulin growth factor 1 receptor (IGF1R) play important roles in the etiology of both diabetes mellitus and breast cancer. We aimed to evaluate the expression of... Show moreThe insulin receptor (INSR) and the insulin growth factor 1 receptor (IGF1R) play important roles in the etiology of both diabetes mellitus and breast cancer. We aimed to evaluate the expression of hormone and insulin-related proteins within or related to the PI3K and MAPK pathway in breast tumors of women with or without diabetes mellitus, treated with or without insulin (analogues).\nImmunohistochemistry was performed on tumor tissue of 312 women with invasive breast cancer, with or without pre-existing diabetes mellitus, diagnosed in 2000-2010, who were randomly selected from a Danish breast cancer cohort. Women with diabetes were 2:1 frequency matched by year of birth and age at breast cancer diagnosis to those without diabetes. Tumor Microarrays were successfully stained for p-ER, EGFR, p-ERK1/2, p-mTOR, and IGF1R, and scored by a breast pathologist. Associations of expression of these proteins with diabetes, insulin treatment (human insulin and insulin analogues) and other diabetes medication were evaluated by multivariable logistic regression adjusting for menopause and BMI; effect modification by menopausal status, BMI, and ER status was assessed using interactions terms.\nWe found no significant differences in expression of any of the proteins in breast tumors of women with (n = 211) and without diabetes (n = 101). Among women with diabetes, insulin use (n = 53) was significantly associated with higher tumor protein expression of IGF1R (OR = 2.36; 95%CI:1.02-5.52; p = 0.04) and p-mTOR (OR = 2.35; 95%CI:1.13-4.88; p = 0.02), especially among women treated with insulin analogues. Menopause seemed to modified the association between insulin and IGF1R expression (p = 0.07); the difference in IGF1R expression was only observed in tumors of premenopausal women (OR = 5.10; 95%CI:1.36-19.14; p = 0.02). We found no associations between other types of diabetes medication, such as metformin, and protein expression of the five proteins evaluated.\nIn our study, breast tumors of women with pre-existing diabetes did not show an altered expression of selected PI3K/MAPK pathway-related proteins. We observed an association between insulin treatment and increased p-mTOR and IGF1R expression of breast tumors, especially in premenopausal women. This observation, if confirmed, might be clinically relevant since the use of IGF1R and mTOR inhibitors are currently investigated in clinical trials. Show less
The aim of this thesis was to gain further insight into the role of glucagon in glucose homeostasis in healthy volunteers and type 2 diabetes mellitus (T2DM) patients, and to explore the novel... Show moreThe aim of this thesis was to gain further insight into the role of glucagon in glucose homeostasis in healthy volunteers and type 2 diabetes mellitus (T2DM) patients, and to explore the novel antisense glucagon receptor antagonist. Chapter 2 showed that the effect of meal replacers containing protein hydrolysate on plasma glucose lowering is limited in T2DM patients due to a collective increase of both insulin and glucagon levels. In chapter 3 and 4 a glucagon challenge test in healthy volunteers and T2DM was studied and showed a good reproducibility and no confounding changes in autonomic nervous system tone. T2DM patients respond profoundly different to a glucagon challenge test compared to healthy volunteers and the response to a glucagon challenge test in T2DM subjects is influenced by the type of therapy. Chapter 5 provided the first proof of pharmacology of a novel antisense glucagon receptor antagonist in humans, ISIS 325568, as reflected by a reduction in glucagon-induced glucose excursion by using a well-characterized glucagon challenge test. In chapter 6 we designed a semi-mechanistic model which simultaneously describes glucagon, plasma glucose, insulin and glucagon receptor internalization using data from glucagon challenges in healthy volunteers. Show less
The thesis contains a large study in which eight male hypocretin deficient narcolepsy with cataplexy patients and eight matched controls were enrolled. Blood was sampled before and on the 5th day... Show moreThe thesis contains a large study in which eight male hypocretin deficient narcolepsy with cataplexy patients and eight matched controls were enrolled. Blood was sampled before and on the 5th day of SXB administration. SXB was taken 2 times 3g per night for 5 consecutive nights. Both groups underwent 24-h blood sampling and many hormones (prolactin, Growth hormone, melatonin, ghrelin, leptin) were measured and compared before and during SXB treatment. A study using the golden standard on insuline sensitivity is decribed to compare insuline sensitivity between patients and controls, and between patients, before and during satisfactory SXB treatment. ANother study describes body and skintemperature differences between narcolepsy patients and controls. Another chapter describes a rarely described, common feature in narcolepsy, in which patients mistake the memory of a dream for a real experience. In another chapter describes that date of birth is not a risk factor for narcolepsy. Show less
This thesis aims to describe the role of glucose metabolism in human longevity and to translate the results to an intervention aimed at improving health in older age in the general population. In... Show moreThis thesis aims to describe the role of glucose metabolism in human longevity and to translate the results to an intervention aimed at improving health in older age in the general population. In the first part, we describe evidence for enhanced glucose metabolism in long-lived families. Middle-aged individuals predisposed to longevity were characterized by enhande insulin action at the level of the skeletal muscle, lower accumulation of intramyocellular lipids and lower nocturnal glucose levels. These results have provided the biological basis upon which health-promoting intervention in older age may be funded. In the second part, we have shonw that an internet-based intervention is effective at increasing physical activity and improving health in an older population. Furthermore, increasing total daily physical activity in sedentary elderly was shown to lead to better metabolic health. Show less
Neonatal health care is provided with medication and protocols for almost all morbidities. Before the use of these medicines is allowed, they are extensively studied and tested for efficacy and... Show moreNeonatal health care is provided with medication and protocols for almost all morbidities. Before the use of these medicines is allowed, they are extensively studied and tested for efficacy and safety. As patient population and knowledge on specific diseases changes with time, repeated evaluation of efficacy and safety of current used policies is of paramount importance. In this thesis six __Neonatal Pearls__ are presented: six relatively rare clinical conditions, of which a retrospective study evaluates the efficacy, safety and/or long term consequences of the current protocol. Despite their retrospective design and relatively small sample size, they are all of significant value and may serve as potential foundations for future protocol adjustments and randomized controlled trials. The evaluated clinical conditions and policies include: -Fetal, neonatal and developmental outcomes of lithium exposed pregnancies -Neonatal outcome in allo-immune thrombocytopenia after maternal treatment with antenatal intravenous immunoglobulin - Repeated courses of ibuprofen for closure of a patent ductus arteriosus - Use of rifampin in persistent coagulase negative staphylococcal bacteremia in neonates - Outcome and management in neonatal thrombocytopenia due to maternal idiopathic thrombocytopenic purpura - Short and long term outcome of neonatal hyperglycemia in very preterm infants Show less
This thesis describes the role of the brain in the regulation of peripheral triglyceride metabolism, in the context of the metabolic syndrome. Based on various pharmacological studies we described... Show moreThis thesis describes the role of the brain in the regulation of peripheral triglyceride metabolism, in the context of the metabolic syndrome. Based on various pharmacological studies we described the role of two hormones, insulin and glucagon-like peptide-1, in the production and clearance of triglycerides. We showed that insulin stimulates the uptake of (triglyceride-derived) fatty acids and that the brain plays an essential role in this process. Additionally, we showed that the glucagon-like peptide-1 receptor analogue exendin-4 decreases triglyceride production by the liver, albeit that the brain does not seem to be involved in this effect. Furthermore, we unraveled the mechanism underlying the effects of metformin, the first-line drug used to treat Type 2 Diabetes, on triglyceride metabolism. We showed that metformin lowers plasma triglyceride levels by stimulating the uptake and subsequent oxidation of triglycerides by the brown adipose tissue, and herewith provided new therapeutical opportunities for this drug. Finally, we showed that apolipoprotein A5, a stimulator of triglyceride hydrolysis and subsequent clearance from the plasma, plays a role in the central regulation of food intake, and herewith described a novel function for this apolipoprotein. Show less
The studies in this thesis contribute to the understanding of the role of the brain in insulin sensitivity. We demonstrate that disturbances in circadian rhythm resulting in alterations in SCN... Show moreThe studies in this thesis contribute to the understanding of the role of the brain in insulin sensitivity. We demonstrate that disturbances in circadian rhythm resulting in alterations in SCN output, can contribute to the development of insulin resistance. We also shown that insulin-stimulated glucose uptake by muscle and insulin-stimulated FA uptake by WAT is in part dependent on insulin action in the brain. These effects of circulating insulin on peripheral organs via the brain are abrogated by high-fat diet. These brain-dependent effects of insulin could reflect a similar situation for other hormones, for instance thyroid hormones. Furthermore, we demonstrate that topiramate improves insulin resistance by restoring insulin sensitivity in the brain, suggesting that therapeutical targets in the brain may offer challenging new approaches to treat insulin resistance of peripheral organs in T2DM. Show less
This thesis discusses the metabolic and endocrine characteristics of long-lived Dutch families. We found that familial longevity is marked by low thyroid function and preserved insulin senitivity.... Show moreThis thesis discusses the metabolic and endocrine characteristics of long-lived Dutch families. We found that familial longevity is marked by low thyroid function and preserved insulin senitivity. The second part of this thesis adresses the Gompertz law of mortality as an estimate of the rate of senescence Show less
The research on 3T3-L1 adipocytes described in this thesis demonstrates how two different types of cellular stress inducing agents, namely the vicinal thiol binding agent arsenite and the... Show moreThe research on 3T3-L1 adipocytes described in this thesis demonstrates how two different types of cellular stress inducing agents, namely the vicinal thiol binding agent arsenite and the conventional PKC-binding and -activating agent PMA act to increase glucose uptake in these cells. Whereas arsenite uses mainly the insulin-responsive GLUT4 transporter, PMA increases basal glucose transport through the GLUT1 transporter. As described in Chapter 3, arsenite-induced glucose uptake illustrates several requirements needed by any agent acting through GLUT4. These are, a tyrosine kinase activity, p38 MAPK activation and PKC-\lambda activity. Though PI-3' kinase activation is an essential step in insulin-signalling, this step is not required for arsenite-induced glucose uptake. Apparently, the need for tyrosine-kinase activity in arsenite induced glucose uptake resides in the ability to tyrosine-phosphorylate Cbl (see Chapter 3 Fig. 5). A further illustration of the importance of Cbl-tyrosine phosphorylation comes from our studies on rottlerin (Chapter 4). The ATP-depletion mediated by this pharmacological compound does not seem to be responsible for the observed inhibition of GLUT4 translocation (as was postulated by Kayali et al.[1]). Rather, aside from acting as an uncompetitive inhibitor of GLUT4, rottlerin hampers Cbl tyrosine phosphorylation, which leads to a 75% reduction in GLUT4 translocation (see Chapter 4, Fig. 3 and 4). Regrettably, the nature of the arsenite-induced tyrosine-kinase activity remains as of yet unidentified. Though the specific ability of arsenite to induce STAT5a tyrosine-phosphorylation in the mature adipocyte, should provide a straightforward tool to enable its identification (J.L Gonz_lez-Galindo, unpublished observations) Previously it had been demonstrated that insulin-induced p38 MAPK was involved in regulating the amount of glucose taken up by the cell without affecting GLUT4 translocation, suggesting some kind of intrinsic effect on the GLUT4 transporter itself [2]. Our observations on arsenite, a potent activator of p38 MAPK, illustrate a similar phenomenon in GLUT4-mediated stress-induced glucose uptake (see Chapter 3, Fig. 6). Subsequent research, described in a recently submitted manuscript, provides a detailed analysis of the involvement of p38 MAPK. These data demonstrate that p38 MAPK is involved in fine-tuning glucose uptake by regulating the turnover capacity of the GLUT4 transporter. A further note on the fine tuning of GLUT4-mediated glucose uptake comes from the observations on genistein, described in Chapter 5. This research suggests that in GLUT4 the turnover capacity for glucose can also be regulated through an intracellular ATP-binding Walker B motif akin to that described for GLUT1 [3]. Though further research is required to elucidate this mechanism, this theoretical resolution constitutes a significant step forwards towards understanding mechanisms in action after GLUT4 membrane translocation. If these observations are mechanistically linked in the cell remains to be elucidated. Aside from leading to enquiries into the mechanisms of insulin-induced glucose uptake, arsenite also opened up an avenue of more physiological research. We observed that arsenite-induced glucose uptake was sensitive to treatment with the insulin-resistance inducing agent dexamethasone. Subsequent analysis (described in Chapter 7) learned that although PI-3' kinase signalling is affected, in 3T3-L1 adipocytes the signalling pathway downstream is able to absorb this impediment. Rather, MKP-1 and -4 are upregulated in response to dexamethasone. Consequentially p38 MAPK activity is lost, leading to a reduction in glucose uptake. Given that MKP-4 is also upregulated in db/db- and ob/ob-mice [4], and that treatment of db/db mice with a glucocorticoid-receptor antagonist improves blood glucose levels [5;6], attenuation of p38 MAPK-signalling could be an important factor in type II diabetes. To enable the studies described in this chapter, a novel tool had to be developed. 3T3-L1 adipocytes have for long been inaccessible to ectopic expression of DNA. By the application of Lentivirus as described in Chapter 6, a large number of cells can be efficiently and reliably transduced. This novel tool will make the 3T3-L1 adipocyte readily amendable to routine molecular biological techniques, which will be of great benefit to the research field. In contrast to arsenite, PMA does not induce GLUT4 translocation, but acts solely through GLUT1. As illustrated in Chapter 8 of this thesis, in 3T3-L1 adipocytes the earliest and most PMA-sensitive PKC isoform is PKC-\betaII. But rather than activation, it is the concomitant degradation of this isoform which induces GLUT1 translocation. Further research (described in Chapter 9) highlighted the processes involved : First transcription of GLUT1, operating through the classical PKC-ERK-GLUT1 pathway. Second, translocation of GLUT1. This translocation is mediated by PKC-\lambda which is found associated with PKC-\betaII in the basal state. Thus upon degradation of the \betaII-isoform (or disruption of this complex by treatment with myristoylated pseudo-substrate peptides against \betaII) PKC-\lambda is released and free to act as a positional queue inducing translocation of the GLUT1 containing vesicles. Show less
