Abstract The atherosclerotic plaques develop with the adhesion of inflammatory cells and lipids onto the innermost layer of the vessel. They may eventually occlude the vessel impairing blood flow. A severe complication is the rupture of a plaque resulting in the formation of a thrombus that can cause myocardial infarction or stroke. Though a large number of risk factors for atherosclerosis have been identified, the pathogenesis of atherosclerosis is far from unravelled. The aim of the present work was to study both traditional as well as potential novel risk factors of atherosclerosis. The first study examined the relationship between IGF-I concentrations and carotid artery atherosclerosis and its metabolic risk factors. Low IGF-I concentrations were associated with several cardiovascular risk factors. A positive association was observed between IGF-I concentrations and carotid artery intima-media thickness in women. The results suggest that IGF-I may be involved in the pathogenesis of atherosclerosis. Interestingly, the effect may manifest differentially in men and women. The second study focused upon the effects of obesity and weight loss on liver gene expression. A global decrease in gene expression was observed. The down-regulated genes included genes involved in the ubiquitin cycle, which may point to a reduction in oxidative stress due to the hypocaloric diet. The down-regulation of peroxisome proliferator-activated receptor gamma cofactor 1 alpha (PGC-1α) may be related to improved insulin sensitivity. Several novel genes not previously linked to obesity and weight loss were also discovered. In the third and fourth studies, the developmental origins of atherosclerosis hypothesis was studied in a rat model of fetal undernutrition. Unfavourable changes in the obesity-related peptide hormones adiponectin and resistin were observed which could predispose to insulin resistance in later life. In addition, total cholesterol levels were elevated in the undernourished offspring. The gene expression changes in the rat pups suggest that the development of pancreas was affected, which might further contribute to disturbances in insulin and glucose metabolism. / Tiivistelmä Ateroskleroosi eli valtimonkovettumatauti on sairaus, joka saa alkunsa verisuonen sisäseinämään kiinnittyvistä tulehdussoluista ja veren rasvapartikkeleista, joista muodostuu pitkän ajan kuluessa ateroskleroottisia plakkeja. Plakit voivat kasvaessaan heikentää veren virtausta valtimoissa ja pahimmillaan jopa tukkia suonen kokonaan. Mikäli plakki repeää, voi muodostua verihyytymä joka sydämessä aiheuttaa sydäninfarktin ja aivoissa aivoinfarktin. Vaikka useita ateroskleroosille altistavia tekijöitä tunnetaan, taudin syntymekanismit ovat vielä suurelta osin selvittämättä. Tämän väitöskirjatyön tarkoituksena oli tutkia sekä ateroskleroosin perinteisiä että mahdollisia uusia riskitekijöitä. Ensimmäisessä osatyössä tutkittiin insuliininkaltaisen kasvutekijä I:n (IGF-I) yhteyttä kaulavaltimon ateroskleroosiin sekä perinteisiin ateroskleroosin riskitekijöihin. Matalat IGF-I pitoisuudet liittyivät moniin ateroskleroosin riskitekijöihin. Naisissa korkeammat IGF-I pitoisuudet kuitenkin yhdistyivät paksumpaan kaulavaltimoon, mikä viittaa ateroskleroosiin. Tulosten perusteella IGF-I saattaa liittyä ateroskleroosin kehitykseen ja mahdollisesti sen vaikutukset ilmenevät naisissa ja miehissa eri tavoin. Toisessa osatyössä tutkittiin maksan geenien ilmentymistä lihavuudessa ja laihdutusjakson jälkeen. Laihduttaneessa ryhmässä 142:n geenin ilmentyminen oli vähentynyt ja vain yhden lisääntynyt suhteessa kontrolliryhmään. Ubikitiini-syklin geenien ilmentymisen väheneminen voi viitata vähentyneeseen oksidatiiviseen stressiin elimistössä dieetin seurauksena. Muun muassa diabetekseen liittyvän geenin, peroxisome proliferator-activated receptor gamma cofactor 1 alpha, väheneminen puolestaan voi liittyä parantuneeseen insuliiniherkkyyteen laihduttaneissa. Lisäksi tässä työssä tuli esiin monia uusia, mielenkiintoisia geenejä, joita ei aiemmin ole yhdistetty lihavuuteen tai ateroskleroosiin. Kolmannessa ja neljännessä osatyössä selvitettiin ns. Barkerin hypoteesia, eli sitä, voisiko sairastumisalttius määräytyä jo sikiökauden ja varhaiskehityksen aikana. Rottakokeemme osoittivat, että sikiöaikaisen aliravitsemuksen seurauksena kolesteroliarvot olivat korkeammat ja että lihavuuteen liittyvien peptidihormonien, adiponektiinin ja resistiinin, pitoisuuksissa oli tapahtunut epäsuotuisia muutoksia, jotka voivat altistaa insuliiniresistenssille Tulokset viittasivat myös siihen, että aliravitsemus oli mahdollisesti vaikuttanut haiman kehitykseen, mikä voi myös osaltaan vaikuttaa mm. insuliini- ja sokeriaineenvaihduntaan. Tämänkaltaiset muutokset saattavat altistaa ateroskleroosille myöhemmällä iällä.

O hormônio da paratireóide (PTH) tem sido utilizado como um agente anabólico ósseo para o tratamento de condições de osteopenia / osteoporose, prevenção e consolidação de fraturas. O papel do fator de crescimento semelhante à insulina I (IGF-I), como um potencial mediador dos efeitos anabólicos do PTH, é controverso. O rato dwarf pode ser adequado para o estudo dessas interações in vivo, uma vez que a os níveis séricos de hormônio do crescimento (GH) encontram-se reduzidos a cerca de 6% dos valores normais em fêmeas e os níveis séricos de IGF-I, a cerca de 10% dos valores normais, mas estes animais são saudáveis e sem malformações esqueléticas. Os objetivos deste estudo foram: 1 - Avaliar o rato dwarf (dw-/dw-) como um modelo animal para o estudo dos efeitos da deficiência do GH e do IGF-I sobre o esqueleto e o metabolismo ósseo; 2 - Comparar os efeitos do tratamento com PTH sobre o esqueleto e formação óssea em ratos dwarf e em ratos Lewis, sua linhagem de origem. A partir de 9 semanas de idade, ratas Lewis e dwarf receberam injeções por via subcutânea, diariamente, por duas semanas, com medicamento placebo ou fragmento de hormônio paratireóideo humano, hPTH 1-34, na dose de 50 g / kg de peso corpóreo (N = 7-13/grupo). Foram realizadas avaliações do peso corpóreo semanalmente e, por ocasião da eutanásia, na 11ª semana, foram coletadas amostras de sangue para realização de dosagens séricas de IGF-I (ELISA). As vértebras lombares e as metáfises proximais das tíbias foram avaliadas por meio de histomorfometria óssea. Os fêmures direitos foram mensurados e analisados por tomografia quantitativa periférica computadorizada (pQCT). Os níveis séricos de IGF-I mostraram-se três vezes menores nas ratas dwarf quando comparados aos observados nas ratas Lewis, a despeito do tratamento com PTH, que não provocou aumento de IGF-I em nenhum dos dois grupos. No entanto, o PTH aumentou significativamente o volume ósseo trabecular em ambos os grupos, dwarf (p<0.003) e Lewis (p < 0.0001) comparados aos seus respectivos grupos controle, efeito associado ao aumento da espessura e da distância trabeculares. As ratas dwarf tratadas com PTH também exibiram aumentos de 7 a 13 vezes na superfície de mineralização e na taxa de formação óssea respectivamente, quando comparadas às ratas dwarf tratadas com placebo, enquanto as ratas Lewis tratadas com PTH mostraram aumentos de 3 e 4 vezes quando comparadas as ratas Lewis tratadas com placebo. A taxa de aposição mineral, indicativa de atividade osteoblástica, estava aumentada nas ratas dwarf e Lewis tratadas com PTH (p<0.0001) comparadas aos seus respectivos grupos controle. As análises pela pQCT das metáfises femorais distais revelaram que todos os parâmetros estruturais do osso trabecular (BMC total, BMD total, BMC trabecular e BMD trabecular) também apresentaram valores significativamente aumentados nas ratas, Lewis e dwarf, tratadas com PTH, quando comparadas às ratas tratadas com placebo (p<0.0001). Ao se considerar os parâmetros para o osso cortical, praticamente todos os valores obtidos nas diáfises femorais (BMC total, BMD total, BMC cortical, BMD cortical, área cortical, espessura cortical, circunferência periosteal e endosteal) não mostraram qualquer efeito do tratamento com PTH nos dois grupos. Em conclusão, o PTH induziu efeitos anabólicos altamente significativos no tecido ósseo trabecular das tíbias e vértebras lombares, a despeito dos baixos níveis circulantes de IGF-I em animais da linhagem dwarf. A resposta positiva ao tratamento com PTH confirma a sua utilização terapêutica como potente agente anabólico ósseo mesmo em face à deficiência no eixo GH/IGF-I / The parathyroid hormone (PTH) has been used as a bone anabolic agent to treat osteopenic/osteoporotic conditions, prevention and healing of fractures. The role of insulin-like growth factor I (IGF-I) as a potential mediator for the bone anabolic effects of PTH is controversial. The dwarf rat (dw-/dw-) may be suitable to study these interactions in vivo, since GH synthesis is selectively reduced to about 6% of normal in females, and serum IGF-I levels are about 10% of normal, but these animals are healthy without skeletal malformations. The objectives of this study were: 1- Evaluate the dwarf rat (dw-/dw-) as an animal model for studies of the effects of GH and IGF-I deficiency on the skeleton and bone metabolism; 2- Compare the skeletal effects of PTH treatment in dwarf rats and their background strain, Lewis rats. At 9 weeks of age, female Lewis and dwarf rats were injected SC daily for 2 weeks with vehicle or human parathyroid hormone fragment, hPTH 1-34, at a dose of 50 g/kg body weight (N=7- 13/group). The body weight was evaluated weekly and at the time of euthanasia, at 11 weeks, blood samples were collected. Serum IGF-I was measured by ELISA, and cancellous bone histomorphometry was performed in the lumbar vertebral body and tibial proximal metaphysis. The right femurs were measured, scanned and analyzed by peripheral quantitative computed tomography (pQCT). Serum levels of IGF-I were nearly 3-fold lower in dwarf rats compared with Lewis rats regardless of treatment, but PTH treatment did not increase serum IGF-I in either Lewis or dwarf rats. However, PTH significantly increased cancellous bone volume in both dwarf (P<0.003) and Lewis rats (P<0.0001) when compared to vehicle-treated rats, which was associated with increased trabecular width and decreased trabecular separation. PTH-treated dwarf rats also exhibited 7- and 13-fold increases in mineralizing surface and bone formation rate respectively, compared to vehicle-treated dwarf rats, while PTH-treated Lewis rats showed 3- and 4-fold increases when compared to vehicle-treated Lewis rats. Mineral apposition rate, an index of osteoblast activity, was increased in PTH-treated dwarf rats (P<0.0001) and in Lewis rats (P<0.0001) compared to their respective control groups. The pQCT analyses of the distal femoral metaphysis revealed that cancellous bone structural parameters (total BMC, total BMD, trabecular BMC, and trabecular BMD) also presented significantly higher values in PTH-treated dwarf and Lewis rats, when compared to vehicle treated rats (P<0.0001). When considering cortical bone parameters, almost all the values obtained at the femoral shafts (total BMC, total BMD, cortical BMC, cortical area, cortical thickness, periosteal and endocortical circumferences) did not show any PTH treatment effect in either groups. In conclusion, PTH induced highly significant anabolic effects in vertebral and tibial cancellous bone despite low circulating levels of IGF-I in dwarf rats. The positive response to PTH treatment confirms its therapeutic use as a potent bone anabolic agent, even in the face of GH/IGF-I deficiency

Objective: Insulin-like growth factor-I (IGF-I) has been suggested to be a prognostic marker for the development of cancer and, more recently, cardiovascular disease. These diseases are closely linked to obesity, but reports of the association of IGF-I with measures of obesity are divergent. In this study, we assessed the association of age-dependent IGF-I standard deviation scores with body mass index (BMI) and intra-abdominal fat accumulation in a large population. Design: A cross-sectional, epidemiological study. Methods: IGF-I levels were measured with an automated chemiluminescence assay system in 6282 patients from the DETECT study. Weight, height, and waist and hip circumference were measured according to the written instructions. Standard deviation scores (SDS), correcting IGF-I levels for age, were calculated and were used for further analyses. Results: An inverse U-shaped association of IGF-I SDS with BMI, waist circumference, and the ratio of waist circumference to height was found. BMI was positively associated with IGF-I SDS in normal weight subjects, and negatively associated in obese subjects. The highest mean IGF-I SDS were seen at a BMI of 22.5–25 kg/m2 in men (+0.08), and at a BMI of 27.5–30 kg/m2 in women (+0.21). Multiple linear regression models, controlling for different diseases, medications and risk conditions, revealed a significant negative association of BMI with IGF-I SDS. BMI contributed most to the additional explained variance to the other health conditions. Conclusions: IGF-I standard deviation scores are decreased in obesity and underweight subjects. These interactions should be taken into account when analyzing the association of IGF-I with diseases and risk conditions.

Objective: IGF1 is associated with metabolic parameters and involved in glucose metabolism. Low-IGF1 has been implicated in the etiology of glucose intolerance and subjects with pathological causes of either low- or high-IGF1 are at risk of diabetes. We hypothesized that both low- and high-IGF1 levels increase the risk of diabetes and aimed to assess the role of IGF1 in the risk of developing diabetes in a large prospective study. Design: An analysis of two prospective cohort studies, the DETECT study and SHIP. Methods: We measured IGF1 levels in 7777 nondiabetic subjects and assessed incident diabetes mellitus during follow-up. Results: There were 464 cases of incident diabetes during 32 229 person-years (time of follow-up in the DETECT study and SHIP: 4.5 and 5 years respectively). There was no heterogeneity between both studies (P>0.4). The hazard ratios (HRs) of incident diabetes in subjects with IGF1 levels below the 10th or above the 90th age- and sex-specific percentile, compared to subjects with intermediate IGF1 levels, were 1.44 (95% confidence interval (CI) 1.07–1.94) and 1.55 (95% CI 1.06–2.06) respectively, after multiple adjustment. After further adjustment for metabolic parameters, the HR for low-IGF1 became insignificant. Analysis of IGF1 quintiles revealed a U-shaped association of IGF1 with risk of diabetes. Results remained similar after exclusion of patients with onset of new diabetes within 1 year or with borderline glucose or HbA1c levels at baseline. Conclusions: Subjects with low- or high-IGF1 level are at increased risk of developing diabetes.

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

Mutations in the insulin-like growth factor-I (IGF-I) receptor (IGF1R) have been associated with prenatal and postnatal growth retardation. However, little is known about potential effects of mutations in the IGF1R on carbohydrate homeostasis. Methods: We investigated clinical, endocrine and metabolic parameters in four family members carrying a novel IGF1R mutation (p.Tyr387X): an 18-year-old male (index case), his sister and two paternal aunts. Results: All family members showed a variable degree of impairment in prenatal growth, with birth weight standard deviation scores (SDS) between –1.65 and –2.37 and birth length SDS between –1.78 and –3.08. Their postnatal growth was also impaired, with height SDS between –1.75 and –4.86. The index case presented high IGF-I levels during childhood and adolescence and delayed bone age. The index case and his two paternal aunts had impaired glucose tolerance (IGT) associated with a variable degree of alterations in insulin sensitivity and secretion. In contrast, the index case’s sister, who had had IGT during pregnancy, showed normal glucose metabolism but reduced insulin sensitivity. Conclusion: This is the first study showing an association between a novel IGF1R mutation and a variable degree of alterations in prenatal and postnatal growth and in carbohydrate metabolism.

Endothelial cells separate the bloodstream from the underlying tissue and play a crucial role in vascular homeostasis. They also form an important barrier for vascular drug delivery. This thesis contains preliminary studies targeted at understanding the mechanisms of binding and transport across endothelial cells cultured in vitro. Specifically, the first study investigates how the recombinant source of Factor IX (FIX), a blood coagulant protein used in the treatment of Hemophilia B, impacts surface ligand binding (FIX to its specific receptors) to bovine aortic endothelial cells (BAECs). Competitive binding experiments between 125I-FIX and FIX were undertaken to quantify the interaction of recombinant and transgenic FIX with BAECs and human collagen IV and determine if there was a measurable difference in binding affinity. Results indicate limited specific binding of 125I-FIX to BAECs and no binding to human collagen IV. Concrete conclusions were not drawn from this data due to technical issues during the experimental process. The second study investigates insulin-like growth factor-I (IGF-I) transport across both BAEC and MAC-T cells, a mammary epithelial cell line, cultured on tissue culture inserts. IGF-I is a circulatory growth factor implicated in the regulation of cell division and tissue proliferation. Competitive binding experiments between 125I-IGF-I and unlabeled protein (IGF-I, Y60L-IGF-I, a mutant of IGF-I, and IGF Binding Protein-3 (IGFBP-3)) were undertaken to quantify the binding and transport of IGF-I under various experimental conditions. Results confirmed earlier work from the Williams' laboratory indicating that 125I-IGF-I transport was enhanced by incubation with its non-receptor-binding analog, Y60L-IGF-I, but cell surface associated 125I-IGF-I was decreased by its presence. Other studies were undertaken but conclusive results could not be drawn. / Master of Science

Insulin-like growth factor-I is a small growth factor known to signal in a variety of mammalian cells through the IGF-I cell surface receptor (IGF-IR). A unique feature of the IGF-I system is the regulation of this binding by soluble IGF binding proteins. Recent studies from our laboratory show that there is a pH dependence in the association of IGF-I with the cell surface in the presence of IGFBP-3 which suggested increased association of IGF-I with IGFBP-3 at low pH. We studied cell free interaction of IGF-I and IGFBP-3 as a function of pH using surface plasmon resonance (SPR) in order to understand the mechanism that causes the increased association. In our studies three different SPR instruments with different surfaces for immobilization of one of the binding partners were used: a Leica Bio-SPR 9000 with a low molecular weight carboxymethylated dextran (CMD) surface, a BIAcore 2000 with a high molecular weight CMD surface and a Leica SPR 2001 Alpha with a planar mixed self-assembled monolayer (mSAM) surface. Since the experimental system we used was transport sensitive, only the mSAM surface, under optimized conditions, produced results that fit to a single site model. Results suggest that use of CMD layers for immobilization of one partner of a high-affinity binding complex can result in transport limited binding for which simple analysis is inappropriate. Future studies are planned to expand the work with the mSAM surface to elucidate whether a significant difference between the binding parameters as a function of pH exists. / Master of Science

Las hormonas recombinantes eritropoyetina (EPO) y hormona de crecimiento (GH), prácticamente iguales a las endógenas y de corta vida media en circulación, son de difícil detección directa en el control antidopaje. Se determinaron los valores poblacionales de los biomarcadores indirectos EPO, receptor soluble de la transferrina, insulin-like growth factor-I (IGF-I) y procolágeno tipo III péptido (P-III-P), en poblaciones seleccionadas de deportistas, y el efecto del ejercicio y los distintos tipos de entrenamiento sobre su concentración sérica. La comparación de resultados obtenidos mediante distintos ensayos demostró la necesidad de una validación exhaustiva previa a su utilización. A excepción del P-III-P, los biomarcadores séricos propuestos para la detección de rhEPO y rhGH no se encuentran directamente afectados por el nivel atlético, el ejercicio o la distinta carga de entrenamiento realizada a lo largo de la temporada deportiva. La edad es la principal influencia sobre las concentraciones séricas de IGF-I y P-III-P. / Direct detection of recombinant peptidic hormones erythropoietin (EPO) and growth hormone (GH), very similar to endogen molecules and with a short half life in blood, is difficult in antidoping control. The main objective of this work is to determine indirect biomarkers' values of EPO, soluble transferrin receptor, insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P), in selected populations of athletes, and the effect of exercise and different types of training on their concentration in serum. The comparison of results obtained by the different assays showed the need of extensive validation of the analytical techniques before their use in the antidoping field. Excepting P-III-P, proposed biomarkers for the detection of rhEPO and rhGH abuse are not directly influenced by the athletic level, exercise or different training workload along the sport season. Age is the main factor affecting IGF-I and P-III-P concentrations in serum.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 6 uppsatser.