The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.

Telomeres are repetitive TTAGGG structures ending each chromosome and thereby protecting its integrity. Due to the end-replication problem, telomeres shorten with each cell division. When reaching a critical telomere length (TL), the cells stop dividing and enter replicative senescence. It has been speculated that telomeres might regulate lifespan at the organism level but this hypothesis is controversial. However, telomeres in human blood cells do shorten with increasing age. Telomerase is an enzyme capable of lengthen telomeres. It consists of a catalytic subunit, hTERT, and a RNA template, hTR. Telomerase is active in germ cells, stem cells, activated lymphocytes and highly proliferating epithelial cells while no activity is found in other somatic cells. One step in order to produce a tumour mass is that cancer cells need to have a limitless replicative potential and this can be achieved by activating telomerase. Most tumour cells express telomerase activity and hence, the enzyme is an interesting target for cancer therapy. Telomere length is in part inherited. Two separate family cohorts were investigated to elucidate the inheritance pattern and a strong paternal inheritance was observed. In the larger, multifamily cohort spanning up to four generations, a weak correlation between the TL of the mother and the child was also found, as well as a significant correlation between grandparent-grandchild pairs. Interestingly, the heritable impact diminished with increasing age, indicating than non-heritable factors might influence TL during life. A functional T to C transition polymorphism in the hTERT promoter was previously reported, showing that the -1327C/C genotype was correlated with shorter TL compared to the alternative genotypes in healthy individuals and in coronary artery disease patients. When investigating 226 myocardial infarction patients and 444 controls separately, no differences were observed regarding mean TL or increased attrition rate between the different genotypes. TL in blood cells is shown to be altered in patients with certain types of solid tumours. In our breast cancer cohort, TL was a strong prognostic marker. Short telomeres were associated with increased survival, especially in young patients and in those with advanced tumours. It has been speculated that cancer patients might have a faster telomere attrition rate than controls but this has not been experimentally proven. Two blood samples from the same individual taken with 9-11 years interval was investigated. Some were diagnosed with a malignancy after the second blood draw. When comparing patients with controls, telomere attrition rate was not correlated to future tumour development. About one third of the individuals elongated their telomeres over a decade and the individual telomere attrition rate was telomere length dependent, showing an inverse correlation to TL at a highly significant level. This strongly suggests that the TL maintenance mechanism shown to provide protection for short telomeres in vitro is important also in human cells in vivo.

Regulation of growth is of fundamental importance for development of the organism and to maintain health. The induction of cell proliferation and matrix production are influenced by several different signaling systems, most importantly by growth factors. The human HER-family of growth factor ligands and receptors is one of the most studied and, at present, one of the most complex including 4 tyrosine kinase receptors and at least 11 different ligands cooperating in the transfer of signals. The HER-family growth responses are also influenced by other intercellular and extracellular signals, including matrix components, cytokines and hormones mediating e.g. inflammation. HER-1 (EGFR) is one of the best known and most extensively studied growth factor receptors. TGF-alpha is possibly the most potent HER-1 ligand and influences wound healing, epidermal maintenance, gastrointestinal function, lactation, pulmonary function and more. Several studies have shown important regulatory functions for some inflammatory cytokines on TGF-alpha production in white blood cells. HER-1 is widespread in epithelial cells but also in mesenchymal cells such as fibroblasts, osteogenic and chondrogenic cells. Consequently, many tumors arising from these cell types express HER family members and often show TGF-alpha and/or HER activation. Indeed, mammary cancer development has been shown when over expressing both TGF-alpha and HER-2 in mouse mammary cells in vivo. In recent years the first HER-1 and HER-2 inhibitors have come into clinical practice for treatment of breast cancer, lung cancer and gastrointestinal cancers, sometimes with great success. However, more knowledge is needed concerning the inflammatory regulation of HER-family expression including where and how the ligands and receptors cooperate. Therefore we were interested in studying the role of TGF-alpha in normal and abnormal growth. First we showed that the acute inflammatory cytokine IL-6 regulates TGF-alpha expression in U-937-1 monocytoid cells. Secondly, we detected a possible long-term enhancing influence of singledose UVR on HER-1 expression in normal human melanocytes. We continued thirdly by revealing TGF-alpha production concomitant with HER-2 in normal human synovia and release of soluble TGF-alpha into the synovial fluid. Both TGF-alpha and HER-2 production were significantly increased in inflammatory joint conditions, e.g. RA. Fourthly, we demonstrated expression of TGF-alpha, HER-1 and HER-2 in synovial sarcoma cells in culture; the observed HER-2 phosphorylation was dependent on ligand induced HER-1 activation. The presented results indicate that TGF-alpha expression can be enhanced by acute inflammatory cytokine IL-6, possibly contributing to growth stimulatory effects assigned to IL-6 itself. The acute effects of UVR on melanocytes mediate up-regulated steady-state expression of HER-1, constituting a potential target for locally produced TGF-alpha that may induce melanocyte proliferation. TGF-alpha and HER-2 seem to have a role in the maintenance of synovial joint tissues. Upregulation of TGF-alpha and HER-2 in inflammatory joint conditions, e.g. RA, represents a novel mechanism for synovial proliferation contributing to joint deterioration. TGF-alpha, HER1 and HER-2 may have a role in synovial sarcoma proliferation; further investigation is needed to evaluate HER-family inhibitors as a possible treatment alternative in this type of cancer.

Platelet-derived growth factor (PDGF) is a potent stimulator of cell growth, survival and motility. PDGF exerts its function by binding to specific tyrosine kinase receptors, initiating receptor auotphosphorylation and initiation of specific signaling pathways that regulates the cellular response. It is critical that these signals can be modulated and terminated, since over-activation of signaling pathways are often found in diseases, such as cancer. Protein tyrosine phosphatases (PTPs) counteract the tyrosine kinases by dephosphorylating proteins, thereby playing a crucial role in the control of signaling events. The aim of this thesis has been to study the regulation of PDGF receptor signaling by the T-cell protein tyrosine phosphatase (TC-PTP). In the first two studies, we demonstrated that loss of TC-PTP specifically redirected the PDGF β-receptor towards a rapid Rab4a-dependent recycling after ligand-induced internalization. Furthermore, we found that the sorting of activated PDGF β-receptor into the recycling pathway was dependent on sequential PKCα and Rab4a activation. Since the PDGF α-receptor did not recycle in the absence of TC-PTP, this study displays the first evidence of differences in trafficking of the PDGF receptor family members. PDGF β-receptor recycling was also induced by activating PKCα through the LPA receptor. The LPA-induced PDGF β-receptor recycling correlated with increased receptor phosphorylation and cell migration at low concentrations of PDGF-BB. The data suggests that PKCα activation could serve as a point of cross-talk between receptor families, regulating the duration and magnitude of PDGF β-receptor signaling. In the last study, we searched for novel substrates for TC-PTP downstream of the PDGF β-receptor, and identified the pyruvate kinase M2, PK-M2, as a possible substrate. PK-M2 is expressed in cells that proliferate rapidly, including tumor cells. Our data suggests that TC-PTP can interact with the glycolytic complex, affecting the activity of PK-M2 and hence, altering the glucose metabolism for proliferating tumor cells.

<p>In type 2 diabetes the concentration of glucose in the blood is increased, and tissues like fat and musclebecome less sensitive to insulin. These two phenomena are interrelated, but since the glucose-insulininterplay is highly complex, many aspects are still not understood. Here, a model-based approachmight help. Nevertheless, also a model-based approach has a limited impact, unless models for thesub-systems can be combined into a model for the whole-body regulation. Such a multi-level,module-based model is referred to as a hierarchical model, and this thesis is a proof-of-principle studyfor the future development of such models.</p><p>We have extended one of the best available models for the whole-body regulations, to include azoomable module for the fat tissue. The first step was to implement the whole-body model in thesoftware MathModelica, which support hierarchical modeling. Second, the originally mergedinsulin-responding module was sub-divided, so that a fat tissue was singled out. Third, a model for theinput-output profile for the fat tissue was developed by combining mechanistic knowledge withexisting and novel data from human fat cells. Finally, this detailed model was fitted to the profile of theoriginal fat model, and inserted in the whole-body model, with negligible effect on the whole-bodysimulations.</p><p>The resulting model has the ability to translate mechanistically oriented simulations on the biochemicallevel, which is the level were drugs act, to the whole-body level, which is of clinical interest. This is aquantum leap forward for modeling, and understanding, glucose homeostasis and type 2 diabetes.</p>

The Sun is the ultimate energy source on Earth. Photosynthetic organisms are able to catalyze the conversion of solar energy to chemical energy by a reaction called photosynthesis. In plants, this process occurs inside a green organelle called the chloroplast. The protein complexes involved in the photosynthetic light reactions are situated in the thylakoid membrane, which encloses a tiny space called lumen. The Peptidyl-Prolyl cis-trans Isomerase (PPIase) family is the most abundant protein family in the thylakoid lumen. The three PPIase subfamilies, cyclophilins, FKBPs (FK506 binding proteins) and parvulins form a group by their enzymatic activity despite lack of sequence similarity between the subfamilies. Cyclophilins and FKBPs, collectively called immunophilins, were originally discovered as the targets of the immunosuppressive drugs cyclosporine A and FK506, respectively. By suppressing the immune response in humans, these immunophilin-drug complexes revolutionized the field of organ transplantation by preventing graft rejection. Cis-trans isomerization of peptide bonds preceding the amino acid proline is the rate-limiting step of protein folding and several immunophilins have been shown to be important for catalysis of protein folding in vivo. PPIases have been found to be part of large protein complexes as well as in functions such as signalling, protein secretion, RNA processing and cell cycle control. A picture is therefore emerging in which the actual interaction between the PPIase and its target is perhaps more important than the PPIase activity. In the present work, PPIases have been characterized in the chloroplast thylakoid lumen of Spinacia oleracea (spinach) and Arabidopsis thaliana (Arabidopsis). The most active PPIase in the spinach lumen was identified as the cyclophilin TLP20. AtCYP20-2, the Arabidopsis homologue of TLP20, was found to be upregulated at high light and attached to the thylakoid membrane, more precisely to the outer regions of photosystem II supercomplexes. In Arabidopsis, up to 5 cyclophilins and 11 FKBPs were predicted to reside in the lumen. Of these 16 immunophilins, only 2 were identified as active PPIases and significant differences were observed between the two plant species. AtCYP20-2, like TLP20, is an active isomerase although AtFKBP13 is the most active PPIase in the lumen of Arabidopsis. Mutant Arabidopsis plants deficient in AtCYP20-2 displayed no phenothypical changes or decrease in total lumenal PPIase activity. Being the only active PPIase in the mutants, the redox sensitive AtFKBP13 is proposed to compensate for the lack of AtCYP20-2 by oxidative activation. In agreement with the experimental data, the sequence analyses of catalytic domains of lumenal immunophilins demonstrate that only AtCYP20-2 and AtFKBP13 possess the amino acids found essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. It is concluded that with the exception of AtCYP20-2 and AtFKBP13 most immunophilins in the lumen of Arabidopsis lost their PPIase activity on peptide substrates and developed other specialized functions.

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells.

Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid. The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation. From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition.

Prevalensen av fetma ökar drastiskt i stora delar av världen och utgör en stor riskfaktor för att utveckla insulinresistens och typ 2 diabetes. Fettväven kan bli mycket stor om för mycket energi tas upp av kroppen. Vid extrem övervikt är fettväven i kroppen i ett stresstillstånd, vilket gör att risken för att utveckla metabola sjukdomar som t.ex. typ 2 diabetes ökar. Fett lagras i olika fettdepåer i kroppen. Inlagringen i djupare kroppsdelar, runt och i inre organ s.k. visceralt fett, skiljer sig från fettväven som lagras direkt under huden s.k. subkutant fett. Nyare rön visar att mer visceral fettväv ökar risken för att utveckla insulinresistens och typ 2 diabetes. Fettcellen är tillsammans med muskel- och leverceller de viktigaste för glukosmetabolismen. Fettcellen är en stor cell, som man lätt kan se med blotta ögat. Storleken på ellerna varierar dock kraftigt i en och samma fettvävnad. Upptag av glukos från maten vi äter regleras av hormonet insulin. Insulinresistens är ett tillstånd då cellerna svarar dåligt på insulin, vilket gör att glukoshalten i blodet ökar. Detta förekommer vid typ 2 diabetes, men även vid andra tillstånd där cellerna blir stressade, t.ex. kirurgiska ingrepp. Insulinsignaleringen i fettcellen är komplex och signalöverföringen inne i cellen sker främst via en kaskad av fosforyleringar, där olika proteiner i en signalkedja fosforyleras eller defosforyleras. Slutligen leder denna fosforyleringskaskad till insulinets sluteffekter som t.ex. upptag av glukos, proteinsyntes och celltillväxt. Efter att insulin bundit till och fosforylerat/aktiverat insulinreceptorn delas signalen upp inne i cellen i två huvudvägar; den metabola signalvägen och den mitogena signalvägen. Insulinreceptorsubstrat 1, IRS1, är ett stort protein som insulinreceptorn verkar direkt på. Fosforylering av aminosyran tyrosin på IRS1 är mycket viktigt för fortsatt insulinsignalering i fettcellen. IRS1 fosforyleras även på aminosyran serin som svar på bl.a. insulin. Serinfosforyleringen av IRS1 hämmar eller stimulerar insulinsignaleringen, ofta genom återkoppling av insulinsignalen. Syftet med den här avhandlingen är att beskriva möjliga cellulära mekanismer i insulinsignaleringen vid insulinresistens som resultat av kirurgisk stress eller vid typ 2 diabetes i fettceller från människa. Häri har upptaget av glukos analyserats och jämförts i fettceller från olika fettdepåer. Viscerala fettceller har högre basalt och insulinstimulerat glukosupptag och mer glucostransportörprotein än subkutana fettceller. Däremot är det ingen skillnad i insulinkänslighet angående glukosupptaget i de olika typerna av fettceller. Vidare fann vi att den kirurgiskt orsakade insulinresistensen hos subkutana fettceller från människa återgår till det normala efter övernattinkubering av cellerna i odlingsmedium. Insulinresistensen vid typ 2 diabetes är däremot permanent och har en annan mekanism än den reversibla, stress-relaterade insulinresistensen. Insulinresistansen vid typ 2 diabetes beror på att signalöverföringen mellan olika proteiner i cellen är defekt. Insulinreceptorns förmåga att fosforylera IRS1 på aminosyran tyrosin är nedsatt hos patienter med typ 2 diabetes. Fosforyleringen av IRS1 på serin 307 (i den humana sekvensen) ökar snabbt hos icke-diabetiska fettceller som svar på insulin. Denna serinfosforylering verkar behövas för att IRS1 effektivt ska tyrosinfosforyleras och därmed leda insulinsignalen vidare inne i cellen. Fosforyleringen av IRS1 på serin 307 är kraftigt nedsatt hos subkutana fettceller från patienter med typ 2 diabetes. Fosforyleringen av IRS1 på serin 312 är däremot liknande i fettceller från icke-diabetiker och diabetiker (Öst et.al. (2007) Faseb.J. doi: 10.1096/fj.07-8173com). Fosforyleringen av IRS1 på serin 312 är mest involverad i insulinsignaleringens negativa återkoppling. Fosforyleringen av serin 307 sker snabbt och vid låga insulinkoncentrationer, medan fosforyleringen på serin 312 sker först efter lång inkubering och vid höga insulinkoncentrationer. Detta är en ny mekanism på cellulär nivå som möjligen kan beskriva insulinresistansen i fettceller från människa. Tillsammans styrs återkopplingen via den stimulerande fosforyleringen (serin 307) eller den hämmande fosforyleringen (serin 312) och kontrollerar insulinsignaleringen i cellen. Fosforyleringarna sker möjligen via samma proteinkinas och/eller proteinfosfatas och kan bli mål för terapeutiska läkemedel mot typ 2 diabetes i framtiden. / The prevalence of obesity is increasing in most parts of the world and is a strong risk factor for the development of insulin resistance and type 2 diabetes. Adipose tissue is important in whole body energy balance and grows in size with excess energy intake. Adipose tissue in different regions of the body has different characteristics and adipocytes coming from intraabdominal fat depots, are more associated with insulin resistance than adipocytes from subcutaneous fat depots. Insulin signalling is complex and consists of two major signalling pathways in the cell; the metabolic signalling pathway and the mitogenic signalling pathway. After insulin binding to the insulin receptor a cascade of protein phosphorylations and dephosphorylations is started, eventually leading to the target effects of the hormone. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), a protein directly downstream of the insulin receptor, is essential for further insulin signalling. Serine phosphorylation of IRS1 also affects insulin signalling through inhibitory or stimulatory effects. Adipocytes are together with muscle cells and liver cells central in the development of type 2 diabetes. The focus of this thesis is to describe mechanisms in insulin signalling in primary human adipocytes in insulin resistant states, surgical stress or type 2 diabetes. Visceral adipocytes from humans were analysed and compared to subcutaneous adipocytes. Visceral adipocytes were slightly bigger than subcutaneous adipocytes. Furthermore, visceral adipocytes had an increased level of the glucose transporterprotein GLUT4 and a higher basal and insulin-stimulated glucose uptake, but the sensitivity to insulin was the same. Here it was found that surgical insulin resistance is reversible after overnight incubation of the adipocytes and the impaired insulin sensitivity is at the level between IRS1 and PKB/Akt in insulin signalling. In contrast, the insulin resistance in type 2 diabetes is irreversible and the impaired insulin sensitivity is at the level of insulin receptor-mediated tyrosine phosphorylation of IRS1. Adipocytes from patients with type 2 diabetes were investigated and it was found that diabetic adipocytes have an attenuated insulin-stimulated phosphorylation of IRS1 at serine 307 (corresponding to serine 302 in the mouse sequence). In adipocytes from non-diabetic individuals, the phosphorylation of IRS1 at serine 307 occurred rapidly at low concentrations of insulin. This phosphorylation was associated with the tyrosine phosphorylation of IRS1. The phosphorylation of IRS1 at serine 312 (corresponding to serine 307 in the mouse sequence) in response to insulin was similar in adipocytes from non-diabetic individuals and from patients with type 2 diabetes (Öst et.al. (2007) Faseb.J. doi: 10.1096/fj.07-8173com) and occurred only at high concentrations after prolonged incubation with insulin. This thesis reports the investigation of mechanisms in insulin signalling at a cellular and molecular level in primary human adipocytes. The insulin resistance resulted from surgical stress is different from that in type 2 diabetes and adipocytes from patients with type 2 diabetes have impaired insulin sensitivity at the level of IRS1. Together, the phosphorylation of IRS1 at serine 307 and serine 312 may control insulin signalling through feedback mechanisms in primary human adipocytes.