Oxidized low-density lipoprotein (LDL) diminishes NO production from activated macrophages. The interaction between LDL and inactivated macrophages is neglected and controversial. This study examines the effect of LDL, 7-oxysterols and iron compounds on NO production in unstimulated J774 macrophages. J774 cells and human umbilical vein endothelial cells (HUVEC) were either incubated for 24 h with native LDL (LDL) or ultraviolet (UV)-oxidized LDL (UVoxLDL), in the absence or presence of an inducible nitric oxide synthase (iNOS)- or an endothelial constitutive nitric oxide synthase (eNOS)-inhibitor. J774 cells were also incubated with lipopolysaccharide (LPS), in the absence or presence of an iNOS- or an eNOS-inhibitor. Nitrite was analysed as a marker of NO production. The mRNA levels of iNOS were evaluated by reverse transcriptase polymerase chain reaction. LDL and UVoxLDL significantly increased NO production from unstimulated J774 macrophages. This increase in NO was accompanied by enhanced expression of iNOS mRNA, and was inhibited by the iNOS inhibitor. Furthermore, NO production was elevated and angiotensin-converting enzyme (ACE) activity was reduced in HUVEC following the exposure to LDL and UVoxLDL. In conclusion, LDL may serve as an important inflammatory activator of macrophages and HUVEC, inducing inducible nitric oxide production but diminishing ACE. After its oxidation, this function of LDL may be further enhanced and may contribute to the regulation and progression of atheroma formation.

The prevalence of obesity is increasing in most parts of the world and is a strong risk factor for the development of insulin resistance, type 2 diabetes and hypertension. Adipose tissue is mainly composed of adipocytes which store energy in the form of triglycerides and release it as free fatty acids. Adipose tissue is one of the major regulators of energy homeostasis in the body. Adipose tissue in different regions of the body has different characteristics and adipocytes in intra-abdominal fat depots are more associated with insulin resistance than adipocytes from subcutaneous fat depots.

Research performed during the past several years has led to an explosion in the understanding of adipose tissue and the active role that it plays in aspects of physiology and pathophysiology. One important discovery has been identification of the nuclear hormone receptor called peroxisome proliferator-activated receptor γ (PPARγ). Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor, which is highly expressed in adipocytes. PPARγ has been shown to affect several genes of importance for lipid metabolism, differentiation of fat cells and insulin sensitivity. The PPARγ receptor can be activated by thiazolidinediones (TZD), a class of insulinsensitising drugs, which promote fatty acid storage in fat depots and decrease glucose levels in plasma, thus, demonstrating the importance of PPARγ activity in insulin resistance and metabolic syndrome.

This thesis has investigated the transcriptional activity of PPARγ in a clinically relevant cell type for insulin resistance and type 2 diabetes; the primary human adipocyte. For this purpose, a method for transfection of primary human adipocytes by electroporation and for measurement of the activity of PPARγ has been developed and optimised. This method has been used to study the effect of saturated and unsaturated fatty acids on the transcriptional activity of PPARγ. Interestingly, it was been found that saturated fatty acids can activate PPARγ, thus promoting a protection against diabetes. The strongest activator was the monounsaturated palmitoleic acid. The transcriptional activity of PPARγ in primary human adipocytes from intra-abdominal and subcutaneous adipose tissues was also examined. It was found that PPARγ activity is considerably lower in adipocytes from visceral compared with subcutaneous fat from the same subject. Another reason for using human tissue to reach clinical relevance shown here was that the same difference in PPARγ activity could not be found between intra-abdominal and subcutaneous fat tissues in mice. This finding may serve as the basis of why excess intraabdominal fat tissue is associated with high risk for development of type 2 diabetes and cardiovascular diseases.

The blood pressure regulating renin-angiotensin system (RAS) in human adipose tissue and in isolated adipocytes was examined and related to PPARγ. It was found that the production of angiotensin II, which is an important hormone for increasing the blood pressure, can be produced by isolated adipocytes and that the production is higher in adipocytes coming from omental than subcutaneous fat tissue. Further, it was shown that angiotensin II inhibits PPARγ activity in omental adipocytes, thus reducing the insulin sensitivity. Therefore, this study connects two of the major risk factors in obesity; diabetes and hypertension, and may also explain how drugs, which inhibit the RAS, can also be protective against diabetes. In conclusion, the findings in this thesis give new knowledge about regulating mechanisms of fat cells and its importance in diabetes and cardiovascular disease.

Prevalensen av fetma ökar drastiskt i stora delar av världen och utgör en stor riskfaktor för att utveckla insulinresistens, typ 2 diabetes och hypertoni. Fett kan lagras i olika fettdepåer i kroppen. Fettet som inlagras inuti kroppen, intraabdominellt fett, skiljer sig från fettväven som lagras direkt under huden (subkutant fett). Nyare rön visar att en stor mängd intra-abdominell fettvävnad är en särskilt stark riskfaktor för att utveckla insulinresistens och typ 2 diabetes, samt att avlägsnande av subkutant fett knappast alls påverkar riskfaktorer för kardiovaskulär sjukdom.

Under de senaste åren har forskningen lett till en djupare förståelse av fettvävnaden och dess aktiva roll i fysiologin och patofysiologin av insulinresistens. En viktig upptäckt har varit identifieringen av en nukleär receptor som kallas för PPARγ (peroxisome proliferator-activated receptor gamma). PPARγ receptorn uttrycks huvudsakligen i fettceller och är viktig för fettcelldifferentieringen och fettcellsfunktionen. Receptorn aktiveras av vissa läkemedel för behandling av insulinresistens och hyperglykemi, de så kallade tiazolidindionerna (avandia och actos finns på den svenska marknaden), som sänker blodsockret och även påverkar blodtrycket samt blodfetterna i gynnsam riktning. Detta utgör ett tydligt bevis för betydelsen av PPARγ aktiviteten vid insulinresistens och det metabola syndromet.

Den här avhandlingen studerar transkriptionsaktiviteten av PPARγ i en klinisk relevant celltyp för insulinresistens och typ 2 diabetes, den mänskliga fettcellen. För detta ändamål har en metod för transfektion av primära humana fettceller utvecklats. Metoden användes för att studera insulinsignaleringen i detalj och också för att mäta aktiviteten hos transkriptionsfaktorer. Aktiviteten av PPARγ i primära humana fettceller påverkades olika av olika mättade och omättade fettsyror, som alltså kan verka som hormoner. Intressant nog visades att mättat fett, som av många anses vara särskilt ”onyttigt”, i form av stearinsyra kan aktivera PPARγ och därmed tänkas medföra ett skydd mot diabetes. Den starkaste aktivatorn var enkelomättad palmoljesyra. Aktiviteten hos PPARγ i fettceller från de två olika fettdepåerna, intra-abdominella och subkutana fettvävnaden, studerades. Aktiviteten av PPARγ i isolerade fettceller från intra-abdominellt fett befanns vara betydligt lägre än i subkutant fett från samma person. Som en ytterligare anledning att använda mänsklig vävnad för att nå klinisk relevans visades också av att möss inte har samma skillnad i PPARγ aktivitet mellan subkutant och intra-abdominellt. Fynden ger underlag till varför stor mängd intraabdominellt fett är förknippat med hög risk för diabetes och därmed kopplad ökad kardiovaskulär risk.

Det blodtrycksreglerande renin-angiotensin systemet (RAS) i human fettvävnad och i isolerade fettceller och relationen till PPARγ studerades. Produktionen av angiotensin II, som är ett viktigt blodtryckshöjande hormon, producerades av isolerade human fettceller och produktionen var högre från fettceller som kommer från mänskligt omentfett än från subkutant fett. Vidare visades att tillsatt angiotensin II hämmade PPARγ aktiviteten i fettceller från omentfettet. Detta fynd kopplar alltså samman två av de stora riskfaktorerna vid fetma; diabetes och högt blodtyck. Det ger också nya intressanta infallsvinklar i hur blodtrycksläkemedel som hämmar reninsystemet kan tänkas skydda mot diabetesuppkomst.

Sammanfattningsvis visar denna avhandling att man kan transfektera primära humana fettceller och studera PPARγ aktivitet i denna celltyp, och att PPARγ aktiviteten kan styras av fettsyror, vilket alltså innebär att matkomponenter (fettsyror) har direkt hormonella effekter i kroppen. Omentfett visades ha särskilt låg PPARγ aktivitet. Slutligen befanns att fettväven och isolerade fettceller kan producera olika komponenter i RAS. Det är tydligt att dessa fynd tillsammans har givit upphov till viktig ny kunskap om fettcellens reglermekanismer och dess betydelse för diabetes och kardiovaskulär sjukdom.

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

AIMS/HYPOTHESIS: The amount of visceral fat mass strongly relates to insulin resistance in humans. The transcription factor peroxisome proliferator activated receptor gamma (PPARG) is abundant in adipocytes and regulates genes of importance for insulin sensitivity. Our objective was to study PPARG activity in human visceral and subcutaneous adipocytes and to compare this with the most common model for human disease, the mouse.

MATERIALS AND METHODS: We transfected primary human adipocytes with a plasmid encoding firefly luciferase controlled by PPARG response element (PPRE) from the acyl-CoA-oxidase gene and measured PPRE activity by emission of light. RESULTS: We found that PPRE activity was 6.6-fold higher (median) in adipocytes from subcutaneous than from omental fat from the same subjects (n = 23). The activity was also 6.2-fold higher in subcutaneous than in intra-abdominal fat cells when we used a PPARG ligand-binding domain-GAL4 fusion protein as reporter, demonstrating that the difference in PPRE activity was due to different levels of activity of the PPARG receptor in the two fat depots. Stimulation with 5 micromol/l rosiglitazone did not induce a PPRE activity in visceral adipocytes that was as high as basal levels in subcutaneous adipocytes. Interestingly, in mice of two different strains the PPRE activity was similar in visceral and subcutaneous fat cells.

CONCLUSIONS/INTERPRETATION: We found considerably lower PPARG activity in visceral than in subcutaneous primary human adipocytes. Further studies of the molecular mechanisms behind this difference could lead to development of drugs that target the adverse effects of visceral obesity.

OBJECTIVE: In clonal animal cells, certain angiotensin receptor blockers (ARB) activate the peroxisome proliferator-activated receptor-gamma (PPARgamma). The aim of this work was to validate that observation in human cells and humans. METHODS: We investigated the induction of in-vitro adipogenesis and the activation of PPARgamma-target genes, adiponectin and lipoprotein lipase, by ARB in human preadipocytes. We also studied PPARgamma response-element-driven luciferase reporter gene activation in human adipocytes. Finally, we treated 14 obese men for 10 days with placebo crossed over with 150 mg/day irbesartan. Subcutaneous fat was analyzed for mRNA expression of adiponectin and lipoprotein lipase. RESULTS: Telmisartan and irbesartan, and to a lesser degree losartan, induced adipogenesis and activated PPARgamma-target genes. This stimulation of PPARgamma-target genes was prevented by the PPARgamma antagonist GW9662. Eprosartan had no effect. Paradoxically, all ARB activated the luciferase reporter gene. PPARgamma activity increased approximately two-fold with pioglitazone and 1.5-fold with the ARB in all assays. In the cross-over clinical study, irbesartan lowered blood pressure but had no effect on adiponectin or lipoprotein lipase mRNA expression. CONCLUSIONS: Our data are the first to show that ARB induce adipogenesis and PPARgamma-target gene expression in human adipocytes. Pharmacokinetic differences may contribute to the heterogeneous effects on metabolism and preadipocyte differentiation. In humans, larger doses of ARB, longer treatments, or both may be required to activate PPARgamma in adipose cells.

OBJECTIVE: We studied the activity and regulation of the peroxisome proliferator-activated receptor-gamma response element (PPRE) in primary human adipocytes.

METHODS: We transfected primary human adipocytes with a plasmid-encoding firefly luciferase cDNA under control of a PPRE from the acyl-coenzyme A oxidase gene by using our newly developed electroporation-based method. Several fatty acids were added to the fat cells to study potential activation of peroxisome proliferator-activated receptor-gamma.

RESULTS: Cells responded maximally to 5 microM of rosiglitazone at a 5.1 +/- 1.4-fold over basal increase in luciferase activity. There was a positive correlation between body mass index and the response to 5 microM of rosiglitazone (r = 0.36, P = 0.03). Patients with type 2 diabetes had similar basal PPRE activity but responded more strongly to 5 microM of rosiglitazone than did non-diabetic subjects (10.2 +/- 5-fold and 5.4 +/- 1-fold over basal increase, respectively, P < 0.0001). Among saturated fatty acids, lauric acid was without effect, but 10 microM of palmitic or stearic acid increased PPRE activity 20% to 35% above basal levels. Monounsaturated palmitoleic acid at 1 microM induced a PPRE transcriptional activity that corresponded to half the therapeutic levels of rosiglitazone.

CONCLUSION: Adipocytes from obese subjects and patients with type 2 diabetes responded particularly strongly to the effect of rosiglitazone on PPRE. Because fatty acids in the diet can affect the transcriptional activity of peroxisome proliferator-activated receptor-gamma over decades, the stimulation induced by stearic and palmitoleic acids can affect insulin sensitivity and, hence, cardiovascular morbidity and mortality in humans.

Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% ± 5 (mean ± S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.

Background/aims: Epidermis forms the protective barrier of the skin by its outermost layer, stratum corneum. The purpose of this study was to investigate the epidermal barrier in view of epidermal calcium release (ECR), phosphate release, transepidermal water loss (TEWL) and skin surface pH. Calcium is mainly an intracellular ion. Calcium was sampled introducing a new and simple washout chamber technique for the study of epidermal release in vivo. Methods: Test sites on forearms of 13 healthy subjects were pre-treated with 24 h water occlusion, 24 h 2% sodium lauryl sulphate (SLS) or tape stripped. Both untreated and pretreated test sites were exposed to a water washout chamber with 200╡ deionized water as a solvent. Water washout chambers were removed after two hours and calcium and phosphate in the water was analyzed. Transepidermal water loss and pH were measured before and after the trial. Results: pH increased after tape stripping and after exposure to SLS. Transepidermal water loss increased significantly at all test sites. Calcium was significantly released from SLS-treated sites but not from tape stripped sites. There was generally a correlation between ECR, phosphate release, TEWL and pH. In this study ECR is showed to be a barrier marker of high reproducibility. Conclusions: Epidermal calcium release or ECR is found useful as an indicator of skin barrier function. Calcium release and increase of pH appear mainly to illustrate direct and corrosive damage to epidermal cells and functions contrasting TEWL, in this experiment probably reflecting intercellular damage of fracturing as exemplified by mechanical damage resulting from surface stripping. This new distinction of skin barrier damage into cellular damage resulting from a corrosive chemical trauma and intercellular damage and fracturing resulting from a mechanical trauma is exemplified in SLS provocative testing and tape stripping, the former characterized by increased ECR. The washout chamber technique was deemed technically reliable and reproducible, and has a major potential in experimental dermatology and skin pharmacology for the study of in vivo epidermal release of a range of endogenous and exogenous substances.

Background. Visceral obesity relates strongly to the metabolic syndrome and hence to hypertension. Although a local renin-angiotensin-system (RAS) in fat tissue is known, very few studies have dealt with RAS in isolated primary human fat cells, in particular from the visceral compartment.

Methods. Measurement of angiotensin II (Ang II) in medium from isolated primary human fat cells from visceral and subcutaneous adipose tissues and analyses of RAS-components in human fat cells and fat tissues.

Results. Primary human fat cells from omental adipose tissue produced more Ang II than subcutaneous cells. Treatment with insulin did not affect Ang II production and body-massindex of the fat-donors was unrelated to Ang II production. The PPAR gamma agonist rosiglitazone inhibited Ang II production in both types of isolated fat cells while addition of the Ang II receptor antagonist eprosartan inhibited the production in only subcutaneous fat cells. Addition of 50 or 200 nM of Ang II inhibited the PPAR gamma response elementactivity (PPRE-activity) in visceral, but not in the subcutaneous adipocytes.

Conclusions. Since high PPRE-activity induced by rosiglitazone inhibited the Ang II production, it is possible that reduced PPRE-activity in the visceral human fat cells, demonstrated by us earlier, can explain the comparatively high Ang II production in these cells. This could form the basis for a local paracrine viscous circle in visceral fat where low PPRE-activity increases Ang II production that is further enhanced by Ang II-mediated inhibition of PPRE-activity which ultimately leads to high concentrations of Ang II in human adipose tissue.