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Insulin signalling in human adipocytes: mechanisms of insulin resistance in type 2 diabetes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

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.

Abstract [en]

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.

Place, publisher, year, edition, pages
Institutionen för klinisk och experimentell medicin , 2007.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1026
Keyword [en]
adipocytes, type 2 diabetes, insulin, insulin resistance, insulin signalling, phosphorylation, insulin receptor substrate 1, feedback control
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-10327ISBN: 978-91-85895-60-1 (print)OAI: oai:DiVA.org:liu-10327DiVA: diva2:17065
Public defence
2007-12-12, Aulan, Hälsans hus, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2013-09-10Bibliographically approved
List of papers
1. Glucose transport is equally sensitive to insulin stimulation, but basal and insulin-stimulated transport is higher, in human omental compared with subcutaneous adipocytes
Open this publication in new window or tab >>Glucose transport is equally sensitive to insulin stimulation, but basal and insulin-stimulated transport is higher, in human omental compared with subcutaneous adipocytes
2005 (English)In: Metabolism, ISSN 0026-0495, Vol. 54, no 6, 781-785 p.Article in journal (Refereed) Published
Abstract [en]

Excess visceral fat has been found to correlate more closely with morbidity than subcutaneous fat. We found that isolated adipocytes from omental fat of nondiabetic women expressed significantly more of the insulin-regulated glucose transporter glucose transporter 4 protein and exhibited a higher basal and insulin-stimulated rate of glucose transport, at all concentrations of insulin, than subcutaneous adipocytes from the same individuals. In contrast, dose-response relationships for insulin stimulation of glucose transport demonstrated identical sensitivity to insulin in adipocytes from the 2 locations. The results demonstrate that there is no relative insulin resistance to stimulate glucose uptake in visceral compared with subcutaneous fat cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12789 (URN)10.1016/j.metabol.2005.01.021 (DOI)
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2013-09-10
2. Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes
Open this publication in new window or tab >>Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes
Show others...
2005 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, no 1, 141-151 p.Article in journal (Refereed) Published
Abstract [en]

Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO2 at 37 °C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.

Keyword
glucose transport, insulin receptor substrate, MAP-kinase, p38, protein kinase B
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12790 (URN)10.1111/j.1432-1033.2004.04396.x (DOI)
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2017-12-14
3. Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes
Open this publication in new window or tab >>Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes
2005 (English)In: Journal of biological chemistry, ISSN 0021-9258, Vol. 280, no 41, 34389-3492 p.Article in journal (Refereed) Published
Abstract [en]

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC50 for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t1/2 of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12791 (URN)10.1074/jbc.C500230200 (DOI)
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2013-09-10Bibliographically approved
4. Phosphorylation of IRS1 at serine 307 and serine 312 in response to insulin in human adipocytes
Open this publication in new window or tab >>Phosphorylation of IRS1 at serine 307 and serine 312 in response to insulin in human adipocytes
2006 (English)In: Biochemical and biophysical research communications, ISSN 0006-291X, Vol. 342, no 4, 1183-1187 p.Article in journal (Refereed) Published
Abstract [en]

Feedback control in insulin signaling involves serine phosphorylation of insulin receptor substrate-1 (IRS1). By analyzing the insulin-induced phosphorylation of IRS1 at serine 307, serine 312, and tyrosine in the same primary human adipocytes, we now report that negative feedback phosphorylation of serine 312 (corresponding to murine serine 307) required relatively high concentrations of insulin (EC50 = 3 nM) for a long time (t1/2 ca. 30 min) and reduced the steady-state tyrosine phosphorylation, without affecting the cellular concentration, of IRS1. In contrast, positive feedback phosphorylation of serine 307 was a rapid (t1/2 ca. 2 min) event at physiological concentrations of insulin (EC50 = 0.2 nM).

Keyword
Insulin; Insulin resistance; Insulin receptor substrate-1; Serine phosphorylation; Tyrosine phosphorylation; Positive feedback; Negative feedback
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-12792 (URN)10.1016/j.bbrc.2006.02.075 (DOI)
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2013-09-10

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