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Lipid Metabolism andInsulin Signalling in Adipocytes: enhanced autophagy in type 2 diabetes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Energy storage in the adipose tissue, to an extent leading to obesity, is associated with local as well assystemic insulin resistance. When insulin-producing beta-cells in the pancreas gradually fail tocompensate, plasma levels of glucose rise and overt type 2 diabetes is diagnosed. Adipocytes are largecells, mostly consisting of one big central lipid droplet, with the surrounding plasma membrane full ofsmall invaginations called caveolae. As caveolae contain the insulin receptor and several other insulinsignallingproteins, we have investigated several aspects of caveolae. We have also mapped mechanismsand defects in the insulin-signalling network in adipocytes from type 2 diabetic patients.

In paper I, we show that a subtype of caveolae has the capability to synthesize triglycerides from fattyacids and glycerol-3-phosphate. The triglyceride-synthesizing caveolae subtype also contains perilipin,suggesting the existence of a mechanism to protect newly made triglycerides from hydrolysis.

In paper II, we demonstrate that adipocytes from patients with type 2 diabetes have an attenuated insulinstimulatedphosphorylation of IRS-1 at Ser-307 (human sequence), which correlates with reduced insulinstimulatedphosphorylation of IRS-1 at tyrosine residues. Insulin-stimulated phosphorylation of IRS-1 atSer-307 is dependent on the nutrient sensor TORC1. This finding indicates that adipocytes from type 2diabetic patients have reduced TORC1 activity.

In paper III, we focus on the mechanisms for RBP4-induced insulin resistance. We also continue ourmapping of insulin-resistance in adipocytes from type 2 diabetes. These cells exhibit, in addition toimpaired insulin-stimulated glucose uptake and the defects presented in paper I, impaired insulinstimulatedphosphorylation of ERK. We do, however, not see any defects in PKB signalling. Neither dowe se any enhanced insulin-stimulated phosphorylation of IRS-1 at Ser-312 (human sequence), a site thatin mice is hyper-stimulated in response to high-fat feeding. Incubation with RBP4 recapitulates all defectswe so far have seen in type 2 diabetes except reduced insulin-stimulated glucose uptake. These results aremirrored by blockade of endogenously produced RBP4 in the incubations with adipocytes from type 2diabetic patients. In other words, RBP4-blocking antibodies restore all insulin-signalling defects we havefound in adipocytes from type 2 diabetic patients, except insulin-stimulated glucose uptake.

In paper IV we show by several approaches that TORC1 activation is down-regulated in adipocytes fromtype 2 diabetic patients. The main finding is that there is enhanced autophagy in those adipocytes.Interestingly, autophagy may be a mechanism to enhance the breakdown of stored triglycerides in theadipocyte.

In conclusion, our data suggest that caveolae, in addition to being micro-domains for insulin-signallingare metabolic platforms. We describe defects in insulin-signalling in adipocytes from type 2 diabeticpatients where the main finding is enhanced autophagy in these obese patients. The perceived starvationin adipose tissue might via secretion of adipokines, such as RBP4, have implications for local as well assystemic insulin-resistance.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 71 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1138
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-20656ISBN: 978-91-7393-575-3 (print)OAI: oai:DiVA.org:liu-20656DiVA: diva2:235454
Public defence
2009-10-09, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-09-16 Created: 2009-09-16 Last updated: 2009-09-17Bibliographically approved
List of papers
1. Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes
Open this publication in new window or tab >>Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 1, 5-8 p.Article in journal (Refereed) Published
Abstract [en]

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20650 (URN)10.1074/jbc.C400429200 (DOI)15537657 (PubMedID)
Available from: 2009-09-16 Created: 2009-09-16 Last updated: 2017-12-13Bibliographically approved
2. 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
3. Retinol-binding protein-4 attenuates insulin-induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes
Open this publication in new window or tab >>Retinol-binding protein-4 attenuates insulin-induced phosphorylation of IRS1 and ERK1/2 in primary human adipocytes
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2007 (English)In: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, ISSN 1530-6860, Vol. 21, no 13, 3696-3704 p.Article in journal (Refereed) Published
Abstract [en]

Reduced sensitivity to insulin in adipose, muscle, and liver tissues is a hallmark of type 2 diabetes. Animal models and patients with type 2 diabetes exhibit elevated levels of circulating retinol-binding protein (RBP4), and RBP4 can induce insulin resistance in mice. However, little is known about how RBP4 affects insulin signaling. We examined the mechanisms of action of RBP4 in primary human adipocytes. RBP4-treated adipocytes exhibited the same molecular defects in insulin signaling, via IRS1 to MAP kinase, as in adipocytes from patients with type 2 diabetes. Without affecting autophosphorylation of the insulin receptor, RBP4 blocked the insulin-stimulated phosphorylation of IRS1 at serine (307) [corresponding to serine (302) in the murine sequence] and concomitantly increased the EC50 (from 0.5 to 2 nM) for insulin stimulation of IRS1 phosphorylation at tyrosine. The phosphorylation of IRS1 at serine (312) [corresponding to serine (307) in the murine sequence] was not affected in cells from diabetic patients and was also not affected by RBP4. The EC50 for insulin stimulation of downstream phosphorylation of MAP kinase ERK1/2 was increased (from 0.2 to 0.8 nM) by RBP4. We show that ERK1/2 phosphorylation is similarly impaired in adipocytes from patients with type 2 diabetes. However, the sensitivity to insulin for downstream signaling to control of protein kinase B and glucose uptake was not affected by RBP4. When insulin-resistant adipocytes from patients with type 2 diabetes were incubated with antibodies against RBP4, insulin-induced phosphorylation of IRS1 at serine (307) was normalized and the EC50 for insulin stimulation of ERK1/2 phosphorylation was reduced. Endogenous levels of RBP4 were markedly reduced in adipocytes from obese or type 2 diabetic subjects, whereas expression levels of RBP4 mRNA were unaffected. These findings indicate that RBP4 may be released from diabetic adipocytes and act locally to inhibit phosphorylation of IRS1 at serine (307), a phosphorylation site that may integrate nutrient sensing with insulin signaling.

Keyword
Insulin resistance, type 2 diabetes, adipokine, protein phosphorylation, MAP kinase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20654 (URN)10.1096/fj.07-8173com (DOI)17575262 (PubMedID)
Available from: 2009-09-16 Created: 2009-09-16 Last updated: 2013-09-10Bibliographically approved
4. Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes
Open this publication in new window or tab >>Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes
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2010 (English)In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 16, no 07-Aug, 235-246 p.Article in journal (Refereed) Published
Abstract [en]

The protein kinase mammalian target of rapamycin (mTOR) mediates insulin control ofprotein synthesis, autophagy, mitochondrial function, and, through feedback signaling tophosphorylation of IRS1 at serine residues, mTOR directly controls insulin signaling. Weshow that in adipocytes from patients with type 2 diabetes (T2D) insulin activation of mTORis attenuated and that the resultant phenotype is compatible with, and can be mimicked by,loss of mTOR activation. In T2D adipocytes mitochondrial function is impaired andautophagy strongly upregulated, with concomitant increased autophagic destruction ofmitochondria and lipofuscin particles, and a dependence on autophagy for ATP production.Conversely, mitochondrial dysfunction attenuates insulin activation of mTOR, enhancesautophagy and attenuates feedback to IRS1. Our findings put mTOR in the driver´s seat of aninsulin resistance that in adipocytes can be fuelled by mitochondrial dysfunction,inflammation, ER-stress, or hypoxia.

Place, publisher, year, edition, pages
Feinstein Institute for Medical Research, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20655 (URN)10.2119/molmed.2010.00023 (DOI)000280048100001 ()20386866 (PubMedID)
Available from: 2009-09-16 Created: 2009-09-16 Last updated: 2017-12-13Bibliographically approved

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