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Regulation of Lipoprotein Lipase Activity: an In-vitro Study of a Complex and Dynamic System
Umeå University, Faculty of Medicine, Department of Medical Biosciences. (Physiological Chemistry)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The progress of human society outpaces our biological evolution, and this brings unexpected health problems. The industrial revolution brought up dramatic changes in nutrition and lifestyle – we are increasingly shifting towards a nutrient-rich Western-patterned diet and to a sedentary lifestyle. Unfortunately, our energy metabolism is not fully capable to adapt to these changes. The excess of nutrients has led to global epidemics in obesity, type 2 diabetes, and cardiovascular diseases. To battle this, medical research has focused on the metabolism of sugars and cholesterol, whereas the fate of triglycerides, the major dietary lipid, has received less attention. Recently it became clear that blood triglycerides are connected to the development of type 2 diabetes and cardiovascular diseases. As a result, triglyceride metabolism became a focus of attention in both basic and clinical research.

Lipoprotein lipase (LPL) is the cornerstone of blood triglyceride metabolism. This means that LPL must be tightly regulated in response to the nutritional state of the body, and to the needs of particular tissues. LPL is produced and secreted by cells that store triglycerides or use them for generation of energy. After secretion, LPL stays attached to the capillary endothelium where it hydrolyses triglycerides from the triglyceride-rich lipoproteins. LPL is relatively unstable and the instability is a key property in its physiological regulation since transcriptional control of LPL does not respond to the metabolic changes fast enough. Instead, LPL is regulated by two groups of proteins – plasma apolipoproteins, which serve as activators or inhibitors of LPL, and angiopoietin-like (ANGPTL) proteins, which irreversibly inactivate LPL in the tissues which do not require triglycerides.

One aim of my thesis was to study the effects of ANGPTL proteins on LPL structure and function. In papers I and II, using various biophysical and biochemical methods, we studied the effects of ANGPTL3, 4 and 8 on LPL structure and function. All data supported the concept that LPL is inactivated by dissociation of active dimers to monomers. Additionally, we describe the molecular basis for complex formation between ANGPTL3 and 8, as well as a novel complex between ANGPTL4 and 8 with unique properties. The other aim of my thesis was to perform an in-depth study of rate-limiting factors that control the activity of LPL in human plasma. In papers III and IV we study LPL activity using an isothermal titration calorimetry-based assay directly in plasma samples. We found that the normal variation in plasma levels of either ANGPTL proteins or apolipoproteins had no significant impact on LPL activity. Instead, the strongest determinant for LPL action was the size of the triglyceride-rich plasma lipoproteins.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2019. , p. 80
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2036
Keywords [en]
Triglyceride Metabolism, Lipoprotein lipase, Angiopoietin-Like Proteins, Apolipoproteins, Isothermal titration calorimetry
National Category
Medical and Health Sciences Medicinal Chemistry Cell and Molecular Biology
Research subject
Biochemistry; Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-159175ISBN: 978-91-7855-050-0 (print)OAI: oai:DiVA.org:umu-159175DiVA, id: diva2:1316868
Public defence
2019-06-13, Lecture hall D, Unod T9, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2019-05-23 Created: 2019-05-21 Last updated: 2019-05-22Bibliographically approved
List of papers
1. On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity
Open this publication in new window or tab >>On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity
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2019 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 60, no 4, p. 783-793Article in journal (Refereed) Published
Abstract [en]

Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2019
Keywords
angiopoietin-like 8, angiopoietin-like 3, angiopoietin-like 4, glycosylphosphatidylinositol-anchored HDL binding protein 1, lipoprotein metabolism, enzymology, enzyme regulation, triglycerides
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:umu:diva-159138 (URN)10.1194/jlr.M088807 (DOI)000466567300011 ()30686789 (PubMedID)2-s2.0-85064197839 (Scopus ID)
Available from: 2019-05-20 Created: 2019-05-20 Last updated: 2019-06-18Bibliographically approved
2. The angiopoietin-like protein ANGPTL4 catalyzes unfolding of the hydrolase domain in lipoprotein lipase and the endothelial membrane protein GPIHBP1 counteracts this unfolding
Open this publication in new window or tab >>The angiopoietin-like protein ANGPTL4 catalyzes unfolding of the hydrolase domain in lipoprotein lipase and the endothelial membrane protein GPIHBP1 counteracts this unfolding
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2016 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 5, article id e20958Article in journal (Refereed) Published
Abstract [en]

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal alpha-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-130455 (URN)10.7554/eLife.20958 (DOI)000390662000001 ()27929370 (PubMedID)
Available from: 2017-01-24 Created: 2017-01-20 Last updated: 2019-05-21Bibliographically approved
3. Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry
Open this publication in new window or tab >>Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry
2017 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 58, no 1, p. 279-288Article in journal (Refereed) Published
Abstract [en]

LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or postheparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride poor. Addition of angiopoietinlike protein (ANGPTL) 3 or ANGPTL4 caused reduction of the activity of LPL via a two-step mechanism.(Jlr) We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system.

Keywords
lipolysis, apolipoproteins, angiopoietin-like proteins, triglycerides, very low density lipoprotein, enzymology
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-131652 (URN)10.1194/jlr.D071787 (DOI)000392408700024 ()27845686 (PubMedID)2-s2.0-85009962352 (Scopus ID)
Available from: 2017-02-27 Created: 2017-02-27 Last updated: 2019-05-21Bibliographically approved
4. Effects of human plasma components on the activity of lipoprotein lipase: a study of samples from the VIPVIZA cohort using isothermal titration calorimetry
Open this publication in new window or tab >>Effects of human plasma components on the activity of lipoprotein lipase: a study of samples from the VIPVIZA cohort using isothermal titration calorimetry
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(English)Manuscript (preprint) (Other academic)
National Category
Medicinal Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-159139 (URN)
Available from: 2019-05-21 Created: 2019-05-21 Last updated: 2019-06-13Bibliographically approved

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