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Mitochondrial form and function in pancreatic β-cells and brown adipocytes
Stockholm University, Faculty of Science, The Wenner-Gren Institute . (Jan Nedergaard)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is focused on the role of mitochondria in pancreatic β-cells and brown adipose tissue (BAT). Two main aspects of mitochondria were explored; mitochondrial functional efficiency and the interrelationship between mitochondrial shape and function.

Mitochondria in β-cells were found to exhibit heterogeneity in mitochondrial membrane potential. This functional diversity decreased when cells were challenged with glucose stimuli, suggesting that at higher fuel levels low-activity mitochondria are recruited into a pool of high-activity mitochondria. Glucolipotoxic conditions increased the functional diversity suggesting that this may be of importance for diabetes pathophysiology.

To examine mitochondrial efficiency in intact islets a high throughput islet respirometry method was developed. Due to increased uncoupling, islets from a diabetic animal model exhibit lower respiratory efficiency. Glucose, free fatty acids and amino acids all decreased respiratory efficiency. A large portion of the respiratory efficiency was mediated by reactive oxygen species and the adenine nucleotide translocase.

In β-cells mitochondria were found to undergo cycles of fusion and fission. During glucolipotoxicity mitochondria fragmented and lost their fusion ability. Knock down of the fission protein Fis1 rescued the β-cells from glucolipotoxic induced cell death. BAT mitochondria also showed fusion and fission. The mitochondrial dynamics proteins Mfn2 and Drp1 were shown to strongly affect BAT mitochondrial morphology. In response to a combination of adrenergic and free fatty acid stimuli mitochondria drastically changed from long filamentous structures to fragmented spheres. Inhibiting fission by the negative form of Drp1 decreased BAT response to adrenergic stimuli by half.

In conclusion, mitochondrial efficiency may be of importance for normal as well as compromised β-cell and islet function. Mitochondrial morphology appears critical for mitochondrial function in β-cells and BAT.

Place, publisher, year, edition, pages
The Wenner-Gren Institute,Stockholm University , 2010. , 70 p.
Keyword [en]
mitochondria, beta cells, brown adipose tissue, diabetes, obesity
National Category
Physiology
Research subject
Physiology
Identifiers
URN: urn:nbn:se:su:diva-39336ISBN: 978-91-7447-095-6 (print)OAI: oai:DiVA.org:su-39336DiVA: diva2:319497
Public defence
2010-06-14, hörsal Fb107, Frescati backe, Svante Arrhenius väg 21 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.Available from: 2010-05-23 Created: 2010-05-18 Last updated: 2010-08-04Bibliographically approved
List of papers
1. β-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels
Open this publication in new window or tab >>β-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels
2007 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 56, 2569-2578 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: beta-Cell response to glucose is characterized by mitochondrial membrane potential (Delta Psi) hyperpolarization and the production of metabolites that serve as insulin secretory signals. We have previously shown that glucose-induced mitochondrial hyperpolarization accompanies the concentration-dependent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the integrated response of a large number of mitochondria within each individual cell. However, it is currently unclear whether all mitochondria within a single beta-cell represent a metabolically homogenous population and whether fuel or other stimuli can recruit or silence sizable subpopulations of mitochondria. This study offers insight into the different metabolic states of beta-cell mitochondria. RESULTS: We show that mitochondria display a wide heterogeneity in Delta Psi and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increasing glucose concentration recruits mitochondria into higher levels of homogeneity, while an in vitro diabetes model results in increased Delta Psi heterogeneity. Exploration of the mechanism behind heterogeneity revealed that temporary changes in Delta Psi of individual mitochondria, ATP-hydrolyzing mitochondria, and uncoupling protein 2 are not significant contributors to Delta Psi heterogeneity. We identified BAD, a proapoptotic BCL-2 family member previously implicated in mitochondrial recruitment of glucokinase, as a significant factor influencing the level of heterogeneity. CONCLUSIONS: We suggest that mitochondrial Delta Psi heterogeneity in beta-cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target.

Place, publisher, year, edition, pages
American Diabetes Association, 2007
Keyword
mitochondria, beta cells, diabetes
National Category
Physiology
Research subject
Physiology
Identifiers
urn:nbn:se:su:diva-39341 (URN)10.2337/db06-0757 (DOI)
Note
12 authorsAvailable from: 2010-05-18 Created: 2010-05-18 Last updated: 2017-12-12Bibliographically approved
2. Mitochondrial networking protects beta cells from nutrient -induced apoptosis
Open this publication in new window or tab >>Mitochondrial networking protects beta cells from nutrient -induced apoptosis
2009 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 58, 2303-2315 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Previous studies have reported that beta-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating beta-cell mitochondrial morphology and function, remains unclear. In this article, we investigate beta-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients. RESEARCH DESIGN AND METHODS: Using matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in beta-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in beta-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on beta-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity. RESULTS: beta-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated beta-cell, a beta-cell within an islet, and an INS1 cell. Under noxious conditions, we find that beta-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis. CONCLUSIONS: These data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced beta-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.

Place, publisher, year, edition, pages
American Diabetes Association, 2009
Keyword
mitochondria, beta cells, diabetes
National Category
Physiology
Research subject
Physiology
Identifiers
urn:nbn:se:su:diva-39342 (URN)10.2337/db07-1781 (DOI)000270776200018 ()
Note
11 authorsAvailable from: 2010-05-18 Created: 2010-05-18 Last updated: 2017-12-12Bibliographically approved
3. Islet bioenergetic efficiency is regulated by nutrients
Open this publication in new window or tab >>Islet bioenergetic efficiency is regulated by nutrients
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background and aims

Excessive or unbalanced nutrient intake may cause diabetes. A growing body of evidence points to that mitochondrial dysfunction plays an important role in

Materials and methods

To enable the study we developed a high-throughput islet respirometry approach based on the XF24 platform, originally designed to study monolayers of cells. By applying drugs that act on the respiratory chain we can estimate the level of fuel-stimulated, uncoupled (reflecting proton leak), maximal as well as non-mitochondrial respiration under various conditions. Islets were derived from wildtype and high fat diet fed C57Bl6/J mice as well as from human donors.

β-cell dysfunction. The reason for this is likely that mitochondria stand in the center of nutrient metabolism. In this study we tested the hypothesis that islets from diabetic animals have defect mitochondrial respiratory function. Results

We found that due to increased proton leak, islets from diabetic high fat diet fed animals exhibit lower respiratory efficiency as compared to animals fed control chow. Examining the regulation of the leak we found that fuels that stimulate insulin secretion also increase uncoupled respiration, and that this may be mediated by reactive oxygen species. Moreover, dissecting the molecular mechanism, we show that the adenine nucleotide transporter contributes to one-third of the leak while uncoupling protein 2 and permeability transition pore appear not to contribute. Finally, we examined a cohort of human islets and found lower levels of uncoupled respiration as compared to mouse islets. However, as in the mouse islets glucose challenge induced increase in uncoupled respiration. Interestingly, there was a trend towards lower oxygen consumption rates in islets from obese donors.

Conclusion:

Islets have relative high levels of proton leak, which is regulated by cellular fuels and reactive oxygen species. Adenine nucleotide transporter but not uncoupling protein 2 or permeability transition pore appears to contribute to the observed uncoupled respiration. Interestingly, levels of uncoupled respiration increase in a diabetes animal model. In theory, tuning islet mitochondrial efficiency may represent a therapeutic target.

Keyword
mitochondria, islets, diabetes
National Category
Physiology
Research subject
Physiology
Identifiers
urn:nbn:se:su:diva-39343 (URN)
Available from: 2010-05-18 Created: 2010-05-18 Last updated: 2010-08-04Bibliographically approved
4. Brown adipocyte activation is characterized by a wave of mitochondrial fission and depolarization that is dependent on β3 receptor stimulation and Drp1, and is characterized by complete, but reversible, arrest of fusion
Open this publication in new window or tab >>Brown adipocyte activation is characterized by a wave of mitochondrial fission and depolarization that is dependent on β3 receptor stimulation and Drp1, and is characterized by complete, but reversible, arrest of fusion
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background and aims:

Mitochondria are dynamic organelles that frequently undergo fusion and fission. Mitochondrial dynamics has been shown to be essential for a variety of cellular functions and its abrogation has been associated with several diseases. However, the role fusion, fission and architecture play in mitochondrial bioenergetics is still not well understood. Brown adipose tissue (BAT) is a mitochondria dense organ that converts cellular fuels into heat by mitochondrial uncoupling. When activated adrenergically, BAT shows a unique increase in mitochondrial respiratory activity. Therefore, BAT may be a good model to study the interdependence between mitochondrial morphology, dynamics and function. To date, mitochondrial dynamics was not studied in BAT. In this study we set out to examine mitochondrial morphology in BAT and test the hypothesis that mitochondrial morphology is of importance for BAT function.

Materials and methods:

BAT was harvested from 3 to 4-week-old wild-type male C57BL6/J mice and from 5-8 day old pups (Mitofusin2 knockout). Brown adipocytes were differentiated in vitro. A LSM 710 laser scanning confocal microscopy (Zeiss) was used for imaging of mitochondrial morphology using several fluorescent dyes and proteins. Oxygen consumption was measured using the XF24 platform (Seahorse Bioscience). The pro fusion protein Mfn2 was knocked out under the AP2 promoter and the pro fission protein Drp1 was inhibited with adenoviral expression of its dominant negative form.

Results:

Mitochondria were found to be highly networked and dependent on mitochondrial dynamics proteins. When stimulating cells with a combination of norepinephrine and free fatty acids we found a synergistic response that included a marked increase in oxygen consumption rates and mitochondrial membrane potential (Δψm) depolarization. Somewhat unexpectedly it was also found that mitochondria in parallel underwent a distinct fragmentation. The fragmented mitochondria appeared sphere-like and had dampened fusion; however cells regained normal function as well as mitochondrial morphology and Δψm within 24h.  Interestingly, Δψm depolarized and mitochondria fragmented in a wave-like fashion where depolarization preceded fragmentation. Inhibition of the pro-fission protein Drp1 was found to inhibit the synergistic response, while knock-out of the pro-fusion protein Mfn2 did not. Thus, mitochondrial fission appeared essential for proper BAT function. Finally, we found the synergistic response to go through the β-adrenergic pathway and be dependent on reactive oxygen species but not on Ca++, permeability transition pore or uncoupling protein 1 expression levels.

Conclusion:

Taken together, these findings suggest that mitochondrial morphology in general and mitochondrial fission in particular may play an important physiological role in BA. Future studies will reveal if this may represent a therapeutic target for manipulating BAT activity.

Keyword
mitochondria, brown adipose tissue, obesity
National Category
Physiology
Research subject
Physiology
Identifiers
urn:nbn:se:su:diva-39344 (URN)
Available from: 2010-05-18 Created: 2010-05-18 Last updated: 2010-08-04Bibliographically approved

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