Thesis
Reference
Generation and characterization of an inducible conditional b-cell-specific glutamate dehydrogenase knockout mouse
VETTERLI, Laurene Marine
Abstract
La glutamate déshydrogénase (GDH) est une enzyme mitochondriale qui convertit l'a-ketoglutarate produit par le cycle de Krebs en glutamate, et vice et versa. Nous avons généré une lignée de souris dont les cellules bêta-pancréatiques n'expriment plus cette enzyme (ßGlud1-/-) et avons observé qu'une sécrétion optimale d'insuline n'est pas requise pour maintenir l'homéostase du glucose lorsque ces animaux sont sous diète normale. Nous avons soumis ces animaux à une diète hautement calorique, situation requérant une demande accrue en insuline, et observé une résistance à l'obésité. Dans le cas ou l'inactivation de la GDH dans les cellules b aurait lieu une fois que les souris sont obèses, la question est de savoir si la perte de cette enzyme pourrait induire une perte de poids ou provoquer le diabète. Nous avons donc généré un modèle de souris inductible permettant d'inactiver la GDH dans les cellules b une fois ces souris rendues obèses. L'inactivation de cette enzyme ne provoque pas de perte de poids, néanmoins, les souris dont les cellules b n'expriment plus la GDH suite à l'injection de [...]
VETTERLI, Laurene Marine. Generation and characterization of an inducible conditional b-cell-specific glutamate dehydrogenase knockout mouse. Thèse de doctorat : Univ.
Genève, 2011, no. Sc. 4298
URN : urn:nbn:ch:unige-158465
DOI : 10.13097/archive-ouverte/unige:15846
Available at:
http://archive-ouverte.unige.ch/unige:15846
Disclaimer: layout of this document may differ from the published version.
U N I V E RSI T E D E G E N E V E
Département de Biologie Moléculaire F A C U L T E D ES SC I E N C ES
Professeur Ueli Schibler
Département de Physiologie Cellulaire et F A C U L T E D E M E D E C I N E
Métabolisme Professeur Pierre Maechler
G ene ration and characte rization of an indu cible conditional E-cell-specifi c glutamate d ehydrogenase knoc kout mouse
T H ESE
3UpVHQWpHjOD)DFXOWpGHVVFLHQFHVGHO¶8QLYHUVLWpGH*HQqYH pour obtenir le grade de Docteur ès sciences, mention biologie
par Laurène Vetterli
de Genève (GE)
Thèse N° 4298
GENEVE
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"Shoot for the moon, even if you miss it you will land among the stars"
Oscar Wilde
Je te dédie ce travail Opa Avec les années ton VRXYHQLUV¶HVWRPSHPDLVUHVWHUD toujours dans ma mémoire
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7RXW G¶DERUG MH YRXGUDLV UHPHUFLHUles Pr. Ueli Schibler et Hindrik Mulder G¶DYRLU DFFHSWp G¶être membres de mon jury de thèse.
Mes plus vifs remerciements reviennent au Pr.Pierre MaechOHU TXL P¶D DFFXHLOOLH GDQV VRQ pTXLSHGHUHFKHUFKHTXLP¶DIDLWFRQILDQFHHWP¶DODLVVpXQHJUDQGHOLEHUWpGDQVPDIDoRQGH WUDYDLOOHU 7X P¶DV GRQQp OD FKDQFH GH WUDYDLOOHU VXU GHV VXMHWV H[WUrPHPHQW VWLPXODQWV intellectuellement dans une atmosphère décontractée. Je recommencerais une thèse demain dans ton laboratoire et je te souhaite une très longue et brillante carrière scientifique.
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Je remercie chaleureusement tous mes collègues de laboratoire, Déborah, Clarissa, Gaëlle, 1LQJ 6DFKLQ 'RPLQLTXH HW $QGUHD SRXU O¶DPELDQFH GH WUDYDLO GpWHQGXH SRXU OHXU DLGH HW OHXUVFRQVHLOV*UkFHjYRXVM¶DLSDVVpGHWUqVERQVPRPHQWVSHQGDQWFHVTXDWUHDQQpHV.
Un immense merci à Francesca pour ses conseils scientifiques, son écoute, mais aussi pour son amitié et les bons moments que nous avons partagés. Tu avais la lourde responsabilité G¶rWUH OD SRVW-GRF GX ODER PDLV WX DV WRXMRXUV IDLW SUHXYH G¶XQH JUDQGH SDWLHQFH HW G¶XQH JUDQGHGLVSRQLELOLWpDYHFOHVRXULUHHQSOXV«(WPrPHVLWXQ¶HVFR-auteure sur aucun de mes SDSLHUVF¶HVWWRLTXLP¶DVDSSULVODSOXSDUWGHVWHFKQLTXHVTXHM¶DLXWLOLVpHVSHQGDQWPDWKqVH -¶HVSqUHXQMRXUSRXYRLUIDLUHODPrPHFKRVHSRXUXQGRFWRUDQW«
0HUFLpJDOHPHQWj0HOLVTXLP¶DpFRXWpHHWFRQVHLOOpHFRQFHUQDQWPHVFKRL[IXWXUV«0HUFL pour ta disponibilité et ta gentillesse, je te souhaite beaucoup de succès dans ton post-doc ! Un merci tout particulier à Thierry, pour toutes les discussions que nous avons eues, et pas seulement les discussions scientifiques 7XDVWRXMRXUVVXP¶pFRXWHUTXDQGM¶HQDYDLVEHVRLQ et par ta personnalité riche, intéressante et optimiste, tu as grandement contribué à rendre O¶DWPRVSKqUHDJUpDEOHHWGpWHQGXHDXODER&¶pWDLWXQSODLVLUGHWUDYDLOOHUDYHFWRLWXYDVPH PDQTXHU«
Merci à Stefania, qui a généré le modèle de souris knock-RXWSRXUOD*'+HWTXLP¶DFRDFKpH pendant les 3 premiers mois de ma thèse.
0HUFL j $VOODQ HW j &KULVWLDQ TXL P¶RQW DLGpH SRXr la chirurgie sur les souris. Christian, SKLORVRSKHU HQ WUDYDLOODQW FH Q¶HVW SDV DYHF WRXW OH PRQGH TXH M¶DL SX OH IDLUH 0HUFL pJDOHPHQW j WRXWHV OHV SHUVRQQHV GX GpSDUWHPHQW HW GX &08 TXL P¶RQW DLGpH VRLW HQ PH prêtant du matériel, soit en me donnant des conseils ou suggestions, en particulier le Dr.
Françoise Assimacopoulos-Jeannet et le Dr. Christelle Veyrat-Durebex.
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Merci égalemenW j 6pEDVWLHQ TXL P¶D VXSHUYLVpH pendant mon master et qui a guidé mes premiers pas dans un labo.
Merci du fond du coeur à mes fidèles amis, Nuria, Christine, Christophe, Steph, Caro, Rachel SRXU OHXU SUpVHQFH TXDQG M¶HQ DYDLV EHVRLQ SRXU WRXV OHV ERQV PRPHQWV HW OHV IRXV ULUHV SDUWDJpV6DQVYRXVMHQHVHUDLVSDVOjDXMRXUG¶KXL«
Julien, tu as suivi ce projet pendDQWWURLVDQV0HUFLG¶DYRLUWRXMRXUVpWpOjSRXUPRLPHUFLGH P¶DYRLUHQFRXUDJpHHWpFRXWpH
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T A B L E O F C O N T E N TS
List of Abreviations ... I Abstract... IV Résumé ... VI
1. Introduction ... 1
1.1 Anatomy and Physiology of the islets of L angerhans ... 1
1.2 Insulin ... 3
1.2.1 Insulin structure and synthesis ...3
1.2.2 Insulin action in the Liver ...5
1.2.3 Insulin action in the Muscle ...5
1.2.4 Insulin action in the Adipose Tissue ...6
1.3 E-cell function and regulation of insulin secretion ... 6
1.3.1 The in vivo regulation of pulsative insulin secretion ...7
1.3.2 The first phase of insulin secretion...8
1.3.3 The amplifying pathway...9
1.3.4 Exocytosis in E-cells ...10
1.3.5 Hormones and factors regulating insulin secretion ...10
1.4 Implication of mitochondria in E-cell function and dysfunction... 12
1.4.1 Mitochondria origin and function...12
1.4.2 Anaplerosis and Cataplerosis ...13
1.4.3 Oxidative phosphorylation ...14
1.4.4 Nucleotides and metabolites involved in glucose-stimulated insulin secretion...15
1.5 Glutamate and other amino acids implicated in insulin secretion ... 17
1.5.1 The role of Glutamate in glucose-stimulated insulin secretion ...18
1.5.2 Glutamate biosynthesis ...19
1.5.3 Glutamate carriers ...21
1.6 A focus on Glutamate dehydrogenase ... 22
1.6.1 Structure and enzymatic regulation of mammalian GDH ...22
1.6.2 Genetics of GDH ...24
1.6.3 GDH function in the brain ...24
1.6.4 GDH function in pancreatic E-cell ...25
1.7 T he Sirtuin family ... 27
1.7.1 Discovery of the Sirtuins ...27
1.7.2 The Mammalian Sirtuins: SIRT1-7 ...27
1.7.3 Mitochondrial Sirtuins-key regulators of metabolism? ...28
1.7.4 A focus on SIRT1...31
1.7.4.1 Implication of SIRT1 in calorie restriction ...31
1.7.4.2 SIRT1 implication in glucose homeostasis in different tissues...32
1.7.4.3 Resveratrol effects on metabolism- is SIRT1 implicated? ...34
1.8 Diseases linked to E-cell dysfunction... 35
1.8.1 Diabetes ...35
1.8.1.1 Type I Diabetes Mellitus (T1DM) ...35
1.8.1.2 Type II Diabetes Mellitus (T2DM) ...35
1.8.1.3 Maturity Onset Diabetes of the Young (MODY) ...37
1.8.1.4 Mitochondrial Diabetes...37
1.8.2 Hyperinsulinism-hyperamonaemia syndrome ...38
2. Aim of the study ... 39
2.1 In vitro cellular characterization of the ßGlud1-/- islets... 39
2.2 In vivo importance of the amplifying pathway in obesity... 40
2.3 In vitro effects of sirtuins and resveratrol in INS-1E cell line and human islets... 41
3. Materials and Methods ... 42
3.1 In vitro cellular characterization of the E-cell G D H-K O islets ... 42
3.2 In vivo importance of the amplifying pathway in obesity... 42
3.2.1 Generation and genotyping of E-cell specific GDH knockout mouse (ßGlud1-/-), and time-specific tamoxifen-inducible in vivo GDH knockout mouse (Rip-CreERT mice) ...42
3.2.2 High fat diet treatment...43
3.2.3 Tamoxifen Treatment ...43
3.2.4 Glucose tolerance test (ipGTT) and Insulin tolerance test (ipITT) ...43
3.2.5 Mouse pancreatic islet isolation ...43
3.2.6 Perifusions on isolated mouse islets ...44
3.2.7 In situ pancreatic perfusion ...44
3.2.8 Static insulin secretion on isolated mouse islets...44
3.2.9 Metabolic analyses ...45
3.2.10 Cold stress test ...45
3.2.11 Body composition ...45
3.2.12 Gene expression analyses by quantitative RT-PCR...45
3.2.13 Immunoblotting on pancreatic islets lysate ...49
3.2.14 Statistical analysis. ...49
3.3 In vitro effects of sirtuins and resveratrol in INS-1E cell line and human islets... 49
4.1 In vitro cellular characterization of the ßGlud1-/- islets... 50
4.2 In vivo importance of the amplifying pathway in obesity... 52
4.2.1 ßGlud1-/- mice are resistant to diet-induced obesity ...52
4.2.2 ßGlud1-/- mice are protected against glucose intolerance, with decreased plasma insulin levels during ipGTT and are more insulin sensitive ...55
4.2.3 ßGlud1-/- mice secrete less insulin than control mice, both on standard and high fat diet ...59
4.2.4 Calorimetric experiments demonstrate a hypermetabolic state in ßGlud1-/- mice ...62
4.2.5 Gene expression profile ...67
4.2.6 The inducible KO mice established once the mice are insulin-resistant seem to protect them against further weight gain ...71
4.3 In vitro effects of sirtuins and resveratrol in INS-1E cell line and human islets... 79
5. Discussion ... 82
5.1 G D H is necessary for the full development of both the triggering pathway and the amplifying pathway of the secretory response ... 82
5.2 Glutamine-induced insulin secretion is completely abrogated in ßGlud1-/- islets but could be rescued by G D H overexpression ... 82
5.3 T he decrease in glucose-stimulated insulin secretion in ßGlud1-/- islets correlates with lower glutamate levels, and is rescued by dimethyl-glutamate... 83
5.4 Aspartate and alanine levels are decreased in ßGlud1-/- mice upon glucose stimulation, and the aminotransferases are not able to compensate the lack of G D H ... 84
5.5 Glutamate levels and glucose-stimulated insulin secretion could be rescued in ßGlud1-/- mice in the presence of glutamine ... 87
5.6 T he expression of the glutamate transporter G C1 was decreased in ßGlud1-/- mice .... 88
5.7 Reduced E-cell secretory response is asymptomatic under normo caloric conditions.. 88
5.8 Insulin secretion impairment protects against high-fat induced obesity ... 89
5.9 ßGlud1-/- mice fed a high-fat diet are hypermetabolic ... 92
5.10 Would limited E-cell function in a situation of insulin resistance promote weight loss or trigger diabetes? ... 94
5.11 Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through Sirt1 dependent mechanism. ... 95
6. Conclusion and Perspectives ... 100
References ... 103
D-K I C D-ketoisocaproate
A C adenyl cyclase
A G C aspartate-glutamate car rier
A L A T/ASA T Alanine/Aspartate aminotransfer ase A M P K A M P-activated protein kinase A O A Amino-oxyacetate
A T P/ADP adenine tri/diphosphate B A T Brown adipose tissue
B C H beta-(+/-)-2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid B EST O Ecellspecific-SI R T1-overexpressingmice
ßGlud1-/- Glutamate dehydrogenase E-cell specific knockout mouse [C a2+]c C ytoplasmic C alcium Concentr ation
[C a2+]i Intracellular C alcium Concentration cA M P cyclic adenosine mono-phosphate
CoA coenzyme A
CPS1 carbamoyl phosphate synthetase 1 CPT-1 carnithine palmitoyltransferase 1
C R calorie restriction
E R endoplasmic reticulum
E T C electron transport chain
F I R K O Fat-specific insulin receptor knockout mouse G AD Glutamate decarboxylase
G AB A J-aminobutyric acid G D H Glutamate dehydrogenase G IP gastric inhibitory polypeptide
G K Glucokinase
Glc Glucose
Gln Glutamine
G L P-1 Glucagon-like peptide 1
Glut Glutamate
G L U T1-4 Glucose transporter 1-4
GSIS Glucose-stimulated insulin secretion
H I/H A hyperinsulinism/hyperammonaemia syndrome I NS-1 insulinoma rat cell line
ip G T T intraperitoneal Glucose Tolerance Test ipI T T intraperitoneal I nsulin Tolerance Test KATP-channel A T P-sensitive potassium-channel K C l Potassium C hloride
K Da kilo D alton DK G D-ketoglutar ate DK I C D-ketoisocaproate
K O knock-out
M I D D M aternally inherited diabetes and deafness M I R K O M uscle-specific insulin receptor knockout mouse M O D Y M aturity-O nset D iabetes of the Young
mR N A messenger Ribonucleic acid mtD N A mitochondrial D N A
O A A oxaloacetate
PC Pyruvate car boxylase
PC1 Prohormone convertase 1 PC2 Prohormone convertase 2 PD H Pyruvate dehydrogenase
PG C-1D Peroxisome proliferator-DFWLYDWHGUHFHSWRUĮFRDFWLYDWRU-Į PP ARJ Peroxisome proliferator-activated receptor J
P K A Protein kinase A
PP Pancreatic polypeptide
Pyr Pyruvate
red.equ. reducing equivalents
R E R rough endoplasmic reticulum R IP Rat I nsulin Promoter
R OS Reactive oxygen species R RP Readily Releasable Pool
RSV Resveratrol
SI R T1 Sirtuin 1
SN AR E soluble N-ethylmaleimide-sensitive factor attachment receptor SU R1 sufonylurea receptor 1
T C A T ricarboxylic acid T1D M Type 1 Diabetes Mellitus T2D M Type 2 Diabetes Mellitus U CP1-2 Uncoupling protein 1-2
V G L U T1-2 Vesicular glutamate tr ansporters 1 and 2
'<m/c M itochondrial or cytoplasmic membr ane potential W A T White adipose tissue
W T Wild type
Abstrac t
Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyses the reversible transformation of glutamate to D-ketoglutarate. GDH might play a role in glucose-induced amplifying pathway through generation of glutamate and/or as an amino acid sensor triggering insulin release upon glutamine stimulation in conditions of GDH allosteric activation. Here, we investigated the role of GDH in a E-cell specific GDH knockout mouse model, named ßGlud1-/-, generated in our laboratory. We observed that ßGlud1-/- mice had limited secretory response to high glucose without apparent in vivo pathological consequences, at least in animals fed a normal diet. However, the secretory response to glucose could be rescued when GDH knockout islets were simultaneously exposed to high glucose and dimethyl-glutamate, added as a cell permeable glutamate precursor. When testing the amplifying pathway by combination of KCl and diazoxide plus high glucose, the sustained insulin release observed in control islets was inhibited in ßGlud1-/- islets. Glutamine stimulation is strictly GDH dependent and elicits insulin release only upon GDH allosteric activation, for instance using BCH. In accordance with this model, ßGlud1-/- islets were totally unresponsive to glutamine plus BCH stimulation. The response to glutamine plus BCH was rescued in GDH knockout islets transduced with an adenovirus encoding for GDH.
Amino acids related to glutamate pathways (glutamate, glutamine, aspartate and alanine) were all reduced in GDH knockout islets stimulated with high glucose. In conclusion, our data show that GDH is essential for sustained insulin secretion at optimal glucose concentration. In these conditions, lower glutamate concentrations seem to be responsible for reduced secretory response as provision of cell permeant glutamate restored the secretory response. The lack of GDH also impacted on related glutamate pathways, pointing to GDH as a master switch linking glucose and amino acid metabolisms.
As our ßGlud1-/- mouse model shows that the full secretory capacity is not required for maintenance of glucose homeostasis when animals are fed a normal diet, this questions the role of the amplifying pathway in insulin secretion. This is why, in the context of diet-induced obesity and E-cell efficiency, we wondered how a primary limitation of E-cell function, prior to both high calorie intake and insulin resistance, would impact on weight gain and eventually obesity. After 20 weeks of high fat diet, control mice developed obesity and were heavier than lean controls maintained on standard chow diet. Remarkably, ßGlud1-/- mice were resistant to diet-induced obesity, as their body weights overlapped lean controls. Metabolic efficiency was reduced in ßGlud1-/- mice compared to control mice, along with unchanged food intake.
The mean RER value over 24h was similar between ßGlud1-/- and control obese mice.
However, when detailed over time, RER was lower in ßGlud1-/- compared to control mice by the end of the night, indicating that whole-body fatty acid oxidation was increased at that time. This was paralleled by higher energy expenditure (VO2)and VCO2 in ßGlud1-/- mice compared to control mice. Since the large majority of whole body O2 consumption and CO2
production in mammals resulted from physical activity, we measured differences in ambulatory activity, and we observed that ßGlud1-/- mice exhibited significant higher activity over a-24h period. However, this was not the case for the ßGlud1-/- mice on a standard chow diet, suggesting that the increased activity is a consequence rather than a cause of the resistance to obesity.
ßGlud1-/- mice did not develop obesity-associated glucose intolerance and glycaemia measured after an overnight fast were lower compared to control mice. This paralleled lower plasma insulin levels in the fast state, and increased insulin sensitivity calculated with the homeostasis model assessment index (HOMA-IR). Glucose-stimulated insulin secretion was much lower in ßGlud1-/- than in control animals on high-fat diet. In conclusion, the absence of GDH in beta-cells preserved a lean phenotype by limiting insulin release up to levels preventing excessive fat storage and the accompanying insulin resistance.
It is well known that obesity is associated to insulin hypersecretion due to insulin resistance and may lead to diabetes in case of E-cell failure. However, only 50% of obese people become diabetic, suggesting that genetic background is important. In case deletion of GDH within E-cells would happen once mice are obese, it is a intriguing question if the loss of this enzyme would promote weight loss or trigger diabetes because of limited E-cell function in a situation of insulin resistance requiring increased E-cell activity. Therefore, we used the inducible Cre-lox system to achieve the recombination of the GDH specifically within the E- cell following a high-fat diet treatment. This system allows temporally-controlled recombination because tamoxifen injection results in a rapid and transient nuclear translocation of the CreERT protein which is maintained in the cytoplasm bound by heat shock proteins in the absence of ligand. When floxed mice became obese and insulin-resistant after 14 weeks of high-fat diet treatment, we injected tamoxifen. Our data show that the loss of GDH did not induce weight loss because of limited E-cell function in a situation of insulin resistance requ were protected against further fat body mass depot and against hyperglycaemia.
Résumé
La glutamate déshydrogénase (GDH) est une enzyme mitochondriale TXL FRQYHUWLW O¶D- ketoglutarate produit par le F\FOH GH .UHEV HQ JOXWDPDWH HW YLFH HW YHUVD /RUV G¶XQH stimulation de la cellule beta par du glucose, il a été suggeré que le glutamate généré agit comme messager intracellulaire et participe à la phase amplificatrice de la sécrétion G¶LQVXOLQH1RXVDvons généré dans le laboratoire une lignée de souris dont les cellules bêta- SDQFUpDWLTXHV Q¶H[SULPHQW SOXV FHWWH HQ]\PH (ßGlud1-/-) et avons observé que ces souris VpFUqWHQW PRLQV G¶LQVXOLQH HQ UpSRQVH DX JOXFRVH P0 VDQV WRXWHIRLV PRQWUHU de consequences pathologiques in vivo, du moins lorsque ces animaux sont sous diète normale.
Toutefois, nous avons pu restaurer la réponse au glucose lorsque les ilots des souris ßGlud1-/- sont exposés à des hautes concentrations de glucose en présence de diméthyl-glutamate, un précurseur du glutamate pérméable à la cellule. Nous avons testé la réponse de la phase DPSOLILFDWULFH GH OD VpFUpWLRQ G¶LQVXOLQH HQ SUpVHQFH GX PpODQJH .&O HW GLD]R[LGH HQ FRPELQDLVRQDYHFGXJOXFRVHHWDYRQVREVHUYpTX¶HOOHHVWGLPLQXpHFKHz les souris ßGlud1-/- par rapport aux contrôles. La réponse à la glutamine est dépendante de la présence de la GDH HW VWLPXOHOD VpFUpWLRQ G¶LQVXOLQHXQLTXHPHQWHQSUpVHQFH G¶DFWLYDWHXUVDOORVWpULTXHV GH FHWWH enzyme, tels que la leucine ou la BCH. En accord avec ce modèle, nous avons observé que les îlots des souris ßGlud1-/- ne pépondent pas du tout aux sécrétagogues glutamine/BCH. En revanche, la réponse au mélange glutamine/BCH a pu être restaurée dans les îlots infectés avec un adénovirus codant pour la GDH. Nous avons mesuré le taux des acides aminés V\QWKpWLVpVjSDUWLUGXJOXWDPDWHJOXWDPLQHDVSDUWDWHHWDODQLQHHWDYRQVREVHUYpTX¶LOVVRQW tous diminués dans les îlots des souris ßGlud1-/- stimulés en présence de glucose (22.8mM).
En conclusion, nos données montrent que la GDH est essentielle pour une sécrétion optimale G¶LQVXOLQH 'DQV FHV FRQGLWLRQV OHV IDLEOHV FRQFHQWUDWLRQV GH JOXWDPDWH VHPEOHQW rWUH UHVSRQVDEOHVGHO¶LQKLELWLRQGHODVpFUpWLRQREVHUYpHFDUODSUpVHQFHGHGLPpWK\OJOXWDPDWHHst VXIILVDQWHSRXUUHVWDXUHUODVpFUpWLRQ G¶LQVXOLQHDXPrPHQLYHDXTXHGDQVOHVvORWVFRQWU{OHV /HPDQTXHGHJOXWDPDWHDpJDOHPHQWXQLPSDFWVXUOHVWDX[G¶DFLGHVDPLQpVJpQpUpVjSDUWLU de celui-ci, suggérant que la GDH joue un rôle pivot reliant le métabolisme du glucose et celui des acides aminés.
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SUpVHQWDYDQWXQHSULVHGHQRXUULWXUHLPSRUWDQWHHQWUDvQDQWODUpVLVWDQFHjO¶LQVXOLQHDXra-t-il un impact sur la prise de poids de ces souris et sur une éventuelle obésité induite? Nous avons donc testé la réponse à une diète hautement calorique de ces souris. Après 20 semaines de diète riche en calories, les souris contrôles sont devenues obèses et pesaient nettement plus que les souris contrôles sous diète normale. Les souris ßGlud1-/- sur diète riche en calories ont maintenu un poids comparable à celui des souris contrôles sur diète normale et présentaient XQH PDVVH JUDLVVHXVH DLQVL TX¶XQHtaille G¶adipocytes moindres; ceci malgré une prise de nourriture semblable. /¶HIILFDFLWp métabolique était diminuée chez les souris ßGlud1-/- comparativement aux souris contrôles. En fin de nuit, les souris ßGlud1-/- présentaient un quotient respiratoire plus bas comparé aux contrôles, indiquant une oxydation lipidique plus élevée. &HOD V¶DFFRPSDJQDLW G¶XQH GpSHQVH HQHUJpWLTXH SOXV LPSRUWDQWH 922 HW G¶XQH consommation de CO2 plus élevée. Nous avons également observé que les souris ßGlud1-/- avaient une aFWLYLWpSK\VLTXHSOXVLPSRUWDQWHVXUXQHSpULRGHGHKFHTXLQ¶pWDLWSDVOHFDV des souris ßGlud1-/- VRXV GLqWH QRUPDOH VXJJpUDQW TXH O¶DXJPHQWDWLRQ GH OHXU DFWLYLWp SK\VLTXHHVWXQHFRQVpTXHQFHSOXW{WTX¶XQHFDXVHGHOHXUUpVLVWDQFHjO¶REpVLWp
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1. Introduction
1.1 A natomy and Physiology of the islets of Langerhans
The pancreas is a gland organ, located next to the stomach, the duodenum and the bladder (Figure 1). It is composed of an exocrine part that secretes digestive enzymes and alkaline fluid, and an endocrine part, which represents only 1 to 2% of the total mass. The endocrine function of the pancreas is mediated by the islets of Langerhans, which are composed of four different cell types: insulin-secreting E-cells, glucagon-secreting D-cells, somatostatin- secreting G-cells, and pancreatic polypeptide-secreting PP-cells (Figure 1).
Fig.1: The pancreas is localized in the upper abdominal cavity, posterior to the stomach with its head enclosed in the duodenum. This organ contains both exocrine and endocrine cells, the latter are named the islets of Langerhans and consist mainly of insulin-VHFUHWLQJ ȕ-cells (green), glucagon-VHFUHWLQJ Į-cells (blue), and somatostatin-secreting į-cells (red), which are visualized by confocal microscopy. Ref [1]
Somatostati
Somatostatin Glucagon Insulin Merge
The islets (there are approximately one million islets in the human pancreas [2]) are highly vascularised by an extensive endothelial network, and innervated by sympathetic, parasympathetic, and sensory nerves [2]. The islet size is variable: it ranges from 40 to 900 Pm [3]. In a normal pancreas, the insulin-containing E-cells constitute 60-80% of the islet and the non E-cells and capillaries fill the remaining 20% [4].
Human islets do not show the anatomical subdivisions like rodent islets where the E-cells are located in the core of the islet, and D- and G-cells are located at the periphery [5]. In fact, the cytoarchitecture of the human islet, where most of the E-cells (71%) showed associations with other endocrine cells, suggest unique paracrine interactions. And human islets contain proportionally fewer E-cells and more D-cells that mouse islets [6, 7]. Along with insulin, E- cells secrete gamma-aminobutyric acid (GABA) [8] and amyline, also known as islet amyloid poplypeptide (IAAP). GABA, co-secreted from E-cells, was proposed to mediate part of the inhibitory action of glucose on glucagon secretion by activating GABA-receptors in D-cells [8]. Recently, ghrelin-producing cells have also been observed in the islet [9].
Ghrehlin is an hormone usually produced in the stomach during fasting, which stimulates appetite and thereby food intake. It has been suggested that ghrelin has a paracrine stimulatory action on glucagon-secreting D-cell during hypoglycaemia [10].
PP and ghrelin are orexigenic hormones, and somatostatin regulates the secretion of insulin and glucagon. Both insulin and glucagon are important to regulate blood glucose homeostasis, and they are secreted in an opposite manner. Glucagon is released during hypoglycemia to induce hepatic glucose output by stimulating glycogenolysis and gluconeogenesis and by decreasing glycogenesis and glycolysis in a concerted fashion via multiple mechanisms, whereas insulin is released when blood glucose concentration increases. In humans, glyceamia is maintained in a narrow range: 80 mg/dl (4.4mM) for the fasting glucose levels, and 120 mg/dl (6.7mM) for the postprandial blood glucose levels.
Insulin lowers blood glucose by stimulating glucose uptake into skeletal muscle and adipose tissue. In addition, insulin both inhibits glucagon secretion and lowers serum free-fatty-acid concentrations, contributing to the sharp decline in hepatic glucose production. Not only insulin and glucagon are implicated in the regulation of blood glucose levels. High blood glucose concentrations also stimulate somatostatin secretion and inhibit PP release [11].
1.2 Insulin
1.2.1 Insulin str ucture and synthesis
Pancreatic E-cells in the islet of Langerhans are the source of insulin, the only hypoglycaemic hormone. Insulin, discovered in 1921, is a peptide hormone released from E-cells, which is delivered to different target tissues: liver, skeletal muscle and adipose tissue. Although insulin circulates in the serum and binds to its receptor as a monomer, it forms dimmers at micromolar concentrations, and in the presence of zinc, is further assembled to hexamers [12].
The insulin monomer itself consists of two chains, an A chain of 21 amino acids and a B chain of 30 amino acids with a molecular weight of 5802 Da and an iso-electric point of pH 5.5. The A chain has an N-terminal helix linked to an anti-parallel C-terminal helix and the B chain has a central helical segment. The two chains are joined by two disulphide bonds (A7- B7 and A20-B19), which clamp the A chain helices at each end of the central B chain helix [13] and are essential for stability and bioactivity (Figure 2.B).
Insulin is encoded on the short arm of chromosome 11 [14] and synthesized in the ȕ cells of the pancreatic islets as its precursor, proinsulin. The proinsulin precursor of insulin is encoded by the INS gene. In contrast to humans, rodents express two proinsulin isoforms, encoded by distinct genes located on chromosome 7 and 19, respectively, in the mouse. The two isoforms differ by two amino acids in the B-chain, three amino acids in C-peptide, and several differences in the leader peptide sequence. One isoform, proinsulin1, is expressed exclusively in islets. The second, proinsulin 2, is expressed in islets and in other tissues, especially the thymus [15]. There are several regulatory sequences in the promoter region of the insulin gene, to which transcription factors bind. In general, the Pdx1 transcription factors bind to the A-boxes, the NeuroD factors to the E-boxes, MafA transcription factors to the C-boxes, and CREB to the cAMP response elements. Insulin is synthesized from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase E. Cleavage occurs at conserved dibasic sites (BC and CA junctions, green in Figure 2.A and B) [16].
Fig.2: A, pathway of insulin biosynthesis beginning with pre-proinsulin (upper): signal peptide (gray), B-domain (blue), dibasic BC junction (green), C-domain (red), dibasic CA junction (green), and A-domain (red). In the ER, the unfolded prehormone undergoes specific disulfide pairing to yield native proinsulin (center). Cleavage of BC and CA junctions by prohormone convertases (PC1 and PC2) and carboxypeptidase E leads to mature insulin and the C-peptide (lower). B, structural model of insulin. The A- and B-domains are shown in red and blue, and the disordered domain is shown by the dashed black line. Cystines are labeled in yellow boxes. C, cellular pathway of insulin biosynthesis. Nascent proinsulin folds as a monomer in the rough ER, where zinc ion concentration is low; in post-Golgi granules, proinsulin is processed to yield mature insulin, and zinc-stabilized hexamers begin to assemble. Zinc-insulin crystals are observed in secretory granules. Upon secretion, hexamers dissociate to yield bioactive insulin monomers. Ref [16]
Proinsulin is synthesised in the ribosomes of the rough endoplasmic reticulum (RER) from mRNA as pre-proinsulin. Pre-proinsulin is formed by sequential synthesis of a signal peptide, the B chain, the connecting (C) peptide and then the A chain comprising a single chain of 100 amino acids. Removal of the signal peptide forms proinsulin, which acquires its characteristic 3 dimensional structure in the endoplasmic reticulum. Secretory vesicles transfer proinsulin from the RER to the Golgi apparatus, whose aqueous zinc and calcium rich environment
favours formation of soluble zinc-containing proinsulin hexamers. When mature granules are secreted into the circulation by exocytosis, insulin, and an equimolar ratio of C-peptide are released (Figure 2.C). Proinsulin and zinc typically comprise no more than 6% of the islet cell secretion [13]. Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset diabetes [17-19]. The mutations are predicted to block folding of the precursor in the ER of pancreatic E-cells.
1.2.2 Insulin action in the Liver
Liver is the major organ with the ability to consume, store, and produce glucose and lipids. In the liver, binding of insulin to its receptor promotes glycogen storage. Glucose transport is not stimulated by insulin in hepatocytes. GLUT2 is the major glucose transporter expressed at the cell surface of hepatocytes, as well as pancreatic E-cells [20]. Glucose uptake in the liver accounts for about 30% of whole body glucose disposal [21]. Liver-specific insulin receptor knockout (LIRKO) mice exhibit insulin resistance, moderate glucose intolerance, and a failure of insulin to suppress hepatic glucose production and regulate hepatic gene expression [22].
1.2.3 Insulin action in the M uscle
Skeletal muscle is the principal site of insulin-stimulated glucose disposal in vivo. It accounts for about 75 % of whole body insulin-stimulated glucose uptake [23]. In the muscle, insulin promotes glucose uptake and stimulates glycogen synthesis. The transport of glucose into the skeletal muscle cells is the rate-limiting step in whole body glucose metabolism in both normal subjects [24] and those with type 2 diabetes. In both skeletal muscle and adipose tissue, GLUT1 (glucose tranporter 1) mediates basal glucose transport, whereas GLUT4 is responsible for insulin-mediated glucose uptake. GLUT4 differs from other glucose transporters in that about 90% of it is sequestered intracellularly in the absence of insulin or other stimuli such as exercise. Insulin regulates translocation of GLUT4 in vectorial transfer, vesicle tethering, vesicle docking and vesicle fusion [23]. Skeletal muscle insulin resistance is among the earliest detectable defects in humans with type 2 diabetes, however, surprisingly, muscle-specific insulin receptor knockout (MIRKO) mice do not show major defects in glucose metabolism [25].
1.2.4 Insulin action in the Adipose Tissue
In the adipose tissue, insulin promotes lipogenesis and inhibits lipolysis. Less glucose is transported in adipose tissue than in the muscle: it only accounts for 10% of the insulin- stimulated whole body glucose uptake [21]. In adipose tissue, like in the muscle, it is the glucose transporter GLUT4 which mediates insulin-stimulated glucose uptake by rapidly moving from intracellular storage sites to the plasma membrane. In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression is decreased in adipose tissue but preserved in the muscle. Adipose selective inactivation of the GLUT4 gene causes glucose intolerance and hyperinsulinemia, and induces secondary alterations in insulin action in muscle and liver [26]. On the other hand, fat-specific insulin receptor knockout (FIRKO) mice have reduced fat mass and whole-body triglycerides stores, and are resistant to obesity [27].
1.3 E-cell function and regulation of insulin secretion
Secreting right amount of insulin at the right moment is the crucial function of pancreatic E- cells that adjust insulin secretion to fluctuations of blood glucose levels. Upon glucose stimulation, insulin secretion is biphasic, with a rapid transient (4-8 min) first phase and a second sustained phase that lasts as long as the glucose stimulation is applied [28]. However, plasma glucose changes during meals are not rapid or large enough to elicit clear biphasic insulin secretion in vivo [29]. And several clonal E-cell models fail to fully replicate the kinetics of biphasic insulin secretion exhibited by an islet [30]. Moreover, the kinetics of in vitro insulin secretion differs between the two most widely used model species [31]. In rats, the second phase of insulin secretion is gradually increasing (Figure 3A). In mouse, the dynamics of insulin secretion is different upon the experimental technique employed. With in situ perfused pancreas, the glucose-stimulated insulin secretion is essentially monophasic (Figure 3B), with a weak second phase following the 4-5 min first phase [32]. In contrast, in freshly isolated islets stimulated with glucose in a perifusion protocol, the secretory response is biphasic with a sustained second phase [33]. Culture of isolated mouse islets did not modify the pattern of secretion kinetics [33], suggesting that inhibitory mechanisms of insulin secrtion, present in the mouse in in situ perfused pancreas are absent once islets are isolated.
Fig.3: Control in situ pancreatic perfusions. The pancreas was perfused at 5 ml/min for rat (A) and 1.5 ml/min for mouse (B). With mouse perfused pancreas, the second phase of insulin secretion is low and flat (B) compared to the first phase, whereas in rats, the second phase is gradually increasing (A). Ref [32].
1.3.1 The in vivo regulation of pulsative insulin secretion
The presence of oscillations in peripheral insulin concentrations has been described long ago, and different studies have shown that equal amounts of insulin presented to target organs have improved actions when delivered in a pulsative manner. The pulsatile secretion of insulin was shown to coincide with islet pulsatile release of glucagon [34], and somatostatin [35]. In addition, impaired (not absent) pulsatility of insulin secretion has been demonstrated in type 2 diabetic patients. The regular rapid oscillations present in normal subjects are replaced by irregular cycles of shorter duration both in type 2 diabetic subjects and in their first-degree relatives [36, 37]. The ultradian oscillations in type 2 diabetic patients are also more irregular and their amplitude is smaller. In addition to the rapid pulsatile insulin release pattern with a periodicity of 8-15 min, an ultradian oscillatory pattern with a period of 1.5-2h has been described [38, 39], and associated with improved insulin action, and in case of impairment, with type 2 [40, 41] and early type 1 diabetes mellitus [42]. The rapid pulses persist in the
isolated perfused pancreas and in isolated islets [43] exposed to a constant gluose concentration, suggesting that they occur independently of oscillations in plasma glucose and that they originate due to intraislet mechanisms. Finally, diurnal oscillations related to meal ingestion are also an important contributor to the complexity of insulin release [44].
1.3.2 The first phase of insulin secretion
The rapid first phase of insulin secretion is induced by the triggering pathway, also called the KATP channel-dependent pathway, because the channel is the key player in the transduction of the glucose effects. The E-cell ATP-sensitive K+ channels are composed of regulatory receptor subunits (sufonylurea receptor 1 [SUR1]) and pore-forming (Kir6.2) subunits arranged in a 4:4 stoichiometry [45-47]. In the presence of non-stimulatory concentrations of glucose, KATP channels are open in the plasma membrane to maintain the membrane potential at values more negative than the threshold necessary to open the voltage-gated Ca2+ channels.
When glucose enters E-cell, it is taken up by a specific glucose transporter of high capacity (GLUT2 in rodents, GLUT1 in humans) localized at the cell membrane [48]. Then, it is phosphorylated by the glucokinase (GK), which has a high Km for glucose [49] and is the first rate-limiting enzyme of glycolysis. Glycolysis requires no oxygen and is also called anaerobic metabolism. The overall reaction of glycolysis is:
Glucose + 2NAD+ + 2Pi + 2ADP Æ 2 pyruvate + 2NADH + 2ATP + 2H+ + 2H2O The pyruvate formed during glycolysis in the cytoplasm then enters mitochondria to feed the TCA cycle, leading to the transfer of reducing equivalents to the respiratory chain complexes (Figure 4). This promotes hyperpolarisation of the mitochondrial membrane and ATP generation, which is then transferred into the cytosol. The subsequent increase in the ATP/ADP ratio leads to closure of KATP channels [46, 50], and the resulting decrease in K+ effluxcauses membrane depolarization and Ca2+ entry trough the voltage-gated Ca2+ channels [51]. The increase in [Ca2+]c then promotes exocytosis of insulin-containing granules [52, 53].
The triggering Ca2+ is essential, as conditions that prevent Ca2+ rise impair glucose-stimulated insulin secretion, whereas physiological or pharmacological agents that increase E-cell [Ca2+]c induce insulin secretion [54, 55].
Fig.4: Model for coupling glucose metabolism to insulin secretion in the E-cell. Glucose equilibrates across the plasma membrane and is phosphorylated by glucokinase (GK), which initiates its conversion to pyruvate (Pyr) by glycolysis. Pyr preferentially enters the mitochondria and fuels the TCA cycle, resulting in the transfer of reducing equivalents (red.equ.) to the respiratory chain, leading to hyperpolarisation of the mitochondrial membrane ('<m) and generation of ATP. ATP is then transferred to the cytosol, raising the ATP/ADP ratio.
Subsequently, closure of KATP channels depolarises the cell membrane ('<c). This opens voltage-gated Ca2+
channels, increasing the cytosolic Ca2+ concentration ([Ca2+]c), which triggers insulin exocytosis. Ref [56].
1.3.3 The amplifying pathway
The elevation of cytosolic Ca2+ in the ȕ-cell is the primary and necessary signal for insulin exocytosis of a small pool of readily releasable granules, but is not sufficient to explain the complete and sustained biphasic response typically observed [57]. The amplifying pathway, also known as the KATP channel-independent pathway, has been identified by using diazoxide to prevent glucose from closing KATP channels, and high K+ to restore membrane depolarization, Ca2+ influx, and [Ca2+]i rise in ȕ-cell [58-60]. Under these conditions, glucose did not further increase [Ca2+]i in mouse or rat islets, but potentiated the stimulatory effect of [Ca2+]i on exocytosis, suggesting that glucose acts on different targets [58, 59]. The second experimental approach to study the amplifying pathway of insulin secretion is to close the KATP channels by a high concentration of sulfonylurea, such as tolbutamide or glibenclamide.
Glucose can still increase insulin secretion under these conditions. Additionally, glucose has
been shown to increase insulin secretion from mouse islets lacking KATP channels [28, 61], and insulin secretagogues that do not affect metabolism, such as potassium or arginine, can trigger the first phase of insulin release and raise [Ca2+]c but do not induce the second phase of insulin secretion.
Altogether, these observations suggest that, on top of glucose, additive factors are required for the amplification of the calcium signal; thereby contributing to the second sustained phase of insulin secretion, originally referred to as the KATP channel-independent pathway [58, 59], and now called the amplifying pathway. The molecular mechanisms of this amplification are still undefined, but could involve a refilling of the readily releasable granules [53, 62]. If it was originally agreed that the amplifying pathway is not involved in the first phase of glucose- stimulated insulin secretion, this paradigm has been recently revised, and amplifying signals are now thought to be implicated in both the first and the second phases [63].
1.3.4 Exocytosis in E-cells
Within each E-cell, insulin is contained in dense core granules. In response to the rise in [Ca2+]c, granules fuse with the plasma membrane in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process [64]. Initial models suggested that the two phases of insulin secretion resulted from intra-islet E-cell heterogeneity or from different populations of E-cell preferentially secreting during the first or the second phase [65]%XWQRZWKH³VWRUDJH-OLPLWHGPRGHO´[66] proposes that biphasic secretion is due to the release from geographically or functionally distinct pools of granules [67]. During the first phase, a small pool of granules pre-docked at the plasma membrane; referred to as the
³UHDGLO\UHOHDVDEOHSRRO´535ZLOOUHOHDVHLQVXOLQ7KHQJUDQXOHVWKDWDUHGHHSHUZLWKLQWKH cell (referred to DVWKH³VWRUDJH-JUDQXOHSRRO´DUHPRELOL]HGWRUHSOHQLVKWKH535DWWKHFHOO surface [68].
1.3.5 Hormones and factors regulating insulin secretion
Glucose is not the only secretagogue that stimulates insulin secretion. Four amino acids were found to be particularly important for stimulating ȕ-cell electrical activity, essential for insulin secretion: leucine, isoleucine, alanine and arginine [69]. Moreover, nutrient-induced secretion is potentiated by the neurotransmitters acetylcholine and pituitary adenylate cyclise-activating polypeptide (PACAP), as well as by the gastrointestinal hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) [70, 71]. By binding on their specific
receptors expressed on the E-cell membrane, GLP-1 and GIP promote the activation of adenyl cyclase (AC), which catalyses the conversion of ATP to cAMP and activation of PKA.
Incretin hormones action on E-cells accelerates Ca2+ signalling and insulin exocytosis [72].
Insulin release from E-cells is directly controlled by the blood glucose level and modulated by circulating hormones and the autonoumous nervous system. In addition, hormone release from E-cells, as well as from the other islet cell types, is regulated by autocrine and paracrine interactions. For example, glucagon release from D-cells has a stimulatory effect on insulin secretion from E-cells [73, 74]. And insulin exocytosis is under the direct negative control of noradrenaline, somatostatin (released by G-cells), and circulating adrenaline [75-77].
Moreover, we have demonstrated that dopamine inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E cell line, fluorescence-activated cell-sorted primary rat E-cells, and pancreatic islets of rat, mouse, and human origin [78]. We identified dopamine D2 receptors in INS-1E insulin-secreting cells as well as in rodent and human isolated islets both by reverse transcription-PCR and immunodetection [78], suggesting that dopamine effect on insulin secretion might be ascribed to D2-like receptors activation.
Sympathetic nervous system Circulating substrates
-Glucose, Fructose -NEFA
-Ketone bodies -Amino acids
Cellule E -
-
-Insulin -IAPP
-Glucagon -Peptide C -Somatostatin
-Dopamine
-Growth Hormone -Leptin
-Cortisol
-Thyroid hormones - Adrenaline
+
-Acetylcholine -VIP -PACAP -GRP
+ -
-Noradrenaline -Neuropeptide Y
-GIP -GLP-1 Parasympathetic
nervous system Peptides and insular hormones
Other hormones
Enteroinsular axis +
+
+ -
Fig.5: Hormones and factors regulating insulin secretion. NEFA, non-esterified fatty acid; VIP, vasoactive intestinal peptide; PACAP, pituary adenylate cyclase activating polypeptide; GRP, gastrin releasing peptide;
GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide 1; IAPP, islet amyloid polypeptide.
1.4 Implication of mitochondria in E-cell function and dysfunction
The mitochondria are the main source of ATP within the cell, which is essential for vital cellular functions. As mitochondria are the power-generating units of the cell, they are abundant in tissues where energy-requiring processes take place, such as skeletal and cardiac muscle [79], as well as brown adipose tissue. Additionally, in the pancreatic E-cell, mitochondria integrate and generate metabolic signals, ensuring efficient coupling of glucose recognition to insulin secretion [80]. Several mitochondrion-derived molecules distinct from ATP have been proposed to act as additive factors participating in the amplifying pathway of insulin secretion. In fact, mitochondrial defects in E-cells, such as mutations and reactive oxygen species (ROS) production, are associated with E-cell failure in the course of diabetes [81], suggesting that this organelle plays an important role in insulin secretion. At the clinical level, one of the most direct indications that mitochondrial dysfunction could impair insulin release comes from the association between mutated mitochondrial genome and maternally inherited diabetes.
1.4.1 M itochondria origin and function
Mitochondria are organelles present in most eukaryotic cells, varying in number from hundreds to thousands [82]. Mitochondria are thought to derive from the endosymbiotic association of oxidative bacteria and glycolytic proto-eukaryotic cells [83]. This hypothesis of mitochondria origin is supported by a unique mitochondrial genome in the form of circular DNA (mtDNA) with bacterial characteristics [83]. mtDNA has the same fundamental role in all eukaryotes that contain it: it encodes a limited number of RNAs and proteins essential for formation of a functional mitochondrion. In contrast to nuclear DNA, mtDNA consists only of coding sequences: human mtDNA carries 37 genes (16569 bp), encoding 22 tRNAs, 2 rRNAs, and 13 polypeptides that are part of the multisubunit enzyme complexes of the respiratory chain [82]. MtDNA is transcribed and translated within the mitochondrion, whereas the nuclear genome encodes the remaining majority of the enzyme subunits and other mitochondrial proteins that are synthesized in the cytosol and imported within the mitochondria. The mtDNA is maternally inherited because of the non-persistence of paternal mitochondria in the zygote after fertilization. The only mammalian cells that lack mitochondria are the peripheral red blood cells, which entirely depend on glycolysis for their energy supply. In contrast to nuclear DNA, mtDNA is more vulnerable to oxidative stress, and consecutives damages are more extensive than those in nuclear DNA due to the lack of
protective histones and low repair mechanisms [84, 85]. Moreover, it is juxtaposed to the respiratory chain compexes, which generate reactive oxygen species (ROS) [86].
1.4.2 A naplerosis and C ataplerosis
The mitochondria can be activated by three classes of fuels: amino acids, fatty acids and carbohydrates, the latter being the most relevant for the E-cell. Importantly, the E-cell has negligible lactate dehydrogenase, so that pyruvate from glycolysis is weakly metabolised into lactate, and the fate of pyruvate-derived carbons is essentially mitochondrial [87]. In fact, pyruvate, formed during glycolysis, enters mitochondria where it is converted to acetyl-CoA by pyruvate dehydrogenase (PDH) or to oxaloacetate by pyruvate carboxylase (PC), thereby ensuring anaplerosis (provision of carbon skeleton) to the tricarboxylic acid (TCA) cycle, also called the Krebs cycle (Figure 6).
Fig.6: The tricarboxylic acid (TCA) cycle with Ca2+-sensitive dehydrogenases (DH). In the mitochondria, pyruvate is a substrate for both pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). Among the TCA cycle enzymes, succinate-DH (SDH) is also part of the respiratory chain (complex II) [56].
This results in the net synthesis of TCA cycle intermediates, that act directly as, or as precursors of, important signals in insulin secretion. These products of anaplerosis are exported from the mitochondria (cataplerosis) and have extramitochondrial signalling actions.
In E-cells, it has been shown, by using 14CO2 ratios method, that about 50% of glucose-
derived pyruvate enters mitochondria via carboxylation catalysed by PC, and the other 50%
enters via decarboxylation to acetylCoA through PDH [88]. Pyruvate carboxylase activity is particularly high in E-cells and is only equalled by gluconeogenic tissues such as liver and kidney [89, 90]. The fact that carboxylation is so active in E-cell compared to other cell types suggests that anaplerosis is important for insulin secretion. In fact, levels of PC are increased in response to high glucose concentrations, and conversely, inhibition of PC has been shown to reduce glucose-stimulated insulin secretion in rat islets [91].
Oxaloacetate, produced by malate dehydrogenase, or alternatively by the anaplerotic enzyme PC, condenses with acetyl-CoA to form citrate, which then undergoes additional oxidation steps and decarboxylation, generating isocitrate, Į-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, and finally oxaloacetate. One complete turn of the cycle results in the formation of two molecules of CO2 and one molecule of GTP, as well as three reducing equivalents NADH and FADH2. The fate of Į-ketoglutarate is influenced by the redox state of mitochondria. Low NADH to NAD+ ratio would favor further oxidative decarboxylation to succinyl-CoA, as NAD+ is required as co-factor for this pathway. On the contrary, high NADH to NAD+ ratio would promote reductive transamination trough glutamate dehydrogenase (GDH) forming glutamate. The latter situation, i.e. high NADH to NAD+ ratio, is observed following glucose stimulation.
1.4.3 O xidative phosphorylation
The final step of aerobic respiration is called oxidative phosphorylation. The electron generated by the reducing equivalents NADH and FADH2 are transported through the complexes I and II of the respiratory chain, also called the electron transport chain (ETC). The electron transport chain is made of different proteins (complexes I-IV): NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome bc1 (complex III), cytochrome c oxidase (Complex IV), (Figure 7).
Energy is released as the electrons flow along the ETC, and this energy is used to pump protons out across the mitochondrial inner membrane trough complexes I, III and IV. This creates an electrochemical gradient of protons. Protons diffusing back along this gradient drive the synthesis of ATP by the enzyme ATP synthase (Complex V). The coupling of substrate oxidation with ATP formation in the mitochondria is called oxidative phosphorylation. The efficiency by which reducing equivalents (from oxidation of acetyl-
CoA) are converted to ATP by oxidative phosphorylation is called the coupling efficiency.
Mitochondria are imperfectly coupled, such that during normal oxidative phosphorylation, some of the energy substrates derived is lost as heat [79]. In addition to energy, oxidative phosphorylation also generates reactive oxygen species (ROS). The pumping of protons across the inner membrane with the accumulation of electrons causes the mitochondria to be the major source of cellular ROS production, which may contribute to a wide variety of pathologic conditions including degenerative diseases, cancer, diabetes and aging [82].
Fig.7: The electron transport chain (ETC) of mitochondria. Ref [79]
1.4.4 Nucleotides and metabolites involved in glucose-stimulated insulin secretion Several metabolites and nucleotides have been proposed to play a role in the amplifying pathway of insulin secretion (listed in Figure 8), but neither the second messenger nor the cellular effector has yet been identified. In contrast to ATP, GTP generated by glucose is able to promote insulin exocytosis in a Ca2+-independent manner [92]. GTP is formed in the mitochondria during the TCA cycle activity. The inhibition of the GTP-generating form of succinyl-CoA synthetase (SCS-GTP) in clonal E-cell and rat islets resulted in impaired GSIS and reduced GTP levels [93]. In contrast, inhibition of the ATP-generating form of this enzyme (SCS-ATP) increased insulin release at stimulatory glucose concentrations [93].
Fig.8: Several putative messengers or additive signals are listed here, which have been proposed to participate in the metabolism-secretion coupling [94].
As described above, cAMP generated at the plasma membrane from ATP, was also demonstrated to potentiate Ca2+-dependent GSIS [95]. Many neurotransmitters and hormones, including glucagon as well as the intestinal hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), increase cAMP levels in E-cell by activating adenyl cyclase [71].
Recent studies also suggest a role of AMP-activated protein kinase (AMPK) [96], reactive oxygen species such as H2O2 [97], and granule translocation by the cytoskeleton [53, 98]. A direct effect of NADPH, generated by glucose metabolism via the pentose phosphate shunt [99] and by mitochondrial shuttles [100], was reported on the release of insulin from isolated secretory granules [101], NADPH being possibly bound or taken up by granules [102]. In pancreatic E-cells, glucose raised the NADPH/NADP+ ratio and stimulated insulin release in parallel, and addition of NADPH was demonstrated to stimulate exocytosis of insulin [103].
Effects of NADPH on exocytosis were proposed to be mediated by the redox proteins glutaredoxin (GRX) and thioredoxin (TRX). Actually, it has been recently demonstrated that glutaredoxin-1 (GRX-1) mediates NADPH-dependent stimulation of Ca2+-dependent insulin secretion in pancreatic E-cells by a local redox reaction that accelerates E-cell exocytosis and, in turn, insulin secretion [104].
