Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance
Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca2+) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin’s action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice.
We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER–mitochondria interactions, inter-organelle Ca2+ exchange, organelle homeostasis and insulin action.
Pharmacological and genetic inhibition of CYPD concomitantly reduced ER–mitochondria interactions, inhibited inter-organelle Ca2+ exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca2+ transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca2+ transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells.
Disruption of IP3R-mediated Ca2+ signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER–mitochondria Ca2+ exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.
KeywordsCalcium signalling Cyclophilin D Endoplasmic reticulum Inositol 1,4,5-triphosphate receptor Insulin resistance Liver Mitochondria Mitochondria-associated endoplasmic reticulum membranes PKCε
Ciclosporin (cyclosporin A)
Eukaryotic translation initiation factor 2α
Glucose-regulated protein 75
Inositol 1,4,5-triphosphate receptor
c-Jun N-terminal kinase
Mitochondrial-associated endoplasmic reticulum membrane
Protein kinase B
Protein kinase C epsilon
Proximity ligation assay
Permeability transition pore
Sarco(endo)plasmic reticulum Ca2+ ATPase 2
Small interfering RNA
Voltage-dependent anion channel
Mitochondria and endoplasmic reticulum (ER) are organised as a network with specific contact points, referred to as mitochondrial-associated ER membranes (MAMs), which play a pivotal role in calcium (Ca2+) signalling and energy metabolism . Efficient Ca2+ transmission from the ER to mitochondria is mediated through the interaction of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane with the inositol-1,4,5-triphosphate receptor (IP3R) on the ER via the chaperone glucose-regulated protein 75 (GRP75) . Mitochondrial Ca2+ uptake is essential for the regulation of both mitochondrial metabolism and ER homeostasis , and alteration of ER–mitochondria cross-talk may result in a disruption of inter-organelle Ca2+ transfer  and subsequent ER stress . Under certain conditions excessive Ca2+ entry into the mitochondrial matrix may be detrimental, causing the opening of the permeability transition pore (PTP) and cell death . Therefore, tight control of Ca2+ exchange between ER and mitochondria is required to regulate vital functions and metabolic homeostasis.
The molecular and functional characterisation of MAMs in physiological and pathological conditions has improved in the last few years, highlighting unexpected roles for MAMs in cellular signalling . Particularly, we recently demonstrated that cyclophilin D (CYPD), a mitochondrial protein known to modulate the opening of the PTP , also interacts with the VDAC–GRP75–IP3R complex at the MAM interface in both heart  and liver . In cardiomyocytes, we found that the loss of CYPD reduced mitochondrial Ca2+ overload by depressing ER–mitochondria interactions and protected cells against lethal reperfusion injury , suggesting that CYPD regulates Ca2+ transfer from ER to mitochondria. In liver, the loss of CYPD reduced organelle interactions and induced hepatic insulin resistance, pointing to a new role of MAM integrity in the control of insulin’s action . Whereas other studies in mice also suggest a role for MAMs in the control of glucose homeostasis [11, 12, 13, 14], the mechanisms by which disruption of MAMs alters insulin signalling are unknown.
Based on the role of MAMs in Ca2+ transfer from ER to mitochondria, we hypothesised that a disruption of Ca2+ transfer between both organelles could contribute to hepatic insulin resistance. To this aim, using pharmacological and genetic loss of function approaches both in vivo and in vitro, we investigated whether the disruption of Ca2+ transfer from ER to mitochondria could link MAM alterations to hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice.
HuH7 cells were a gift from G. Mithieux’s laboratory (Inserm U855, Lyon, France) and were free of mycoplasma. They were cultured as previously described  and stimulated either with CYPD (ciclosporin [cyclosporin A, CsA] or NIM811) or with IP3R (2-APB and Xestospongin C) pharmacological inhibitors. Inhibition of CYPD was also achieved by silencing of CYPD. See electronic supplementary material (ESM) Methods for further details. For measurement of insulin signalling, cells were depleted in serum for 3 h before incubation with insulin (10−7 mol/l, 15 min).
CYPD-KO mice on a C57Bl/6/SV129 genetic background were a gift from S. J. Korsmeyer’s laboratory (Boston, MA, USA) . Both male wild-type (WT) and CYPD-KO mice were obtained by homozygous intercross in our laboratory. All experiments were performed on mice of 18–22 weeks of age, and were conducted in accordance with institutional guidelines for the care and use of laboratory animals, and a regional ethics committee has approved all procedures. No randomisation or blinding were performed. No data samples or animals were excluded from the study.
Using confocal fluorescence imaging, we evaluated variations in the time course of mitochondrial Ca2+ content simultaneously with intra-ER Ca2+ in a native cell environment in the presence of extracellular calcium (1.8 mmol/l CaCl2) . To measure mitochondrial Ca2+, cells were loaded with Rhod-2 AM (3 μmol/l) whereas the measurement of ER Ca2+ was performed using a low-affinity Ca2+ indicator, Fluo-5N (5 μmol/l; ThermoFisher Scientific, MA, USA). For measurement of absolute cytosolic Ca2+ levels, cells were loaded with 1 μmol/l of Fura2-AM and pluronic acid (both from ThermoFisher Scientific). See ESM Methods for further details.
ER–mitochondria interactions were measured both by subcellular fractionation and by in situ proximity ligation assay (PLA), as previously described and thoroughly validated . See ESM Methods for further details.
mRNA levels were measured by real-time RT-PCR. See ESM Methods for further details.
Protein expression was analysed by SDS-PAGE. See ESM Methods for further details.
Insulin sensitivity of mice was measured during a hyperinsulinaemic–euglycaemic clamp. See ESM Methods for further details.
Mitochondrial respiration was measured in intact or permeabilised primary hepatocytes. See ESM Methods for further details.
Hepatic lipid content
Hepatic triacylglycerol (TG) and diacylglycerol (DAG) content were measured by spectrophotometry. See ESM Methods for further details.
PKCε activity in liver was determined by western blot based on the membrane translocation. See ESM Methods for further details.
Results are expressed as mean ± SEM. Student’s t-test was used to analyse the difference between control and experimental groups. Statistically significant differences were assessed with a one-way ANOVA with a Newman–Keuls post hoc test when three or more groups were compared. A p value <0.05 was considered as statistically significant.
Pharmacological and genetic inhibition of CYPD inhibits Ca2+ transfer from ER to mitochondria in HuH7 cells
Inhibition of CYPD function induces ER stress and alters insulin response in HuH7 cells
Loss of CYPD in mice alters Ca2+ transfer from ER to mitochondria in isolated hepatocytes
We also measured cytoplasmic Ca2+ in response to a discharge of ER store by thapsigargin, both in the presence and absence of extracellular Ca2+. In both conditions, thapsigargin-sensitive Ca2+ stores were significantly higher in CypD-KO mice compared with WT mice (Fig. 5e, f), indicating that the reduced Ca2+ transfer from ER to mitochondria in CypD-KO hepatocytes was not due to a defect of ER Ca2+ storage. In addition, resting cytosolic Ca2+ was significantly increased in CypD-KO hepatocytes in the presence of extracellular Ca2+, whereas this effect was absent in the absence of extracellular Ca2+ (ESM Fig. 3b, c). The cytosolic Ca2+ peak after histamine stimulation did not differ significantly between WT and CypD-KO mice, in either the presence or absence of extracellular Ca2+ (ESM Fig. 3d).
Loss of CYPD in mice induces ER stress and mitochondrial dysfunction in liver
As Ca2+ import into mitochondria affects mitochondrial bioenergetics [21, 22], we studied mitochondrial respiration in either permeabilised (glutamate/malate) or intact (20 mmol/l glucose) primary hepatocytes of both WT and CypD-KO mice, in order to maintain ER mitochondria cross-talk. As shown in Fig. 6c, d, oxygen consumption was significantly reduced in CypD-KO hepatocytes, compared with WT hepatocytes.
Loss of CYPD in mice increases lipid accumulation and PKC activity and induces hepatic insulin resistance
We also examined lipid metabolism in both WT and CypD-KO mice. Circulating NEFA were not modified in CypD-KO mice compared with WT mice (0.19 ± 0.05 vs 0.16 ± 0.05 mmol/l, respectively, n = 10), whereas blood TG levels were significantly increased (0.90 ± 0.03 vs 0.60 ± 0.05 g/l, respectively, p < 0.05, n = 10). Interestingly, the lipid staining (Fig. 7d), as well as TG and total DAG levels (Fig. 7e), were significantly increased in the liver of CypD-KO mice. As PKCε was shown to be involved in DAG-mediated hepatic insulin resistance , we analysed its activity by measuring its translocation from cytosol to membrane. Consistent with DAG accumulation, PKCε activity was induced in the livers of CypD-KO mice (Fig. 7f). Furthermore, the mRNA levels of genes related to β-oxidation (Cpt1a) were decreased in liver of CypD-KO mice, whereas the expression of both lipogenic genes (Srebp1c [also known as Srebf1], Srebp1a, Fasn, Acaca) and genes involved in lipid export (Apob, Mttp) were increased (Fig. 7g). The expression of neither Mlycd nor Dgat2 was modified in liver of CypD-KO mice compared with WT mice. Altogether, these results suggest that lipid accumulation in liver of CypD-KO mice is likely related to both a reduction in lipid oxidation and an increase in lipid storage, the latter being mainly associated with increased de novo lipogenesis.
Role of PKCε in CYPD-related alteration of insulin signalling
Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis are only partly known. We recently proposed a role for MAM integrity in the control of hepatic insulin action and demonstrated that disruption of MAM integrity in the liver of CypD-KO mice can induce hepatic insulin resistance . In the present study, we further investigated the underlying mechanisms and showed that the disruption of Ca2+ transfer from ER to mitochondria in the liver of CypD-KO mice is likely the molecular consequence of MAM disruption, accounting for defective insulin action in the liver.
ER–mitochondria contact points are known to be enriched in Ca2+-handling proteins and chaperones and to generate microdomains with a high Ca2+ concentration . We provide in vivo and in vitro evidence that pharmacological or genetic inhibition of the mitochondrial chaperone CYPD results in modifications of ER–mitochondria Ca2+ transfer: (1) via CYPD binding to the mitochondrial inner membrane since its actions are inhibited by CsA and NIM811 and (2) via an interaction with the VDAC1–GRP75–IP3R1 Ca2+-channelling complex. These data are consistent with our recent study demonstrating that inhibition of CYPD in cardiomyocytes decreased the Ca2+ transfer from ER to mitochondria through IP3R under normoxic conditions . Furthermore, CYPD-mediated disruption of ER–mitochondria cross-talk seems related rather to a dissociation of organelle than to a modification of protein expression at MAM interface, and seems independent of a modification of both Mcu and Serca2b mRNA levels. This new physiological role of CYPD in ER–mitochondria communication should now be considered as clinically relevant because inhibitors of CYPD function are either already used in clinical practice (e.g. CsA in transplanted patients) or are being developed (e.g. for treatment of hepatitis C virus or HIV infections) .
Our findings also point to a fundamental mechanism by which reduced structural and functional ER–mitochondria interactions alter organelle function and subsequently inhibit insulin’s action, leading to hepatic insulin resistance. We thus propose a model by which the loss of CYPD can induce liver insulin resistance (Fig. 8c). Invalidation of CYPD alters ER–mitochondria interactions and Ca2+ exchange, leading to increased ER Ca2+ storage and ER stress. It is likely that the loss of CYPD-mediated uncoupling of the ER from the mitochondria is compensated by increased levels of the ER protein-folding machinery and Ca2+ to re-establish ER homeostasis, as previously reported [4, 5, 21]. At the same time, lack of Ca2+ transfer to mitochondria in CypD-KO mouse hepatocytes can reduce mitochondrial respiration, as also seen in a recent study demonstrating that constitutive low-level IP3R-mediated Ca2+ delivery to mitochondria is essential to maintain normal cellular bioenergetics . The chronic disturbance of Ca2+ homeostasis likely maintains the activation of unfolded protein response in liver of CypD-KO mice, despite increased GRP78 expression. Subsequently, both ER stress and mitochondrial dysfunction may contribute to an increase in hepatic lipid levels. Indeed, fat accumulation in the liver of CypD-KO mice seems to be related to both a reduction in lipid oxidation (based on reduction in Cpt1 expression) and an induction of de novo lipogenesis (based on induction of lipogenic genes), whereas esterification of lipid (based on the absence of change in circulating NEFA levels) and export of lipids (based on induction of ApoB and Mttp genes) from liver should not be altered. The induction of de novo lipogenesis is in agreement with the activation of both PERK and IRE1 branches of the unfolded protein response in the liver of CypD-KO mice, as both pathways were shown to activate the lipogenic transcription factor SREBP-1c [25, 26, 27]. Particularly, accumulation of DAG likely contributes to PKCε activation in the liver of CypD-KO mice. Together, these metabolic stresses would consequently result in alterations of hepatic insulin signalling and in the deterioration of glucose homeostasis in CypD-KO mice. Indeed, both induction of ER stress and accumulation of intracellular lipids have been involved in hepatic insulin resistance. Activation of JNK has been shown in ER stress-mediated hepatic insulin resistance  and activation of PKCε has been related to DAG-induced hepatic insulin resistance . In CypD-KO mice, hepatic insulin resistance appears to be mainly secondary to ER stress modulation of hepatic lipogenesis and subsequent DAG-mediated activation of PKCε rather than to ER stress-induced JNK activation, at least in vitro. Whereas an unspecific effect of inhibitors could not be excluded, this result fits well with the mechanisms recently proposed for another mouse model of insulin resistance , as well as with recent data in human liver . Nevertheless, we cannot exclude the possibility that other players could be involved in the insulin-resistant phenotype of CypD-KO mice. Particularly, as absolute cytosolic Ca2+ levels were increased in CypD-KO mouse hepatocytes, we cannot exclude the participation of Ca2+-sensitive kinases and/or phosphatases in the alteration of insulin signalling, as suggested by others studies [29, 30]. As disruption of MAM integrity has been observed in liver of different mouse models of obesity and type 2 diabetes , it is likely that the unexpected role of inter-organelle Ca2+ exchange in triggering hepatic insulin resistance might be extrapolated to these models. In agreement, the direct inhibition of IP3R, independently of CYPD, also induced ER stress and altered hepatic insulin signalling, suggesting that the proposed mechanism is a more general model and not restricted to the CypD-KO mouse model. Nevertheless, as controversy exists in this topic , further studies are required to clarify the role of inter-organelle Ca2+ exchange in triggering hepatic insulin resistance in obesity.
In conclusion, our data demonstrate that CYPD is an important regulator of MAM integrity and subsequently of Ca2+ exchange at the MAM interface, and provide the first evidence that IP3R-mediated Ca2+ transfer from ER to mitochondria is an essential cellular process involved in the control of hepatic insulin action. Therefore, modulation of ER–mitochondria Ca2+ exchange may provide an exciting new avenue for treating hepatic insulin resistance.
The authors would like to thank the Lyon-Est imaging center (CIQLE) and SFR Santé Lyon Est (CNRS UMS3453 – INSERM US7, Université Lyon 1, France) for access to technological platforms and Physiogenex (Labege, France) for providing hyperinsulinaemic–euglycaemic clamps.
This work was supported by Servier Laboratories, Inserm, the ANR (ANR-07-PHYSIO-020-01 and ANR-11-BSV1-033-02 from MO and ANR-09-JCJC-0116 from JR) and the FRM (MO, AL).
Duality of interest
The authors declare that there is no duality of interested associated with this manuscript.
JR and MO conceived and designed the study. JF, MP, EBe, ET, MAC, AD, AB, GT, BB, MM, PT, GV, MD, EBl and LG contributed to the collection of data. JR, JF and MP analysed and interpreted the data. JR and MO wrote the paper. FZ, HV and AL contributed to study conception and reviewed the manuscript for important intellectual concept. All authors contributed to critical revisions and have read and approved the final version to be published. JR is responsible for the integrity of the work as a whole.
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