Disruption of mitochondria-associated endoplasmic ......2018/01/08 · Transmission electronic analysis (TEM) Fixation and post-treatment of muscle fractions or myotubes, as well

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1

Disruption of mitochondria-associated endoplasmic reticulum membranes (MAMs)

integrity contributes to muscle insulin resistance in mice and humans.

Emily Tubbs1, Stéphanie Chanon1*, Maud Robert1,2*, Nadia Bendridi1*, Gabriel Bidaux1,

Marie-Agnès Chauvin1, Jingwei Ji-Cao1, Christine Durand1, Daphné Gauvrit-Ramette1,

Hubert Vidal1,2, Etienne Lefai1 and Jennifer Rieusset1,2

1 Lyon University, Laboratoire CarMeN, INSERM U1060, INRA U1235, Université Claude Bernard

Lyon1, INSA-Lyon, , F-69600 Oullins, France.

2 Hospices Civils de Lyon, Lyon-Sud Hospital, Endocrinology, diabetology and nutrition service, F-

69310 Pierre Bénite.

* Contributed equally to this work

Corresponding author: Jennifer RIEUSSET

Address: UMR INSERM U1060, Faculté de médecine Lyon-Sud, 165

chemin du grand Revoyet, BP12, 69921 Oullins cedex

Phone number: 33 (0)4 26 23 59 20, Fax: 33 (0)4 26 23 59 16

E-mail: [email protected]

Word count: 5007

Number of Figures: 8

Short running title: Organelle miscommunication and muscle insulin resistance

Page 1 of 44 Diabetes

Diabetes Publish Ahead of Print, published online January 11, 2018

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ABSTRACT

Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria,

defined as mitochondria-associated membranes (MAMs), were recently involved in the

control of hepatic insulin action and glucose homeostasis, but with conflicting results.

Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main

target for alterations in insulin resistant states, the relevance of MAM integrity in muscle

insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin

signaling could help to clarify this controversy. Here, we show in skeletal muscle of different

mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria

interactions, as an early event preceding mitochondrial dysfunction and insulin resistance.

Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a

reduction of structural and functional ER-mitochondria interactions. Importantly,

experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-

induced alterations of insulin signaling and action, whereas disruption of MAM integrity

alters the action of the hormone. Lastly, we found an association between altered insulin

signaling and ER-mitochondria interactions in human myotubes from obese subjects with or

without T2D, compared to healthy lean subjects. Collectively, our data reveal a new role of

MAM integrity in insulin action of skeletal muscle, and highlight MAM disruption as an

essential subcellular alteration associated with muscle insulin resistance in mice and humans.

Therefore, reduced ER-mitochondria coupling could be a common alteration of several

insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of

obesity and T2D.

Page 2 of 44Diabetes

3

INTRODUCTION

Whereas mitochondria and endoplasmic reticulum (ER) dysfunction were largely

shown to contribute independently to insulin resistance (1; 2), it has been now demonstrated

that alterations in their physical interactions is also involved (3). Indeed, mitochondria and ER

interact at contact points, called mitochondria-associated endoplasmic reticulum membranes

(MAMs), in order to exchange calcium (Ca2+) and lipids, thus regulating cell metabolism and

fate (4; 5). Few years ago, we indentified that MAM integrity controled hepatic insulin

signaling and that disruption of organelle coupling contributed to hepatic insulin resistance

(6). This was recently confirmed by an independent group (7). Accordingly, mice models with

liver-specific invalidation of proteins located at MAM proteins, such as mitofusin 2 (MFN2)

(8) and mammalian target of rapamycin complex 2 (mTORC2) (9), as well as mutation of

inositol 1,4,5-triphosphate receptor 1 (IP3R1) (10), display hyperglycemia, glucose

intolerance and increased neoglucogenesis. In addition, alterations of mitochondrial Ca2+

uptake from the ER can also disrupt insulin signaling in cardiomyocytes (11). However, the

literature is still equivocal regarding the exact role of MAMs in hepatic insulin resistance, as

altered hepatic insulin sensitivity can be associated with enrichment of ER-mitochondria

contacts (12), and that reduced IP3R levels can be associated with decreased glucose

production by the liver (13).

Another tissue crucial for glucose homeostasis is the skeletal muscle. This tissue is

indeed the primary site of insulin-stimulated glucose uptake and therefore the main target for

alterations in insulin resistant states (14). Interestingly, interactions of mitochondria with the

sarco-endoplasmic reticulum (SR/ER) have been demonstrated in skeletal muscle, although

the molecular components of the tethers remain to be characterized (15). Furthermore, a local

transfer of Ca2+ from SR/ER to mitochondria was illustrated upon caffein stimulation in

skeletal muscle (16), which seems important for the control of oxidative metabolism (17).

Accordingly, insulin-dependent Ca2+ mobilization participates to the translocation of the

glucose transporter GLUT4 to the cell surface and to glucose uptake (18), suggesting a

potential role of organelle coupling in muscle insulin action. However, up to now, the

relevance of MAM in skeletal muscle insulin resistance has not been invastigated.

The present study was conducted to gain more insight in this important issue. We

show here that SR/ER-mitochondria interactions in skeletal muscle are altered in insulin

resistant states, in both mice and humans, and that the modulation of organelle coupling

regulates insulin signaling and action in human myotubes.


4

MATERIALS AND METHODS

Animal studies

Animal studies were performed in accordance with the French guidelines for the care and use

of animals and were approved by a regional ethic committee. Male C57BL/6J and ob/ob mice

(12 weeks-old) were purchased from Harlan and adapted to the facility for one week before

study. Five weeks-old C57Bl/6J males were either fed with a standard diet (SD) or with a

high-fat and high-sucrose diet (HFHSD, prepared by the Unit of Experimental Food

Preparation in Jouy-en-Josas, France) for 16 weeks, as previously described (6; 19).

Metabolic characteristics of these mice were reported previously (6). For this study,

gastrocnemius muscles were removed and freshly used for either MAM purification or ex-

vivo insulin signaling studies (± 10-7M insulin, 15 minutes). An independent group of mice (5

weeks-old) was fed with a SD or HFHSD for 1, 4, 8, 12 or 16 weeks (n=3 mice/group); their

gastrocnemius muscles were either fixed in formaldehyde for paraffin inclusion or frozen.

Laslty, 12 weeks-old male C57BL/6J mice were used to infect gastrocnemius muscles

(intramuscular injection, 109 infection forming units/muscle) with recombinant adenoviruses

encoding for GFP or FATE1 proteins (Ad-GFP and Ad-FATE1, respectively), for 7 days.

MAM purification

Isolation of MAM fractions were performed by differential ultracentrifugation as previously

described (6).

Transmission electronic analysis (TEM)

Fixation and post-treatment of muscle fractions or myotubes, as well as the observation of

ultrathin sections, were performed as previously described (6). For analysis of SR/ER-

mitochondria contacts in human myotubes, we delimitate both organelles using Image J, and

the fraction of mitochondria in contact with SR/ER within a 50nm range was calculated and

normalized to mitochondria perimeter.

Human myotubes

Primary culture of human myotubes were initiated from satellite cells of Vastus lateralis

muscle biopsies obtained from donors during surgery at Edourd Herriot hospital (Lyon,

France). Myotubes from 7 donors (1M/6W; age: 68.8 ± 5.3 years old; body mass index: 25.3

± 0.7 kg/m2) were used for palmitate/oligomycin treatments. Myotubes from 10 lean subjects,


5

15 obese non-diabetic subjects and 12 obese type 2 diabetic patients were used to investigate

whether organelle interactions are altered during obesity and type 2 diabetes (T2D) (Table 1).

Patients with T2D were treated with oral antidiabetic drug and/or with insulin and did not

interrupt their treatment before the surgery. All participants gave their written consent after

being informed of the nature and purpose of the study. The experimental protocol

(DIOMEDE, agreement number 2012-111/A13-06) was approved by the Ethical Committees

Sud-EST IV and performed according to the French legislation. The myoblasts were purified

and differentiated myotubes were prepared according to the procedure previously described

(20). Human myotubes were either incubated with palmitate (500µM) for 24 hours to induce

insulin resistance (21), or treated with oligomycin (1 et 10µM, 16 hours) to alter mitochondria

function. For the analysis of insulin signaling, myotubes were depleted for 3 hours in serum

and further incubated with or without 10-7M of insulin for 20 minutes in serum-free medium.

Modulation of MAM protein expression

Human myotubes were transfected in 6-well plates, for 48 hours, with either 2µg expression

plasmids or siRNA (25 or 50nM, Qiagen) for specific targets, using Exgen 500 transfection

reagent (Roche Diagnostics) or High Perfect transfection reagent (Qiagen) respectively, as

previously reported (6).

FATE1 expression vectors and recombinant adenovirus

pcDNA4/TO and pcDNA4/FATE1 (fetal and adult testis expressed 1) expression vectors were

a generous gift from Enzo Lalli (22). FATE1 was subcloned into pcDNA-IRES-GFP vector,

obtained as previously described (23), in order to obtain pcDNA-FATE1-IRES-GFP vector,

allowing the simultaneous expression of both FATE1 and GFP proteins into cells under the

control of the same promoter. Recombinant adenoviruses expressing either GFP (as a control)

or FATE1 were generated by homologous recombination in the VmAdcDNA3 plasmid,

amplified and purified, as previously described (24; 25).

Western-blotting and real-time PCR

Protein expression was analyzed by SDS-PAGE and mRNA levels were measured by real-

time RT-PCR. In order to compare inter-gel analysis of insulin-stimulated PKB

phosphorylation in human myotubes from lean, obese and type 2 diabetic subjects, a pooled

sample of human myotubes was used as an internal standard on each SDS-PAGE gels and

used to normalize protein expression. For that, the intensity (in pixel) of each targeted protein


6

(i.e. phosphorylated PKB or total PKB) is normalized by the intensity of the internal standard,

before performing the ratio of phosphorylated vs. total PKB.

In situ Proximity Ligation Assay (PLA)

Voltage-dependant anion channel 1 (VDAC1) and IP3R1 proximity were measured by in situ

proximity ligation assay (PLA) (Olink Bioscience) to detect and quantify ER-mitochondria

interactions, as previously described and throughly validated (6; 26). PLA was also used here

to analyze PKB phosphorylation by using separate primary antibodies (Cell signaling) against

PKB protein and the S473 phosphorylation site of PKB. In muscle tissues, in situ PLA were

performed on paraffin-embedded gastrocnemius sections, after an antigen retrieval at pH 6,

using a bright-field revelation, as previously described (27).

Wide-field Ca2+ imaging

The concentration of cytosolic calcium ([Ca2+

]c) was measured using ratiometric dye fura2-

AM (5µM) loaded in human myotubes at 37°C for 1 hour, whereas that of mitochondria

([Ca2+]m) was measured using ratiometric 4mtD3cpv biosensor expressed after a 2-days

adenovirus infection of human myotubes at 37°C. After 3 washes with a Ca2+

-free Tyrode

solution (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 10 mM Glucose and

100 µM EGTA at pH 7.4), dishes were set on a Leica DMI6000 B microscope equipped with

a x20 objective, an ORCA-Flash 4.0 Scientific CMOS camera (Hamamatsu) and a Lambda

DG-4+ illumination system (Sutter Instrument). Myotubes were selected with the following

the criterions: elongated (crossing the field of vision), branched and polynucleated. Cells were

treated with 200 µM Na-ATP and 5 µM tapsigargin (Tg). Since not all myotubes responded to

Tg by an increase in free Ca2+ either in cytosol or in mitochondria, we selected only cells

showing a significant difference in the average fluorescence between the 40 sec prior and

after Tg treatment. These cells were considered as having a good probe load and sufficient

Ca2+ stores to enable measurement of variations in free [Ca2+]. Four independent experiments

were analyzed. Fluorescence ratio was normalized by the fluorescence at the origin (F/F0) and

all cells were gathered for statistical analysis.

Glucose transport

Glucose transport were measured in human myotubes as previously described (29).

Mitochondrial DNA quantification:


7

Total DNA was extracted from gastrocnemius muscle of mice and both mitochondrial and

nuclear DNAs were quantified by real-time PCR, as previously described (30).

COX and CS activities:

Cytochrome C oxidase (COX) and citrate synthase (CS) activities were measured

spectrophotometrically in total lysates from gastrocnemius muscle of mice, as previously

described (31 and 32, respectively).

Statistical analysis

Data are expressed as the mean ± SEM, and statistical significance was defined as a value of

p<0.05. Normal distribution of the data was tested using Shapiro-Wilk. Comparisons between

more than 2 groups were analyzed by one-way ANOVA followed by Turkey post hoc tests.

For all other analysis, data were compared by Student t test. For Ca2+ imaging, statistical error

(α), statistical power (β) and effect size (d) were calculated in function of the number of cells,

and statistical difference was defined as α <0.05 and β >0.8.


8

RESULTS

ER-mitochondria interactions are altered in skeletal muscle of obese and diabetic mice

We performed subcellular fractionation of gastrocnemius muscle from both genetically

(ob/ob) and diet-induced insulin resistant mice. The skeletal muscles were sampled on the

mice that have been previously used to study MAM integrity in the liver, and these mice were

glucose intolerant and insulin resistant (6). Both ob/ob (Figure 1A) and HFHSD (Figure 1B)

mice showed altered muscle insulin signaling, as illustrated by the significant reduction of

insulin-stimulated PKB and GSK3β phosphorylations in gastrocnemius explants.

Interestingly, in both models we found a marked reduction of MAM amount in gastrocnemius

muscle assessed by subcellular fractionation (Figure 1C and 1D, respectively). This reduction

of MAMs in diabetic muscle was also observed when MAM amounts were normalized

relative to mitochondria amounts. The purity of MAM fractions was validated by both

Western blotting and TEM (Supplementary Figures 1A-B), and the expression levels of

75kDa glucose regulated protein (GRP75), VDAC1, MFN2 and cyclophilin D were not

modified in muscle MAM fractions by obesity and type 2 diabetes (Supplementary Figure 2A

and 2B). We then used in situ PLA as an additional strategy to quantify the number of MAM

contact points in paraffin-embedded gastrocnemius muscle, by measuring the proximity

between VDAC1 and IP3R1 as previously described (6; 26). Both ob/ob (Figure 1E) and

HFHSD (Figure 1F) mice showed a significant reduction of VDAC1/IP3R1 interactions,

compared to respective control mice, illustrating a marked disruption of organelle coupling in

skeletal muscles of insulin resistant mice.

Palmitate-induced insulin resistance in human myotubes is associated with altered

organelle coupling

Next, we induced insulin resistance in primary cultures of human myotubes from

control subjects, using palmitate treatment (500µM, 24 hours). As expected, palmitate-treated

myotubes displayed a reduction of insulin-mediated PKB and GSK3β phosphorylations

(Figure 2A) and an inhibition of insulin-stimulated glucose transport (Figure 2B).

Importantly, they also showed a dramatic reduction of VDAC1-IP3R1 interactions measured

by in situ PLA (Figure 2C), as well as a decrease of the percentage of mitochondrial

membrane in contact with SR/ER, analyzed by TEM (Figure 2D), indicating a disruption of

organelle coupling.


9

Then, we analyzed organelle Ca2+ exchange in palmitate-treated human myotubes

using Ca2+ videomicrofluoremetry. Cells were either infected with a 4mtD3cpv-endocing

adenovirus for 48h to measure free [Ca2+

] in the mitochondrial matrix ([Ca2+

]m) or incubated

with 5µm Fura2-AM for 1h to measure free [Ca2+] in cytosol ([Ca2+]c). 200 µM ATP were

added in the medium to induce the IP3-mediated mobilization of Ca2+ stores. Conversely,

cells were treated with 5µM Tg to release Ca2+ from ER store independently of MAMs, as

Ca2+ leak from ER stores could be carried by several types of channels which have not been

described at MAMs (33). As shown in the Figures 2E-F, ATP induced a transient increase in

both [Ca2+]c and [Ca2+]m in BSA-treated cells, while it only induced increased [Ca2+]c in

palmitate-treated myotubes, suggesting that palmitate induced either a decrease of ATP-

mediated Ca2+

mobilization from ER stores or a decreased Ca2+

uptake by mitochondria. We

thus compared both ATP- and Tg-mediated variations in [Ca2+]m and [Ca2+]c. Tg induced a

similar increase in the free [Ca2+]c in BSA- and palmitate-treated myotubes suggesting no

modification in the ER Ca2+

stores. A slight but non-significant (Figure 2G and

Supplementary Table 5) decrease in [Ca2+]m was observed in palmitate-treated myotubes,

suggesting that a part of Tg-mediated Ca2+ could be released in MAMs.

SR/ER-mitochondria miscommunication is idependent of mitochondrial dysfunction

and insulin resistance

As mitochondrial physiology is markedly altered in skeletal muscle during obesity and

T2D, it is difficult to determine whether alterations of muscle insulin action result from

organelle miscommunication and/or mitochondria dysfunction. To get more insight into this

issue, we examined the relationship between organelle communication and mitochondria

physiology in insulin resistant states, both in vivo and in vitro. Firstly, we analyzed insulin

sensitivity (insulin tolerance test), SR/ER-mitochondria interactions (in situ PLA) and

mitochondrial biology (mtDNA amount and COX/CS activity) in muscle of SD and HFHSD

mice after 1, 4, 8, 12 or 16 weeks of diet. Diet-induced insulin resistance appeared in HFHSD

mice after 12 weeks of diet (Supplementary Table 6). SR/ER-mitochondria interactions are

decreased in skeletal muscle of HFHSD mice as soon as 1 week of feeding, and remains

diminished during all the feeding period (Figure 3A). Conversely, mtDNA amount and

COX/CS activity are reduced only after 16 weeks of HFHSD feeding (Figure 3B and 3C,

respectively), whereas we found a transient increase of COX/CS activity during the first

weeks of HFHSD feeding (Figure 3C). Secondly, we thought to alter mitochondria function

by oligomycin (an ATPase inhibitor) treatment (1µM or 10µM for 16 hours) in human


10

myotubes and to analyze the repercussions on both organelle contacts and insulin signaling.

Oligomycin treatment markedly induced mitochondria fragmentation (Figure 3D), without

altering cell viability (data not shown). Importantly, 1µM and 10µM oligomycin treatments

markedly induced VDAC1-IP3R1 interactions (Figure 3E), whereas only 1µM oligomycin

significantly increased insulin-stimulated PKB phosphorylation (Figure 3F).

Palmitate-induced insulin resistance and ER stress is prevented by increasing MAMs in

human myotubes

Next, we investigated whether the inhibitory action of palmitate on insulin signaling

could be prevented by increasing organelle coupling through Grp75 or Mfn2 overexpression,

as previously performed in hepatocytes (6; 26). Using a fluorescent vector, we estimated that

46.7 ± 2.3% of myotubes (n=20 images) were transfected in our conditions (data not shown),

and sufficient to observe an increase of Grp75 and Mfn2 protein expression (Figure 4B). As

expected, transient Grp75 or Mfn2 overexpression increased VDAC1-IP3R1 interactions in

human myotubes (Figure 4A). Interestingly, increasing organelle coupling by overexpressing

Grp75 or Mfn2 restored palmitate-induced alterations of insulin signaling, as illustrated by the

increase of insulin-mediated PKB and GSK3β phosphorylations (Figure 4B). In order, to

evaluate the impacts of MAM reinforcement on other parameters related to insulin action, we

analyzed the effect of the hormone on the regulation of GLUT4, hexokinase II (HKII) and

sterol regulatory element-binding protein 1c (SREBP1c) mRNA, as the regulation of these

genes by insulin is altered in muscle of type 2 diabetic patients (34), and on glucose transport.

We found that insulin induced expression of GLUT4, HKII and SREBP1c in BSA-treated

myotubes transfected with an empty vector, whereas palmitate treatment completely abolished

these regulations (Figure 4C). Importantly, both Grp75 and Mfn2 overexpression restored

insulin-mediated regulation of these 3 genes in palmitate-treated myotubes (Figure 4C).

Similarly, we found that palmitate treatment altered insulin-mediated glucose uptake in

human myotubes transfected with an empty vector, whereas insulin-mediated glucose uptake

was restored in palmitate-treated myotubes overexpressing Grp75 or Mfn2 (Figure 4D).

Altogether, these data demonstrate that increasing ER-mitochondria tethering in muscle cells

is sufficient to restore metabolic insulin signaling and metabolic action.

In hepatocytes, reduction of MAM integrity has been associated with ER stress (35)

which may contribute to insulin resistance. In order to determine whether the same

phenomenon also occurs in human myotubes, we evaluated the effects of different treatments

on the unfolded protein response (UPR) markers. Palmitate treatment increased mRNA levels


11

of the 78 kDa glucose-regulated protein (GRP78), the spliced X-box-binding protein 1 (Xbp1-

s) and the CCAAT-enhancer-binding protein homologous protein (CHOP) (Figure 4E),

confirming palmitate-mediated ER stress in human myotubes (21). Interestingly,

overexpression of Grp75 or Mfn2 counteracted palmitate-induced ER stress, as illustrated by

the significant reduction of all UPR markers (Figure 4E), indicating that reinforcement of

MAM can improve palmitate-induced ER stress in muscle cells.

Disruption of organelle coupling alters insulin signaling and action in human myotubes

We next explored whether the reduction of organelle coupling, by reducing GRP75 or

MFN2 protein levels by specific siRNA, can alter insulin action in muscle cells, as observed

previously in hepatocytes (6). We validated the efficiency of siRNA targeting in human

myotubes and found that 51 ± 4 % of human mytubes (n=20 images) were targeted by

fluorescent siRNA (data not shown). Silencing of Grp75 and Mfn2 using specific siRNA

(Figure 5A) reduced VDAC1-IP3R1 interactions in human myotubes (Figure 5B).

Importantly, in both conditions of disrupted ER-mitochondria interactions, insulin action was

altered in muscle cells, as illustrated 1) by the reduction of insulin-mediated PKB

phosphorylation (Figure 5C), 2) by a reduction of Glut4 mRNA expression (Figure 5D), and

3) by an inhibition of insulin-mediated glucose uptake (Figure 5E). Lastly, co-treatment of

human myotubes with siRNA (Grp75 or Mfn2) and palmitate did not lead to additive effects

on insulin-stimulated PKB phosphorylation (Supplementary Figure 3), suggesting that the two

treatments may affect the same regulatory process.

FATE1-mediated disruption of organelle coupling alters insulin signaling

Even if genetic manipulation of MAM proteins is a good approach to modulate SR/ER

mitochondria interactions, none of these proteins are exclusively expressed at MAMs and

their manipulation may result in alterations of cellular functions outside of MAMs. Therefore,

we sought to overexpress FATE1, a cancer-testis antigen recently identifed as an uncoupler of

MAMs (22) and normally not expressed in skeletal muscle. For that, we constructed a

pcDNA-FATE1-IRES-GFP vector, which simulteaneously overexpresses FATE1 and GFP

proteins, and allows us to analyze organelle interactions by in situ PLA only in transfected

cells (green cells), compared to untransfected cells (unfluorescent cells). Firstly, we

confirmed that FATE1 is not expressed in human myotubes and that its overexpression

increased its protein levels (Figure 6A). More importantly, transient FATE1 overexpression

reduced VDAC1-IP3R1 interactions in FATE1-transfected myotubes, compared to cells either


12

transfected with an empty vector or to untransfected cells in FATE1-transfected myotubes

(Figure 6B), validating the uncloupling activity of FATE1 in non testis cells. In order to

analyze the repercussions on insulin signaling similarly in both transfected and untransfected

myotubes, we analyzed insulin-stimulated PKB phosphorylation by in situ PLA, using

separate primary antibodies against PKB protein and the S473 phosphorylation site of PKB.

As expected, insulin increased pS473 phosphorylation of PKB in myotubes transfected with

an empty vector (Figure 6C), validating the use of PLA for the analysis of insulin signaling.

Importantly, insulin-stimulated PKB phosphorylation is markdly and specifically reduced in

FATE1-overexpressing myotubes (Figure 6C), indicating that disrupting organelle

interactions independently of endogenous MAM proteins also alters insulin action.

Next, we constructed an adenovirus starting from the pcDNA-FATE1-IRES-GFP

vector and we used an adenovirus overexpressing only GFP as control. Firstly, we validated

that the use of Ad-FATE1 in human myotubes reduced ER-mitochondria interactions

(Supplementary Figure 4A) and altered insulin-stimulated PKB phosphorylation

(Supplementary Figure 4B), as in transient transfection experiments. Interestingly, the same

experiment performed in HuH7 cells hepatic cell line also showed a clear reduction of both

organelle interactions (Supplementary Figure 5A) and insulin-stimulated IRS2 and PKB

phosphorylations (Supplementary Figure 5B), validating that FATE1-mediated organelle

uncoupling also alters insulin signaling in hepatocytes, in agreement with our previous

observations when we modulated endogenous MAM proteins (6). Therefore, we tested in vivo

the injection of Ad-FATE1 in gastrocnemius muscle of mice. We validated that infected

muscle fibers of both Ad-GFP and Ad-FATE1 mice express the GFP protein in muscle fibers

(Supplementary Figure 6). Importantly, FATE1 overexpression reduced VDAC1-IP3R1

interactions (Figure 6D) and altered the effect of insulin on PKB phosphorylation (Figure 6E)

in gastrocnemius muscle of infected mice.

ER-mitochondria interactions are altered in human myotubes of obeses and type 2

diabetic patients.

As myotubes in primary culture are known to maintain insulin sensitivity of the donor,

(36; 37), we investigated whether ER-mitochondria interactions are altered in primary

myotubes established from obese and type 2 diabetic donors, compared to cells from healthy

lean controls. The diabetic patients were BMI- and age-matched with the obese subjects, and

for age with the lean donors (Table 1). We observed a reduction of insulin-mediated PKB

phosphorylation in myotubes from obese and diabetic patients, compared to lean donors


13

(Figure 7A), whereas no significant difference was observed between obese non-diabetic

donors and obese type 2 diabetic patients, indicating similar degree of insulin resistance.

Importantly, VDAC1-IP3R1 interactions, measured by in situ PLA, were decreased in

myotubes from non-diabetic and diabetic obese subjects, compared to healthy donors (Figure

7B). There was no significant difference between myotubes from non-diabetic and diabetic

obese donors. Lastly, VDAC1-IP3R1 interactions were significantly and positively correlated

with the effect of insulin on PKB phosphorylation (Figure 7C, P=0.33, R2=0.11, p=0.04)

when all the subjects were analyzed.


14

DISCUSSION

We have recently shown that MAM integrity contributes to insulin action in liver, and

is altered in situations of hepatic insulin resistance (6). Here, we investigated whether this

mechanism is restricted to liver or could be generalized to other insulin sensitive tissues,

particularly skeletal muscles. This study in muscle cells is particularly important as the few

reported studies on this issue in the hepatocytes are highly controversial (6; 12). We

demonstrate both in vitro and in vivo, from mice to humans, that the disruption of organelle

coupling may contribute to muscle insulin resistance and that reinforcing MAM increases

insulin action, at least in human myotubes. Therefore, targeting MAM could be a novel

strategy to improve insulin sensitivity and restore glucose homeostasis.

As previously observed in the liver (6; 27), we found that ER-mitochondria

interactions are reduced in skeletal muscle of both genetically and diet-induced obese and

diabetic mice, in palmitate-treated human myotubes, and in myotubes from obese and T2D

patients, indicating a close relationship between MAM integrity and muscle insulin

sensitivity. These structural analyses were systematically performed by in situ PLA both in

vivo and in vitro, whereas subcellular fractionation or TEM analysis confirm PLA results in

vivo and in vitro, respectively. We tried to analyze ER-mitochondria interactions in skeletal

muscle by TEM, but we were not able to perform a reliable analysis due to the architecture of

muscle fibers. We feel that TEM is not really able to reveal fine structural details because of

overlapping density/noisy background in muscle tissues. This issue notwithstanding,

cumulative evidence supports the use of in situ PLA as a reliable method to study in situ ER-

mitochondria interactions in skeletal muscle, as previously validated in the liver (6; 26; 27).

Furthermore, the functionality of MAMs is also altered in insulin resistant states, since Ca2+

transfer from ER to mitochondria is reduced in palmitate-treated myotubes, supporting a

potential implication of organelle miscommunication in muscle insulin resistance.

Next, we found that MAM integrity is required for an efficient insulin action in

skeletal muscle, as the experimental disruption of MAM dampens insulin signaling and action

in human myotubes, whereas the experimental induction of organelle interactions prevents

palmitate-induced insulin resistance. Although interesting, these genetic experiments present

two weaknesses. Firstly, all myotubes are not transfected and therefore we analyzed organelle

tethering on a pool of untransfected and transfected cells, reducing the robustness of the

measurements. Secondly, none of MAM proteins is exclusively expressed at MAMs, their

modifications may therefore result in alterations of cellular function outside of MAMs. To get


15

around these two problems, we sought to overexpress FATE1, an organelle uncoupler not

expressed in skeletal muscle (22), using the pCDNA-FATE1-IRES-GFP vector allowing the

simultaneous expression of both FATE1 and GFP proteins. Using this strategy, we are able to

analyze by in situ PLA both VDAC1-IP3R1 interactions and PKB phosphorylation in both

transfected and untransfected cells, as a negative control. We validated that FATE1-mediated

reduction in organelle coupling is specific to transfected myotubes, indicating that FATE1

overexpression is a good strategy to reduce ER-mitochondria interactions in non-testis cells.

Furthermore, we found that overexpression of FATE1 specifically altered insulin-stimulated

PKB phosphorylation in both transfected or Ad-FATE1-infected myotubes, and in

gastrocnemius muscle of mice. Altogether, these data demonstrate that FATE1-mediated

organelle uncoupling is sufficient to alter insulin signaling both in vitro and in vivo.

As mitochondria physiology was previously reported to be altered in skeletal muscle

of obese and diabetic mice (30; 38) and in palmitate-induced insulin resistant myotubes (21),

the reduction of ER-mitochondria interactions in all these models could be related to

mitochondrial alterations. Nevertheless, we firstly confirmed that the reduction of MAM

amount persists after normalization by mitochondria amount in subcellular fractionation of

mouse liver. Furthermore, we demonstrated that organelle miscommunication is an early

event in diet-induced insulin resistance preceding both mitochondrial dysfunction and insulin

resistance. Lastly, oligomycin-induced mitochondrial dysfunction in human myotubes rather

increased SR/ER-mitochondria interactions and potentiated insulin signaling, probably as an

adaptive mechanism to improve mitochondrial function and cell homeostasis. Altogether,

these results suggest that muscle organelle miscommunication in diabetic state is not a

consequence of mitochondrial alterations or of the insulin resistance state, supporting its

participation in the development of muscle insulin resistance. Nevertheless, future

experiments are required to demonstrate that preventing MAM disruption could improve

muscle insulin sensitivity in vivo.

The mechanisms linking organelle miscommunication to muscle insulin resistance is

currently unknown. Modulation of Ca2+ signaling and exchange between organelle could

participate, as previously found in the liver (35). Activation of the UPR into the ER could also

play a role, as we found here that the reinforcement of MAMs through Mfn2 or Grp75

overexpression prevented the regulation of UPR markers by palmitate. Nevertheless, as Mfn2

was shown to directly modulate UPR (39), we cannot exclude an effect independent from

MAMs. Furthermore, the involvement of ER stress in muscle insulin resistance is more

controversial than in liver (38), and we previously found that reducing ER stress either by


16

genetic or pharmacological approaches is not sufficient to improve palmitate-induced insulin

resistance in myotubes (21), suggesting that ER stress is probably not the link between MAM

disruption and muscle insulin resistance. Alternatively, intramyocellular lipid accumulation

has been associated to reduced muscle insulin sensitivity (40). In this context, mitochondria

dysfunction, and the subsequent impaired ability to oxidize fatty acids, could play an

important role in muscle insulin resistance, although the causality of this association is still

controversial (1). We previously demonstrated (30) and confirmed here that mitochondrial

alterations were not an early event in the diet-induced development of insulin resistance,

contrary to MAM disruption. Therefore, organelle miscommunication could participate to

mitochondrial dysfunction in skeletal muscle, subsequently leading to insulin resistance.

Finally, boosting mitochondrial function might be beneficial to muscle insulin sensitivity and

patient health (41). As organelle coupling is known to control mitochondrial bioenergetics

(42), MAM-mediated improvement of insulin action in human myotubes may be related to

improved mitochondria function. However, further investigations are required to clarify this

hypothesis.

Whereas organelle miscommunication recently emerged as a new mechanism of

insulin resistance, the few reported studies on this issue are highly controversial. Indeed, we

and others found that insulin resistance is associated with reduced ER-mitochondria contacts

in the liver (6, 7) and in adipose tissue (43), whereas another group found excessive organelle

coupling in the liver of obese and diabetic mice (12). Of course, MAMs are dynamic

structures strongly dependent of nutritional and metabolic status (27), as well as of

environmental conditions and stress factors (44). In addition, their precise quantitative

measurement is particularly tricky, potentially depending of the methodology, and most

importantly whether the measurement takes into account the number, length or thickness of

the organelle coupling (45). It is likely that several types of mitochondria-ER contact sites

exist, with potentially different protein complexes and functions, and it is plausible that these

different tethers could be differentially regulated, as recently suggested (46). Consequently,

all these factors probably participate to the contrasting conclusions in this exciting topic.

Lastly, there is currently no good in vivo models of MAM disruption or reinforcement, and no

optimal reporter in order to follow dynamically ER-mitochondria interactions, making studies

more difficult. Further studies are thus required to solve this controversy, and the future use of

the very recent SPLICS tool to follow narrow and wide organelle coupling (46) could be a

relevant strategy. In the meantime and as a first step in this quest, we confirmed here that

FATE1-mediated organelle uncoupling reduced insulin-stimulated IRS2 and PKB


17

phosphorylation in HuH7 cells, as we previously observed when we modulated endogenous

MAM proteins (6). In this context, the present study in skeletal muscle confirms that

disruption of organelle interactions is also associated with insulin resistance, in another

insulin-sensitive tissue, and further extends the results to humans, an aspect not investigated

until now.

In summary, we demonstrated both in vivo and in vitro that defective ER-mitochondria

coupling is closely associated with impaired muscle insulin sensitivity, in both mice and

humans. Furthermore, organelle miscommunication is an early event in diet-induced insulin

resistance preceding mitochondrial dysfunction. Lastly, disruption of MAM integrity alters

insulin signaling both in vitro and in vivo, whereas reinforcing organelle coupling improves

palmitate-induced insulin resistance in human myotubes. Taking into account that similar

observations were done previously in liver (6), and although additional works are required,

especially to demonstrate that MAM reinforcement in muscle of insulin resistant patients

improves insulin action, the presented data pave the way for considering targeting MAM

interface in insulin-sensitive tissues as a potential attractive strategy to improve glucose

homeostasis.


18

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ACKNOWLEDGMENTS

This work was supported by INSERM, the national research agency (ANR-09-JCJC-0116

from JR), and by the “Fondation pour la Recherche Médicale” (DRM20101220461). E.T was

supported by a research fellowship from french governement of higher education and

research, and then by a scholarship from Lund University.

We thank Enzo Lalli (CNRS UMR7275, Valbonne, France) for the gift of pcDNA-T0 and

pcDNA-FATE1 vectors, Elisabeth Errazuriz for her technical help at the CIQLE Imaging

Center (Lyon, France), Kassem Makki (INSERM U1060, Lyon, France) for his help with

statistical analyses, Elsa Hoibian (INSERM U1060, Lyon, France) for her help with mice,

Aurélie Vieille Marchiset (INSERM U1060, Lyon, France) for her help with culture of human

myotubes, and Mélanie Paillard for her helpful discussion on FATE1.

E.T and J.R. designed the experiments, researched data, contributed to discussion, and wrote

the manuscript. S.C., N.B., G.B., M-A.C., J.J., C. D., and D.G-R. researched data. M.R. and

E.L. constituted the DIOMEDE biobank. G.B., E.L. and H.V. contributed to discussion and

reviewed/edited the manuscript. J.R. is the guarantor of this work and, as such, had full access

to all the data in the study and takes responsibility for the integrity of the data and the

accuracy of the data analysis.

Prior presentation: Some of the data in the article were presented at the 2016 annual meeting

of the French Society of Diabetes (SFD) and at the 2016 francophone day of nutrition (JFN).

The authors have no conflict of interest for this work.


23

Table 1: Donor Characteristics for primary myotubes from lean, obese non-diabetic

(obese) and obese type 2 subjects (T2D). Mean±SD are presented. *** p<0.001 compared to

lean subjects. BMI: body mass index.

Subjects n Gender ratio Age (years) BMI (kg/m2)

Lean 10 3M/8W 65 ± 4.9 24 ± 0.8

Obese 15 8M/7W 44 ± 3.4*** 45 ± 1.8***

T2D 12 3M/9W 54 ± 1.9 41 ± 2.3***


24

FIGURE LEGENDS

Figure 1: Disruption of MAM integrity in skeletal muscle of genetically and diet-induced

obese and diabetic mice. A-B) Representative Western Blots (at top) and quantitative

analysis (below) of insulin-stimulated PKB and GSK3β in gastrocnemius muscle of ob/ob (A)

and high-fat and high-sucrose diet (HFHSD)-fed mice (B). * p<0.0001, n=3. Basal and

insulin-stimulated values are illustrated in Supplementary Table 1 and 2, respectively. C-D)

Quantitative analysis of protein levels in MAM fractions following subcellular fractionation

of gastrocnemius muscles of ob/ob (C) and HFHSD-fed mice (D). MAM protein levels were

normalized either by muscle weight or by protein in pure mitochondria (pM) fraction. *

p<0.05, n=5 E-F) Representative images (at top) and quantitative analysis (below) of the

VDAC1-IP3R1 interactions (brown dots) measured by in situ PLA in paraffin-embedded

gastrocnemius muscle of ob/ob (E) and HFHSD-fed mice (F). *** p<0.00001, n=3-5/group,

5-10 pictures/mice.

Figure 2: Palmitate-induced alterations of insulin signaling and action are associated

with disruption of MAM integrity and function in human myotubes. Human myotubes

were treated with BSA or palmitate (500 µM) for 24 h. A) Representative Western Blots (at

top) and quantitative analysis (below) of insulin-stimulated PKB and GSK3β following BSA

or palmitate treatments. * p<0.0001, n=3. Basal and insulin-stimulated values are illustrated in

Supplementary Table 3. B) Effect of palmitate treatment on insulin-mediated glucose uptake

in human myotubes. * p<0.05, n=5. Basal and insulin-stimulated values are illustrated in

Supplementary Table 4. C) Representative PLA images (at left, x63 and scale bar=20µM) and

quantitative analysis (right) of VDAC1-IP3R1 interactions in human myotubes treated with

BSA or palmitate (500 µM) for 24 h. * p<0.05, n=3. D) Representative images (at top) and

quantitative analysis (below) of the percentage of mitochondria membrane in contact with ER

in human myotubes treated with BSA or palmitate (500 µM) for 24 h. *p<0.05, n=30 images

per condition in 3 independent experiments. E-G) Average time traces of the normalized

fluorescence (F/F0) reporting either the mitochondrial matrix Ca2+ concentration, [Ca2+]m (E)

or the cytosolic Ca2+ concentration, [Ca2+]c (F) in human myotubes incubated with either 500

µM BSA or 500 µM palmitate (PALM) for 24h. [Ca2+

]c was measured with fura2-AM while

[Ca2+]m was measured with 4mtD3cpv biosensor as explain in the method section.

Experiments were initiated in absence of extracellular Ca2+. 200 µM ATP was applied for 5

min prior to 5 µM Thapsigargin (Tg), to induce ER Ca2+

release. Values from 4 independent


25

human samples were pooled and presented as Mean±95%CI. Statistics and cell numbers are

presented in the Supplementary Table 5. G) Histogram reported the average variation in F/F0

over 40sec after drug treatment. Values: mean±SD. Significant difference between BSA-

treated and PALM-treated cells: ***. More detailed statistics are presented in the

Supplementary Table 5.

Figure 3: MAM alterations in insulin resistance states are independent of mitochondrial

dysfunction in skeletal muscle. A) Quantitative analysis of VDAC1-IP3R1 interactions in

gastrocnemius muscles of SD and HFHSD mice, after 1, 4, 8, 12 and 16 weeks of feeding. *

p<0.05 vs SD, n=30 images in 3 mice/group. B) Quantification of mitochondrial DNA

amount measured by real-time PCR in gastrocnemius muscle of SD and HFHSD mice, after

1, 4, 8, 12 and 16 weeks of feeding. *p<0.05 vs SD, n=3 mice/group. C) COX/CS activity

measured by spectrophotometry in gastrocnemius muscle of SD and HFHSD mice, after 1, 4,

8, 12 and 16 weeks of feeding. *p<0.05, ** p<0.01 vs SD, n=3-15 mice/group. D)

Geometrical analysis and representative images of MitoTracker-labeled mitochondria after a

16 hour oligomycin treatment (1 and 10µM). AR: aspect ratio, FF: form factor. * p<0.05, **

p<0.01, p<0.001 vs ethanol, n=30 images in 3 independent experiments. E) Representative

PLA images (at top, x63 and scale bar=20µM) and quantitative analysis (below) of VDAC1-

IP3R1 interactions in human myotubes incubated with ethanol or oligomycin (10 and 10µM)

for 16 hours. *** p<0.0005 vs ethanol, n=30 images in 3 independent experiments. F)

Representative Western Blots (at top) and quantitative analysis (below) of basal and insulin-

stimulated PKB phosphorylation in human myotubes treated with ethanol or 1µM oligomycin

for 16 hours. ** p<0.01 vs Co, $ p<0.05 vs respective ethanol, n=3.

Figure 4: Experimental reinforcement of MAM prevents palmitate-induced alterations

of insulin signaling and action in human myotubes. Human myotubes were transfected

with empty (Co), Grp75 or Mfn2 expressing vectors and treated post-24 h with BSA or

palmitate (200µM) for additional 24 h. When required, cells were depleted for 3 h in serum at

the end of the treatment, and incubated with insulin (10-7 mol/L, 15 minutes for insulin

signaling, 1 hour for glucose transport). A) Representative PLA images (left, x63 and scale

bar=20µM) and quantitative analysis (right) of VDAC1-IP3R1 interactions in transfected

human myotubes treated with BSA or palmitate (200 µM) during 24 hours. * p<0.01 vs. BSA

pcDNA3 Co, # p<0.05 vs. palmitate pcDNA3 Co, n=3 B) Representative Western Blots (at

top) and quantitative analysis (below) of insulin-stimulated PKB and GSK3β following


26

transfection and BSA or palmitate treatments. Basal and insulin-stimulated values are

illustrated in Supplementary Table 7. Validations of Grp75 and Mfn2 overexpression were

also shown. * p<0.05 vs. BSA pcDNA3 Co, # p<0.05 vs. palmitate pcDNA3 Co, n=3 C)

Effect of 6 h insulin treatment on Glut4, HKII and SREBP1c mRNA levels measured by real-

time PCR in transfected mytubes treated with BSA or palmitate for 24 h. * p<0.05 vs. BSA

pcDNA3 Co, # p<0.05 vs. palmitate pcDNA3 Co, n=3. Basal and insulin-stimulated values

are illustrated in Supplementary Table 8. D) Measurement of insulin-mediated glucose uptake

in MFN2-overexpressing human myotubes treated with BSA or palmitate for 24 h. * p<0.01

vs. BSA pcDNA3 Co, # p<0.05 vs. palmitate pcDNA3 Co, n=4. Basal and insulin-stimulated

values are illustrated in Supplementary Table 9. E) mRNA levels of ER stress markers,

measured by real-time PCR in transfected mytubes treated with BSA or palmitate for 24 h. *

p<0.05 vs. BSA pcDNA3 Co, # p<0.01 vs. palmitate pcDNA3 Co, n=3.

Figure 5: Experimental disruption of MAM integrity alters insulin action in human

myotubes. Human myotubes were transfected with specific siRNA for Grp75 or Mfn2

during 48 h. When required, cells were depleted for 3 h in serum, and treated with insulin (10-

7 mol/L, 15 minutes for insulin signaling, 1 hour for glucose transport). A) Validation of the

silencing of Grp75 or Mfn2 in human myotubes. B) Representative PLA images (at top, x63

and scale bar=20µM) and quantitative analysis (below) of VDAC1-IP3R1 interactions in

human myotubes silenced for Grp75 or Mfn2. * p<0.0001 vs. siRNA Co, n=3. C)

Representative Western Blots (at top) and quantitative analysis (below) of insulin-stimulated

PKB in human myotubes silenced for Grp75 or Mfn2. Basal and insulin-stimulated values are

illustrated in Supplementary Table 10.* p<0.0001 vs siRNA Co, n=3. D) Effect of 6 h insulin

treatment on Glut4, mRNA levels measured by real-time PCR in myotubes silenced for Grp75

or Mfn2. * p<0.05 and ** p<0.01 vs. siRNA Co, n=3. E) Measurement of insulin-mediated

glucose uptake in human myotubes silenced for Mfn2. Basal and insulin-stimulated values are

illustrated in Supplementary Table 11.** p<0.01 vs. control, n=4.

Figure 6: FATE1-induced disruption of MAMs is sufficient to alter insulin action in

skeletal muscle. A) Validation by Western blot that FATE1 is not expressed in human

myotubes and that its overexpression increases its protein levels. B) Representative PLA

images (at top, x63 and scale bar=20µM) and quantitative analysis (below) of VDAC1-IP3R1

interactions in human myotubes transfected with pcDNA-TO and pcDNA-FATE1-IRES-GFP

vectors. ** p<0.005, *** p<0.0001, n= 30 images in 3 independent experiments. C) Analysis


27

of insulin-stimulated PKB phosphorylation by in situ PLA (pS473 PKB-PKB proximity) in

human myotubes infected with Ad-GFP or Ad-FATE1 for 48 hours, and stimulated or not

with insulin (10-7

M, 15 minutes). ***p<0.0001 vs Co, ## p<0.001 vs T0 insulin, n=30 images

in 3 independent experiments. D) Representative PLA images (at top) and quantitative

analysis (below) of VDAC1-IP3R1 interactions in gastrocnemius muscle of Ad-GFP or Ad-

FATE1 infected mice 7 days post-infection. *** p<0.0005, n=30 images in 3 independent

mice. E) Representative Western Blots (at left) and quantitative analysis (at right) of insulin-

stimulated PKB phosphorylation in gastrocnemius muscle of Ad-GFP or Ad-FATE1 infected

mice 7 days post-infection. Basal and insulin-stimulated values are illustrated in

Supplementary Table 12.* p<0.05, n=5/group.

Figure 7: MAM integrity is altered in myotubes from insulin resistant subjects.

Myotubes from lean healthy subjects (lean), obese non-diabetic (obese) and obese type 2

diabetic patients (T2D) were analyzed for ER-mitochondria interactions (A) and insulin-

stimulated PKB phosphorylation (B). * p<0.05, ** p< 0.01, *** p<0.001 compared to lean

subjects. C) Correlation between VDAC1-IP3R1 interactions and insulin effect on PKB

phosphorylation in myotubes from lean, obese and T2D subjects (n=37).


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0.20

0.40

0.60

0.80

1.00

1.20

wt ob/ob

VD

AC

1-I

P3R

1

dots

/fie

ld (

a.u

.)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

SD16w HFHSD 16w

VD

AC

1-I

P3R

1

dots

/fie

ld (

a.u

.)

***

VDAC1-IP3R1 VDAC1-IP3R1

PKB GSK3b

* *

*

PKB GSK3b

*

*

Su

bce

llu

lar

fract

ion

ati

on

(a.u

.)

MAM MAM/Mp

p=0.07

MAM MAM/Mp

* *

Su

bce

llu

lar

fract

ion

ati

on

(a.u

.)


0

0.2

0.4

0.6

0.8

1

1.2

BSA Palmitate

Co

nta

ct/m

ito

cho

nd

ria

(a.u

)

A - - - + + + - - - + + +

BSA Palmitate

Insulin:

pS473-PKB

PKB

pS9-GSK3b

GSK3b

BSA Palm

VDAC-IP3R1

B

Figure 2

D

VD

AC

1-I

P3R

1 d

ots

/myotu

be

surf

ace

(a.u

.)

*

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.0 BSA Palmitate

C

E

F

G

M

M ER

ER

Palm

itate

M

M

ER

ER

ER

ER

BS

A

*

Insu

lin

eff

ect

on

pro

tein

ph

osp

hory

lati

on

PKB GSK3b

*

p=0.07

*

Insu

lin

eff

ect

on

glu

cose

up

tak

e (f

old

)


D

F

E 0

0.2

0.4

0.6

0.8

1

1.2

Ave

rage

val

ue

(a.

u.)

AR FF AR FF

1µM 10µM

* ** *** ***

EtOH Oligomycin 10µM

EtOH Oligomycin 1µM

0

0.5

1

1.5

2

1µM 10µM

VD

AC

1-I

P3R

1

dots

/myotu

be

surf

ace

(a.u

.)

Ethanol Oligo 10µM

Ethanol Oligo 1µM

VDAC1-IP3R1

Ethanol

Oligomycin

Ethanol

Oligomycin

Figure 3

0

0.2

0.4

0.6

0.8

1

1.2

1w 4w 8w 12w 16w

VD

AC

1-I

P3R

1 d

ots

/fie

ld

(a.u

.)

SD HFHSDA

* * * * *

B C

OX

/CS

act

ivit

y

C

*

p=0.07

p=0.07

**

1w 4w 8w 12w 16w

SD HFHSD

Insulin: - - - + + + - - - + + +

Ethanol Oligomycin 1µM

pSer473PKB

PKB

**

** $

Co

Insulin

pS

47

3-P

KB

/PK

B

Ethanol Oligomycin 1µM

*

SD HFHSD

1w 4w 8w 12w 16w

mtD

NA

/nu

clea

r D

NA

*** ***


A

VD

AC

1-I

P3

R1

do

ts/m

yo

tub

e

su

rfa

ce (

a.u

.)

*

#

#

0.0

0.2

0.4

0.6

0.8

1.0

1.2 BSA pcDNA3 Co Palm pcDNA3 Co

Palm pcDNA3 GRP75 Palm pcDNA3 MFN2

VDAC1-IP3R1

B BSA

pcDNA3-

Co

Palmitate

pcDNA3-

Co

Palmitate

pcDNA3-

Grp75

Palmitate

pcDNA3-

Mfn2

Insu

lin

eff

ect

on

pro

tein

ph

osp

hory

lati

on

PKB

p=0.08

GSK3

*

#

0.0

0.5

1.0

1.5

2.0

2.5

3.0

*

#

pS473-PKB

PKB

pS9-GSK3b

GSK3b

MFN2

GRP75-myc

- + - + - + - + Insulin:

C

*

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Glut4

#

Insu

lin

eff

ect

on

mR

NA

lev

els

#

HKII SREBP1c

*

p=0.06

*

# p=0.08

#

BSA pcDNA3 Co

Palmitate pcDNA3 Co

Palmitate pcDNA3 Grp75

Palmitate pcDNA3 Mfn2

D

E

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

*

# #

*

# #

*

#

CHOP Xbp1S Grp78

mR

NA

/TB

P (

fold

)

#

BSA pcDNA3 Co

Palmitate pcDNA3 Co



BSA pcDNA3 Co

Palmitate pcDNA3 Co



Figure 4

BSA pcDNA3 Co

Palmitate pcDNA3 Co



BSA

pcDNA3-Co

Palmitate

pcDNA3-Co

Palmitate

pcDNA3-Mfn2

Insu

lin

eff

ect

on

glu

cose

up

tak

e (f

old

)

#

*


Figure 5

siRNA Co

siRNA Grp75

siRNA Mfn2

A B

C

0.0

0.2

0.4

0.6

0.8

1.0

1.2

siRNA

Co

siRNA

Grp75

siRNA

Mfn2

VD

AC

1-I

P3R

1 d

ots

/myotu

be

Su

rface

(a.u

.) *

*

PKB

pS473-PKB

- + - + - +

siRNA Co siRNA Grp75 siRNA Mfn2

Insulin :

D E

MFN2

GRP75

Actin

siRNA

Co

siRNA

Mfn2

siRNA

Grp75

VDAC1-IP3R1

siRNA Co siRNA Grp75 siRNA Mfn2

Insu

lin

ef

fect

on

PK

B

ph

osp

hory

lati

on

(fo

ld)

* *

**

**

Glu

t4/T

BP

Insu

lin

eff

ect

on

glu

cose

up

tak

e (f

old

) p=0.07


A C

D

Figure 6

Insulin : - + - + - +

pSer473-PKB

PKB

Insu

lin

ef

fect

on

PK

B

ph

osp

hory

lati

on

(fo

ld)

*

0

0.2

0.4

0.6

0.8

1

1.2

Ad-GFP Ad-FATE1

VD

AC

1-I

P3R

1 d

ots

/fie

ld a

.u.)

***

Ad-GFP Ad-FATE1

VDAC1-IP3R1

0.00

0.20

0.40

0.60

0.80

1.00

1.20

T0 tranfected cells untransfected

cells

VD

AC

1-I

P3

R1

do

ts/m

yo

tub

e

surf

ace

(a

.u.)

*** **

pcDNA-FATE1-IRES-GFP

E D

0

2

4

6

8

10

12

pS

473 P

KB

-PK

B d

ots

/myotu

be s

urf

ace

(a.u

.)

Co Insulin

T0 transfected

cells

untransfected

cells

pcDNA-FATE1-IRES-GFP

*** *** ##

##

pcD

NA

-FA

TE

1-

IRE

S-G

FP

p

cDN

A-T

0

Co Insulin

B pcDNA-TO pcDNA-FATE1-IRES-GFP

VDAC1-IP3R1

pcDNA-T0

Tubulin

FATE1

pcDNA-FATE1

-IRES-GFP

pS473 PKB-PKB


Figure 7

VD

AC

1-I

P3R

1 d

ots

Lean Obese DT20

100

200

300

400

500

***

Eff

ect

of

insu

lin

on

PK

B p

ho

sp

ho

ryla

tio

n

Lean Obese DT20

2

4

6

8***

***

A B

C

0

1

2

3

4

5

6

7

8

0 100 200 300 400 500

Eff

ect

of

insu

lin

on

PK

B

ph

osp

ho

ryla

tion

VDAC1-IP3R1 dots


ER

MAM

pM VDAC1

GRP75

H ER MAM pM

Mfn2

Hsp60

CypD

IP3R1

B A

Supplementary Figure 1: Validation of MAM fractions purified from gastrocnemius muscle. A-B)

Mouse gastrocnnemius homogenate (H) was fractioned into pure mitochondria (pM), mitochondria-

associated endoplasmic reticulum membranes (MAM) and endoplasmic reticulum (ER) fractions,

which were analyzed either by Western-blot (A) or transmission electronic microscopy (B).


SD16w HFHSD16w

GRP75

wt ob/ob

VDAC

GRP75

VDAC

MAM fractions from

gastrocnemius muscle

MAM fractions from

gastrocnemius muscle

CYPD

MFN2

CYPD

MFN2

B

A

Supplementary Figure 2: Protein expression levels in MAM fractions purified from gastrocnemius muscle. A) Expression levels of GRP75, VDAC1, cyclophilin D (CYPD) and MFN2 in MAMs of

gastrocnemius muscle from SD and HFHSD mice after 16 weeks of feeding. B) Expression levels of

GRP75, VDAC1, CYPD and MFN2 in MAMs of gastrocnemius muscle from wt and ob/ob mice.

N=3-4 mice/group. NS: non significant.

NS

NS

GRP75 VDAC1 CYPD MFN2

SD HFHSD

Pro

tein

lev

els

(a.u

.)

NS

NS

NS

NS

wt ob/ob

Pro

tein

lev

els

(a.u

.)

Grp75 VDAC1 CYPD MFN2


- + - + - + - + Insulin :

+ + - - - - - - BSA : - - + + + + + + Palm. :

siRNA Co siRNA-Mfn2 siRNA-Grp75

pS473-PKB

PKB

Supplementary Figure 3: Effect of silencing of Grp75 or Mfn2 on insulin-stimulated PKB phosphorylation in palmitate-treated myotubes. Representative Western Blots (at top) and

quantitative analysis (below) of insulin-stimulated PKB in palmitate-treated human myotubes

following silencing of Grp75 or Mfn2. * p<0.05. ** p<0.01 vs respective Co, n=3. NS: non

significant. No additive effects of silencing of MAM proteins on palmitate-induced alterations of

insulin signaling.

**

*

* *

NS

BSA

si RNA Co

Palmitate

siRNA Co

Palmitate

siRNA Grp75

Palmitate

siRNA Mfn2

P-P

KB

/PK

B

Co Insulin


A

B

Supplementary Figure 4: Effects of Ad-FATE1 infection on ER-mitochondria interactions and insulin signaling in human myotubes. Almost all myotubes express GFP and are green (data not shown). A) Representative PLA images (at top, x63 and scale bar=20µM) and quantitative analysis (below) of VDAC1-IP3R1 interactions in human myotubes infected with Ad-GFP or Ad-FATE1 for 48 hours. ** p<0.01, n=30 images in 3 independent experiments. B) Analysis of insulin-stimulated PKB phosphorylation by in situ PLA in human myotubes infected with Ad-GFP or Ad-FATE1 for 96 hours. ** p<0.001, n=30 images in 3 independent experiments, # p<0.001 vs AD-GFP Co.

0

0.2

0.4

0.6

0.8

1

1.2

Ad-GFP Ad-FATE1

VD

AC

1-I

P3

R1

do

ts

(a.u

.)

**

VDAC1-IP3R1

Ad-GFP Ad-FATE1

0

5

10

15

20

25

30

35

40

Ad-GFP Ad-FATE1

pS

473 P

KB

/PK

B

dots

/su

rface

Co

Insulin

Co Insulin

Ad

-GF

P

Ad

-FA

TE

1

*

#

pS473 PKB-PKB


pSer473PKB

PKB

a-tubulin

Co Ins Co Ins

Ad-GFP Ad-FATE1

FATE1

pIRS2

IRS2

0

50

100

150

200

250

Ad-GFP Ad-FATE1

VD

AC

1-I

P3

R1

do

ts

B

A

Supplementary Figure 5: Effects of FATE1 overexpression using an adenovirus on insulin signaling in HuH7 cells. A) Representative images (at left) and quantitative analysis (at righ) of VDAC1-IP3R1 interactions, measured by in situ PLA in infected HuH7 cells. Adenoviral-mediated overexpression of FATE1 disrupts ER-mitochondria interactions. *** p<0.0001 vs Ad-GFP, n=30 images on 3 independent experiments. B) Representative Western blots (at left) and quantitative analysis (at righ) of insulin-stimulated PKB and IRS2 phosphorylations in infected HuH7 cells. Adenoviral-mediated overexpression of FATE1 disrupts insulin signaling. * p<0.05, ** p<0.005 vs respective Co; $ p<0.05 vs respective Ad-GFP.

***

**

*

$

**

$

P-P

KB

/PK

B

Co Insulin

Ad-GFP Ad-FATE1

Ad-GFP Ad-FATE1

P-I

RS

2/I

RS

2

Co Insulin

VDAC1-IP3R1

Ad-GFP Ad-FATE1


Supplementary Figure 6: Validation of adenoinfection of gastrocnemius muscle of mice. Fluorescent (at the top), bright-field (at the medium) and merged (below) acquisitions of muscle fibers of Ad-GFP- and Ad-FATE1-infected mice. We made these pictures on muscle sections used for in situ PLA experiments, explaining why we observed black dots (corresponding to PLA dots) on fluorescent images. Infected muscle fibers express in both cases the GFP protein, validating the adenoinfection of muscle fibers. VDAC1-IP3R1 dots quantified in Figure 6D are analyzed in infected muscle fibers (green ones).

Ad-GFP Ad-FATE1 Page 40 of 44Diabetes

Supplementary Table 1: Basal and insulin-stimulated PKB and GSK3β phosphorylations in

skeletal muscle of wt and ob/ob mice. * p<0.05, *** p<0.0001 vs respective basal. $$$ p<

0.0001 vs respective wt.

P-PKB/PKB P-GSK3 β /GSK3 β

wt ob/ob wt ob/ob

Basal 0.17 ± 0.02 0.26 ± 0.02 0.69 ± 0.17 0.66 ± 0.03

Insulin-stimulated 0.91 ± 0.08***

0.25 ± 0.02$$$

1.16 ± 0.18* 0.80 ± 0.04


skeletal muscle of SD16w and HFHSD16w mice. * p<0.05, *** p<0.0001 vs respective basal. $

p<0.05, $$ p<0.005 vs respective wt.


SD16w HFHSD16w SD16w HFHSD16w

Basal 0.15 ± 0.01 0.20 ± 0.01 0.62 ± 0.02 0.61 ± 0.06


0.57 ± 0.04*$$

0.76 ± 0.05* 0.61 ± 0.04

$


BSA- and palmitate-treated human myotubes. * p<0.05, *** p<0.0001 vs respective basal. $

p<0.05, $$ p<0.01, $$$ p<0.0001 vs respective BSA.


BSA Palmitate BSA palmitate

Basal 0.15 ± 0.01 0.11 ± 0.02 0.65 ± 0.004 0.46 ± 0.02$$


0.36 ± 0.02***$$$

1.036 ± 0.19* 0.48 ± 0.07

$

Supplementary Table 4: Basal and insulin-stimulated glucose transport in BSA- and

palmitate-treated human myotubes. * p<0.05 vs respective basal; $ p<0.05 vs respective

BSA.

Glucose uptake (pmol/mg.min) Basal Insulin-stimulated

BSA 2612 ± 847 3130 ± 808*

Palmitate 2432 ± 917 2656 ± 885*$

Supplementary Table 5: Statistical analysis of the variation in mitochondrial (∆[Ca2+

]m) or cytosolic

Ca2+ (∆[Ca2+

]c) concentration in BSA- or Palmitate-treated human myotubes (BSA and PALM,

respectively). Comparison was made on the 20 time-points (covering 40 sec) preceding or following

ATP or tapsigargin (Tg) treatment for ether BSA-treated cells or for PALM-treated cells. A

comparison was also achieved on BSA- and PALM-treated cells (BSA vs. PALM) after ATP or Tg

treatment (20 time-points). Mean and SD were calculated and statistical significance was assessed by a

student t-test. When the t-test was conclusive (error α < 0.05), a statistical power analysis (error β >

0.8) was performed with G*Power 3.1 to withdraw type-II error. The last column indicates when

hypothesis H1 was found to be different to H0 (YES).

ATP-mediated ∆[Ca

2+]m

BSA (N=31) Mean±SD t-test αααα ββββ d H0≠H1

Before 1.02±0.04 1.31E-05 1.00% 90.00% 1.01 YES


After 1.11±0.12

PALM (N=35) Mean±SD t-test αααα ββββ d H0≠H1

Before 1.01±0.04 9.07E-02 NO

After 1.02±0.04

BSA vs. PALM Mean±SD t-test αααα ββββ d H0≠H1

BSA 1.11±0.12 3.14E-05 0.10% 91.00% 0.936 YES

PALM 1.02±0.04

Tg-mediated ∆[Ca2+

]m


Before 1.04±0.04 2.42E-12 0.10% 99.00% 1.71 YES

After 1.17±0.10


Before 1.02±0.03 1.74E-11 0.10% 99.00% 1.65 YES

After 1.12±0.08


BSA 1.17±0.10 9.75E-03 5.00% 59.00% 0.55 NO

PALM 1.12±0.08

ATP-mediated ∆[Ca2+

]c


Before 1.01±0.04 1.65E-03 5.00% 80.00% 0.81 YES

After 1.11±0.17


Before 1.02±0.05 1.41E-03 5.00% 80.00% 1.12 YES

After 1.13±0.13

BSA vs. PALM Mean t-test αααα ββββ d H0≠H1

BSA 1.11±0.17 7.16E-01 NO

PALM 1.13±0.13

Tg-mediated ∆[Ca2+

]c


Before 1.01±0.06 1.07E-04 5.00% 95.00% 1.05 YES

After 1.26±0.33


Before 1.00±0.07 8.55E-04 5.00% 90.00% 1.37 YES

After 1.27±0.38


BSA 1.26±0.33 7.75E-01 NO

PALM 1.27±0.38

Supplementary Table 6: Area under curve (a.u.) of insulin tolerance tests performed in SD

and HFHSD mice after different times of feeding (4, 8, 12 et 16 weeks). ** p<0.005 vs SD.

ND: not determined.

Week of feeding SD mice HFHSD mice

1w ND ND

4w 1 ± 0.05 1.25 ± 0.1

8w 1 ± 0.14 1.13 ± 0.13

12w 1 ± 0.02 1.70 ± 0.13**

16w 1 ± 0.01 1.68 ± 0.09**


transfected human myotubes.* p<0.05 vs respective basal.


Basal Insulin-stimulated

P-PKB/PKB

BSA pcDNA3-Co 0. 51 ± 0.1 1.49 ± 0.01*

Palmitate pCDNA-Co 0.61 ± 0,18 1 ± 0.001

Palmitate pCDNA-Grp75 0.59 ± 0.2 1.26 ± 0.18*

Palmitate pCDNA-Mfn2 0.60 ± 0.2 1.14 ± 0.22*

P-GSK3β/GSK3β

BSA pcDNA3-Co 0. 85 ± 0.15 1.49 ± 0.19*

Palmitate pCDNA-Co 0.58 ± 0.02 0.62 ± 0.03


Palmitate pCDNA-Mfn2 1.06 ± 0.2 1.36 ± 0.25

Supplementary table 8: Basal and insulin-stimulated Glut4, HKII and SREBP1c gene

expression in transfected human myotubes. *p<0.05 vs respective basal.


Glut4 mRNA levels

BSA pcDNA3-Co 71.1 ± 4,1 108.9 ± 9.5*

Palmitate pCDNA-Co 122 ± 37.3 116.5 ± 28.7


Palmitate pCDNA-Mfn2 75. 5 ± 15 108.2 19.9*

HKII mRNA levels

BSA pcDNA3-Co 258.12 ± 21.5 375.24 ± 19.2*



Palmitate pCDNA-Mfn2 186.5 ± 21 240.4 ± 32.6*

SREBP1c mRNA levels

BSA pcDNA3-Co 118.01 ± 2.9 170. 2 ± 34.8*



Palmitate pCDNA-Mfn2 155.5 ± 23.6 214.8 ± 4.3*

Supplementary Table 9: Basal and insulin-stimulated glucose transport in transfected

human myotubes. * p<0.05 vs respective basal.


BSA pcDNA3-Co 2271.35 ± 486 3065.85 ± 531*

Palmitate pCDNA-Co 2785 ± 657 2825.5 ± 642

Palmitate pCDNA-Mfn2 2282.1 ± 233 2792.9 ± 296*

Supplementary Table 10: Basal and insulin-stimulated PKB phosphorylation in silenced

human myotubes. *** p<0.0001 vs respective basal. $$$ p<0.0001 vs respective siRNA Co.


siRNA Co 1.56 ± 0.05 2.47 ± 0.16***

siRNA Grp75 1.63 ± 0.06 1.61 ± 0.06$$$

siRNA Mfn2 1.48 ± 0.03 1.43 ± 0.02$$$

Supplementary Table 11: Basal and insulin-stimulated glucose transport in silenced human

myotubes. * p<0.05 vs respective basal.


Glucose transport (pmol/mg.min) Basal Insulin-stimulated

siRNA Co 2371.6 ± 659 2919.5 ± 683*

siRNA Mfn2 2508.1 ± 690 2675.3 ± 623

Supplementary Table 12: Basal and insulin-stimulated PKB phosphorylation in

gastrocnemius muscle of mice infected with Ad-GFP and Ad-FATE1. ** p<0.001, ***

p<0.0001 vs respective basal. $ p<0.05 vs respective Ad-GFP.

P-PKB/PKB Basal Insulin-stimulated

Ad-GFP 0.26 ± 0.01 1.4 ± 0.15***

Ad-FATE1 0,34 ± 0.03$ 1.20 ± 0.04

**


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Disruption of mitochondria-associated endoplasmic ......2018/01/08 · Transmission electronic analysis (TEM) Fixation and post-treatment of muscle fractions or myotubes, as well