Mitochondrial dysfunction induces aberrant insulin signalling and glucose utilisation in murine C2C12 myotube cells
- First Online:
- Cite this article as:
- Lim, J.H., Lee, J.I., Suh, Y.H. et al. Diabetologia (2006) 49: 1924. doi:10.1007/s00125-006-0278-4
- 1k Downloads
Mitochondrial dysfunction is considered a critical component in the development of diabetes. The aim of this study was to elucidate the molecular mechanisms involved in the development of insulin resistance and diabetes through investigation of mitochondrial retrograde signalling.
Materials and methods
Mitochondrial function of C2C12 myotube cells was impaired by genetic (ethidium bromide) and metabolic (oligomycin) stress, and changes in target molecules related to insulin signalling were analysed.
Concomitant with reductions in mitochondrial membrane potential (ΔΨm) and ATP synthesis, production of IRS1 and solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) were reduced. Moreover, serine phosphorylation of IRS1 increased, resulting in decreased tyrosine phosphorylation. This indicates that mitochondrial dysfunction decreases insulin-stimulated SLC2A4 translocation and glucose uptake. Mitochondrial stress activated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) signalling in a Ca2+-dependent manner, and removal of free Ca2+ by BAPTA-AM, as well as inhibition of JNK and p38 MAPK, abrogated mitochondrial stress-induced reductions in IRS1 and SLC2A4 production. Mitochondrial dysfunction after oligomycin treatment significantly increased levels of activating transcription factor 3 (ATF3), which represses Irs1 promoter activity. Removal of the 5′ flanking region of Irs1 demonstrated that the promoter region within 191 bases from the transcription site may be involved in the transcriptional repression of Irs1 by mitochondrial stress.
We show distinct mitochondrial retrograde signalling, where Irs1 is downregulated through ATF3 in a Ca2+-, JNK- and p38 MAPK-dependent manner, and IRS1 is inactivated. Therefore, mitochondrial dysfunction causes aberrant insulin signalling and abnormal utilisation of glucose, as observed in many insulin resistance states.
KeywordsInsulin resistanceInsulin signallingMitochondrial dysfunctionRetrograde signalling
activating transcription factor 3
1,2-bis(o-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid, tetraacetoxymethyl ester
calcium/calmodulin-dependent serine/threonine protein kinase
cAMP response element-binding
green fluorescent protein
c-Jun N-terminal kinase
protein kinase C
- p38 MAPK
p38 mitogen-activated protein kinase
solute carrier family 2 (facilitated glucose transporter), member 4
The cluster of pathologies known as the metabolic syndrome, including obesity, insulin resistance and type 2 diabetes, has become one of the most serious threats to human health. However, the molecular mechanisms underlying these individual disorders and their links with each other have not yet been elucidated. A number of studies have suggested that prominent features of type 2 diabetes are attributable to dysfunctional mitochondria [1–3]. Mutations in mitochondrial DNA (mtDNA) are associated with various disease states [4–7], a few of which are strongly associated with diabetes, including the A3243G mutation in the mitochondrial DNA-encoded mt-Tl1 [8–10]. In addition, maternally inherited defects in mtDNA that disrupt mitochondrial functioning are known to cause an insulin-deficient form of diabetes resembling type 1 diabetes . Studies using the proton magnetic resonance spectroscopy technique have shown decreased mitochondrial activity and increased intramyocellular fat content in insulin-resistant children of parents with type 2 diabetes, a group with a strong tendency to develop diabetes later in life .
It has been shown that mitochondrial dysfunction can greatly modify nuclear gene expression . Retrograde regulation, a general term for mitochondrial signalling, is broadly defined as cellular responses to changes in the functional status of mitochondria [14, 15]. Much of our understanding of the regulation of the retrograde response has been derived from studies with Saccharomyces cerevisiae [16–18]. In mammalian systems, mitochondrial retrograde signalling has been described in C2C12 skeletal myoblasts and in human lung carcinoma A549 cells [19, 20]. Mitochondrial dysfunction in both cell types resulted in elevated cytosolic free Ca2+ and enhanced expression of genes involved in Ca2+ transport and storage, including ryanodine receptors I and II [14, 19]. Furthermore, increased Ca2+ correlated with activation of several Ca2+-dependent protein kinases, including protein kinase C (PKC) and calcium/calmodulin-dependent serine/threonine protein kinase (CaMK) [21, 22], and Ca2+-responsive transcription factors .
Activating transcription factor 3 (ATF3) is a member of the ATF/cAMP-response element-binding (CREB) protein family of transcription factors, which recognise a consensus DNA sequence, TGACGTCA, and have structurally similar basic region/leucine zipper domains. They interact selectively with each other to form hetero- or homodimers through their leucine zipper regions . Despite overwhelming evidence indicating that ATF3 is a stress-inducible gene in a variety of tissues and acts as a transcriptional repressor, the physiological consequences of Atf3 expression are not well understood. However, it was recently reported that Atf3 is expressed in primary sensory neurons of diabetic mice  and ATF3 is expressed in the islets of patients with type 1 or type 2 diabetes [25, 26].
Here, we investigate the effects of mitochondrial dysfunction on the development of insulin resistance in C2C12 myotube cells with functionally inactive mitochondria. Our findings show that mitochondrial dysfunction represses IRS1 through ATF3 in a Ca2+- and c-Jun N-terminal kinase (JNK)-dependent manner. Additionally, inactivation of IRS1 by increased serine phosphorylation and decreased tyrosine phosphorylation results in the aberrant insulin signalling and abnormal glucose utilisation observed in many insulin resistance states.
Materials and methods
Oligomycin and the somatic cell ATP assay kit were purchased from Sigma (St Louis, MO, USA). The kinase inhibitors SP600125, SB203580, Calphostin C and KN-93 were from Calbiochem (San Diego, CA, USA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) was obtained from Molecular Probes (Eugene, OR, USA).
Induction of mitochondrial dysfunction
Murine C2C12 skeletal myoblast cells (ATCC CRL 1772; American Type Culture Collection, Manassas, VA, USA) were grown in DMEM supplemented with 2% horse serum to induce differentiation into myotubes. Cells with functionally inactivated mitochondria were obtained by growing C2C12 cells in serum-supplemented DMEM containing 1 mmol/l pyruvate, 50 μg/ml uridine and 200 or 500 ng/ml ethidium bromide (EtBr) for 4 weeks to selectively inhibit mtDNA replication and transcription without detectable effects on the nuclear DNA [19, 20]. To disturb mitochondrial electron transport, differentiated C2C12 myotubes were treated with 20 or 40 μmol/l oligomycin, an inhibitor of mitochondrial ATPase, for 24 h.
To reverse mitochondrial dysfunction, cells were grown for at least 1 (oligomycin-treated) or 7 (EtBr-treated) days after removal of either oligomycin or EtBr from media.
Measurement of mitochondrial membrane potential (ΔΨm) and cellular calcium concentration
Mitochondrial membrane potential was determined using MitoTracker, a mitochondrially selective probe. Oligomycin- or EtBr-treated and untreated C2C12 cells in serum-free medium were incubated with 100 nmol/l MitoTracker Green FM (Molecular Probes) for 30 min, and fluorescent signals were detected using a confocal microscope (Radiance 2000; Bio-Rad, Hercules, CA, USA) at 490/516 nm (excitation/emission).
Cellular steady-state Ca2+ levels were measured by confocal microscopy (Bio-Rad) at 494/516 nm (excitation/emission) using Fluo-4 AM (Molecular Probes) loaded into C2C12 cells to a final concentration of 5 μmol/l for 1 h.
Western blot analysis
The cell lysates were subjected to electrophoresis through 10% SDS–PAGE and blotted with antibodies for IRS1, solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) and β-actin purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and phospho-IRS1, JNK, phospho-JNK, p38 mitogen-activated protein kinase (p38 MAPK) and phospho-p38 MAPK purchased from Cell Signaling Technology (Beverly, MA, USA). The immunoblots were visualised by chemiluminescence using the ECL Western Blotting System (Amersham Biosciences, Freiburg, Germany).
Determination of IRS1 tyrosine phosphorylation by immunoprecipitation
Endogenous IRS1 (1 mg) was immunoprecipitated with 10 μg of anti-IRS1 antibody (Santa Cruz Biotechnology) from the lysates of control or oligomycin-treated (40 μmol/l) cells for 24 h. Immune complexes were immobilised by the addition of 30 μl of protein A–Sepharose (Amersham Biosciences) and then eluted by the addition of 40 μl sample buffer. The amounts of tyrosine-phosphorylated and total IRS1 were determined by Western blotting using specific phosphotyrosine (Cell Signaling Technology) and IRS1 (Santa Cruz Biotechnology) antibodies respectively.
Measurement of triglyceride accumulation
Cells were completely lysed at 4°C using ultrasonication (Sonics & Materials, Newtown, CT, USA). After centrifugation at 3,500 rpm for 5 min, the supernatant was used for measurement of triglyceride. The amount of triglyceride was quantitated colorimetrically as glycerol using an enzymatic assay kit (Asan Pharm, Seoul, South Korea).
Determination of SLC2A4-GFP translocation
Control pGFP vectors were generated by removing Slc2a4 cDNA sequences from pSLC2A4-GFP at the EcoRI and NotI sites, followed by DNA PolI (Klenow fragment)-mediated filling and blunt-end ligation. Each construct was transiently transfected into C2C12 myotubes using Lipofectamine 2000 Transfection Reagent (Invitrogen, Karlsruhe, Germany). Cells were treated with 40 μmol/l oligomycin for 24 h to induce mitochondrial dysfunction, starved for 20 h, and then fixed as single cells for examination under confocal microscopy. The time at which insulin (100 nmol/l) was applied to the cells was set to zero, and the movements of SLC2A4-GFP or green fluorescent protein (GFP) were chased over time.
Measurement of 2-deoxy-[3H]-d-glucose uptake
After induction of mitochondrial dysfunction, cells were starved for 24 h and washed twice with HEPES buffer (20 mmol/l HEPES [pH 7.4], 140 mmol/l NaCl, 2.5 mmol/l MgSO4, 5 mmol/l KCl, 1 mmol/l CaCl2). Cells were preincubated with HEPES buffer for 1 h and incubated for 10 min in the presence or absence of 100 nmol/l insulin, followed by treatment with 2-deoxy-[3H]-d-glucose (37,000 Bq/ml; Amersham Biosciences) for 10 min. The uptake was stopped by adding 10 μmol/l cytochalasin B. After washing with ice-cold 0.9% NaCl three times, total cells were lysed with 0.1 mol/l NaOH. Non-specific uptake was measured in the presence of 10 μmol/l cytochalasin B and was subtracted from all the values.
Using the internal pBlue-IRS1 restriction enzymes KpnI and XhoI, we generated the Irs1 promoter, pGL3-IRS1 L (2,535 bp), with the pGL3 basic vector (Promega, Madison, WI, USA). To generate the deletion construct, pGL3-IRS1 S, an internal region of 2,084 bp from the EcoRV and SmaI fragment was removed from the pGL3-IRS1 L. Cells were transfected using the Lipofectamine 2000 Transfection Reagent (Invitrogen), and luciferase activities were measured with the luciferase assay system (Promega) according to the manufacturer’s instructions. The pcDNA3.1-β-galactosidase (pcDNA3.1-LacZ) expression vector (Invitrogen) was used as an internal control to normalise firefly luciferase activity. In certain experiments, cells were treated for 30 min with BAPTA-AM (20 μmol/l) prior to oligomycin (40 μmol/l) treatment for 24 h.
All the data are expressed as means±SEM. Differences between the groups were determined by one-way analysis of variance (ANOVA) using the SAS statistical analysis program (SAS Institute, Cary, NC, USA). Results were considered significant if the value of p was less than 0.05. Duncan’s multiple range test was performed to evaluate any differences between the groups.
Mitochondrial dysfunction represses IRS1 and SLC2A4
Mitochondrial dysfunction increases IRS1 serine phosphorylation and attenuates insulin-stimulated tyrosine phosphorylation
Mitochondrial dysfunction impairs glucose utilisation in C2C12 myotubes
Next, the effects of mitochondrial dysfunction on glucose uptake were investigated. As Fig. 5b reveals, basal glucose uptake was significantly reduced, by about 60%, in oligomycin-treated C2C12 cells compared with control cells. Moreover, there was no insulin stimulated-glucose uptake in oligomycin-treated C2C12 cells, whereas insulin-stimulated glucose uptake increased 1.8-fold in control cells. The decreased glucose uptake in oligomycin-treated C2C12 cells may have resulted from both reduced SLC2A4 content and translocation potential together with impaired insulin signalling. Interestingly, when C2C12 cells were pretreated with BAPTA-AM, the basal glucose uptake was restored to 90% of normal, and insulin-stimulated glucose uptake also recovered. From these results, it was postulated that a Ca2+-dependent signalling pathway may be involved in the abnormal glucose utilisation and aberrant insulin signalling induced by mitochondrial dysfunction.
Elevated cytosolic Ca2+ and activation of JNK and p38 MAPK induced by mitochondrial dysfunction repress IRS1
ATF3 induced by mitochondrial dysfunction plays an important role in transcriptional repression of Irs1
Mitochondrial dysfunction contributes to several human diseases, such as obesity, hyperlipidaemia and type 2 diabetes. In this study, we correlated mitochondrial dysfunction and the development of insulin resistance, and we also characterised retrograde signalling using two different C2C12 cell models of mitochondrial dysfunction. The mitochondrial dysfunctions observed in C2C12 cells reveal marked decreases in IRS1 and SLC2A4 production (Fig. 2). Furthermore, mitochondrial dysfunction also increases the serine phosphorylation of IRS1 at critical sites (e.g. Ser307 or Ser636/639) and attenuates insulin signalling (Fig. 3). Therefore, basal and insulin-stimulated SLC2A4 translocation and glucose uptake decreased significantly in C2C12 cells with dysfunctional mitochondria, suggesting that mitochondrial dysfunction can develop into insulin resistance in C2C12 myotube cells (Fig. 5). In humans, diminished SLC2A4 and IRS1 protein levels can predict the development of type 2 diabetes . Moreover, low levels of IRS1 have been reported in 30% of subjects at high risk of type 2 diabetes, such as first-degree relatives of type 2 diabetics and obese subjects [29, 38], suggesting that dysregulation of the production of these proteins may be an early step towards the development of type 2 diabetes.
Here, we propose possible molecular mechanisms for how mitochondrial dysfunction causes reductions in IRS1 and insulin resistance, which influences numerous cellular and organismic activities under normal and pathophysiological conditions. In cells with functionally inactivated mitochondria, elevated steady-state cytosolic free Ca2+ and the subsequent activation of JNK and p38 MAPK were responsible for downregulating IRS1 and SLC2A4 (Figs. 6 and 7). The mitochondrion is a known major Ca2+ storage organelle, and important mechanisms for mitochondrial Ca2+ regulation are achieved primarily via the mitochondrial Ca2+ uniporters, whereby Ca2+ is taken up by means of ΔΨm. Therefore, we believe that disruption of ΔΨm by metabolic and genetic stress in C2C12 cells impairs Ca2+ uptake, thereby elevating cytosolic free Ca2+. Additionally, the upregulation of genes involved in Ca2+ transport and storage, such as ryanodine receptor I or II, by mitochondrial stress [19, 20] may also be involved in elevating cytosolic free Ca2+. Because it has been demonstrated that Ca2+ activates JNK and p38 MAPK [39–41], and we have shown that activation of JNK and p38 MAPK is dependent on Ca2+, the elevation of intracellular Ca2+ induced by mitochondrial dysfunction may trigger the subsequent activation of JNK and p38 MAPK. Actually, a similar pattern of retrograde signalling has been reported whereby respiratory deficiency and its associated increase in cytosolic free Ca2+ activates Ca2+-responsive calcineurin and CaMKIV, which in turn activate their respective transcription factors [19, 22].
In an effort to determine which transcription factors function in IRS1 downregulation downstream of Ca2+, JNK or p38 MAPK activation, we found that ATF3 represses IRS1 in a Ca2+- or JNK-dependent manner (Fig. 8). In fact, expression of Atf3 in the liver represses the expression of genes encoding gluconeogenic enzymes , and the expression of Atf3 in the pancreas leads to reduced numbers of hormone-producing cells . Although the exact binding sites for ATF3 on the Irs1 promoter remain unverified, as we have shown in Fig. 8f, the region within 191 bases from the transcription site may be responsible for repression of Irs1 by mitochondrial dysfunction. However, the biological significance of this and the precise mechanism of ATF3 regulation by Ca2+ and JNK remains to be elucidated.
Another possible explanation for how mitochondrial dysfunction induces insulin resistance in vivo comes from reports by Petersen et al. [12, 33]. They demonstrated that insulin resistance in the elderly is related to increases in intramyocellular fatty acid metabolites that may be a result of an age-associated reduction in mitochondrial oxidation and phosphorylation. Hence, dysregulation in intracellular fatty acid metabolism associated with mitochondrial dysfunction may play a critical role in the development of insulin resistance. Coincidentally, we also observed abnormal triglyceride accumulation in C2C12 cells with dysfunctional mitochondria (Fig. 4), which might also contribute to impairment of insulin signalling leading to insulin resistance, in agreement with a previous report .
Recently, Park et al.  showed that reductions in Irs1 expression and insulin-stimulated phosphorylation of IRS1 and Akt2/protein kinase B are associated with insulin resistance in L6 Glut4myc myocytes, where 95% of mtDNA was depleted by EtBr treatment. They showed no discernible changes in total SLC2A4, whereas we observed reduced SLC2A4 production in oligomycin- or EtBr-treated C2C12 cells. The different cells used in the experiments may explain this discrepancy. While we employed C2C12 cells, they used L6Glut4myc cells in which Slc2a4 was highly expressed using enhancers not dependent on intracellular signalling, and consequently total SLC2A4 may be unaffected by mitochondrial stress despite influencing endogenous SLC2A4 production. Furthermore, our investigation found that induction of insulin resistance occurs in dysfunctional mitochondrial C2C12 myotube cells without drastic genetic defects in mtDNA, as produced by Park et al. Therefore, subtle mtDNA defects leading to mitochondrial dysfunction can be sufficient to trigger the pathogenesis of insulin resistance and type 2 diabetes.
In conclusion, we demonstrate here that mitochondrial dysfunction causes inactivation of IRS1 and a drastic reduction in IRS1 and SLC2A4 production in a Ca2+-dependent manner, thereby inducing aberrant insulin signalling and glucose utilisation, as observed in type 2 diabetes. Additionally, we observed that JNK and p38 MAPK function as potential candidates for modulating the loss of IRS1 and SLC2A4 production, and that ATF3 is involved in repressing Irs1 gene expression. We characterised one mechanism underlying the molecular sensing of genetic and environmentally induced stress by mitochondria and the resulting inhibition of insulin signalling, ultimately leading to insulin resistance.
This work was supported by research grants from the Korean National Institutes of Health (347-6111-211-207). J. H. Lim and J. I. Lee contributed equally to this work. We would like to acknowledge J. E. Pessin (Medical Center at Stony Brook) for his generous gift of pSLC2A4-GFP, C. R. Kahn (Harvard Medical School) for pBlue-IRS1, T. Hai (Indiana University School of Medicine) for pCG-ATF3, and S. Sarsfield (Johns Hopkins University School of Medicine) for pcDNA-CREB and pcDNA-ACREB.