Age-Related Mitochondrial DNA Depletion and the Impact on Pancreatic Beta Cell Function
Age-Related Mitochondrial DNA Depletion and the Impact on Pancreatic Beta Cell Function
Donna L. Nile 0 5 6 7
Audrey E. Brown 0 5 6 7
Meutia A. Kumaheri 0 5 6 7
Helen R. Blair 0 5 6 7
Alison Heggie 0 5 6 7
Satomi Miwa 1 5 6 7
Lynsey M. Cree 2 5 6 7
Brendan Payne 3 5 6 7
Patrick F. Chinnery 3 5 6 7
Louise Brown 4 5 6 7
David A. Gunn 4 5 6 7
Mark Walker * 0 5 6 7
0 Institute of Cellular Medicine, Newcastle University , Newcastle upon Tyne, NE2 4HH , United Kingdom,
1 Newcastle University Institute for Ageing, Newcastle University , Newcastle upon Tyne, NE4 5PL , United Kingdom,
2 Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland , Auckland 1142 , New Zealand,
3 Wellcome Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University , Newcastle upon Tyne, NE1 3BZ , United Kingdom,
4 Unilever R&D , Colworth Science Park, Sharnbrook, Bedford, MK44 1LQ , United Kingdom
5 Funding: This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) Industrial CASE Studentship (grant reference BB/G529991/1) and as such, financial support was provided by Unilever. The authors acknowledge support of the Newcastle National Institute for Health Research Biomedical Research Centre (NIHR BRC). Co-authors Dr. David Gunn and Dr. Louise Brown are employed by Unilever R&D. Unilever R&D provided support in the form of salaries for authors Dr. David Gunn and Dr. Louise Brown, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section
6 Editor: Feng Ling, RIKEN Advanced Science Institute , Japan
7 Current address: Radiation Oncology, Wolfson-Wohl Cancer Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary & Life Sciences, University of Glasgow , Garscube Estate, Switchback Road, Bearsden, G61 1QH , United Kingdom
Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA
depletion in human islets is not simply a biomarker of the aging process, but will
contribute to the age-related risk of type 2 diabetes.
The prevalence of type 2 diabetes has been found to increase with advancing age
. This is thought to be due in part to the age-related progressive decline in
pancreatic beta cell function , resulting in an age-related decrease in insulin
secretion  and abnormal glucose tolerance . But it is still unknown what
mechanisms contribute to this age-related decline in pancreatic beta cell function.
Mitochondrial DNA (mtDNA) is a circular double stranded DNA molecule of
16.6 kb in length in humans  and encodes 13 polypeptides essential for
mitochondrial oxidative phosphorylation [8, 9]. Insulin secretion is heavily
dependent upon the ATP produced following glucose metabolism and
mitochondrial oxidative phosphorylation, and occurs following ATP-gated K+
channel closure, subsequent membrane depolarisation and Ca2+-stimulated
insulin exocytosis [8, 10]. Inherited mutations in the mitochondrial genome have
been estimated to account for approximately 1% of all diabetes cases . It is
known that certain mtDNA abnormalities are strongly associated with diabetes,
particularly the A3243G mutation, which has been shown to result in impaired
insulin secretion [12, 13].
An age-related decline in mtDNA copy number has been reported in numerous
human tissues . In human pancreatic islets, it has been shown that
mtDNA copy number was significantly reduced in non-diabetic islet donors aged
$50 years compared to donors aged #50 years; and that the mean mtDNA copy
number decreased by 50% in individuals aged between 17 and 75 years . The
study assessed mtDNA copy number in hand-picked whole islets, obtained from
15 non-diabetic pancreas donors; there was a significant negative correlation
between mtDNA copy number and advancing age. Although the 50% reduction in
mtDNA copy number reported by Cree et al. was from whole islets and was not
exclusively beta cells, it has been well documented in vitro that severe (.90%)
mtDNA depletion in insulin secreting beta cell lines results in decreased insulin
secretion . Mutations of the thymidine kinase 2 (TK2) gene result in
mtDNA depletion in skeletal muscle, but cytochrome c oxidase (COX) deficiency
as a marker of mitochondrial dysfunction was only seen in the presence of severe
($95%) mtDNA depletion . So the question arises as to whether the 50%
mtDNA depletion observed in aged human islets is sufficient to impair
mitochondrial function and insulin secretion, or whether it is functionally well
tolerated as seen in skeletal muscle and is simply a biomarker of the aging process.
To address this question, we developed a model of partial mtDNA depletion to
replicate that seen in aged human islets using the approach of targeted knock
down of TFAM gene expression in MIN6 cells. TFAM is an important nuclear
encoded mtDNA transcription factor found to play a key role in mtDNA
transcription [23, 24], as well as mtDNA copy number  and maintenance .
Using our model we were able to replicate a similar degree of mtDNA copy
number depletion that had been observed in the human islets with aging. Under
these conditions, we found that partial mtDNA depletion significantly impaired
mitochondrial gene transcription and translation, as well as mitochondrial
oxidative respiration and ultimately, glucose-stimulated insulin secretion. The
age-related decline in mtDNA copy number observed in human islets could well
contribute directly to the increased prevalence of type 2 diabetes with increasing
Materials and Methods
MIN6 cells, a mouse pancreatic beta cell line established by Miyazaki et al. ,
were donated by Dr Susan Campbell and Dr Catherine Arden (Diabetes Research
Group, Newcastle University, UK). All experiments were conducted between
passages 23 and 31. Unless otherwise stated, cell culture reagents were
manufactured by Gibco, and supplied by Life Technologies (Paisley, UK). MIN6
cells were cultured in DMEM supplemented with 25 mM D-glucose, L-glutamine,
15% filter sterilised FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and
0.0005% b-mercaptoethanol (Sigma, Dorset, UK). Cells were incubated at 37C
5% CO2 in a humidified incubator and were passaged every 57 days.
TFAM was transcriptionally silenced in MIN6 cells using the Neon
electroporation transfection system (Life Technologies, Paisely, UK). When cells reached
,80% confluency, they were trypsinised and washed with PBS prior to cell
counting using trypan blue exclusion. Cells were resuspended in Resuspension
Buffer R, from the 10 ml Neon transfection kit (Life Technologies, Paisley, UK).
Cells were transfected in solution at a density of 200,000 cells per well in 24 well
plates using MIN6 growth medium without additional antibiotics. Two TFAM
siRNA probes were used at a concentration of 20 mM per well: TFAM-193
(59CCUCGUCUAUCAGUCUUGUCUGUAU -39; 39- AUACAGACAA
GACUGAUAGACGAGG -59) and TFAM-429
(59UACAAAGAAGCUGUGAGCAAGU AUA -39;
39UAUACUUGCUCACAGCUUCUUUGUA -59), both were Stealth duplex siRNA
synthesised by Life Technologies (Paisley, UK). A Scrambled medium GC content
siRNA probe (Life Technologies, Paisley, UK) was used as a transfection control;
cells that were electroporated (shocked) in the absence of any siRNA were used to
control for siRNA toxicity. Transfected cells were incubated at 37C 5% CO2 in a
humidified incubator and were harvested 48 h and 72 h post transfection.
Transfection efficiency was based on the degree of target TFAM gene knock down;
transfections were only accepted for analysis if TFAM knock down was $80%.
Total DNA and RNA were extracted simultaneously using the AllPrep DNA/RNA
mini extraction kit (Qiagen, Crawley, UK), or separately using the GenElute
Mammalian Total RNA miniprep kit (Sigma, Dorset, UK) or the DNeasy Blood &
Tissue DNA extraction kit (Qiagen, Crawley, UK). Data presented in this
manuscript utilise DNA extracted using both the DNeasy and AllPrep kits, but
despite a slightly reduced DNA yield using the AllPrep kit, the quality of extracted
DNA was the same for both kits. RNA was quantified using the Agilent 2100
BioAnalyzer (Agilent Technologies UK Limited) and 150 ng reverse transcribed
using the First Strand cDNA Synthesis Kit (Life Technologies, Paisley, UK).
Messenger RNA for the TFAM, COX1 and Ins1 genes were detected using TaqMan
hydrolysis probes obtained from Applied Biosystems (Life Technologies, Paisley,
UK), and normalised to the reference gene b2-microglobulin (B2M). DNA was
used to determine mtDNA copy number, as described below. Real-time PCR was
conducted using the Roche LightCycler 480 thermo cycler (Roche Diagnostics
Ltd) and PCR products were quantified fluorometrically using the LightCycler 480
Master I (Roche, Welwyn Garden City, UK) kit and TaqMan probes for RNA or
the LightCycler 480 SYBR Green I Master (Roche, Welwyn Garden City, UK) kit
for DNA. Quantification of gene expression was performed using the Delta Ct
(DCt) method .
mtDNA Copy Number Assay
mtDNA levels were measured by relative real-time PCR, calculating the ratio of
the mtDNA encoded target gene ND5  to the nuclear DNA encoded reference
gene GAPDH. DNA was extracted as described above, quantified using the
NanoDrop ND-1000 Spectrophotometer (Labtech International Ltd) and 50 ng
was amplified per PCR reaction using 300 nM ND5 primers (forward:
59CTGGCAGACGAACAAGAC -39; reverse: 59- GAGGCTTCCGATTACTAGG -39)
or 500 nM GAPDH primers (forward: 59- CAATGTGTCCGTCGTGGATCT -39;
reverse: 59- GTCCTCAGTGTAGCCCAAGAT -39). Each reaction was optimized
and confirmed linear over an appropriate concentration range (S1 Fig.). Gene
quantification was performed by DCt  (S1 Table). The relative gene expression
ratio obtained was then multiplied by two on account of GAPDH being diploid.
Measurement of Mitochondrial Respiration
Mitochondrial respiration was measured in MIN6 cells 72 h after transfection
using the Seahorse XF24 Analyzer (Seahorse Biosciences). Cells were transfected at
a density of 200,000 per well as described above and seeded in a 24 well plate.
Media was replaced with basic media containing FBS (3%), pyruvate (10 mM),
Lglutamine (2 mM) and glucose (25 mM) and cells incubated in a CO2-free
environment for 1 h prior to the experiment. Oxygen consumption rates (OCR)
were measured in live cells in the absence and then presence of various
compounds inhibiting specific mitochondrial complexes in order to assess
mitochondrial activity. Oligomycin (1 mg/ml) was injected to inhibit Complex V
(ATP Synthase), followed by sequential addition of carbonyl cyanide
ptrifluoromethoxy-phenylhydrazone (FCCP) (2 mM and 3.5 mM) to uncouple
respiration and promote maximal respiration, and finally antimycin A (2.5 mM)
was injected to inhibit Complex III (Ubiquinol-Cytochrome c Reductase). Basal
respiration was measured as the area under the curve prior to injection of
oligomycin; maximal respiration was measured as the area under the curve
following the first FCCP injection and prior to the antimycin injection. ATP
synthesis by oxidative phosphorylation was calculated by multiplying the ATP
turnover by 2.3 as described previously ; where ATP turnover was (basal
OCR non mitochondrial respiration) (oligomycin-inhibited OCR non
mitochondrial respiration), and 2.3 was the established phosphate/oxygen ratio.
Data were normalised to total protein content.
Glucose-Stimulated Insulin Secretion (GSIS)
Method adapted from Ishihara et al. . Briefly, cells were washed twice with
Krebs-Hepes buffer (119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM
MgCl2, 1.19 mM KH2PO4, 25 mM NaHCO3, 10 mM HEPES, 0.5% BSA, pH 7.4)
before pre-incubating with Krebs-Hepes buffer at 37C for 30 min. Cells were
then washed again before stimulating for 1 h at 37C with either basal 3 mM or
high 25 mM glucose, with or without 0.1 mM glibenclamide (Sigma, Dorset, UK)
in Krebs-Hepes buffer. Cell medium was harvested and insulin secretion
determined using the high range rat insulin ELISA kit (Mercodia AB, Upsala,
Sweden). Insulin concentration of unknown samples was calculated using a
standard curve of known insulin concentrations and was normalised to protein
After GSIS, cells were harvested in 100 ml distilled water and were sonicated for
10 sec. Insulin was liberated from cells by acid-ethanol extraction; 50 ml sample
volume was added to 100 ml of 0.18 M HCl in 96% ethanol before incubating
overnight at 4C. Samples were then briefly vortexed and cell debris was removed
following centrifugation at 1000 rpm for 5 min at 4C. Insulin content was
determined by insulin ELISA, after diluting samples at least 1:100, and was
normalised to whole cell protein content.
Whole cell protein lysates were harvested from MIN6 cells in ice cold protein
extraction buffer (100 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EDTA, 25 mM
Kf, 0.1% Triton X-100, 0.5 mM sodium orthovanadate, 1X protease inhibitor
cocktail). Extracted proteins were quantified using the Coomassie Plus (Bradford)
Protein Assay (Pierce, Thermo Fisher Scientific, Cramlington, UK) and 25 mg
protein was resolved on a 12% SDS-polyacrylamide gel after denaturing at 37C
for 10 min. Resolved proteins were then transferred electrophoretically onto a
nitrocellulose membrane before blocking with 5% Marvel solution (5% Marvel
milk powder in TBS-T wash buffer; 65 mM Tris pH 7.4, 150 mM NaCl, 0.1%
Tween-20) for 1 h at room temperature with agitation. Membranes were then
incubated at 4C overnight in the presence of either a mouse COX1 antibody
diluted 1:10,000 (MitoSciences, Abcam, Cambridge, UK; Catalogue No. MS404);
a mouse SDH70 antibody diluted 1:10,000 (MitoSciences, Abcam, Cambridge,
UK; Catalogue No. MS204); or a mouse b-Actin antibody diluted 1:10,000
(Sigma, Dorset, UK; Catalogue No. A5441). A goat anti-mouse IgG antibody
conjugated to horse radish peroxidase (1:2000) (Sigma, Dorset, UK; Catalogue
No. A3673) was then added and the membrane incubated for 1 h at room
temperature. Bound antibodies were detected by addition of an enhanced
chemiluminescent solution (SuperSignal West Pico, Thermo Fisher Scientific,
Cramlington, UK) and immunoreactive products were visualised following
exposure to blue X-ray film (Thermo Fisher Scientific, Cramlington, UK) for less
than 1 min. Protein bands were quantified using the GS-800 Calibrated
Densitometer (BioRad Laboratories, Bath, UK) and the Quantity One 4.2.3
BioRad software. b-Actin was used as a loading control.
All statistical analyses were performed using GraphPad Prism version 5.01
(GraphPad Software, San Diego, California, USA). Data presented as means
standard error of the mean (SEM), unless otherwise stated, with the number of
experimental repeats provided in the figure legend. Significance was tested with
one-way ANOVA followed by an unpaired t test. A probability (p) value ,0.05
was considered statistically significant and ,0.01 highly significant.
TFAM transcriptional silencing causes partial mtDNA depletion
In order to partially deplete mtDNA levels, we used siRNA technology to
transcriptionally silence the TFAM gene. TFAM was chosen as a target gene
through its known roles in regulating mtDNA copy number , mtDNA
transcription [23, 24], as well as mtDNA stability . MIN6 cells were
transfected using electroporation and two TFAM siRNA duplexes targeting
nucleotides 193 and 429 of the TFAM mRNA molecule. Transfected cells were
Fig. 1. TFAM mRNA silencing induces mtDNA depletion 72 h post transfection. MIN6 cells were transfected with TFAM-193, TFAM-429 or Scrambled
siRNA probes, or with no siRNA (Shocked). TFAM mRNA expression was quantified relative to reference gene B2M by real-time PCR at 48 h (A) and 72 h
(B) post transfection. mtDNA depletion was also measured by real-time PCR, using mitochondrial encoded ND5 relative to nuclear encoded GAPDH at 48 h
(C) and 72 h (D) post transfection. All results normalised to Scrambled negative control. Experiment repeated once (C), twice (A) or 4 times (B, D) in
triplicate. Data presented are means SEM (SD in (C)). * p,0.05, *** p,0.001.B2M, b2 Microglobulin; GAPDH, Glyceraldehyde-3-Phosphate
Dehydrogenase; ND5, NADH Dehydrogenase 5; TFAM, Mitochondrial Transcription Factor A.
harvested at 48 and 72 h post transfection. After 48 h (Fig. 1A) and 72 h
(Fig. 1B), TFAM mRNA levels were markedly decreased by.80% with both the
TFAM-193 and TFAM-429 probes when compared with the Scrambled negative
control (p50.0001). DNA was extracted and used to calculate mtDNA levels by
quantifying mtDNA encoded target gene ND5 relative to nuclear encoded
reference gene GAPDH. Interestingly, after 48 h post transfection mtDNA levels
seem unaffected by the TFAM transcriptional silencing (Fig. 1C). However, after
72 h post transfection, there was around a 40% reduction in mtDNA levels with
both the TFAM-193 and TFAM-429 probes when compared with the Scrambled
negative control (p,0.001) (Fig. 1D). We validated this mtDNA depletion using a
second nuclear encoded reference gene, CDKN2A (cyclin-dependant kinase
inhibitor 2A) (S2 Fig.), and also found that TFAM transcriptional silencing with
both probes resulted in the same 40% reduction in mtDNA levels 72 h post
transfection. Therefore, using both the TFAM-193 and TFAM-429 siRNA probes,
we successfully silenced TFAM mRNA by.80% 72 h post transfection, and this
resulted in a 40% decrease in mtDNA levels.
Partial mtDNA depletion affects mitochondrial gene expression
To investigate whether decreased mtDNA levels following TFAM gene silencing
affected mitochondrial gene expression, we looked at transcription and translation
of the mtDNA encoded gene COX1, cytochrome c oxidase 1. COX1 is one of the
three mtDNA encoded subunits that comprise the catalytic holoenzyme of
Complex IV, Cytochrome c Oxidase (COX), of the respiratory chain [26, 34]. As
before, MIN6 cells were transfected and incubated for 72 h after which, either
whole cell RNA or protein was harvested. COX1 mRNA expression relative to
reference gene B2M was determined by real-time PCR. As shown in Fig. 2A,
COX1 mRNA levels were significantly decreased by 24% (p,0.01) and 33%
(p,0.001) in cells transfected with the TFAM-193 and TFAM-429 probes
respectively, compared with the Scrambled control. Because the TFAM-429 siRNA
probe produced the greatest decrease in COX1 mRNA levels, we used this probe
to examine the effect of mtDNA depletion on COX1 protein levels. We found that
the decrease in COX1 mRNA led to a 25% decrease in COX1 protein expression
(p50.034) (Fig. 2B and 2D). We also investigated the nuclear encoded protein
SDH70, a 70 kDa component of Complex II, succinate dehydrogenase, of the
respiratory chain. Unlike the other respiratory complexes, succinate
dehydrogenase is entirely nuclear encoded, and should not be affected by mtDNA
depletion. This was confirmed as SDH70 protein levels were unaffected by TFAM
silencing-induced mtDNA depletion (Fig. 2C and 2D).
Partial mtDNA depletion impairs mitochondrial function
The Seahorse XF24 Analyzer was used to measure mitochondrial respiratory
capacity in transfected cells (Fig. 3A). Both basal and maximal respiration were
significantly impaired in siRNA TFAM transfected cells compared to scrambled
control (p50.0005 and p50.005, respectively) (Fig. 3B). Basal respiratory
capacity is represented by the area under the curve prior to oligomycin injection,
whereas maximal respiratory capacity is defined as the area under the curve
between the first FCCP injection and the antimycin injection (Fig. 3A) and is
presented graphically in Fig. 3B. Consistent with a reduced basal respiration,
Fig. 2. The effect of partial mtDNA depletion on mitochondrial gene transcription and protein translation. Mitochondrial DNA was depleted following
TFAM silencing and COX1 mRNA expression was quantified relative to reference gene B2M 72 h post transfection (A). Protein was extracted 72 h post
transfection and analysed by western blotting, probing for COX1, SDH70, and b-Actin proteins. Protein bands were quantified by densitometry, and optical
density readings used to calculate the ratio of COX1 (B) and SDH70 (C) mitochondrial proteins relative to b-Actin loading control. A representative blot is
shown in (D). Data in (A) are normalised to Scrambled control cells. Both experiments repeated 3 times, with each experimental repeat performed in
triplicate (A) or duplicate (B, C). Data presented are means SEM. * p,0.05, ** p,0.01, *** p,0.001. COX1, Cytochrome c Oxidase 1; SDH70, Succinate
Dehydrogenase 70 kDa subunit.
siRNA TFAM transfected cells also showed significantly reduced ATP synthesis
due to oxidative phosphorylation (p,0.05) (Fig. 3C).
Having demonstrated that partial mtDNA depletion following TFAM gene
silencing directly affects mitochondrial gene expression and mitochondrial
function, we were keen to establish whether this in turn affects glucose-stimulated
Fig. 3. The effect of partial mtDNA depletion on mitochondrial function. Cells were harvested 72 h post transfection and oxygen consumption rate
(OCR) was measured using the Seahorse XF24 Analyzer. OCR in TFAM-429 cells (n58) was severely impaired compared to that of Scrambled (n58) and
Shocked (n58) control cells (A). Mitochondrial activity was measured following injection of oligomycin, an inhibitor of Complex V (ATP Synthase), followed
by two sequential injections of FCCP to uncouple respiration and induce maximal respiration, and finally antimycin, an inhibitor of Complex III
(UbiquinolCytochrome c Reductase) preventing electron transfer and subsequently abolishing the proton gradient required for ATP synthesis. Basal and maximal
respiratory capacity (B) and ATP synthesis by oxidative phosphorylation (OXPHOS) (C) were calculated as described previously . Data were
normalised to protein concentration and are presented means SEM. * p,0.5, ** p,0.001, *** p,0.0001.
insulin secretion. Seventy-two hours post transfection, MIN6 cells were stimulated
with either basal 3 mM glucose or high 25 mM glucose for a period of 1 h. As
shown in Fig. 4A, both TFAM-429 transfected cells and Scrambled control cells
both responded to the higher 25 mM glucose stimulation by secreting
significantly more insulin, when compared with basal 3 mM stimulation
(p50.0004 and p,0.0001 respectively). What is interesting however, is that
insulin secretion was significantly impaired in TFAM silencing-induced mtDNA
depleted cells following 25 mM glucose stimulation (p50.003). The impaired
insulin secretion observed in mtDNA depleted cells was not a consequence of
Fig. 4. The effect of partial mtDNA depletion on glucose-stimulated insulin secretion. Seventy two hours post transfection cells were stimulated with
basal (3 mM) or high (25 mM) glucose concentrations. Insulin secretion (A) and insulin content (B) were determined by insulin ELISA and normalised to
protein content. Data normalised to 3 mM glucose stimulated Scrambled control cells. mtDNA levels (C) and Ins1 insulin gene expression (D) were
quantified and normalised to the Scrambled control. Data shown are from 9 (A) or 3 (B, C, D) separate experiments, each performed in triplicate. Data
presented are means SEM. ** p#0.01, *** p,0.001.
altered insulin content following TFAM knock down (Fig. 4B). We confirmed
that partial mtDNA depletion was achieved as before under these conditions
(Fig. 4C). Fig. 4D shows that mtDNA depletion did not alter Ins1 insulin gene
Glibenclamide restores the impaired insulin secretion seen
following mtDNA depletion
Glibenclamide is a second generation sulphonylurea. By targeting the
sulphonylurea 1 (SUR1) protein on the beta cell K+ channel, it promotes channel closure
and subsequent pancreatic beta cell membrane depolarisation and insulin
secretion . Consequently, glibenclamide bypasses the step of ATP generation
by the mitochondria and is therefore a useful drug to help determine whether or
not a deficit in insulin secretion is mitochondrial in origin. To see whether
glibenclamide treatment had any effect on insulin secretion in mtDNA depleted
cells, transfected cells were stimulated with either 3 mM or 25 mM glucose for
1 h, with or without 0.1 mM glibenclamide. The siRNA data corroborate what we
have seen previously, that insulin secretion (in the absence of glibenclamide) is
impaired in TFAM-429 cells after 25 mM glucose stimulation compared with
Scrambled control cells (p,0.05) (Fig. 5). At 3 mM glucose, 0.1 mM
glibenclamide increased insulin secretion to a comparable degree in TFAM-429 cells and
Scrambled control cells (Fig. 5) compared to no glibenclamide (both p,0.01). At
25 mM glucose, glibenclamide fully restored insulin secretion to normal in the
TFAM-429 cells compared with Scrambled control cells, indicating that the
impact of mtDNA depletion is focused at the step of mitochondrial respiration.
The primary objective of our work was to generate a model of partial mtDNA
depletion by genetically silencing mitochondrial transcription factor A, TFAM.
Indeed, tissue specific [19, 36, 37] and global knockdown  of the TFAM gene
in mouse animal models has proven an effective means of depleting mtDNA
levels. After transcriptionally silencing the TFAM gene in MIN6 cells by.80%, we
achieved a 40% reduction in mtDNA levels, which was comparable to the degree
of mtDNA depletion seen in aged human islets .
COX1, cytochrome c oxidase subunit 1, is one of the three mtDNA encoded
subunits of Cytochrome c Oxidase (COX, Complex IV), and is essential during
the assembly of the Cytochrome c Oxidase complex [26, 34]. Partial mtDNA
depletion significantly decreased COX1 mRNA and protein expression by 33%
and 25%, respectively. Our finding that mtDNA depletion results in decreased
mtDNA transcription and protein translation has also been shown in previous
studies in rodent clonal beta cells [20, 21, 39] and rodent pancreatic islets . We
have also demonstrated that partial mtDNA depletion had a direct impact on
mitochondrial function. We found a significant decrease in basal and maximal
respiratory capacity, as well as a significant reduction in ATP production by
oxidative phosphorylation in TFAM-silenced cells compared to control cells.
However, these results were founded on the assumption that the phosphate/
oxygen ratio remained constant, which may not have been the case under
conditions of mtDNA depletion .
Previous studies have observed decreased insulin secretion after severe (around
90%) depletion of mtDNA depleted pancreatic beta cells [1821, 39]. However,
this magnitude of severe mtDNA depletion does not accurately reflect the degree
of age-related mtDNA depletion observed in human islets. In human pancreatic
islets, it was noted that there was an average 50% decrease in mtDNA copy
number in non-diabetic islet donors aged between 17 and 75 years . In this
study mtDNA levels were depleted relative to the control by an average of 40%,
close to the degree of depletion observed in human islets. This level of mtDNA
depletion resulted in a significant decrease in insulin secretion following 25 mM
Fig. 5. The effect of glibenclamide on insulin secretion following TFAM silencing-induced mtDNA depletion. Seventy two hours post transfection
cells were stimulated with 3 mM or 25 mM glucose, supplemented with or without 0.1 mM glibenclamide. Insulin secretion was determined by insulin ELISA
and normalised to whole cell protein content. Data shown are from 4 separated experiments performed in triplicate, and are normalised to Scrambled
negative control cells stimulated with 3 mM glucose without glibenclamide. Data presented are means SEM. * p,0.05, ** p,0.01.
high glucose stimulation. This supports the idea that the age-related depletion in
mtDNA has a direct impact on beta cell function and is not simply a biomarker of
beta cell aging. Ihm et al. correlated the decline in glucose-stimulated insulin
secretion and decreased ATP content to islet donor age . Gauthier et al.
observed partial mtDNA depletion to similar levels produced by ourselves
following transcriptional silencing of the beta cell regulatory transcription factor
Pdx1 . By silencing Pdx1 gene expression by.90%, the group found this
depleted mtDNA levels by 40% via TFAM suppression, which resulted in
impaired insulin secretion at high glucose stimulation only . This model
differs from our targeted knockdown of TFAM in that Pdx1 is a key transcription
factor that regulates the expression of multiple genes involved in pancreatic
function beyond the mitochondria .
Gene variants in other mitochondrial transcription factors, namely TFB1M,
have been found to correlate with reduced insulin secretion, elevated postprandial
glucose levels and increased risk of developing type 2 diabetes . TFB1M
actually functions as a methyltransferase as opposed to a transcription factor
[45, 46], but this work serves to show that mutations in nuclear encoded
mitochondrial genes can be diabetogenic.
Taking these data together it would suggest that partial mtDNA depletion to
levels observed in aging has a direct and detrimental effect on pancreatic beta cell
function. This seems to be different to the findings in other tissues. Specifically,
COX deficiency as a marker of mitochondrial dysfunction was only seen when the
degree of mtDNA depletion was $95% in human skeletal muscle . This
implies that the degree of mtDNA depletion required to impair mitochondrial
dysfunction differs between tissues. The reason for this is not clear. It may be that
the absolute amounts of mtDNA differ between cells types, so that those with
comparatively lower levels are more susceptible to the effects of depletion. It may
be that mechanisms to maintain mitochondrial function and ATP production in
the face of mtDNA depletion differ between cells types. Whatever the reason, it
appears that pancreatic islet cells are particularly sensitive to mtDNA depletion
and altered mitochondrial function. This is in keeping with our earlier
observation that comparatively low levels of mutated to wild type mtDNA were
found in pancreatic islet cells in a patient with diabetes secondary to the A3243G
mtDNA mutation . Interestingly, a mouse model of marked peripheral insulin
resistance was characterised by an age-related development of diabetes that was
linked to a decrease in both pancreatic beta cell mtDNA content and
mitochondrial function, and decreased glucose-stimulated insulin secretion .
What might be causing the reduced insulin secretion seen in TFAM silenced
mtDNA depleted cells? We found no change in the Ins1 insulin gene expression in
TFAM silenced mtDNA depleted cells, so the defect does not appear to alter
insulin gene transcription. This is supported by our finding that total insulin
content remained relatively unchanged in TFAM silenced cells compared to
control cells, after 3 mM and 25 mM glucose stimulation. Nonetheless insulin
secretion, when expressed as a percentage of total insulin content, was still
reduced in TFAM-transfected cells (S2 Table).
Glibenclamide is currently an effective treatment for type 2 diabetes patients
with impaired insulin secretion , although its clinical application has been
generally superseded by sulphonylureas with shorter half-lives. By targeting the
sulphonylurea receptor (SUR1) protein of the ATP-gated K+ channel situated on
the beta cell plasma membrane, it promotes membrane depolarisation, and
subsequent insulin secretion following Ca2+-stimulated exocytosis  and
therefore, acts downstream to the step of mitochondrial respiration and ATP
production. We found that insulin secretion is fully restored to normal in the
mtDNA depleted cells following addition of glibenclamide, so confirming that the
key impact of mtDNA depletion is at the level of mitochondrial respiration.
A potential limitation of our work could be that the effects of TFAM gene
silencing on mtDNA are transient and so, future studies into the effect of chronic
partial mtDNA depletion might more accurately reflect mtDNA depletion in a
clinical setting. Silva et al. produced pancreatic beta cell specific TFAM knock
down and severe mtDNA depletion in mice . The mice initially developed
diabetes due to impaired glucose-stimulated insulin secretion just as we saw in
our model, but as the animals aged there was a concurrent decrease in beta cell
mass that sustained the diabetes phenotype. We found no evidence of decreased
cell mass in our cell line model, based upon comparable DNA, RNA and protein
levels between TFAM knock down cells and the Scrambled control cells. This does
not accurately reflect what is seen in vivo or clinically in type 2 diabetes patients
and so, our studies would need to be repeated using mouse models to further
elucidate the effects of partial mtDNA depletion on diabetes pathogenesis. Finally,
we have demonstrated that the partial mtDNA depletion produced in our MIN6
cell line model contributes to reduced mitochondrial function in terms of oxygen
consumption; however, further characterisation of mitochondrial dysfunction
following evaluation of COX activity would be beneficial for future studies.
In conclusion, we found that mtDNA depletion in MIN6 cells to levels seen in
human islets with aging has a direct effect on insulin secretion when depleting
mtDNA via TFAM gene silencing. This effect on insulin secretion may be due to a
defective electron transport chain following a decrease in the mtDNA encoded
components, resulting in impaired mitochondrial respiration and ATP
production. We found that the impaired insulin secretion was restored following
treatment with the insulin secretagogue glibenclamide, suggesting that the deficit
in insulin secretion occurs upstream of the K+ channel closure and is
mitochondrial in origin. Strategies to slow or even prevent islet mtDNA depletion
in man could help to preserve insulin secretion and delay the development of type
S1 Fig. Optimisation of mtDNA Copy Number Assay. Real-time PCR reactions
for GAPDH and ND5 primers were first optimised using a linear standard curve:
50 ng DNA was serially diluted 1:5, before amplification with GAPDH, ND5 or
CDKN2A primers. Standard curves were confirmed linear over an appropriate
concentration range. Reaction efficiencies were 92.61% (GAPDH), 98.87% (ND5)
and 95.07% (CDKN2A). Reaction specificity was tested by dissociation curve as
well as agarose gel electrophoresis of PCR products.
S2 Fig. Validation of mtDNA assay using a second nuclear encoded gene. The
mtDNA copy number assay was validated by quantifying ND5 relative to a second
nuclear encoded reference gene, CDKN2A, cyclin-dependent kinase inhibitor 2A
(QuantiTect Assay ID Mm_Cdkn2a_va.1_SG; Qiagen, Crawley, UK). Cells were
harvested 72 hrs post transfection and mtDNA depletion determined by relative
real-time PCR and normalisation to Scrambled control cells. Experiment repeated
twice in triplicate, error bars represent SEM. * p,0.05.
S1 Table. Mitochondrial encoded ND5 expression relative to nuclear encoded
GAPDH gene content as determined by differences in Ct values. mtDNA copy
number was determined as the ratio of target mtDNA gene ND5 relative to
reference nDNA gene GAPDH using the Delta-Ct (DCt) method . The cycle
threshold, or Ct, was determined by real-time PCR and DCt was calculated as the
difference in Ct values between the target gene and the reference gene. Change in
gene expression was calculated by 2(22DCt).
S2 Table. Glucose-stimulated insulin secretion normalised to total insulin
content. The table above represents the data values used to construct Fig. 4A and
4B. Percentage insulin secretion was calculated by normalising insulin secreted by
total insulin content.
Conceived and designed the experiments: DLN MW AEB DAG LB. Performed the
experiments: DLN AEB MAK HRB AH SM. Analyzed the data: DLN MW AEB
MAK. Contributed reagents/materials/analysis tools: LMC BP PFC SM AH. Wrote
the paper: DLN MW AEB DAG.
1. Cowie CC , Rust KF , Byrd-Holt DD , Eberhardt MS , Flegal KM , et al. ( 2006 ) Prevalence of diabetes and impaired fasting glucose in adults in the U .S. population: National Health And Nutrition Examination Survey 1999 - 2002 . Diabetes Care 29 : 1263 - 1268 .
2. Cowie CC , Rust KF , Ford ES , Eberhardt MS , Byrd-Holt DD , et al. ( 2009 ) Full accounting of diabetes and pre-diabetes in the U.S. population in 1988-1994 and 2005-2006 . Diabetes Care 32 : 287 - 294 .
3. Harris MI , Hadden WC , Knowler WC , Bennett PH ( 1987 ) Prevalence of diabetes and impaired glucose tolerance and plasma glucose levels in U.S. population aged 20-74 yr. Diabetes 36 : 523 - 534 .
4. Rudenski AS , Hadden DR , Atkinson AB , Kennedy L , Matthews DR , et al. ( 1988 ) Natural history of pancreatic islet b-cell function in type 2 diabetes mellitus studied over six years by homeostasis model assessment . Diabet Med 5 : 36 - 41 .
5. Chang AM , Halter JB ( 2003 ) Aging and insulin secretion . Am J Physiol Endocrinol Metab 284 : E7 - E12 .
6. Basu R , Breda E , Oberg AL , Powell CC , Dalla Man C , et al. ( 2003 ) Mechanisms of the AgeAssociated Deterioration in Glucose Tolerance . Contribution of Alterations in Insulin Secretion, Action, and Clearance . Diabetes. 52 , 1738 - 1748 .
7. Attardi G , Schatz G ( 1988 ) Biogenesis of mitochondria . Annu Rev Cell Biol 4 : 289 - 333 .
8. Maechler P , Wollheim C ( 2001 ) Mitochondrial function in normal and diabetic b-cells . Nature 414 : 807 - 812 .
9. Taylor RW , Turnbull DM ( 2005 ) Mitochondrial DNA mutations in human disease . Nat Rev 6 : 389 - 402 .
10. Wollheim CB ( 2000 ) Beta-cell mitochondria in the regulation of insulin secretion: a new culprit in Type II diabetes . Diabetologia 43 : 265 - 277 .
11. Maassen JA , van Essen E , van den Ouweland JM , Lemkes HH ( 2001 ) Molecular and clinical aspects of mitochondrial diabetes mellitus . Exp. Clin. Endocrinol. Diabetes 109 : 127 - 134 .
12. Lynn S , Borthwick GM , Charnley RM , Walker M , Turnbull DM ( 2003 ) Heteroplasmic ratio of the A3243G mitochondrial DNA mutation in single pancreatic beta cells . Diabetologia 46 : 296 - 299 .
13. Maassen JA , 't Hart LM , van Essen E , Heine RJ , Nijpels G , et al. ( 2004 ) Mitochondrial Diabetes , Molecular Mechanisms and Clinical Presentation. Diabetes 53 : S103 - S109 .
14. Cree LM , Patel SK , Pyle A , Lynn S , Turnbull DM , et al. ( 2008 ) Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets . Diabetologia 51 : 1440 - 1443 .
15. Kaaman M , Sparks LM , van Harmelen V , Smith SR , Sjolin E , et al. ( 2007 ) Strong association between mitochondrial DNA copy number and lipogenesis in human white adipose tissue . Diabetologia 50 : 2526 - 2533 .
16. Laderman KA , Penny JR , Mazzucchelli F , Bresolin N , Scarlato G , et al. ( 1996 ) Aging-dependant functional alterations of mitochondrial DNA (mtDNA) from human fibroblasts transferred in mtDNA-less cells . J Biol Chem 271 : 15891 - 15897 .
17. Short KR , Bigelow ML , Kahl J , Singh R , Coenen-Schimke JC , et al. ( 2005 ) Decline in skeletal muscle mitochondrial function with aging in humans . PNAS 102 : 5618 - 5623 .
18. Kennedy ED , Maechler P , Wollheim CB ( 1998 ) Effects of Depletion of Mitochondrial DNA in Metabolism Secretion Coupling in INS-1 Cells . Diabetes 47 : 374 - 380 .
19. Silva JP , K ohler M , Graff C , Oldfors A , Magnuson MA , et al. ( 2000 ) Impaired insulin secretion and bcell loss in tissue-specific knockout mice with mitochondrial diabetes . Nat Gen 26 : 336 - 340 .
20. Soejima A , Inoue K , Takai D , Kaneko M , Ishihara H , et al. ( 1996 ) Mitochondrial DNA Is Required for Regulation of Glucose-stimulated Insulin Secretion in a Mouse Pancreatic Beta Cell Line, MIN6 . J Biol Chem 271 : 26194 - 16199 .
21. Tsuruzoe K , Araki E , Furukawa N , Shirotani T , Matsumoto K , et al. ( 1998 ) Creation and characterization of a mitochondrial DNA-depleted pancreatic beta-cell line: impaired insulin secretion induced by glucose , leucine, and sulfonylureas. Diabetes 47 : 621 - 631 .
22. Durham SE , Bonilla E , Samuels DC , DiMauro S , Chinnery PF ( 2005 ) Mitochondrial DNA copy number threshold in mtDNA depletion myopathy . Neurology 65 : 453 - 455 .
23. Campbell CT , Kolesar JE , Kaufman BA ( 2012 ) Mitochondrial transcription factor A regulates mitochondrial transcription initiation, DNA packaging, and genome copy number . Biochim Biophys Acta 1819 : 921 - 929 .
24. Ohgaki K , Kanki T , Fukuoh A , Kurisaki H , Aoki Y , et al. ( 2007 ) The C-terminal tail of mitochondrial transcription factor a markedly strengthens its general binding to DNA . J Biochem 141 : 201 - 211 .
25. Ekstrand MI , Falkenberg M , Rantanen A , Park CB , Gaspari M , et al. ( 2004 ) Mitochondrial transcription factor A regulates mtDNA copy number in mammals . Hum Molec Gen 13 : 935 - 944 .
26. Larsson NG ( 2010 ) Somatic mitochondrial DNA mutations in mammalian aging . Annu Rev Biochem 79 : 683 - 706 .
27. Miyazaki JI , Araki K , Yamoto E , Ikegami H , Asano T , et al. ( 1990 ) Establishment of a Pancreatic b Cell Line That Retains Glucose-Inducible Insulin Secretion: Special Reference to Expression of Glucose Transporter Isoforms . Endocrin 127 : 126 - 132 .
28. Schmittgen TD , Livak KJ ( 2008 ) Analyzing real-time PCR data by the comparative C(T) method . Nat Protoc 3 : 1101 - 1108 .
29. Cree LM , Samuels DC , de Sousa Lopes SC , Rajasimha HK , Wonnapinij P , et al. ( 2008 ) A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes . Nat Gen 40 : 249 - 254 .
30. Bonnen PE , Yarham JW , Besse A , Wu P , Faqeih EA , et al. ( 2013 ) Mutations in FBXL4 cause mitochondrial encephalopathy and a disorder of mitochondrial DNA maintenance . Am J Hum Genet 93 : 471 - 481 .
31. Brand MD ( 2005 ) The efficiency and plasticity of mitochondrial energy transduction . Biochem Soc Trans 33 : 897 - 904 .
32. Brand MD and Nicholls DG ( 2011 ) Assessing mitochondrial dysfunction in cells . Biochem J 435 : 297 - 312 .
33. Ishihara H , Asano T , Tsukuda K , Katagiri H , Inukai K , et al. ( 1994 ) Overexpression of Hexokinase 1 but not GLUT1 Glucose Transporter Alters Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic b-Cell Line MIN6 . J Biol Chem 269 : 3081 - 3087 .
34. Fontanesi F , Soto IC , Horn D , Barrientos A ( 2006 ) Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process . Am J Physiol Cell Physiol 291 : C1129 - C1147 .
35. Proks P , Reimann F , Green N , Gribble F , Ashcroft F ( 2002 ) Sulfonylurea stimulation of insulin secretion . Diabetes 51 Suppl 3 :: S368 - S376 .
36. Sorensen L , Ekstrand M , Silva JP , Lindqvist E , Xu B , et al. ( 2001 ) Late-Onset Corticohippocampal Neurodepletion Attributable to Catastrophic Failure of Oxidative Phosphorylation in MILON Mice . J Neurosci 21 : 8082 - 8090 .
37. Wang J , Wilhelmsson H , Graff C , Li H , Oldfors A , et al. ( 1999 ) Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression . Nat Gen 21 : 133 - 137 .
38. Larsson NG , Wang J , Wilhelmsson H , Oldfors A , Rustin P , et al. ( 1998 ) Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice . Nat Gen 18 : 231 - 236 .
39. Hayakawa T , Noda M , Yasuda K , Yorifuji H , Taniguchi S , et al. ( 1998 ) Ethidium Bromide-induced Inhibition of Mitochondrial Gene Transcription Suppresses Glucose-stimulated Insulin Release in the Mouse Pancreatic b-Cell Line bHC9 . J Biol Chem 273 : 20300 - 20307 .
40. Gauthier BR , Wiederkehr A , Baquie M , Dai C , Powers AC , et al. ( 2009 ) PDX1 deficiency causes mitochondrial dysfunction and defective insulin secretion through TFAM suppression . Cell Metab 10 : 110 - 118 .
41. Liesa M and Shirihai OS ( 2013 ) Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure . Cell Metab 17 : 491 - 506 .
42. Ihm SH , Matsumoto I , Sawada T , Nakano M , Zhang HJ , et al. ( 2006 ) Effect of donor age on function of isolated human islets . Diabetes 55 : 1361 - 1368 .
43. Babu DA , Deering TG , Mirmira RG ( 2007 ) A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis . Mol Gen Metab 92 : 43 - 55 .
44. Koeck T , Olsson AH , Nitert MD , Sharoyko VV , Ladenvall C , et al. ( 2011 ) A Common Variant in TFB1M Is Associated with Reduced Insulin Secretion and Increased Future Risk of Type 2 Diabetes . Cell Metab 13 : 80 - 91 .
45. Metodiev MD , Lesko N , Park CB , Camara Y , Shi Y , et al. ( 2009 ). Methylation of 12S rRNA is necessary for in vivo stability of the small subunit of the mammalian mitochondrial ribosome . Cell Metab 9 : 386 - 397 .
46. Litonin D , Sologub M , Shi Y , Savkina M , Anikin M , et al. ( 2010 ). Human mitochondrial transcription revisited: only TFAM and TFB2M are required for transcription of the mitochondrial genes in vitro . J Biol Chem 285 : 18129 - 18133 .
47. Lu H , Koshkin V , Allister EM , Gyulkhandanyan AV , Wheeler MB ( 2010 ) Molecular and Metabolic Evidence for Mitochondrial Defects Associated With b-Cell Dysfunction in a Mouse Model of Type 2 Diabetes . Diabetes 59 : 448 - 459 .
48. Groop LC ( 1992 ) Sulfonylureas in NIDDM . Diabetes Care 15 : 737 - 754 .