An epigenetic mechanism for peripheral insulin resistance

ResearchBlogging.org

PGC-1alpha is my favorite gene/protein to study, as it is essential for mitochondrial regulation, influential on many diseases and ageing.  I also am fascinated by the relatively new field of epigenetics and its relation to nutrition and health.  So you can understand my geeky giddiness when I found that a study by Barrès et al. (1) shows a link between them.  (Warning this is technically dense, I provide a summary at the end with my perspective)

Because the skeletal muscle is the primary site of insulin mediated glucose disposal, it was the logical choice to study since this field is relatively uncharted.  The investigators biopsied the vastus lateralis from male normal glucose tolerant (NGT) subjects, type II diabetics (T2DM), and impaired glucose-tolerant (IGT) subjects and performed a genome-wide promoter analysis of DNA methylation.  Of genes that were differentially methylated between NGT and T2DM subjects (25,500 analyzed, 838 different), 44 were related to mitochondrial function, and the PGC-1alpha promoter was hypermethylated in T2DM compared to NGT.  Since PGC-1alpha is well studied in its role as a master regulator of mitochondrial function, it was a logical choice to continue with.

After bisulfite sequencing (converts cytosine to uracil, but not methylated cytosine), a 2.7 and 2.2-fold increase in unconverted cytosines was observed on the promoter region of PGC-1alpha in IGT and T2DM, respectively compared to NGT of portion of promoter -337 to -37 relative to +1 transcription site of PGC-1alpha gene.  Another promoter of PGC-1alpha was recently identified: -243 to -47 relative to +1 transcription start site, but the methylation was similar in NGT and T2DM.

Further sequencing of 2 other genes proximal to PGC-1alpha showed that hypermethylation in T2DM was not broadly altered on a wide portion of chromatin, rather specific to the canonical PGC-1alpha promoter.

Interestingly, most methylated cytosines were found within non-CpG dinucleotides; non-CpG methylation is almost exclusively found in plants and embryonic stem cells prior to this.

Also, the hypermethylation pattern was unrelated to family history of T2DM, further supporting (in my opinion) that gene variations are not/will not be a good predictor for lifestyle related disease risk, though this of course will need to be studied much more.

Because DNA methylation located within or close to the 5′ region of genes is associated with regulation of gene expression, they examined whether PGC-1alpha mRNA expression was altered from the skeletal muscle biopsies from NGT and T2DM subjects.  A reduction in PGC-1alpha mRNA content by 38% in T2DM was discovered, which was negatively correlated with promoter methylation.

Next, a gene reporter assay was used to investigate the role of the PGC-1alpha promoter methylation on gene activity.  In vitro methylation of single cytosine residue (-260 relative to +1 transcription start site) markedly reduced PGC-1alpha gene activity.

They also found that PGC-1alpha promoter methylation negatively correlated with mitochondrial DNA (mtDNA) content (mtDNA: nDNA ratio).  Additionally, proteins from the mitochondrial respiratory chain were measured: succinate-ubiquinol reductase (complex II), core I (complex III), and cytochrome C were significantly decreased in T2DM compared with NGT. Mitochondrial transcription factor A, a key protein in regulation of mtDNA quantity, was significant decreased in T2DM.  The ratio of mtDNA per nucleus was decreased 22% in T2DM subjects.  Further, ultrastructural analysis of skeletal mucsle found reduced mitochondrial number and area in T2DM subjects.

Next, they wanted to see if extracellular milieu associated with peripheral insulin resistance (hyperglycemia, hyperinsulinemia, elevated free fatty acids, and cytokines) directly and/or acutely affects methylation.

Myocytes from the biopsies were incubated with factors known to induce insulin resistance: TNF-alpha (TNF-alpha and free fatty acids stimulate accumulation of sphingolipid ceramide and various ceramide metabolites), palmitate, and oleate all induced hypermethylation of the PGC-1alpha promoter, whereas high glucose and insulin concentrations did not. A majority of methylated cytosines were located off CpG nucelotides, as found in diabetic skeletal muscle.  Then, they checked to see if these were consequence of whole-genome methylation: with palmitate treatment, both global CpA and CpT methylation within 5′-CCA/TGG-3′ was increased from 1.6% to 4.2%, but CpG methylation within 5′-CCGG-3′ sequence was unaltered in the assay.  Similar results had been found for CpG and non-CpG methylation levels in the human myocytes, lending further evidence of high non-CpG methylation in skeletal muscle.  And, because global methylation were unchanged in T2DM subjects, it suggests that the hypermethylation is gene specific.  Together, the evidence shows that free fatty acids acutely induce non-CpG methyation at PGC-1alpha promoter and at whole-genome level in myocytes

Next was to check what mediates the palmitate-induced DNA methylation.  In mammals, there are 3 functional DNA methyltransferase isoforms: DNMT1, DNMT3A, and DNMT3B.  The investigators silenced each selectively; silencing of DNMT1 or DNMT3A did not prevent palmitate-induced downregulation of PGC-1alpha mRNA and mitochondrial gene expressions, whereas silencing of DNMT3B prevented promoter methylation by 43%, and mtDNA content reduction was prevented.  To detect any variation in expression of genes related to mitochondrial function and biogenesis, quantitative PCR was performed and found that silencing of DNMT3B prevented palmitate-induced downregulated mRNA expression of PGC-alpha, TFAM, citrate synthase, and carnitine palmitoyltranferase -2, while upregulation of mRNA expression of CPT-1 and cytochrome C was unaltered by silencing.  PGC-1alpha may not directly regulate the measured subset of genes (direct, methylation, reduction in PGC-1alpha expression…); TFAM and CPT-2 were hypermethylated in skeletal muscle in T2DM subjects.

An analysis of protein content of DNMT isoforms with 48 hrs exposure of myocytes to high concentrations of glucose, insulin, palimtate, oleate, or TNF-alpha showed no change; from the biopsied samples, DNMT3B mRNA expression was increased in T2DM vs NGT, and the other isoforms unchanged.  The unaltered protein suggests enzymatic activation of DNMT3B rather than changes in protein expression involved in hypermethyation of promoter.

Other studies show changes in DNA methylation and associations with changes in expression of genes involved in mitochondrial function: cytochrome c oxidase subunit VIIa polypeptide 1, NADH dehydrogenase (ubiquinone) 1 beta subcomplex 6, and PGC-1alpha. lysine (K)-specific demethylase 3A implicated in transcriptional regulation of PPARalpha.  In this study, MeDIP screen of biopsies showed numerous genes with differing methylation status in skeletal muscle between T2DM and NGT, including those involved in primary metabolic processes and mitochondrial function. This leaves us with exciting prospects for future study.

What does this all mean and why is it important?

Mitochondrial dysfunction results in impaired oxidative capacity and excess lipid storage.  It is also associated with many diseases, including insulin resistance and type II diabetes.  Reductions in mitochondrial density has been proposed as a primary cause of peripheral insulin resistance.

PGC-1alpha coordinately regulates expression of a subset of mitochondrial genes and participates in overall mitochondrial function in cells.  The finding that the methylation of the promoter of PGC-1alpha is associated with its decreased expression and reduction in mitochondria and mitochondrial enzymes in diabetics, and that incubation with milieu associated with insulin resistance (TNF-alpha and fatty acids) can replicate the findings lend strong evidence that this epigenetic mechanism is a mediator between environmental factors (nutrition, lifestyle) and specific gene/proteins implicated in disease.  PGC-1alpha expression/activity can be upregulated mainly during fasting/calorie restriction, endurance exercise, and to certain compounds from food, mostly specific polyphenols at high doses.

Of note, methylation of the promoter of PGC-1alpha to TNF-alpha or fatty acids occured in time-dependent manner.  This may be important to consider in future studies using meal or lifestyle patterns.  In this study, insulin resistant but not diabetic subjects showed similar a methylation pattern of the promoter compared to the diabetics, suggesting that this may an early cause of the full blown disease.  After all, they are only different labels for different severities of insulin resistance.  Perhaps future evidence will find methylation could be an earlier marker than glucose challenges (which can vary with accuracy).

In other research, a reduced skeletal muscle PGC-1alpha mRNA expression is noted in some but not all nondiabetic family history-positive subjects, despite impairments in mitochondrial function.  There are likely many more factors to find, as well as the question if methylation is causually related to the pathogenesis of insulin resistance or a consequence?

And last but not least, the findings that DNA hypermethylation occurs in differentiated, nondividing cells, and that non-CpG methylation seems to be the influential epigenetic change, are of huge importance.

This opens the door for further study with dietary and activity factors, to link food and lifestyle to health on another level.

References

1.  Barrès R, Osler ME, Yan J, Rune A, Fritz T, Caidahl K, Krook A, & Zierath JR (2009). Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density. Cell metabolism, 10 (3), 189-98 PMID: 19723495