Blood glucose levels, pIns-c-MycERTAM
For the purpose of our experiment, data from at least three replicate experiments per time-point are plotted in Fig. 1. On Day 0, blood glucose levels were around 5 mmol/l, with a clear decrease down to 2.5 mmol/l (and no lower) during the first 24 h of c-Myc activation (brief hypoglycaemia window). Blood glucose levels increased from Day 2, peaking at >25 mmol/l on Day 7. Blood glucose levels fell rapidly following Myc deactivation and were largely normalised at 3 weeks, by which time glucose levels were identical to those of wild-type littermates. Glucose levels remained slightly above mean pre-treatment levels, which, as previously shown, are generally slightly lower than average for wild-type mice . Importantly, transgenic and wild-type mice had an essentially normal response to glucose tolerance tests on Days 26 and 57 (3 weeks after c-Myc was ‘switched off’) (ESM Fig. 1).
As expected, c-Myc-induced suppression of insulin expression and destruction of beta cells was mirrored by the acute onset and maintenance of hyperglycaemia , with similar kinetics in female and male mice (data not shown for males). In line with this, the results obtained here were not influenced by the oestrous cycle in female mice. Wild-type mice that were treated by tamoxifen displayed blood glucose levels ranging from about 6 to 9 mmol/l (ESM Fig. 2).
Cholesterol, triacylglycerol and NEFA levels
HDL, total cholesterol, triacylglycerol and NEFA were measured in serum from Day 0 to Day 57 (n = 3 per time-point) (Fig. 2). The first three remained unchanged when Day 0 was compared with Day 7, while NEFA concentrations were marginally affected at peak hyperglycaemia.
Global GeneChip arrays
Using Genespring GX 10.0 (Agilent Technologies, Wokingham, UK) we identified changes in gene expression by a magnitude of twofold on Day 7 (peak of glycaemia) and the presence/lack of ability to reverse by Day 26 (the longest recovery time-point available for GeneChip Array analysis). Gene expression profiles for total, reversed and non-reversed genes (Fig. 3a–c) were generated. Array data were submitted to NCBI’s GEO (Accession No. GSE15401; www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15401, accessed 9 August 2010).
Genes regulated by tamoxifen in normoglycaemic control mice
In control wild-type mice treated with tamoxifen, 12 genes were differentially expressed when compared with c-MycERTAM up to Day 7 (ESM Fig. 3); for full list of genes, see ESM Table 2. Of the genes affected by tamoxifen, 20 were common to those altered by hyperglycaemia (18 genes) and hypoglycaemia (two genes) (ESM Table 3). No genes from this group appear in of the rest of our data analyses.
Gene changes caused by hypoglycaemia
Changes in 95 genes were attributed to the brief hypoglycaemic phase (ESM Fig. 4; Pathway analysis).
Gene changes at the peak of hyperglycaemia (Day 7)
At Day 7 the expression of 769 genes was altered by more than twofold (up- or downregulated) due to hyperglycaemia (p ≤ 0.05) (Tables 1 and 2). Importantly, among the 769 altered genes at Day 7 vs Day 0, (Fig. 3a), the changes were reversed in 576 (74.9%) genes by Day 26 (Fig. 3b), whereas in the 67 (25.1%) other genes they were not reversed within the same time (Fig. 3c).
Enrichment analyses identified the following genes that changed substantially and closely mirrored changes in blood glucose levels: (1) exclusively upregulated on Day 7: cytoskeletal calmodulin, titin (Ttn), desmoplakin, thrombospondin 2 (Thbs2), protocadherin7 (Pcdh7), phosphodiesterase 1A (Pde1a), retinoic acid receptor responder 2 (Rarres2), adenylate kinase 3 (Ak3), integrin β5 (Itgb5), endothelin receptor A (Ednra), laminin γ1 (Lamγ1 [also known as Lamc1]), osteoglycin (Ogn), caspase 12 (Casp12), angiopoietin 2 (Angpt2); (2) downregulated: myosin VB (Myo5a), myosin light chain kinase (Mylk3), troponin I (TnnI [also known as Tnni1]), myosin heavy polypeptide (Myh [also known as Mutyh]), keratin 13 (Krt13).
Gene ontology enrichment analysis using PatternViewer (GenoSyst, Turku, Finland) revealed that the above list includes genes involved in lipid metabolism (ESM Fig. 5, ESM Table 4). These genes were downregulated by up to twofold from Day 2 through to Day 26.
Genes involved in arterial calcification
A total of 13 genes implicated in arterial calcification and premature atherosclerosis were differentially expressed due to hyperglycaemia, but all reverted to basal levels by Day 26 (listed in Table 1 and ESM Fig. 6).
Genes that recover after restoration of normoglycaemia
In addition to the calcification-related genes, examples of other genes that recovered after normalisation of blood glucose levels are shown in Tables 1 and 2 (576 genes in total). Upregulated genes on Day 7 included: (1) cell cycle-related cyclin D2 (Ccnd2) and insulin growth factor 1 (Igf1); (2) cell adhesion molecules vascular cell adhesion molecule (Vcam1) and matrix metalloproteinases (Mmp3 and Mmp9); and (3) atherosclerosis-related von Willebrand factor (Vwf), apolipoprotein D (Apod), Jun, Myh6 and heat-shock protein Hsp90b1. Downregulated genes on Day 7 included: fatty acid binding protein 7 (Fabp7) and potassium voltage-gated channels (Kcnh3 and Kcnq2). Pathway analysis showed a pivotal role in biological processes for the oncogene Jun in chondrogenesis, supporting an osteogenic profile, which was caused by transient hyperglycaemia. Also, Igf1 leads to tissue growth and proliferation; other processes affected were those involving the potentially atheroprotective phospholipase Pla2g4a, which is responsive to hypoxia (ESM Fig. 7).
A group of genes (n = 67) that were altered on Day 7 due to hyperglycaemia failed to return to basal expression levels by Day 26 (Tables 1 and 2). Examples of non-reversed genes by Day 26 were: angiogenin (Ang1), prostaglandin-associated genes Hpdg and Alox15, the apolipoprotein B (Apobec1) gene and cell turnover-associated proteins such as: (1) the upregulated FBJ osteosarcoma gene Fos, creatine kinase (Ckm), cardiac troponins Tnn2 and Tnn3, and early growth response 3 (Egr3); and (2) the downregulated fatty acid binding proteins 1 and 4 (Fabp1 and Fabp4), and plasminogen (Plg) (Tables 1 and 2). The expression of some genes was dramatically elevated at the peak of hyperglycaemia compared with others. Examples are the cardiac troponin 2 and 3 (Tnn2, Tnn3) genes (upregulated by more than 255- and 56-fold, respectively), Ttn (122-fold upregulation) and small muscle protein (Smpx) with a 43-fold increase on Day 7 (Tables 1 and 2, ESM Fig. 8).
The networks of genes that changed at the peak of hyperglycaemia included pro-inflammatory genes such as S100, the chemokine C-X-C motif and prostaglandin H synthase-2 (Pghs-2 [also known as Ptgs2]) (ESM Figs 6–8), as well as genes related to oxidative stress such as paraoxonase 1 (Pon1) and apolipoprotein d (Apod). Many of these changes due to hyperglycaemia were related to genes involved in endothelial cell dysfunction such as Vwf, Vcam1 and smooth muscle cell-related genes like Tnn2 and myozenin 2 (Myoz2) (Table 1).
Examination of the non-reversed genes suggested on-going tissue remodelling, with Egr1 contributing to a wound-healing and protective mechanism, while Egr3 is involved in cell turnover (apoptosis or proliferation). Furthermore, plasminogen (Plg) promotes angiogenesis, while Fos is potentially involved in tissue modification towards an osteogenic profile of the artery (ESM Fig. 8). The different patterns of gene expression changes are summarised in Table 2.
Day 0 to Day 2: inclusion of the transient hypoglycaemia ‘window’
A set of 95 genes were differentially regulated at Day 2 following a transient window of hypoglycaemia that is first observed 24 h after c-Myc activation and extends for around 24 h. These 95 genes included peroxisome proliferator-activating factor receptor γ (Pparγ [also known as Pparg], down by twofold), Vegfc (up by 2.8-fold), endothelial differentiation gene 3 (Edg3) (down by 2.3-fold), Apoa1 (down by 3.3-fold), Egr (up by twofold), immunoglobin joining chain (Igj, upregulated by 3.5-fold), cytochrome P450 (Cyp3a11, downregulated by threefold), albumin (Alb, downregulated by 4.9-fold) and fatty acid binding protein 1 (Fabp1, downregulated by 4.29-fold). The 69 of these genes that had recovered by the immediate time-point (Day 4) are regarded as exclusively regulated by the hypoglycaemia ‘window’. These genes were included in the 95 genes that changed between Days 0 and 2. Among the hypoglycaemia-regulated genes are the putative homeodomain transcription factor 1 (Phtf1, down by twofold), collagen XII a1 (Col12a1, down by 2.41-fold), the genes encoding solute carrier families Slc35a5 and Slc9a3r2 (down by two- and 2.2-fold, respectively), and the chemokine C-X-C motif (Cxcl2, up by tenfold).
Day 2 to Day 4: recovery from hypoglycaemia and early effects of hyperglycaemia
During this period, the mice have recovered from the brief hypoglycaemia and on Day 4 have just started to become hyperglycaemic. Of the genes that were altered on Day 2, 69 (including those that specifically changed due to hypoglycaemia) had recovered by this time point, suggesting that the effects of transient hypoglycaemia are short-lived and recovered from within 48 h. As only 26 of these genes had not recovered at Day 4, we can confidently exclude any significant residual effect of hypoglycaemia on the genes affected by hyperglycaemia.
On Day 4, a different subset of genes is now being affected, most probably due to onset of hyperglycaemia. Thus 169 genes not previously involved were deregulated at Day 4, including cell cycle- and growth-associated genes. Other genes included the upregulated apolipoprotein E (Apoe), collagen 1a1 (Cola1a1), angiogenin (Ang), Pparγ and the downregulated S100 protein, Cxcl2.
Day 4 to Day 7: effects of peak hyperglycaemia
As the effects of hyperglycaemia become established, dramatic upregulation of genes encoding cytoskeletal proteins occurs, e.g. α-actin (by 100-fold), Myh6, cardiac troponin c 1 (Tnnc1) and i 3 (Tnni3), and myozenin (Myoz). Other genes, e.g. apolipoprotein B (Apobec1), muscle creatine kinase (Ckm), Wif1 and prostaglandin endoperoxide synthase (Ptgs2) were also increased between early and peak hyperglycaemia. In total, 510 genes were altered during this phase of the experiment. Further comparative analyses also revealed a small group of 30 genes that may have been affected by hypoglycaemia and hyperglycaemia (Table 3). Examples of these are apolipoprotein D (Apod, upregulated), Pparα (also known as Ppara, downregulated) and fatty acid binding protein 4 (Fabp4, downregulated).
Day 7 to Day 11: early events immediately after initial reduction in blood glucose levels
During this period blood glucose levels are declining, but have not yet normalised. Interestingly, a large proportion of genes (400) were downregulated from Day 7 to 11 (within 4 days of switching c-Myc off and during the process of reduction in blood glucose). Included in this group are Ccnd2, Mmp3, Vegfc, Myh6, Apobec2, Apod and Ckm. By Day 11, although the mice were still hyperglycaemic, all ‘osteogenic’ genes potentially involved in arterial calcification had returned to basal levels (Table 1 ‘Calcification genes’).
Some genes remained altered on Day 11 and Day 26, when compared with Day 0. These are the non-reversed genes such as the upregulated Egr1 and Fos, and the downregulated Plg, Pparγ c1a (also known as Ppargc1a), leptin (Lep) and Fabp1.
Day 11 to day 26: early to established recovery
This is a potentially very important group of genes (n = 331), as they include those that did not recover by the end of the study period. Examples are the upregulated T-box 20, prostaglandin F receptor (Ptger3) and heat shock protein 90 (Hsp90ab1), and the downregulated beta globin and interferon regulatory factor 4 (Irf4) (see also next section). As mentioned in the previous section, some genes already changed on Day 11 vs Day 0 continued to be altered on Day 26. More altered genes (non-reversed by Day 26) are Pthr1 (upregulated), Apoa1 and Apobec1 (downregulated).
Genes differentially expressed for the first time on Day 11 (early recovery)
The expression of 57 genes, not previously altered by hyperglycaemia, changed after Day 11. This group probably includes genes involved in the recovery from hyperglycaemia. Pathway analysis showed that the following processes are involved: apoptosis and cell cycle, fatty acid oxidation and gluconeogenesis (Pparγ c1a), glucose import (Fabp3) and atherosclerosis (Isl1) (ESM Fig. 9).
Genes differentially expressed for the first time on Day 26 (established recovery)
As the mice fully recovered from hyperglycaemia, a new group of genes changed for the first time on Day 26 (n = 69), such as cell cycle-related Rbm3 and cyclin D1 (Ccnd1), glutamate transporter (Slc1a3), retinol dehydrogenase 11 (Rdh11), which is involved in cell growth, and Socs6 (involved in lipid biosynthesis) (ESM Fig. 10).
Verification of gene array data
Quantitative reverse transcriptase PCR was used to verify expression patterns of a selection of genes altered by glycaemia according to the array data. Examples were genes encoding key molecules implicated in cell cycle (Ccnd2), diabetes/insulin resistance (Pparγ) and cell adhesion (Vcam1) (Fig. 4). All quantitative reverse transcriptase PCR data confirmed the gene array results. Where necessary, later recovery time-points were analysed (Day 140).
We used standard fluorescence immunohistochemical techniques to study protein levels of vascular cell adhesion molecule (VCAM) 1 (cell adhesion-related), the atherosclerosis-related vascular endothelial growth factor (VEGF) and α-actin (smooth muscle cell-related). Immunofluorescent signals increased in parallel to high glucose levels (Fig. 5).