Prostate cancer (PCa) is the most frequently diagnosed cancer type and the second major cause of cancer deaths amongst men . Several factors have been found to contribute to disease susceptibility, progression, and prognosis including familial history and genetics , obesity , aging  and ethnicity .
It is clear that a link exists between obesity and cancer risk . The increased adipose tissue, associated with obesity, produces over 20 hormones and cytokines that can disturb the delicate balance of the cellular environment .
Obesity also increases susceptibility to type 2 diabetes (T2D) . One key characteristic associated with T2D is hyperglycaemia. As glucose is the main energy source for cells, an increase in its supply can cause increased proliferative potential .
Insulin-like growth factor II (IGF-II) is a growth factor expressed in high quantities during early embryonic development . Expression continues throughout adulthood, with the liver being the primary site of synthesis . IGF-II plays a critical role in a number of cellular events, including proliferation and survival .
In obese subjects, circulating serum levels of IGF-II are elevated and show a positive correlation with increased body mass index (BMI) . Conversely, after weight loss, circulating levels of IGF-II have been shown to drop .
The IGF-II/H19 gene is located on the short arm of chromosome 11p. 15.5 . It is composed of 10 exons and contains 5 promoters. Exons 1–4 and 6 are non-coding. Control of expression takes place from promoters 0 to 4 and is strictly regulated. 128 kb downstream from IGF-II is a long non-coding RNA, H19, which is linked to IGF-II by an imprinting control region (ICR) ,
IGF-II was the first gene identified to be ‘imprinted’ . This is a heritable epigenetic event whereby, through changes to the DNA structure (such as methylation or histone modification) one parental copy will be silenced or imprinted . The loss of this natural silencing phenomenon (loss of imprinting – LOI) has been identified across a number of cancers, including prostate , breast , colorectal  and lung .
Models for the imprinting mechanism have been proposed, with the most widely accepted being the ‘enhancer competition model’ , in which the presence of a CCCTC-binding factor (CTCF) – a multi-zinc finger protein (and a transcriptional repressor) - is able to bind to the unmethylated imprint control region (ICR) embedded within the maternal IGF-II / H19 allele. This impedes IGF-II transcription. Conversely, on the paternal allele, the ICR is hypermethylated. This blocks CTCF from binding, which permits IGF-II to be transcribed. LOI occurs when both parental copies of IGF-II / H19 are hypermethylated at the ICR, resulting in the bi-allelic expression of IGF-II .
The function of H19 is unclear. In some cancers it is oncogenic: in gastric cancer, H19 and an embedded micro-RNA (miRNA-675) within its first exon, were found to be increased in tissue and cell lines. This over-expression led to increased cell proliferation and the inhibition of apoptosis . In non-small-cell lung cancer, H19 expression was significantly higher in malignant lung tissue compared with normal, being at its highest in those from stage III and IV tumours . In contrast, H19 behaves as a tumour suppressor in some cancer types, such as colorectal  and prostate . The mechanism that dictates H19 behaviour differs between cancer types.
One study, published in 2012, analysed circulating IGF-II protein levels  in patients with a history of PCa. Of 106 (41 patients - radical prostatectomised (RPE) - and 65 controls) IGF-II levels were significantly elevated in the RPE cohort. LOI was also significantly higher in the RPE group (39%) compared to the control (20%). Despite the link between LOI and elevated serum IGF-II in the RPE group, the two were found to be uncoupled. Of the control cohort with LOI (20%), circulating IGF-II levels were found to be very similar to those of the RPE cohort with LOI (39%). The authors suggested that, under normal conditions, only approximately 35% of the total serum IGF-II is regulated by imprinting .
A previous report focused on promoter methylation as a factor contributing to IGF-II transcription . IGF-II mRNA and peptide levels were decreased in 80% of PCa, compared to non-neoplastic adjacent prostate and were independent of LOI status. IGF-II expression in both tumour and adjacent tissue depended on usage of the IGF-II promoters P3 and P4; decreased IGF-II expression in tumour tissue was strongly related to hypermethylation of these two promoters. The cause of hyper-methylation in this cohort was attributed to cumulative DNA damage, due to aging.
In this study we examined, in vitro, the effects of altering metabolic conditions on IGF-II imprinting status (IS), IGF-II / H19 mRNA, and IGF-II peptide levels in the PC3 PCa cell line. In addition, a clinical cohort of PCa tissue was analysed for expression of IGF-II and H19 mRNA using digital droplet polymerase chain reaction (ddPCR) and compared with two larger publicly available patient cohorts from the Cancer Genome Atlas (TGCA). IGF-II IS status was also analysed, using pyrosequencing, along with IGF-II localisation and abundance using immunohistochemistry (IHC).