Following assessment of impaired glucose tolerance ( Figures 3 A and 3B ; Figures S2 G and S2H), hyperinsulinemic-euglycemic clamp studies were performed on Control, Undernourished, and R2 rats ( Figures 3 C and 3D; Figures S2 I–S2J) to confirm insulin resistance. Significantly lower glucose infusion rates supported maintenance of clamped glucose concentrations in the Undernourished and R2 rats, confirming that the insulin resistance observed in the Undernourished rats was not restored following two generations of Control diet restoration. To understand whether undernutrition over generations altered the susceptibility to diabetes, we carried out a streptozotocin (STZ) dose response (see Experimental Procedures Figure S3 A). STZ, a pancreatic beta cell toxin, is routinely used to induce diabetes in Wistar rats. Undernourished rats died following exposure to a dose of STZ (200 mg/kg b.w.) that rendered ≥90% Control rats ( Figure 3 E) diabetic (fasting blood glucose > 11 mmol/l by day 8 post-STZ). An 8-fold lower dose (25 mg/kg b.w.) offered 100% survival in Undernourished ( Figure 3 F) as well as R2 rats ( Figure 3 G) but all developing diabetes with fasting blood glucose > 11 mmol/l by day 8 from STZ injection; none of the Control animals became diabetic at this (low) dose. We observed that Undernourished and R2 rats injected with this high dose (200 mg/kg b.w.) of STZ died with hypoglycemic convulsions in 12–14 hr ( Figures 3 F and 3G). Serial circulating insulin measurements following STZ injection (200 mg/kg b.w.) in Undernourished and R2 rats ( Figure 3 H) demonstrated that the increased mortality at 200 mg/kg dose was indeed associated with a significant increase in circulating insulin within 3–6 hr after STZ injection, which resulted in hypoglycemic convulsions and death. As Undernourished and R2 rats showed 100% survival with fasting plasma glucose > 11 mmol/l by day 8, at a dose that is eight times lower than the diabetogenic dose in Control rats (200 mg/kg b.w), Undernourished and R2 rats had eight times more susceptibility to STZ-induced diabetes.

Undernourished rats also showed other markers of metabolic disorder. Elevated levels of circulating tHcy ( Table S1 D) is related to higher risk of coronary disease, stroke, and peripheral vascular disease and atherosclerosis in man (). Electrocardiograms ( Figures S3 B–S3F) revealed inverted P and T waves in R2 rats, with elevated Q and ST-segments, consistent with myocardial infarction and associated with higher early mortality and morbidity (), in man. A lower circulating concentration of folate ( Table S1 D) may itself be an atherogenic factor () that could promote hyperhomocysteinemia seen in these Undernourished and R2 rats. Cardiac histology revealed multiple morphological abnormalities in R2 rats ( Figure S3 G) and higher cardiac tissue levels of the DNA methyl transferase dnmt3a1 in Undernourished and R2 rats ( Figure S3 H), which may be associated with epigenetic silencing in cardiac tissue as well ().

American College of Cardiology American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines for the Management of Patients With Unstable Angina/Non-ST-Elevation Myocardial Infarction) American College of Emergency Physicians Society for Cardiovascular Angiography and Interventions Society of Thoracic Surgeons American Association of Cardiovascular and Pulmonary Rehabilitation Society for Academic Emergency Medicine ACC/AHA 2007 guidelines for the management of patients with unstable angina/non-ST-Elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines for the Management of Patients With Unstable Angina/Non-ST-Elevation Myocardial Infarction) developed in collaboration with the American College of Emergency Physicians, the Society for Cardiovascular Angiography and Interventions, and the Society of Thoracic Surgeons endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation and the Society for Academic Emergency Medicine.

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Figure 4 Epigenetic Modifications following Multigeneration Undernutrition and Nutrient Transition Show full caption (A) Pancreatic Insulin-2 gene transcript abundance. (B and C) Epigenetic signatures of histone modifications (relative to control, see Experimental Procedures ). Areas between the rays of the radar plot were assigned as belonging to “activated” or “suppressed” parts of the epigenetic signature based on the antibodies used for IP. The Shoelace formula was then used to measure the areas of these fragments of the signature. Results were compared using t test and plotted mean ± SD. (D and E) Areas for “active” and “suppressed” modifications before and after nutrient transition. (F–H) Recruitment of histone modifying enzymes (F and G) and the transcription factor PDX1 (H) at rat insulin 2 promoter region.

Undernourished and R2 rat pancreas contained significantly fewer (pro-)insulin 2 gene transcripts ( Figure 4 A; Figures S4 A and S4B), indicating that multigenerational undernutrition affected insulin gene transcription with no recovery. This may be a result of epigenetic repression of insulin gene transcription, although active degradation of insulin gene transcripts, which have a long half-life () of ∼30–36 hr (in man), or both, is also a possibility. Indeed, the relative abundance of KMT1A, a histone-3 lysine 9-specific methyl transferase, which trimethylates H3K9me leading to suppression of gene transcription (), was increased in Undernourished and R2 pancreas ( Figures S4 C and S4D). To test whether the pro-insulin gene was epigenetically modified, we carried out chromatin immunoprecipitation (ChIP) for five different histone modifications: H3Ac, H4Ac, and H3K4me3, three modifications associated with transcriptionally activated gene promoters, and H3K9me3 and H3K20me3, two modifications associated with suppressed/silenced gene promoters. TaqMan-based real-time PCR was carried out on immunoprecipitated DNA to quantify the insulin promoter content in each of the IP fractions. Data comparing Undernourished and R2 islets to Control islets ( Figure S4 E) were logarithmically transformed to create radar plots of the epigenetic signature for insulin promoter ( Figures 4 B and 4C; Figures S4 F and S4G). Areas between the rays of these radar plot were assigned as belonging to “activated” (green) or “suppressed” (orange) profiles and measured to quantify differences in overall epigenetic profiles ( Figures 4 D and 4E). These analyses demonstrate that epigenetic signatures leading to suppression of pro-insulin gene transcription were markedly increased (relative to Control; Figures S4 E–S4H) in Undernourished rat islets and were not restored to Control levels following two generations of unrestricted access to “Control” chow (R2 rats). However, relative to the Undernourished epigenetic profiles, we observed a significant increase in activated marks (p < 0.0001) and a decline in suppressed marks (p < 0.0001) in the R2 epigenetic profile, indicating that two generations of normal feeding, significantly, but only partially, improved epigenetic repressive modifications within the insulin promoter region ( Figures 4 D and 4E). In order to understand the underlying molecular mechanisms leading to these histone modifications and metabolic alterations, we examined the recruitment of histone modulators and transcription factors at the insulin-2 gene promoter region. H3K9 methyl transferase KMT1A and the corepressor LSD1 were specifically recruited at the insulin gene promoter region in Undernourished and R2 rats, respectively ( Figures 4 F and 4G; Figures S4 I–S4L). Intriguingly, such an epigenetically modified chromosomal conformation significantly diminished the spatial occupancy/recruitment of the pancreatic transcription factor PDX1, at the insulin-2 gene promoter region ( Figure 4 H; Figures S4 I–S4L). These analyses indicate that dietary and lifestyle adaptations/choices are associated with and possibly regulatory in recruiting histone modifying enzymes at the gene promoter region. The overall chromosomal conformation seen in Undernourished rats is inhibitory to efficient binding of the transcription factor PDX1, at the insulin-2 gene promoter region.