Andrew L. Siebel

Normal Lactational Environment Restores Nephron Endowment and Prevents Hypertension after Placental Restriction in the Rat

Uteroplacental insufficiency in the rat restricts fetal growth, impairs mammary development, compromising postnatal growth; and increases adult BP. The roles of prenatal and postnatal nutritional restraint on later BP and nephron endowment in offspring from mothers that underwent bilateral uterine vessel ligation (restricted) on day 18 of pregnancy were examined. Sham surgery (control) and a group of rats with reduced litter size (reduced; litter size reduced at birth to five, equivalent to restricted group) were used as controls. Offspring (control, reduced, and restricted) were cross-fostered on postnatal day 1 onto a control (normal lactation) or restricted (impaired lactation) mother. BP in male offspring was determined by tail cuff at 8, 12, and 20 wk of age, with glomerular number and volume (Cavalieri/Physical Dissector method) and renal angiotensin II type 1 receptor (AT(1)R) mRNA expression (real-time PCR) determined at 6 mo. Restricted-on-restricted male offspring developed hypertension (+16 mmHg) by 20 wk together with a nephron deficit (-26%) and glomerular hypertrophy (P < 0.05). In contrast, providing a normal lactational environment to restricted offspring improved postnatal growth and prevented the nephron deficit and hypertension. Reduced-on-restricted pups that were born of normal weight but with impaired growth during lactation subsequently grew faster, developed hypertension (+16 mmHg), had increased AT(1A)R and AT(1B)R mRNA expression (P < 0.05), but had no nephron deficit. Our study identifies the prenatal and postnatal nutritional environments in the programming of adult hypertension, associated with distinct renal changes. It is shown for the first time that a prenatally induced nephron deficit can be restored by correcting growth restriction during lactation.

Growth restriction before or after birth reduces nephron number and increases blood pressure in male rats

Impaired growth in utero predicts a low nephron number and high blood pressure later in life as does slowed or accelerated growth after a normal birth weight. We measured the effects of early postnatal growth restriction, with or without prenatal growth restriction, on blood pressure and nephron number in male rat offspring. Bilateral uterine artery and vein ligation were performed to induce uteroplacental insufficiency (Restricted) on day 18 of pregnancy. Postnatal growth restriction was induced in a subset of sham operated control animals by reducing the number of pups at birth to that of the Restricted group (Reduced Litter). Compared to Controls, Restricted pups were born smaller while Reduced Litter pups weighed less by postnatal day 3 and both groups remained lighter throughout lactation. By 10 weeks of age all animals were of similar weight but the Reduced Litter rats had elevated blood pressure. At 22 weeks, Restricted but not Reduced Litter offspring were smaller and the blood pressure was increased in both groups. Restricted and Reduced Litter groups had fewer glomeruli and greater left ventricular mass than Controls. These results suggest that restriction of both perinatal and early postnatal growth increase blood pressure in male offspring. This study also demonstrates that the early postnatal period is a critical time for nephron endowment in the rat.

Uteroplacental insufficiency causes a nephron deficit, modest renal insufficiency but no hypertension with ageing in female rats

In rats, uteroplacental insufficiency induced by uterine vessel ligation restricts fetal growth and impairs mammary development compromising postnatal growth. In male offspring, this results in a nephron deficit and hypertension which can be reversed by improving lactation and postnatal growth. Here, growth, blood pressure and nephron endowment in female offspring from mothers which underwent bilateral uterine vessel ligation (Restricted) on day 18 of pregnancy were examined. Sham surgery (Control) and a reduced litter group (Reduced at birth to 5, equivalent to Restricted group) were used as controls. Offspring (Control, Reduced, Restricted) were cross‐fostered on postnatal day 1 onto a Control (normal lactation) or Restricted (impaired lactation) mother. Restricted‐on‐Restricted offspring were born small but were of similar weight to Control‐on‐Control by postnatal day 35. Blood pressure was not different between groups at 8, 12 or 20 weeks of age. Glomerular number was reduced in Restricted‐on‐Restricted offspring at 6 months without glomerular hypertrophy. Cross‐fostering a Restricted pup onto a Control dam resulted in a glomerular number intermediate between Control‐on‐Control and Restricted‐on‐Restricted. Blood pressure, along with renal function, morphology and mRNA expression, was examined in Control‐on‐Control and Restricted‐on‐Restricted females at 18 months. Restricted‐on‐Restricted offspring did not become hypertensive but developed glomerular hypertrophy by 18 months. They had elevated plasma creatinine and alterations in renal mRNA expression of transforming growth factor‐β1, collagen IV (α1) and matrix matelloproteinase‐9. This suggests that perinatally growth restricted female offspring may be susceptible to onset of renal injury and renal insufficiency with ageing in the absence of concomitant hypertension.

Improved lactational nutrition and postnatal growth ameliorates impairment of glucose tolerance by uteroplacental insufficiency in male rat offspring

Intrauterine growth restriction and accelerated postnatal growth predict increased risk of diabetes. Uteroplacental insufficiency in the rat restricts fetal growth but also impairs mammary development and postnatal growth. We used cross fostering to compare the influence of prenatal and postnatal nutritional restraint on adult glucose tolerance, insulin secretion, insulin sensitivity, and hypothalamic neuropeptide Y content in Wistar Kyoto rats at 6 months of age. Bilateral uterine vessel ligation (restricted) to induce uteroplacental insufficiency or sham surgery (control) was performed on d-18 gestation. Control, restricted, and reduced (reducing litter size of controls to match restricted) pups were cross fostered onto a control or restricted mother 1 d after birth. Restricted pups were born small compared with controls. Restricted males, but not females, remained lighter up to 6 months, regardless of postnatal environment. By 10 wk, restricted-on-restricted males ate more than controls. At 6 months restricted-on-restricted males had increased hypothalamic neuropeptide Y content compared with other groups, and together with reduced-on-restricted males had increased retroperitoneal fat weight (percent body weight) compared with control-on-controls. Restricted-on-restricted males had impaired glucose tolerance, reduced first-phase insulin secretion, but unaltered insulin sensitivity, compared with control-on-controls. In males, being born small and exposed to an impaired lactational environment adversely affects adult glucose tolerance and first-phase insulin secretion, but improving lactation partially ameliorates this condition. This study identifies early life as a target for intervention to prevent later diabetes after prenatal restraint.

Glycemic memory associated epigenetic changes

It is evident that metabolic memory, whereby diabetic complications continue to develop and progress in individuals who returned to normal glycemic control after a period of transient hyperglycemia, can have long lasting effects. We have primary findings that transient hyperglycemia causes profound transcriptional changes in vascular endothelial cells. We hypothesized that ambient hyperglycemia triggers gene-activating events of the NFκB p65 promoter that are mediated by changes in epigenetic modifications. In a follow-up study we identified two histone-specific writing and erasing enzymes involved in the underlying regulation of gene expression during transient hyperglycemia and subsequent return to normoglycemia. Experimental evidence indicates that previous hyperglycemia is associated with persistent expression of the NFκB p65 gene, which activates NFκB-dependent proteins, such as MCP-1, which are implicated in diabetes-associated vascular injury. Increased gene transcription is correspondent with H3K4m1, but not H3K4m2 and H3K4m3, on the NFκB p65 gene. In vascular endothelial cells the histone methyltransferase Set7 can write the mono-methylation mark H3K4m1 and this methyl-writing enzyme is recruited as a gene co-activator in response to glucose. Furthermore, Set7 knockdown prevents glucose-induced p65 expression. We hypothesize that these molecular events represent an integrated response of the epigenome that lead to changes in the expression of genes and proteins that regulate the development and progression of diabetic vascular complications. Further characterisation of these glucose-induced epigenetic events and the identification of key enzymes involved will improve our understanding of the pathways implicated in diabetic vascular injury.

Uteroplacental insufficiency and reducing litter size alters skeletal muscle mitochondrial biogenesis in a sex-specific manner in the adult rat

Uteroplacental insufficiency has been shown to impair insulin action and glucose homeostasis in adult offspring and may act in part via altered mitochondrial biogenesis and lipid balance in skeletal muscle. Bilateral uterine vessel ligation to induce uteroplacental insufficiency in offspring (Restricted) or sham surgery was performed on day 18 of gestation in rats. To match the litter size of Restricted offspring, a separate cohort of sham litters had litter size reduced to five at birth (Reduced Litter), which also restricted postnatal growth. Remaining litters from sham mothers were unaltered (Control). Offspring were studied at 6 mo of age. In males, both Restricted and Reduced Litter offspring had reduced gastrocnemius PPARgamma coactivator-1alpha (PGC-1alpha) mRNA and protein, and mitochondrial transcription factor A (mtTFA) and cytochrome oxidase (COX) III mRNA (P < 0.05), whereas only Restricted had reduced skeletal muscle COX IV mRNA and protein and glycogen (P < 0.05), despite unaltered glucose tolerance, homeostasis model assessment (HOMA) and intramuscular triglycerides. In females, only gastrocnemius mtTFA mRNA was lower in Reduced Litter offspring (P < 0.05). Furthermore, glucose tolerance was not altered in any female offspring, although HOMA and intramuscular triglycerides increased in Restricted offspring (P < 0.05). It is concluded that restriction of growth due to uteroplacental insufficiency alters skeletal muscle mitochondrial biogenesis and metabolic characteristics, such as glycogen and lipid levels, in a sex-specific manner in the adult rat in the absence of impaired glucose tolerance. Furthermore, an adverse postnatal environment induced by reducing litter size also restricts growth and alters skeletal muscle mitochondrial biogenesis and metabolic characteristics in the adult rat.

Short-term exercise training early in life restores deficits in pancreatic β-cell mass associated with growth restriction in adult male rats

Fetal growth restriction is associated with reduced pancreatic β-cell mass, contributing to impaired glucose tolerance and diabetes. Exercise training increases β-cell mass in animals with diabetes and has long-lasting metabolic benefits in rodents and humans. We studied the effect of exercise training on islet and β-cell morphology and plasma insulin and glucose, following an intraperitoneal glucose tolerance test (IPGTT) in juvenile and adult male Wistar-Kyoto rats born small. Bilateral uterine vessel ligation performed on day 18 of pregnancy resulted in Restricted offspring born small compared with sham-operated Controls and also sham-operated Reduced litter offspring that had their litter size reduced to five pups at birth. Restricted, Control, and Reduced litter offspring remained sedentary or underwent treadmill running from 5 to 9 or 20 to 24 wk of age. Early life exercise increased relative islet surface area and β-cell mass across all groups at 9 wk, partially restoring the 60-68% deficit (P < 0.05) in Restricted offspring. Remarkably, despite no further exercise training after 9 wk, β-cell mass was restored in Restricted at 24 wk, while sedentary littermates retained a 45% deficit (P = 0.05) in relative β-cell mass. Later exercise training also restored Restricted β-cell mass to Control levels. In conclusion, early life exercise training in rats born small restored β-cell mass in adulthood and may have beneficial consequences for later metabolic health and disease.

Effects of High-Density Lipoprotein Elevation With Cholesteryl Ester Transfer Protein Inhibition on Insulin Secretion

Rationale: High-density lipoprotein cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which also may have relevance for type 2 diabetes mellitus. Objective: The current study assessed the effects of a CETP inhibitor on postprandial insulin, ex vivo insulin secretion, and cholesterol efflux from pancreatic &bgr;-cells. Methods and Results: Healthy participants received a daily dose of CETP inhibitor (n=10) or placebo (n=15) for 14 days in a randomized double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 &bgr;-cells were determined after incubation with treated plasma. CETP inhibition increased plasma high-density lipoprotein cholesterol, apolipoprotein AI, and postprandial insulin. MIN6N8 &bgr;-cells incubated with plasma from CETP inhibitor–treated individuals (compared with placebo) exhibited an increase in both glucose-stimulated insulin secretion and cholesterol efflux over the 14-day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo &bgr;-cell glucose-stimulated insulin secretion, potentially via enhanced &bgr;-cell cholesterol efflux.Rationale: High-density lipoprotein (HDL) cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which may also have relevance for type 2 diabetes. Objective: The current study assessed the effects of a CETP inhibitor (CETPi) on postprandial insulin, ex vivo insulin secretion and cholesterol efflux from pancreatic β-cells. Methods and Results: Healthy participants received a daily dose of CETPi (n=10) or placebo (n=15) for 14 days in a randomized, double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 β-cells was determined following incubation with treated plasma. CETP inhibition increased plasma HDL cholesterol, apoAI and postprandial insulin. MIN6N8 β-cells incubated with plasma from CETPi-treated individuals (vs placebo) exhibited an increase in both glucose-stimulated insulin secretion (GSIS) and cholesterol efflux over the 14 day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo β-cell GSIS, potentially via enhanced β-cell cholesterol efflux.

Normal lactational environment restores cardiomyocyte number after uteroplacental insufficiency: implications for the preterm neonate

A reduced complement of cardiomyocytes in early life can adversely affect life-long cardiac functional reserve. In the present study, using a cross-fostering approach in rats, we examined the contributions of the prenatal and postnatal environments in the programming of cardiomyocyte growth. Rat dams underwent either bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation. One day after birth, Control and Restricted pups were cross-fostered onto Control (normal lactation) or Restricted (impaired lactation due to impaired mammary gland formation) mothers. In male offspring, genes involved in cardiomyocyte differentiation, proliferation, hypertrophy and apoptosis were examined at gestational day 20 and postnatal days 1 and 7 to assess effects on cardiomyocyte growth. At postnatal day 7 cardiomyocyte number was determined stereologically. Offspring were examined at age 6 mo for evidence of hypertension and pathological cardiac gene expression. There was an increase in Igf1 and Igf2 mRNA expression in hearts of Restricted pups at gestational day 20. At postnatal day 7, Agtr1a and Agtr1b mRNA expression as well as Bcl2 and Cmyc were elevated in all hearts from offspring that were prenatally or postnatally growth restricted. There was a significant reduction (-29%) in cardiomyocyte number in the Restricted-on-Restricted group. Importantly, this deficit was prevented by optimization of postnatal nutrition (in the Restricted-on-Control group). At 6 mo, blood pressure was significantly elevated in the Restricted-on-Restricted group, but there was no difference in expression of the cardiac hypertrophy, remodeling or angiogenic genes across groups. In conclusion, the findings reveal a critical developmental window, when cardiomyocytes are still proliferating, whereby improved neonatal nutrition has the capacity to restore cardiomyocyte number to normal levels. These findings are of particular relevance to the preterm infant who is born at a time when cardiomyocytes are immature and still dividing.

Effects of HDL Elevation with CETP Inhibition on Insulin Secretion

Rationale: High-density lipoprotein cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which also may have relevance for type 2 diabetes mellitus. Objective: The current study assessed the effects of a CETP inhibitor on postprandial insulin, ex vivo insulin secretion, and cholesterol efflux from pancreatic &bgr;-cells. Methods and Results: Healthy participants received a daily dose of CETP inhibitor (n=10) or placebo (n=15) for 14 days in a randomized double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 &bgr;-cells were determined after incubation with treated plasma. CETP inhibition increased plasma high-density lipoprotein cholesterol, apolipoprotein AI, and postprandial insulin. MIN6N8 &bgr;-cells incubated with plasma from CETP inhibitor–treated individuals (compared with placebo) exhibited an increase in both glucose-stimulated insulin secretion and cholesterol efflux over the 14-day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo &bgr;-cell glucose-stimulated insulin secretion, potentially via enhanced &bgr;-cell cholesterol efflux.Rationale: High-density lipoprotein (HDL) cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which may also have relevance for type 2 diabetes. Objective: The current study assessed the effects of a CETP inhibitor (CETPi) on postprandial insulin, ex vivo insulin secretion and cholesterol efflux from pancreatic β-cells. Methods and Results: Healthy participants received a daily dose of CETPi (n=10) or placebo (n=15) for 14 days in a randomized, double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 β-cells was determined following incubation with treated plasma. CETP inhibition increased plasma HDL cholesterol, apoAI and postprandial insulin. MIN6N8 β-cells incubated with plasma from CETPi-treated individuals (vs placebo) exhibited an increase in both glucose-stimulated insulin secretion (GSIS) and cholesterol efflux over the 14 day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo β-cell GSIS, potentially via enhanced β-cell cholesterol efflux.