Alice S Green, Paul J Rozance, and Sean W Limesand
Alice S Green, Paul J Rozance, and Sean W Limesand
Low birth weight is an important risk factor for impaired glucose tolerance and diabetes later in life. One hypothesis is that fetal β-cells inherit a persistent defect as a developmental response to fetal malnutrition, a primary cause of intrauterine growth restriction (IUGR). Our understanding of fetal programing events in the human endocrine pancreas is limited, but several animal models of IUGR extend our knowledge of developmental programing in β-cells. Pathological outcomes such as β-cell dysfunction, impaired glucose tolerance, and diabetes are often observed in adult offspring from these animal models, similar to the associations of low birth weight and metabolic diseases in humans. However, the identified mechanisms underlying β-cell dysfunction across models and species are varied, likely resulting from the different methodologies used to induce experimental IUGR, as well as from intraspecies differences in pancreas development. In this review, we first present the evidence for human β-cell dysfunction being associated with low birth weight or IUGR. We then evaluate relevant animal models of IUGR, focusing on the strengths of each, in order to define critical periods and types of nutrient deficiencies that can lead to impaired β-cell function. These findings frame our current knowledge of β-cell developmental programing and highlight future research directions to clarify the mechanisms of β-cell dysfunction for human IUGR.
Brit H Boehmer, Sean W Limesand, and Paul J Rozance
Placental insufficiency is a primary cause of intrauterine growth restriction (IUGR). IUGR increases the risk of developing type 2 diabetes mellitus (T2DM) throughout life, which indicates that insults from placental insufficiency impair β-cell development during the perinatal period because β-cells have a central role in the regulation of glucose tolerance. The severely IUGR fetal pancreas is characterized by smaller islets, less β-cells, and lower insulin secretion. Because of the important associations among impaired islet growth, β-cell dysfunction, impaired fetal growth, and the propensity for T2DM, significant progress has been made in understanding the pathophysiology of IUGR and programing events in the fetal endocrine pancreas. Animal models of IUGR replicate many of the observations in severe cases of human IUGR and allow us to refine our understanding of the pathophysiology of developmental and functional defects in islet from IUGR fetuses. Almost all models demonstrate a phenotype of progressive loss of β-cell mass and impaired β-cell function. This review will first provide evidence of impaired human islet development and β-cell function associated with IUGR and the impact on glucose homeostasis including the development of glucose intolerance and diabetes in adulthood. We then discuss evidence for the mechanisms regulating β-cell mass and insulin secretion in the IUGR fetus, including the role of hypoxia, catecholamines, nutrients, growth factors, and pancreatic vascularity. We focus on recent evidence from experimental interventions in established models of IUGR to understand better the pathophysiological mechanisms linking placental insufficiency with impaired islet development and β-cell function.
Clyde J. Wright, Sarah McKenna, Robyn De Dios, Brit H. Boehmer, Leanna Nguyen, Sankar Ghosh, Jeryl Sandoval, and Paul J Rozance
The β-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we use streptozotocin (STZ) to injure the β-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8mM, 0-24h) ±TNFα (100ng/mL) and ±IκBβ siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBβ and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBβ expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBβ-/- and wild type (WT) mice were exposed to STZ and monitored for diabetes. The IκBβ-/- mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBβ levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent β-cell injury and the development of diabetes.
Asghar Ali, Callie M Swanepoel, Quinton A Winger, Paul J Rozance, and Russell V Anthony
Chorionic somatomammotropin (CSH) is a placenta-specific hormone associated with fetal growth, and fetal and maternal metabolism in both humans and sheep. We hypothesized that CSH deficiency could impact sheep fetal liver glucose utilization. To generate CSH-deficient pregnancies, day 9 hatched blastocysts were infected with lentiviral particles expressing CSH-specific shRNA (RNAi) or scramble control shRNA (SC) and transferred to synchronized recipients. CSH RNAi generated two distinct phenotypes at 135 days of gestational age (dGA); pregnancies with IUGR (RNAi-IUGR) or with normal fetal weight (RNAi-NW). Fetal body, fetal liver and placental weights were reduced (P < 0.05) only in RNAi-IUGR pregnancies compared to SC. Umbilical artery plasma insulin and insulin-like growth factor 1 (IGF1) concentrations were decreased, whereas insulin receptor beta (INSR) concentration in fetal liver was increased (P < 0.05) in both RNAi phenotypes. The mRNA concentrations of IGF1, IGF2, IGF binding protein 2 (IGFBP2) and IGFBP3 were decreased (P < 0.05) in fetal livers from both RNAi phenotypes. Fetal liver glycogen concentration and glycogen synthase 1 (GYS1) concentration were increased (P < 0.05), whereas fetal liver phosphorylated-GYS (inactive GYS) concentration was reduced (P < 0.05) in both RNAi phenotypes. Lactate dehydrogenase B (LDHB) concentration was increased (P < 0.05) and IGF2 concentration was decreased (P < 0.05) in RNAi-IUGR fetal livers only. Our findings suggest that fetal liver glucose utilization is impacted by CSH RNAi, independent of IUGR, and is likely tied to enhanced fetal liver insulin sensitivity in both RNAi phenotypes. Determining the physiological ramifications of both phenotypes, may help to differentiate direct effect of CSH deficiency or its indirect effect through IUGR.
Brit H Boehmer, Peter R Baker II, Laura D Brown, Stephanie R Wesolowski, and Paul J Rozance
A 9-day infusion of leucine into fetal sheep potentiates fetal glucose-stimulated insulin secretion (GSIS). However, there were accompanying pancreatic structural changes that included a larger proportion of β-cells and increased vascularity. Whether leucine can acutely potentiate fetal GSIS in vivo before these structural changes develop is unknown. The mechanisms by which leucine acutely potentiates GSIS in adult islets and insulin-secreting cell lines are well known. These mechanisms involve leucine metabolism, including leucine oxidation. However, it is not clear if leucine-stimulated metabolic pathways are active in fetal islets. We hypothesized that leucine would acutely potentiate GSIS in fetal sheep and that isolated fetal islets are capable of oxidizing leucine. We also hypothesized that leucine would stimulate other metabolic pathways associated with insulin secretion. In pregnant sheep we tested in vivo GSIS with and without an acute leucine infusion. In isolated fetal sheep islets, we measured leucine oxidation with a [1-14C] l-leucine tracer. We also measured concentrations of other amino acids, glucose, and analytes associated with cellular metabolism following incubation of fetal islets with leucine. In vivo, a leucine infusion resulted in glucose-stimulated insulin concentrations that were over 50% higher than controls (P < 0.05). Isolated fetal islets oxidized leucine. Leucine supplementation of isolated fetal islets also resulted in significant activation of metabolic pathways involving leucine and other amino acids. In summary, acute leucine supplementation potentiates fetal GSIS in vivo, likely through pathways related to the oxidation of leucine and catabolism of other amino acids.
Susan M Soto, Amy C Blake, Stephanie R Wesolowski, Paul J Rozance, Kristen B Barthel, Bifeng Gao, Byron Hetrick, Carrie E McCurdy, Natalia G Garza, William W Hay Jr, Leslie A Leinwand, Jacob E Friedman, and Laura D Brown
Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass and insulin resistance, suggesting muscle growth may be restricted by molecular events that occur during fetal development. To explore the basis of restricted fetal muscle growth, we used a sheep model of progressive placental insufficiency-induced IUGR to assess myoblast proliferation within intact skeletal muscle in vivo and isolated myoblasts stimulated with insulin in vitro. Gastrocnemius and soleus muscle weights were reduced by 25% in IUGR fetuses compared to those in controls (CON). The ratio of PAX7+ nuclei (a marker of myoblasts) to total nuclei was maintained in IUGR muscle compared to CON, but the fraction of PAX7+ myoblasts that also expressed Ki-67 (a marker of cellular proliferation) was reduced by 23%. Despite reduced proliferation in vivo, fetal myoblasts isolated from IUGR biceps femoris and cultured in enriched media in vitro responded robustly to insulin in a dose- and time-dependent manner to increase proliferation. Similarly, insulin stimulation of IUGR myoblasts upregulated key cell cycle genes and DNA replication. There were no differences in the expression of myogenic regulatory transcription factors that drive commitment to muscle differentiation between CON and IUGR groups. These results demonstrate that the molecular machinery necessary for transcriptional control of proliferation remains intact in IUGR fetal myoblasts, indicating that in vivo factors such as reduced insulin and IGF1, hypoxia and/or elevated counter-regulatory hormones may be inhibiting muscle growth in IUGR fetuses.
Eileen I Chang, Paul J Rozance, Stephanie R Wesolowski, Leanna M Nguyen, Steven C Shaw, Robert A Sclafani, Kristen K Bjorkman, Angela K Peter, William W Hay Jr, and Laura D Brown
Intrauterine growth-restricted (IUGR) fetuses are born with reduced skeletal muscle mass. We hypothesized that reduced rates of myogenesis would contribute to fewer and smaller myofibers in IUGR fetal hindlimb muscle compared to the normally growing fetus. We tested this hypothesis in IUGR fetal sheep with progressive placental insufficiency produced by exposing pregnant ewes to elevated ambient temperatures from 38 to 116 days gestation (dGA; term = 147 dGA). Surgically catheterized control (CON, n = 8) and IUGR (n = 13) fetal sheep were injected with intravenous 5-bromo-2′-deoxyuridine (BrdU) prior to muscle collection (134 dGA). Rates of myogenesis, defined as the combined processes of myoblast proliferation, differentiation, and fusion into myofibers, were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. Total myofiber number was determined for the entire cross-section of the FDS muscle. In IUGR fetuses, the number of BrdU+ myonuclei per myofiber cross-section was lower in BF, TA, and FDS (P < 0.05), total myonuclear number per myofiber cross-section was lower in BF and FDS (P < 0.05), and total myofiber number was lower in FDS (P < 0.005) compared to CON. mRNA expression levels of cyclins, cyclin-dependent protein kinases, and myogenic regulatory factors were lower (P < 0.05), and inhibitors of the cell cycle were higher (P < 0.05) in IUGR BF compared to CON. Markers of apoptosis were not different in IUGR BF muscle. These results show that in IUGR fetuses, reduced rates of myogenesis produce fewer numbers of myonuclei, which may limit hypertrophic myofiber growth. Fewer myofibers of smaller size contribute to smaller muscle mass in the IUGR fetus.