Decreased Pyruvate but Not Fatty Acid Driven Mitochondrial Respiration in Skeletal Muscle of Growth Restricted Fetal Sheep.

Fetuses with intrauterine growth restriction (FGR) have impaired oxidative and energy metabolism, with persistent consequences on their postnatal development. In this study, we test the hypothesis that FGR skeletal muscle has lower mitochondrial respiration rate and alters the transcriptomic profiles associated with energy metabolism in an ovine model. At late gestation, mitochondrial oxygen consumption rates (OCRs) and transcriptome profiles were evaluated in the skeletal muscle collected from FGR and control fetuses. The ex vivo mitochondrial OCRs were reduced (p < 0.01) in permeabilized FGR soleus muscle compared to the control muscle but only with pyruvate as the metabolic substrate. Mitochondrial OCRs were similar between the FGR and control groups for palmitoyl-carnitine (fatty acid-driven) or pyruvate plus palmitoyl-carnitine metabolic substrates. A total of 2284 genes were differentially expressed in the semitendinosus muscle from growth restricted fetuses (false discovery rate (FDR) ≤ 0.05). A pathway analysis showed that the upregulated genes (FGR compared to control) were overrepresented for autophagy, HIF-1, AMPK, and FOXO signaling pathways (all with an FDR < 0.05). In addition, the expression of genes modulating pyruvate's entry into the TCA cycle was downregulated, whereas the genes encoding key fatty acid oxidation enzymes were upregulated in the FGR muscle. These findings show that FGR skeletal muscle had attenuated mitochondrial pyruvate oxidation, possibly associated with the inability of pyruvate to enter into the TCA cycle, and that fatty acid oxidation might compensate for the attenuated energy metabolism. The current study provided phenotypic and molecular evidence for adaptive deficiencies in FGR skeletal muscle.

Elevated Norepinephrine Stimulates Adipocyte Hyperplasia in Ovine Fetuses With Placental Insufficiency and IUGR.

Prevailing hypoxemia and hypoglycemia in near-term fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR) chronically increases norepinephrine concentrations, which lower adrenergic sensitivity and lipid mobilization postnatally, indicating a predisposition for adiposity. To determine adrenergic-induced responses, we examined the perirenal adipose tissue transcriptome from IUGR fetuses with or without hypercatecholaminemia. IUGR was induced in sheep with maternal hyperthermia, and hypercatecholaminemia in IUGR was prevented with bilateral adrenal demedullation. Adipose tissue was collected from sham-operated control (CON) and IUGR fetuses and adrenal-demedullated control (CAD) and IUGR (IAD) fetuses. Norepinephrine concentrations were lower in IAD fetuses than in IUGR fetuses despite both being hypoxemic and hypoglycemic. In IUGR fetuses, perirenal adipose tissue mass relative to body mass was greater compared with the CON, adrenal-demedullated control, and IAD groups. Transcriptomic analysis identified 581 differentially expressed genes (DEGs) in CON vs IUGR adipose tissue and 193 DEGs in IUGR vs IAD adipose tissue. Integrated functional analysis of these 2 comparisons showed enrichment for proliferator-activated receptor signaling and metabolic pathways and identified adrenergic responsive genes. Within the adrenergic-regulated DEGs, we identified transcripts that regulate adipocyte proliferation and differentiation: adipogenesis regulatory factor, C/CCAAT/enhancer binding protein α, and sterol carrier protein 2. DEGs associated with the metabolic pathway included pyruvate dehydrogenase kinase 4, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4, IGF-binding proteins (IGFBP-5 and IGFBP-7). Sex-specific expression differences were also found for adipogenesis regulatory factor, pyruvate dehydrogenase kinase 4, IGFBP5, and IGFBP7. These findings indicate that sustained adrenergic stimulation during IUGR leads to adipocyte hyperplasia with alterations in metabolism, proliferation, and preadipocyte differentiation pathways.

Prenatal Oxygen and Glucose Therapy Normalizes Insulin Secretion and Action in Growth-Restricted Fetal Sheep.

Placental insufficiency (PI) lowers fetal oxygen and glucose concentrations, which disrupts glucose-insulin homeostasis and promotes fetal growth restriction (FGR). To date, prenatal treatments for FGR have not attempted to correct the oxygen and glucose supply simultaneously. Therefore, we investigated whether a 5-day correction of oxygen and glucose concentrations in PI-FGR fetuses would normalize insulin secretion and glucose metabolism. Experiments were performed in near-term FGR fetal sheep with maternal hyperthermia-induced PI. Fetal arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction and glucose was infused into FGR fetuses (FGR-OG). FGR-OG fetuses were compared with maternal air insufflated, saline-infused fetuses (FGR-AS) and control fetuses. Prior to treatment, FGR fetuses were hypoxemic and hypoglycemic and had reduced glucose-stimulated insulin secretion (GSIS). During treatment, oxygen, glucose, and insulin concentrations increased, and norepinephrine concentrations decreased in FGR-OG fetuses, whereas FGR-AS fetuses were unaffected. On treatment day 4, glucose fluxes were measured with euglycemic and hyperinsulinemic-euglycemic clamps. During both clamps, rates of glucose utilization and production were greater in FGR-AS than FGR-OG fetuses, while glucose fluxes in FGR-OG fetuses were not different than control rates. After 5 days of treatment, GSIS increased in FGR-OG fetuses to control levels and their ex vivo islet GSIS was greater than FGR-AS islets. Despite normalization in fetal characteristics, GSIS, and glucose fluxes, FGR-OG and FGR-AS fetuses weighed less than controls. These findings show that sustained, simultaneous correction of oxygen and glucose normalized GSIS and whole-body glucose fluxes in PI-FGR fetuses after the onset of FGR.

Augmented glucose production is not contingent on high catecholamines in fetal sheep with IUGR.

Fetuses with intrauterine growth restriction (IUGR) have high concentrations of catecholamines, which lowers the insulin secretion and glucose uptake. Here, we studied the effect of hypercatecholaminemia on glucose metabolism in sheep fetuses with placental insufficiency-induced IUGR. Norepinephrine concentrations are elevated throughout late gestation in IUGR fetuses but not in IUGR fetuses with a bilateral adrenal demedullation (IAD) at 0.65 of gestation. Euglycemic (EC) and hyperinsulinemic-euglycemic (HEC) clamps were performed in control, intact-IUGR, and IAD fetuses at 0.87 of gestation. Compared to controls, basal oxygen, glucose, and insulin concentrations were lower in IUGR groups. Norepinephrine concentrations were five-fold higher in IUGR fetuses than in IAD fetuses. During the EC, rates of glucose entry (GER, umbilical + exogenous), glucose utilization (GUR), and glucose oxidation (GOR) were greater in IUGR groups than in controls. In IUGR and IAD fetuses with euglycemia and euinsulinemia, glucose production rates (GPR) remained elevated. During the HEC, GER and GOR were not different among groups. In IUGR and IAD fetuses, GURs were 40% greater than in controls, which paralleled the sustained GPR despite hyperinsulinemia. Glucose-stimulated insulin concentrations were augmented in IAD fetuses compared to IUGR fetuses. Fetal weights were not different between IUGR groups but were less than controls. Regardless of norepinephrine concentrations, IUGR fetuses not only develop greater peripheral insulin sensitivity for glucose utilization but also develop hepatic insulin resistance because GPR was maintained and unaffected by euglycemia or hyperinsulinemia. These findings show that adaptation in glucose metabolism of IUGR fetuses are independent of catecholamines, which implicate that hypoxemia and hypoglycemia cause the metabolic responses.

Chronically elevated norepinephrine concentrations lower glucose uptake in fetal sheep.

Fetal conditions associated with placental insufficiency and intrauterine growth restriction (IUGR) chronically elevate plasma norepinephrine (NE) concentrations. Our objective was to evaluate the effects of chronically elevated NE on insulin-stimulated glucose metabolism in normally grown, non-IUGR fetal sheep, which are independent of other IUGR-related reductions in nutrients and oxygen availability. After surgical placement of catheters, near-term fetuses received either a saline (control) or NE intravenous infusion with controlled euglycemia. In NE fetuses, plasma NE concentrations were 5.5-fold greater than controls, and fetal euglycemia was maintained with a maternal insulin infusion. Insulin secretion was blunted in NE fetuses during an intravenous glucose tolerance test. Weight-specific fluxes for glucose were measured during a euinsulinemic-euglycemic clamp (EEC) and a hyperinsulinemic-euglycemic clamp (HEC). Plasma glucose and insulin concentrations were not different between groups within each clamp, but insulin concentrations increased 10-fold between the EEC and the HEC. During the EEC, rates of glucose uptake (umbilical uptake + exogenous infusion) and glucose utilization were 47% and 35% lower (P < 0.05) in NE fetuses compared with controls. During the HEC, rates of glucose uptake were 28% lower (P < 0.05) in NE fetuses than controls. Glucose production was undetectable in either group, and glucose oxidation was unaffected by the NE infusion. These findings indicate that chronic exposure to high plasma NE concentrations lowers rates of net glucose uptake in the fetus without affecting glucose oxidation rates or initiating endogenous glucose production. Lower fetal glucose uptake was independent of insulin, which indicates insulin resistance as a consequence of chronically elevated NE.

Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction.

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower hindlimb oxygen consumption rates (OCRs), indicating depressed mitochondrial oxidative phosphorylation capacity in their skeletal muscle. We hypothesized that OCRs are lower in skeletal muscle mitochondria from IUGR fetuses, due to reduced electron transport chain (ETC) activity and lower abundances of tricarboxylic acid (TCA) cycle enzymes. IUGR sheep fetuses (n = 12) were created with mid-gestation maternal hyperthermia and compared with control fetuses (n = 12). At 132 ± 1 days of gestation, biceps femoris muscles were collected, and the mitochondria were isolated. Mitochondria from IUGR muscle have 47% lower State 3 (Complex I-dependent) OCRs than controls, whereas State 4 (proton leak) OCRs were not different between groups. Furthermore, Complex I, but not Complex II or IV, enzymatic activity was lower in IUGR fetuses compared with controls. Proteomic analysis (n = 6/group) identified 160 differentially expressed proteins between groups, with 107 upregulated and 53 downregulated mitochondria proteins in IUGR fetuses compared with controls. Although no differences were identified in ETC subunit protein abundances, abundances of key TCA cycle enzymes [isocitrate dehydrogenase (NAD+) 3 noncatalytic subunit ß (IDH3B), succinate-CoA ligase ADP-forming subunit-ß (SUCLA2), and oxoglutarate dehydrogenase (OGDH)] were lower in IUGR mitochondria. IUGR mitochondria had a greater abundance of a hypoxia-inducible protein, NADH dehydrogenase 1α subcomplex 4-like 2, which is known to incorporate into Complex I and lower Complex I-mediated NADH oxidation. Our findings show that mitochondria from IUGR skeletal muscle adapt to hypoxemia and hypoglycemia by lowering Complex I activity and TCA cycle enzyme concentrations, which together, act to lower OCR and NADH production/oxidation in IUGR skeletal muscle.

Postnatal ß2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle.

KEY POINTS: Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity. Although whole-body glucose clearance is normal, 1-month-old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation. Impaired glucose-stimulated insulin secretion in IUGR lambs is due to lower intra-islet insulin availability and not from glucose sensing. We investigated adrenergic receptor (ADR) ß2 desensitization by administering oral ADRß modifiers for the first month after birth to activate ADRß2 and antagonize ADRß1/3. In IUGR lambs ADRß2 activation increased whole-body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies. IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose-stimulated insulin secretion and insulin-stimulated glucose oxidation, providing new insights into potential mechanisms for this risk. ABSTRACT: Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated ß adrenergic receptor (ADRß) desensitization. Our objectives were to measure insulin-sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRß2 agonist and ADRß1/ß3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose-stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR-AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole-body GUR were not different from controls. Of importance, ADRß2 stimulation with ß1/ß3 inhibition increases both insulin sensitivity and whole-body glucose utilization in IUGR lambs. In IUGR and IUGR-AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR-AR skeletal muscle than in controls but GLUT1 was greater in IUGR-AR. ADRß2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR-AR lambs heart rates were greater, which was independent of cardiac ADRß1 activation. We conclude that targeted ADRß2 stimulation improved whole-body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch-up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.

Increased pyruvate dehydrogenase activity in skeletal muscle of growth-restricted ovine fetuses.

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower fractional rates of glucose oxidation and greater gluconeogenesis, indicating lactate shuttling between skeletal muscle and liver. Suppression of pyruvate dehydrogenase (PDH) activity was proposed because of greater pyruvate dehydrogenase kinase (PDK) 4 and PDK1 mRNA concentrations in IUGR muscle. Although PDK1 and PDK4 inhibit PDH activity to reduce pyruvate metabolism, PDH protein concentrations and activity have not been examined in skeletal muscle from IUGR fetuses. Therefore, we evaluated the protein concentrations and activity of PDH and the kinases and phosphatases that regulate PDH phosphorylation status in the semitendinosus muscle from placenta insufficiency-induced IUGR sheep fetuses and control fetuses. Immunoblots were performed for PDH, phosphorylated PDH (E1α), PDK1, PDK4, and pyruvate dehydrogenase phosphatase 1 and 2 (PDP1 and PDP2, respectively). Additionally, the PDH, lactate dehydrogenase (LDH), and citrate synthase (CS) enzymatic activities were measured. Phosphorylated PDH concentrations were 28% lower (P < 0.01) and PDH activity was 67% greater (P < 0.01) in IUGR fetal muscle compared with control. PDK1, PDK4, PDP1, PDP2, and PDH concentrations were not different between groups. CS and LDH activities were also unaffected. Contrary to the previous speculation, PDH activity was greater in skeletal muscle from IUGR fetuses, which parallels lower phosphorylated PDH. Therefore, greater expression of PDK1 and PDK4 mRNA did not translate to greater PDK1 or PDK4 protein concentrations or inhibition of PDH as proposed. Instead, these findings show greater PDH activity in IUGR fetal muscle, which indicates that alternative regulatory mechanisms are responsible for lower pyruvate catabolism.

Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling.

BACKGROUND: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. METHODS: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of ß-cell function and RNA sequencing to elucidate transcriptomic changes. RESULTS: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. CONCLUSIONS: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved ß cell function.

Chronic Adrenergic Signaling Causes Abnormal RNA Expression of Proliferative Genes in Fetal Sheep Islets.

Intrauterine growth restriction (IUGR) increases the risk of developing diabetes in later life, which indicates developmental programming of islets. IUGR fetuses with placental insufficiency develop hypoxemia, elevating epinephrine and norepinephrine (NE) concentrations throughout late gestation. To isolate the programming effects of chronically elevated catecholamines, NE was continuously infused into normally grown sheep fetuses for 7 days. High plasma NE concentrations suppress insulin, but after the NE infusion was terminated, persistent hypersecretion of insulin occurred. Our objective was to identify differential gene expression with RNA sequencing (RNAseq) in fetal islets after chronic adrenergic stimulation. After determining the NE-regulated genes, we identified the subset of differentially expressed genes that were common to both islets from NE fetuses and fetuses with IUGR to delineate the adrenergic-induced transcriptional responses. A portion of these genes were investigated in mouse insulinoma (Min6) cells chronically treated with epinephrine to better approximate the ß-cell response. In islets from NE fetuses, RNAseq identified 321 differentially expressed genes that were overenriched for metabolic and hormone processes, and the subset of 96 differentially expressed genes common to IUGR islets were overenriched for protein digestion, vitamin metabolism, and cell replication pathways. Thirty-eight of the 96 NE-regulated IUGR genes changed similarly between models with functional enrichment for proliferation. In Min6 cells, chronic epinephrine stimulation slowed proliferation and augmented insulin secretion after treatment. These data establish molecular mechanisms underlying persistent adrenergic stimulation in hyperfunctional fetal islets and identify a subset of genes dysregulated by catecholamines in IUGR islets that may represent programming of ß-cell proliferation capacity.

RNA Sequencing Exposes Adaptive and Immune Responses to Intrauterine Growth Restriction in Fetal Sheep Islets.

The risk of type 2 diabetes is increased in children and adults who exhibited fetal growth restriction. Placental insufficiency and intrauterine growth restriction (IUGR) are common obstetrical complications associated with fetal hypoglycemia and hypoxia that reduce the ß-cell mass and insulin secretion. In the present study, we have defined the underlying mechanisms of reduced growth and proliferation, impaired metabolism, and defective insulin secretion previously established as complications in islets from IUGR fetuses. In an IUGR sheep model that recapitulates human IUGR, high-throughput RNA sequencing showed the transcriptome of islets isolated from IUGR and control sheep fetuses and identified the transcripts that underlie ß-cell dysfunction. Functional analysis expanded mechanisms involved in reduced proliferation and dysregulated metabolism that include specific cell cycle regulators and growth factors and mitochondrial, antioxidant, and exocytotic genes. These data also identified immune responses, wnt signaling, adaptive stress responses, and the proteasome as mechanisms of ß-cell dysfunction. The reduction of immune-related gene expression did not reflect a change in macrophage density within IUGR islets. The present study reports the islet transcriptome in fetal sheep and established processes that limit insulin secretion and ß-cell growth in fetuses with IUGR, which could explain the susceptibility to premature islet failure in adulthood. Islet dysfunction formed by intrauterine growth restriction increases the risk for diabetes.

Intrauterine growth-restricted sheep fetuses exhibit smaller hindlimb muscle fibers and lower proportions of insulin-sensitive Type I fibers near term.

Intrauterine growth restriction (IUGR) reduces muscle mass and insulin sensitivity in offspring. Insulin sensitivity varies among muscle fiber types, with Type I fibers being most sensitive. Differences in fiber-type ratios are associated with insulin resistance in adults, and thus we hypothesized that near-term IUGR sheep fetuses exhibit reduced size and proportions of Type I fibers. Placental insufficiency-induced IUGR fetuses were ∼54% smaller (P < 0.05) than controls and exhibited hypoxemia and hypoglycemia, which contributed to 6.9-fold greater (P < 0.05) plasma norepinephrine and ∼53% lower (P < 0.05) plasma insulin concentrations. IUGR semitendinosus muscles contained less (P < 0.05) myosin heavy chain-I protein (MyHC-I) and proportionally fewer (P < 0.05) Type I and Type I/IIa fibers than controls, but MyHC-II protein concentrations, Type II fibers, and Type IIx fibers were not different. IUGR biceps femoris muscles exhibited similar albeit less dramatic differences in fiber type proportions. Type I and IIa fibers are more responsive to adrenergic and insulin regulation than Type IIx and may be more profoundly impaired by the high catecholamines and low insulin in our IUGR fetuses, leading to their proportional reduction. In both muscles, fibers of each type were uniformly smaller (P < 0.05) in IUGR fetuses than controls, which indicates that fiber hypertrophy is not dependent on type but rather on other factors such as myoblast differentiation or protein synthesis. Together, our findings show that IUGR fetal muscles develop smaller fibers and have proportionally fewer Type I fibers, which is indicative of developmental adaptations that may help explain the link between IUGR and adulthood insulin resistance.

Adrenal Demedullation and Oxygen Supplementation Independently Increase Glucose-Stimulated Insulin Concentrations in Fetal Sheep With Intrauterine Growth Restriction.

In pregnancies complicated by placental insufficiency and intrauterine growth restriction (IUGR), fetal glucose and oxygen concentrations are reduced, whereas plasma norepinephrine and epinephrine concentrations are elevated throughout the final third of gestation. Here we study the effects of chronic hypoxemia and hypercatecholaminemia on ß-cell function in fetal sheep with placental insufficiency-induced IUGR that is produced by maternal hyperthermia. IUGR and control fetuses underwent a sham (intact) or bilateral adrenal demedullation (AD) surgical procedure at 0.65 gestation. As expected, AD-IUGR fetuses had lower norepinephrine concentrations than intact-IUGR fetuses despite being hypoxemic and hypoglycemic. Placental insufficiency reduced fetal weights, but the severity of IUGR was less with AD. Although basal plasma insulin concentrations were lower in intact-IUGR and AD-IUGR fetuses compared with intact-controls, glucose-stimulated insulin concentrations were greater in AD-IUGR fetuses compared with intact-IUGR fetuses. Interestingly, AD-controls had lower glucose- and arginine-stimulated insulin concentrations than intact-controls, but AD-IUGR and AD-control insulin responses were not different. To investigate chronic hypoxemia in the IUGR fetus, arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction. Oxygenation of IUGR fetuses enhanced glucose-stimulated insulin concentrations 3.3-fold in intact-IUGR and 1.7-fold in AD-IUGR fetuses but did not lower norepinephrine and epinephrine concentrations. Together these findings show that chronic hypoxemia and hypercatecholaminemia have distinct but complementary roles in the suppression of ß-cell responsiveness in IUGR fetuses.

A Synthetic Heterobivalent Ligand Composed of Glucagon-Like Peptide 1 and Yohimbine Specifically Targets ß Cells Within the Pancreas.

PURPOSE: ß Cell specificity for a heterobivalent ligand composed of glucagon-like peptide-1 (GLP-1) linked to yohimbine (GLP-1/Yhb) was evaluated to determine its utility as a noninvasive imaging agent. PROCEDURES: Competition binding assays were performed on ßTC3 cells and isolated rat islets. Immunostaining for insulin was used to co-localized intravenously injected Cy5-labeled GLP-1/Yhb in ß cells of Sprague-Dawley rats. Rats were intravenously injected with In-111-labeled GLP-1/Yhb to determine clearance rates and tissue biodistribution. Tissue-specific binding was confirmed by competition with pre-administration of unlabeled GLP-1/Yhb and in Streptozotocin-induced diabetic rats. RESULTS: In ßTC3 cells, high affinity binding of GLP-1/Yhb required interactions with both receptors because monovalent competition or receptor knockdown with RNAi lowered specificity and avidity of the heterobivalent ligand. Binding specificity for isolated islets was 2.6-fold greater than that of acinar tissue or islets pre-incubated with excess unlabeled GLP-1/Yhb. Immunofluorescent localization of Cy5-labeled GLP-1/Yhb was restricted to pancreatic islets. Within 30 min, ~90% of the In-111-labeled GLP-1/Yhb was cleared from blood. Tissue-specific accumulation of radiolabeled ligand was apparent in the pancreas, but not in other tissues within the abdominal imaging field. Pancreas specificity was lost in Streptozotocin-induced diabetic rats. CONCLUSIONS: The GLP-1/Yhb exhibits high specificity for ß cells, rapid blood clearance rates, and low non-specific uptake by other tissues within the abdominal imaging field. These characteristics of GLP-1/Yhb are desirable for application to ß cell imaging in vivo and provide a basis for developing additional multivalent ß cell-specific targeting agents to aid in the management of type 1 diabetes.

Fetal adrenal demedullation lowers circulating norepinephrine and attenuates growth restriction but not reduction of endocrine cell mass in an ovine model of intrauterine growth restriction.

Placental insufficiency is associated with fetal hypoglycemia, hypoxemia, and elevated plasma norepinephrine (NE) that become increasingly pronounced throughout the third trimester and contribute to intrauterine growth restriction (IUGR). This study evaluated the effect of fetal adrenal demedullation (AD) on growth and pancreatic endocrine cell mass. Placental insufficiency-induced IUGR was created by exposing pregnant ewes to elevated ambient temperatures during mid-gestation. Treatment groups consisted of control and IUGR fetuses with either surgical sham or AD at 98 days gestational age (dGA; term = 147 dGA), a time-point that precedes IUGR. Samples were collected at 134 dGA. IUGR-sham fetuses were hypoxemic, hypoglycemic, and hypoinsulinemic, and values were similar in IUGR-AD fetuses. Plasma NE concentrations were ~5-fold greater in IUGR-sham compared to control-sham, control-AD, and IUGR-AD fetuses. IUGR-sham and IUGR-AD fetuses weighed less than controls. Compared to IUGR-sham fetuses, IUGR-AD fetuses weighed more and asymmetrical organ growth was absent. Pancreatic ß-cell mass and α-cell mass were lower in both IUGR-sham and IUGR-AD fetuses compared to controls, however, pancreatic endocrine cell mass relative to fetal mass was lower in IUGR-AD fetuses. These findings indicate that NE, independently of hypoxemia, hypoglycemia and hypoinsulinemia, influence growth and asymmetry of growth but not pancreatic endocrine cell mass in IUGR fetuses.

Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets.

The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.

Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres.

Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.

Reductions in insulin concentrations and ß-cell mass precede growth restriction in sheep fetuses with placental insufficiency.

In pregnancy complicated by placental insufficiency (PI) and intrauterine growth restriction (IUGR), the fetus near term has reduced basal and glucose-stimulated insulin concentrations and reduced ß-cell mass. To determine whether suppression of insulin concentrations and ß-cell mass precedes reductions in fetal weight, which would implicate insulin deficiency as a cause of subsequent IUGR, we measured basal and glucose-stimulated insulin concentrations and pancreatic histology at 0.7 gestation in PI fetuses. Placental weights in the PI pregnancies were 40% lower than controls (265 ± 26 vs. 442 ± 41 g, P < 0.05), but fetal weights were not different. At basal conditions blood oxygen content, plasma glucose concentrations, and plasma insulin concentrations were lower in PI fetuses compared with controls (2.5 ± 0.3 vs. 3.5 ± 0.3 mmol/l oxygen, P < 0.05; 1.11 ± 0.09 vs. 1.44 ± 0.12 mmol/l glucose; 0.12 ± 0.01 vs. 0.27 ± 0.02 ng/ml insulin; P < 0.05). During a steady-state hyperglycemic clamp (~2.5 ± 0.1 mmol/l), glucose-stimulated insulin concentrations were lower in PI fetuses than controls (0.28 ± 0.02 vs. 0.55 ± 0.04 ng/ml; P < 0.01). Plasma norepinephrine concentrations were 3.3-fold higher (P < 0.05) in PI fetuses (635 ± 104 vs. 191 ± 91 pg/ml). Histological examination revealed less insulin area and lower ß-cell mass and rates of mitosis. The pancreatic parenchyma was also less dense (P < 0.01) in PI fetuses, but no differences were found for pancreatic progenitor cells or other endocrine cell types. These findings show that hypoglycemia, hypoxemia, and hypercatecholaminemia are present and potentially contribute to lower insulin concentrations and ß-cell mass due to slower proliferation rates in early third-trimester PI fetuses before discernible reductions in fetal weight.

Hypoxaemia-induced catecholamine secretion from adrenal chromaffin cells inhibits glucose-stimulated hyperinsulinaemia in fetal sheep.

Abstract Hypoxaemia elicits adrenergic suppression of fetal glucose-stimulated hyperinsulinaemia. We postulate that this effect is mediated by catecholamines, exclusively, from fetal adrenal chromaffin cells. To investigate this hypothesis, square-wave hyperglycaemic clamp studies were performed under normoxaemic (26 ± 0.9 mmHg) and hypoxaemic (14 ± 0.3 mmHg) steady-state conditions in near-term fetal sheep that had undergone either surgical sham or bilateral adrenal demedullation (AD), values mentioned are ± SEM. Under normoxaemic conditions plasma noradrenaline concentrations were lower in AD fetuses than in sham-operated fetuses (457 ± 122 versus 1073 ± 103 pg ml(-1), P < 0.05). Plasma insulin concentrations were not different at euglycaemia between shams (0.46 ± 0.07 ng ml(-1)) and AD fetuses (0.44 ± 0.04 ng ml(-1)) and increased (P < 0.05) with hyperglycaemia in both groups although to a lesser extent in AD fetuses (0.94 ± 0.19 ng ml(-1)) compared to shams (1.31 ± 0.15 ng ml(-1); P < 0.05). Hypoxaemia increased plasma adrenaline (26-fold) and noradrenaline (5-fold) in shams but elicited no change in AD fetuses. Under hypoxaemic conditions, euglycaemic plasma insulin concentrations were reduced (P < 0.05) in both sham and AD fetuses to 0.30 ± 0.05 ng ml(-1) and 0.27 ± 0.01 ng ml(-1) respectively, and the insulin response to hyperglycaemia was abolished in shams but not affected in AD fetuses (0.33 ± 0.06 versus 0.73 ± 0.02 ng ml(-1), P < 0.05). Hypoxaemia also induced hyperlactacaemia and hypocarbia to a greater extent in shams than in AD fetuses, indicating that catecholamines potentiate reductions in oxidative metabolism independently of insulin. These findings demonstrate that the fetal adrenal chromaffin cells are the source for acute hypoxaemia-induced elevations in fetal plasma catecholamines and suppression of glucose-stimulated hyperinsulinaemia, but other factors reduce plasma insulin at euglycaemia.

Chronic pulsatile hyperglycemia reduces insulin secretion and increases accumulation of reactive oxygen species in fetal sheep islets.

Children from diabetic pregnancies have a greater incidence of type 2 diabetes. Our objective was to determine if exposure to mild-moderate hyperglycemia, by modeling managed diabetic pregnancies, affects fetal ß-cell function. In sheep fetuses, ß-cell responsiveness was examined after 2 weeks of sustained hyperglycemia with 3 pulses/day, mimicking postprandial excursions, and compared to saline-infused controls (n = 10). Two pulsatile hyperglycemia (PHG) treatments were studied: mild (mPHG, n = 5) with +15% sustained and +55% pulse; and moderate (PHG, n = 10) with +20% sustained and +100% pulse. Fetal glucose-stimulated insulin secretion and glucose-potentiated arginine insulin secretion were lower (P < 0.05) in PHG (0.86 ± 0.13 and 2.91 ± 0.39  ng/ml plasma insulin) but not in mPHG fetuses (1.21 ± 0.08 and 4.25 ± 0.56  ng/ml) compared to controls (1.58 ± 0.25 and 4.51 ± 0.56  ng/ml). Islet insulin content was 35% lower in PHG and 35% higher in mPHG vs controls (P < 0.01). Insulin secretion and maximally stimulated insulin release were also reduced (P < 0.05) in PHG islets due to lower islet insulin content. Isolated PHG islets also had 63% greater (P < 0.01) reactive oxygen species (ROS) accumulation at 11.1  mmol/l glucose than controls (P < 0.01), but oxidative damage was not detected in islet proteins. PHG fetuses showed evidence of oxidative damage to skeletal muscle proteins (P < 0.05) but not insulin resistance. Our findings show that PHG induced dysregulation of islet ROS handling and decreased islet insulin content, but these outcomes are independent. The ß-cell outcomes were dependent on the severity of hyperglycemia because mPHG fetuses had no distinguishable impairments in ROS handling or insulin secretion but greater insulin content.