Hindlimb Glucose Research Articles

442 Awardee Talk: Adaptive Programming of Poor Skeletal Muscle Growth and Metabolism Associated with Heat Stress-Induced Intrauterine Growth Restriction

Abstract Intrauterine growth restriction (IUGR) is associated with reduced β2 adrenergic sensitivity, which contributes to poor postnatal muscle growth and metabolic inefficiency. Thus, our objective was to determine if stimulating β2 adrenergic activity would rescue deficits in muscle growth, body composition, and indicators of metabolic homeostasis in IUGR offspring. Maternal heat stress (40°C, 35% humidity, THI = 85) from d 40 to 95 of gestation was used to produce IUGR lambs. From birth, IUGR lambs received daily IM injections of saline (IUGR; n = 12) or 0.8 μg/kg clenbuterol HCl (IUGR+CLEN; n = 11). Placebo-injected controls (n = 13) were born to pair-fed thermoneutral ewes. Body composition was estimated by ultrasound and bioelectrical impedance analyses (BIA) at d 58. Lambs were catheterized at d 55 and necropsied at d 60. Square-wave hyperglycemic clamps and indirect calorimetry were performed at d 57. Hyperinsulinemic-euglycemic clamps were performed at d 58. Bodyweights were lighter (P ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls at birth, d 30, and d 60. Average daily gain was less (P ≤ 0.05) for IUGR compared with controls and was intermediate for IUGR+CLEN lambs. BIA-estimated fat-free mass, ultrasound-estimated loin eye area (LEA), actual LEA, and flexor digitorum superficialis (FDS) muscle weight were less (P ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Semitendinosus from IUGR lambs had less (P ≤ 0.05) β2 adrenoreceptor content, fewer (P ≤ 0.05) proliferating myoblasts, tended to have fewer (P = 0.08) differentiated myoblasts, and had smaller (P ≤ 0.05) muscle fibers than controls. Proliferating myoblasts and fiber size but not β2 adrenoreceptor content or differentiated myoblasts were rescued (P ≤ 0.05) in IUGR+CLEN lambs compared with IUGR lambs. Basal plasma insulin did not differ, but glucose-stimulated insulin secretion was 44% less (P ≤ 0.05) for IUGR lambs than for controls and was intermediate for IUGR+CLEN lambs. Basal and insulin-stimulated hindlimb glucose uptake and basal glucose oxidation did not differ among groups. Insulin-stimulated glucose oxidation was 54% less (P ≤ 0.05) for IUGR lambs but not IUGR+CLEN lambs compared with controls. Ex vivo glucose uptake was 25% less (P ≤ 0.05) for FDS from IUGR and IUGR+CLEN lambs than from controls. Ex vivo glucose oxidation was less (P ≤ 0.05) for FDS and soleus from IUGR lambs compared with controls and was intermediate (P ≤ 0.05) for FDS but not soleus from IUGR+CLEN lambs. Whole-body O2 consumption was 22% less (P ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls. Whole-body CO2 production was 17% less (P ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. These findings show that IUGR muscle remained responsive to β2 adrenergic stimulation despite reduced β2 adrenoreceptor content, and thus β2 adrenergic pathways may be a strategic target for improving muscle growth, body composition, and metabolic efficiency in IUGR-born offspring.

Maternofetal inflammation induced for 2 wk in late gestation reduced birth weight and impaired neonatal growth and skeletal muscle glucose metabolism in lambs.

Intrauterine stress impairs growth and metabolism in the fetus and offspring. We recently found that sustained maternofetal inflammation resulted in intrauterine growth-restricted (MI-IUGR) fetuses with asymmetric body composition, impaired muscle glucose metabolism, and β-cell dysfunction near term. These fetuses also exhibited heightened inflammatory tone, which we postulated was a fetal programming mechanism for the IUGR phenotype. Thus, the objective of this study was to determine whether poor growth and metabolism persisted in MI-IUGR lambs after birth. Polypay ewes received serial lipopolysaccharide or saline injections in the first 2 wk of the third trimester of pregnancy to produce MI-IUGR (n = 13) and control (n = 12) lambs, respectively. Lambs were catheterized at 25 d of age. β-Cell function was assessed at 29 d, hindlimb glucose metabolism at 30 d, and daily blood parameters from day 26 to 31. Glucose metabolism was also assessed in flexor digitorum superficialis (FDS) muscle isolated at necropsy on day 31. Asymmetric body composition persisted in MI-IUGR neonates, as these lambs were lighter (P < 0.05) than controls at birth and 31 d, but body and cannon bone lengths did not differ at either age. FDS muscles from MI-IUGR lambs were smaller (P < 0.05) and exhibited reduced (P < 0.05) glucose oxidation and Akt phosphorylation but similar glucose uptake compared with controls when incubated in basal or insulin-spiked media. Similarly, hindlimb glucose oxidation was reduced (P < 0.05) in MI-IUGR lambs under basal and hyperinsulinemic conditions, but hindlimb glucose utilization did not differ from controls. Circulating urea nitrogen and cholesterol were reduced (P < 0.05), and triglycerides, high-density lipoprotein cholesterol, and glucose-to-insulin ratios were increased (P < 0.05) in MI-IUGR lambs. Glucose and insulin concentrations did not differ between groups during basal or hyperglycemic conditions. Although circulating monocyte and granulocyte concentrations were greater (P < 0.05) in MI-IUGR lambs, plasma tumor necrosis factor α (TNFα) was reduced (P < 0.05). FDS muscle contained greater (P < 0.05) TNF receptor 1 (TNFR1) and IκBα protein content. These findings indicate that maternofetal inflammation in late pregnancy results in fetal programming that impairs growth capacity, muscle glucose oxidation, and lipid homeostasis in offspring. Inflammatory indicators measured in this study appear to reflect heightened cytokine sensitivity in muscle and compensatory systemic responses to it.

Postnatal β2 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) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 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. Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin‐sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 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, ADRβ2 stimulation with β1/β3 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. ADRβ2, 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 ADRβ1 activation. We conclude that targeted ADRβ2 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.

Sustained maternal inflammation during the early third-trimester yields intrauterine growth restriction, impaired skeletal muscle glucose metabolism, and diminished β-cell function in fetal sheep1,2.

Maternal inflammation causes fetal intrauterine growth restriction (IUGR), but its impact on fetal metabolism is not known. Thus, our objective was to determine the impact of sustained maternal inflammation in late gestation on fetal inflammation, skeletal muscle glucose metabolism, and insulin secretion. Pregnant ewes were injected every third day from the 100th to 112th day of gestation (term = 150 d) with saline (controls) or lipopolysaccharide (LPS) to induce maternal inflammation and IUGR (MI-IUGR). Fetal femoral blood vessels were catheterized on day 118 to assess β-cell function on day 123, hindlimb glucose metabolic rates on day 124, and daily blood parameters from days 120 to 125. Fetal muscle was isolated on day 125 to assess ex vivo glucose metabolism. Injection of LPS increased (P < 0.05) rectal temperatures, circulating white blood cells, and plasma tumor necrosis factor α (TNFα) concentrations in MI-IUGR ewes. Maternal leukocytes remained elevated (P < 0.05) and TNFα tended to remain elevated (P < 0.10) compared with controls almost 2 wk after the final LPS injection. Total white blood cells, monocytes, granulocytes, and TNFα were also greater (P < 0.05) in MI-IUGR fetuses than controls over this period. MI-IUGR fetuses had reduced (P < 0.05) blood O2 partial pressures and greater (P < 0.05) maternofetal O2 gradients, but blood glucose and maternofetal glucose gradients did not differ from controls. Basal and glucose-stimulated insulin secretion were reduced (P < 0.05) by 32% and 42%, respectively, in MI-IUGR fetuses. In vivo hindlimb glucose oxidation did not differ between groups under resting conditions but was 47% less (P < 0.05) in MI-IUGR fetuses than controls during hyperinsulinemia. Hindlimb glucose utilization did not differ between fetal groups. At day 125, MI-IUGR fetuses were 22% lighter (P < 0.05) than controls and tended to have greater (P < 0.10) brain/BW ratios. Ex vivo skeletal muscle glucose oxidation did not differ between groups in basal media but was less (P < 0.05) for MI-IUGR fetuses in insulin-spiked media. Glucose uptake rates and phosphorylated-to-total Akt ratios were less (P < 0.05) in muscle from MI-IUGR fetuses than controls regardless of media. We conclude that maternal inflammation leads to fetal inflammation, reduced β-cell function, and impaired skeletal muscle glucose metabolism that persists after maternal inflammation ceases. Moreover, fetal inflammation may represent a target for improving metabolic dysfunction in IUGR fetuses.

Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth-restricted fetal sheep.

Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass, which may contribute to insulin resistance and the development of diabetes. We demonstrate slower hindlimb linear growth and muscle protein synthesis rates that match the reduced hindlimb blood flow and oxygen consumption rates in IUGR fetal sheep. These adaptations resulted in hindlimb blood flow rates in IUGR that were similar to control fetuses on a weight-specific basis. Net hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was significantly lower in IUGR fetal sheep. Among all fetuses, blood O2 saturation and plasma glucose, insulin and insulin-like growth factor-1 were positively associated and norepinephrine was negatively associated with hindlimb weight. These results further our understanding of the metabolic and hormonal adaptations to reduced oxygen and nutrient supply with placental insufficiency that develop to slow hindlimb growth and muscle protein accretion. Reduced skeletal muscle mass in the fetus with intrauterine growth restriction (IUGR) persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake and protein accretion rates in skeletal muscle, late gestation control (CON) (n=8) and IUGR (n=13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate and fractional synthetic rate were lower in IUGR compared to CON (P<0.05). Absolute hindlimb blood flow was reduced in IUGR (IUGR: 32.9±5.6mlmin-1 ; CON: 60.9±6.5mlmin-1 ; P<0.005), although flow normalized to hindlimb weight was similar between groups. Hindlimb oxygen consumption rate was lower in IUGR (IUGR: 10.4±1.4μmolmin-1 100g-1 ; CON: 14.7±1.3μmolmin-1 100g-1 ; P<0.05). Hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was lower in IUGR (IUGR: 1.3±0.5μmolmin-1 100g-1 ; CON: 2.9±0.2μmolmin-1 100g-1 ; P<0.05). Blood O2 saturation (r2 =0.80, P<0.0001) and plasma glucose (r2 =0.68, P<0.0001), insulin (r2 =0.40, P<0.005) and insulin-like growth factor (IGF)-1 (r2 =0.80, P<0.0001) were positively associated and norepinephrine (r2 =0.59, P<0.0001) was negatively associated with hindlimb weight. Slower hindlimb linear growth and muscle protein synthesis rates match reduced hindlimb blood flow and oxygen consumption rates in the IUGR fetus. Metabolic adaptations to slow hindlimb growth are probably hormonally-mediated by mechanisms that include increased fetal norepinephrine and reduced IGF-1 and insulin.

Pregnancy augments hepatic glucose storage in response to a mixed meal

Studies were carried out on conscious female non-pregnant (NP) and pregnant (P; third-trimester) dogs (n 16; eight animals per group) to define the role of the liver in mixed meal disposition with arteriovenous difference and tracer techniques. Hepatic and hindlimb substrate disposal was assessed for 390 min during and after an intragastric mixed meal infusion labelled with [¹⁴C]glucose. The P dogs exhibited postprandial hyperglycaemia compared with NP dogs (area under the curve (AUC; change from basal over 390 min) of arterial plasma glucose: 86 680 (sem 12 140) and 187 990 (sem 33 990) mg/l in NP and P dogs, respectively; P < 0·05). Plasma insulin concentrations did not differ significantly between the groups (AUC: 88 230 (sem 16 314) and 69 750 (sem 19 512) pmol/l in NP and P dogs, respectively). Net hepatic glucose uptake totalled 3691 (sem 508) v. 5081 (sem 1145) mg/100 g liver in NP and P dogs, respectively (P = 0·38). The AUC of glucose oxidation by the gut and hindlimb were not different in NP and P dogs, but hepatic glucose oxidation (84 (sem 13) v. 206 (sem 30) mg/100 g liver) and glycogen synthesis (0·4 (sem 0·5) v. 26 (sem 0·7) g/100 g liver) were greater in P dogs (P < 0·05). The proportion of hepatic glycogen deposited via the direct pathway did not differ between the groups. Hindlimb glucose uptake and skeletal muscle glycogen synthesis was similar between the groups, although final glycogen concentrations were higher in NP dogs (9·6 (sem 0·6) v. 70 (sem 0·6) mg/g muscle; P < 0·05). Thus, hepatic glucose oxidation and glycogen storage were augmented in late pregnancy. Enhanced hepatic glycogen storage following a meal probably facilitates the maintenance of an adequate glucose supply to maternal and fetal tissues during the post-absorptive period.

Diet-induced impaired glucose tolerance and gestational diabetes in the dog

Glucose metabolism was compared in dogs consuming a chow/meat diet throughout pregnancy (P group, n = 6) and dogs switched to a high-fat/high-fructose (HFF) diet during the 4th-5th gestational week (gestation ≃9 wk; P-HFF group; n = 6). An oral glucose tolerance test (OGTT; 0.9 g/kg) was administered in the 6th-7th gestational week, and a hyperinsulinemic [0-120 min: 1.8 pmol·kg(-1)·min(-1) (low insulin); 120-240 min: 9 pmol·kg(-1)·min(-1) (high insulin)] euglycemic clamp was performed the following week. Nonpregnant (NP) female dogs underwent OGTTs but not clamp studies. All P-HFF dogs exhibited impaired glucose tolerance (IGT) or gestational diabetes (GDM), but only one P dog had IGT. Insulin concentrations in P and P-HFF dogs were significantly lower than in NP dogs 30 and 60 min after the OGTT. Therefore, mean islet size and area were evaluated in P and NP dogs. These values did not differ between groups, and proliferating endocrine cells were rare in pregnancy. During exposure to high insulin, glucose infusion rate and hindlimb glucose uptake were ∼30% greater (P < 0.05) and net hepatic glucose output was more suppressed (-5.5 ± 6.1 vs. 7.8 ± 2.8 mg·100 g liver(-1)·min(-1), P < 0.05) in P than in P-HFF dogs. In conclusion, in the 2nd trimester the canine pancreas does not exhibit islet hypertrophy, hyperplasia, or neogenesis. Combined with the lack of pancreatic adaptation, a HFF diet during late pregnancy produces a canine model of IGT and GDM without hyperinsulinemia but exhibiting liver and muscle insulin resistance.

Role of adrenergic receptors in the caffeine-induced increase in glucose uptake by the canine hindlimb

The present study was designed to investigate the effects of alpha and beta adrenergic receptor blockers on caffeine-induced increase in canine hindlimb glucose uptake. The study was carried out on fasted male anaesthetized dogs divided into five groups [5 dogs per group]. Each dog was given a bolus injection of normal saline, caffeine [6 mg/kg] caffeine after pretreatment with prazosin [0.2 mg/kg], caffeine after pretreatment with propranolol [0.5 mg/kg] or caffeine after pretreatment with a combination of prazosin [0.2 mg/kg] and propranolol [0.5 mg/kg]. The experiments were carried out under resting and exercising conditions of the hindlimb. Hindlimb glucose uptake [HGU] was calculated as the product of blood flow and arterio-venous glucose difference. Blood glucose was determined by the glucose oxidase method and blood flow to the hindlimb was determined by time-collection method. The results showed that pretreatment of the animal with either prazosin or propranolol significantly reduced caffeine-induced hyperglycemia, glucose extraction and hindlimb glucose uptake at rest. The two blockers also separately reduced caffeine-induced hyperglycemia during contraction of the hindlimb. Prazosin or propranolol did not however influence the effect of caffeine on glucose extraction and hindlimb glucose uptake during contraction of the hindlimb. It was therefore concluded that alpha and beta adrenergic receptors are involved in caffeine induced responses at rest and not during hindlimb contraction.

Effects Of Caffeine And Ethanolic Extract Of Kolanut On Glucose Uptake In The Canine Hindlimb At Rest And During Contraction

The study investigated the effects of caffeine and ethanolic extract of kolanut (EEK) on glucose uptake in the canine hindlimb at rest and during contraction. Thirty male anaesthetized mongrel dogs [11-13 kg] were divided into six groups [5 dogs/group] Caffeine [6 mg/kg], EEK [5 mg/kg], or normal saline [control] was administered intravenously to each group at rest. Arterial and venous blood samples were collected at 0, 5, 10, 15, 20, 25, 30, 45, 60, 75 and 90 minutes after drug administration. Blood glucose was measured by glucose oxidase method. Arteriovenous [A-V] glucose difference was calculated and venous blood flow [VBF] was measured during the sampling period. Hindlimb glucose uptake [HGU] was calculated as the product of [A-V] glucose and blood flow. After sampling at rest, the experiments were repeated with the right femoral nerve stimulated using electrical stimulator at 5 Hz. At rest, A-V glucose increased significantly [P<0.05] from 4.2+/-0.2 mg/dl to 29.8+/-8.6, and 24.4+/-2.6 for caffeine and EEK respectively. VBF decreased to 2.0+/-0.9 and 6.0+/-0.6 ml/min for caffeine and EEK respectively. However, HGU significantly increased from 34.8+/-0.1 mg/min to 74.5+/-3.2 mg/min and 175.8+/-3.4 mg/min for caffeine and EEK, respectively. Contraction of the hindlimb muscle alone significantly increased the [A-V] glucose [68%], VBF [26%] and HGU [120%] when compared with the control. During contraction, [A-V] glucose increased from 4.3 +/-1.5 mg/dl to 35.6+/-3.0 mg/dl, and 27.0+/-2.2 mg/dl for caffeine and EEK respectively. VBF also increased from 8.4+/-0.3 ml/min to 12.8+/-0.3 ml/min for EEK. Although, contraction improves VBF [7.3+/-0.5 ml/min] to the hindlimb in response to caffeine, the value was significantly [P<0.05] lower than that of control [8.4+/-0.5 ml/min]. Contraction also significantly increased HGU from 35.8+/-3.6 mg/min to 249.0+/-3.3 and 286.72+/-2.0 mg/min for caffeine and EEK, respectively. The results showed that caffeine and EEK significantly increased HGU and that these effects are due to the increases in glucose extraction [A-V glucose] caused by caffeine and EEK.

Direct Administration of Insulin Into Skeletal Muscle Reveals That the Transport of Insulin Across the Capillary Endothelium Limits the Time Course of Insulin to Activate Glucose Disposal

Intravenous insulin infusion rapidly increases plasma insulin, yet glucose disposal occurs at a much slower rate. This delay in insulin's action may be related to the protracted time for insulin to traverse the capillary endothelium. An increased delay may be associated with the development of insulin resistance. The purpose of the present study was to investigate whether bypassing the transendothelial insulin transport step and injecting insulin directly into the interstitial space would moderate the delay in glucose uptake observed with intravenous administration of the hormone. Intramuscular injections of saline (n = 3) or insulin (n = 10) were administered directly into the vastus medialis of anesthetized dogs. Injections of 0.3, 0.5, 0.7, 1.0, and 3.0 units insulin were administered hourly during a basal insulin euglycemic glucose clamp (0.2mU x min(-1) x kg(-1)). Unlike the saline group, each incremental insulin injection caused interstitial (lymph) insulin to rise within 10 min, indicating rapid diffusion of the hormone within the interstitial matrix. Delay in insulin action was virtually eliminated, indicated by immediate dose-dependent increments in hindlimb glucose uptake. Additionally, bypassing insulin transport by direct injection into muscle revealed a fourfold greater sensitivity to insulin of in vivo muscle tissue than previously reported from intravenous insulin administration. Our results indicate that the transport of insulin to skeletal muscle is a rate-limiting step for insulin to activate glucose disposal. Based on these results, we speculate that defects in insulin transport across the endothelial layer of skeletal muscle will contribute to insulin resistance.

Inhalation of Human Insulin (Exubera) Augments the Efficiency of Muscle Glucose Uptake In Vivo

This study assessed the site of increased glucose uptake resulting from insulin inhalation, quantified its effect under steady-state glucose concentrations, and identified the time to onset of effect. Human insulin was administered to 13 beagles via inhalation (Exubera [insulin human (rDNA origin)] Inhalation Powder; n = 7) or infusion into the inferior vena cava (Humulin R; n = 6) using an algorithm to match plasma insulin levels and kinetics for both groups. Somatostatin and glucagon were infused. Glucose was delivered into the portal vein (4 mg x kg(-1) x min(-1)) and a peripheral vein, as needed, to maintain arterial plasma glucose levels at 180 mg/dl. Hepatic exposure to insulin and glucose and liver glucose uptake were similar in both groups. Despite comparable arterial insulin and glucose levels, hind-limb glucose uptake increased 2.4-fold after inhalation compared with infusion due to increased muscle glucose uptake. Glucose infusion rate, nonhepatic glucose uptake, and tracer-determined glucose disposal were about twice as great compared with intravenous insulin. The effect appeared after 1 h, persisting at least as long as arterial insulin levels remained above basal. Pulmonary administration of insulin increases nonhepatic glucose uptake compared with infusion, and skeletal muscle is the likely site of that effect.

Hepatic and muscle insulin action during late pregnancy in the dog

We evaluated the effects of physiologic increases in insulin on hepatic and peripheral glucose metabolism in nonpregnant (NP) and pregnant (P; 3rd trimester) conscious dogs (n = 9 each) using tracer and arteriovenous difference techniques during a hyperinsulinemic euglycemic clamp. Insulin was initially (-150 to 0 min) infused intraportally at a basal rate. During 0-120 min (Low Insulin), the rate was increased by 0.2 mU x kg(-1) x min(-1), and from 120 to 240 min (High Insulin) insulin was infused at 1.5 mU x kg(-1) x min(-1). Insulin concentrations were significantly higher in NP than P during all periods. Matched subsets (n = 5 NP and 6 P) were identified. In the subsets, insulin was 7 +/- 1, 9 +/- 1, and 28 +/- 3 microU/ml (basal, Low Insulin, and High Insulin, respectively) in NP, and 5 +/- 1, 7 +/- 1, and 27 +/- 3 microU/ml in P. Net hepatic glucose output was suppressed similarly in both subsets (> or =50% with Low Insulin, 100% with High Insulin), as was endogenous glucose rate of appearance. During High Insulin, NP dogs required more glucose (10.8 +/- 1.5 vs. 6.2 +/- 1.0 mg x kg(-1) x min(-1), P < 0.05), and hindlimb (primarily skeletal muscle) glucose uptake tended to be greater in NP than P (18.6 +/- 2.5 mg/min vs. 13.6 +/- 2.0 mg/min, P = 0.06). The normal canine liver remains insulin sensitive during late pregnancy. Differing insulin concentrations in pregnant and nonpregnant women and excessive insulin infusion rates may explain previous findings of hepatic insulin resistance in healthy pregnant women.

Impact of glutamine supplementation on glucose homeostasis during and after exercise

The interaction of glutamine availability and glucose homeostasis during and after exercise was investigated, measuring whole body glucose kinetics with [3-3H]glucose and net organ balances of glucose and amino acids (AA) during basal, exercise, and postexercise hyperinsulinemic-euglycemic clamp periods in six multicatheterized dogs. Dogs were studied twice in random treatment order: once with glutamine (12 micromol.kg(-1).min(-1); Gln) and once with saline (Con) infused intravenously during and after exercise. Plasma glucose fell by 7 mg/dl with exercise in Con (P < 0.05), but it did not fall with Gln. Gln further stimulated whole body glucose production and utilization an additional 24% above a normal exercise response (P < 0.05). Net hepatic uptake of glutamine and alanine was greater with Gln than Con during exercise (P < 0.05). Net hepatic glucose output was increased sevenfold during exercise with Gln (P < 0.05) but not with Con. Net hindlimb glucose uptake was increased similarly during exercise in both groups (P < 0.05). During the postexercise hyperinsulinemic-euglycemic period, glucose production decreased to near zero with Con, but it did not decrease below basal levels with Gln. Gln increased glucose utilization by 16% compared with Con after exercise (P < 0.05). Furthermore, net hindlimb glucose uptake in the postexercise period was increased approximately twofold vs. basal with Gln (P < 0.05) but not with Con. Net hepatic uptake of glutamine during the postexercise period was threefold greater for Gln than Con (P < 0.05). In conclusion, glutamine availability modulates glucose homeostasis during and after exercise, which may have implications for postexercise recovery.

Effects of acute hyperinsulinemia on insulin signal transduction and glucose transporters in ovine fetal skeletal muscle

To test the effects of acute fetal hyperinsulinemia on the pattern and time course of insulin signaling in ovine fetal skeletal muscle, we measured selected signal transduction proteins in the mitogenic, protein synthetic, and metabolic pathways in the skeletal muscle of normally growing fetal sheep in utero. In experiment 1, 4-h hyperinsulinemic-euglycemic clamps were conducted in anesthetized twin fetuses to produce selective fetal hyperinsulinemia-euglycemia in one twin and euinsulinemia-euglycemia in the other. Serial skeletal muscle biopsies were taken from each fetus during the clamp and assayed by Western blot for selected insulin signal transduction proteins. Tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1, and the p85 subunit of phosphatidylinositol 3-kinase doubled at 30 min and gradually returned to control values by 240 min. Phosphorylation of extracellular signal-regulated kinase 1,2 was increased fivefold through 120 min of insulin infusion and decreased to control concentration by 240 min. Protein kinase B phosphorylation doubled at 30 min and remained elevated throughout the study. Phosphorylation of p70 S6K increased fourfold at 30, 60, and 120 min. In the second experiment, a separate group of nonanesthetized singleton fetuses was clamped to intermediate and high hyperinsulinemic-euglycemic conditions for 1 h. GLUT4 increased fourfold in the plasma membrane at 1 h, and hindlimb glucose uptake increased significantly at the higher insulin concentration. These data demonstrate that an acute increase in fetal plasma insulin concentration stimulates a unique pattern of insulin signal transduction proteins in intact skeletal muscle, thereby increasing pathways for mRNA translation, glucose transport, and cell growth.

Inhibiting NOS blocks microvascular recruitment and blunts muscle glucose uptake in response to insulin.

We examined the effects of inhibiting nitric oxide synthase with Nomega-nitro-l-arginine-methyl ester (l-NAME) on total hindlimb blood flow, muscle microvascular recruitment, and hindlimb glucose uptake during euglycemic hyperinsulinemia in vivo in the rat. We used two independent methods to measure microvascular perfusion. In one group of animals, microvascular recruitment was measured using the metabolism of exogenously infused 1-methylxanthine (1-MX), and in a second group contrast-enhanced ultrasound (CEU) was used. Limb glucose uptake was measured by arterial-venous concentration differences after 2 h of insulin infusion. Saline alone did not alter femoral artery flow, glucose uptake, or 1-MX metabolism. Insulin (10 mU.min-1.kg-1) significantly increased hindlimb total blood flow (0.69 +/- 0.02 to 1.22 +/- 0.11 ml/min, P < 0.05), glucose uptake (0.27 +/- 0.05 to 0.95 +/- 0.08 micromol/min, P < 0.05), 1-MX uptake (5.0 +/- 0.5 to 8.5 +/- 1.0 nmol/min, P < 0.05), and skeletal muscle microvascular volume measured by CEU (10.0 +/- 1.6 to 15.0 +/- 1.2 video intensity units, P < 0.05). Addition of l-NAME to insulin completely blocked the effect of insulin on both total limb flow and microvascular recruitment (measured using either 1-MX or CEU) and blunted glucose uptake by 40% (P < 0.05). We conclude that insulin specifically recruits flow to the microvasculture in skeletal muscle via a nitric oxide-dependent pathway and that this may be important to insulin's overall action to regulate glucose disposal.

Hindlimb glucose and lactate metabolism during umbilical cord compression and acute hypoxemia in the late-gestation ovine fetus

The metabolic adaptation of the hindlimb in the fetus to a reversible period of adverse intrauterine conditions and, subsequently, to a further episode of acute hypoxemia has been examined. Sixteen sheep fetuses were chronically instrumented with vascular catheters and transit-time flow probes. In nine of these fetuses, umbilical blood flow was reversibly reduced by 30% from baseline for 3 days (umbilical cord compression), while the remaining fetuses acted as sham-operated, age-matched controls. Acute hypoxemia was subsequently induced in all fetuses by reducing maternal fractional inspired oxygen concentration for 1 h. Paired hindlimb arteriovenous blood samples were taken at appropriate intervals during cord compression and acute hypoxemia, and by using femoral blood flow and the Fick principle, substrate delivery, uptake, and output were calculated. Umbilical cord compression reduced blood oxygen content and delivery to the hindlimb and increased hindlimb oxygen extraction and blood glucose and lactate concentration in the fetus. However, hindlimb glucose and oxygen consumption were unaltered during umbilical cord compression. In contrast, hindlimb oxygen delivery and uptake were significantly reduced in all fetuses during subsequent acute hypoxemia, but glucose extraction, oxygen extraction, and hindlimb lactate output significantly increased in sham-operated control fetuses only. Preexposure of the fetus to a temporary period of adverse intrauterine conditions alters the metabolic response of the fetal hindlimb to subsequent acute stress. Additional data suggest that circulating blood lactate may be derived from sources other than the fetal hindlimb under these circumstances. The lack of hindlimb lactate output during acute hypoxemia in umbilical cord-compressed fetuses, despite a significant fall in oxygen delivery to and uptake by the hindlimb, suggests that the fetal hindlimb may not respire anaerobically after exposure to adverse intrauterine conditions. hypoxia

Resistance training increases glucose uptake and transport in rat skeletal muscle.

The aim of this investigation was to determine if resistance training exercise improved glucose uptake and transport in rodent skeletal muscle. Sprague-Dawley rats were assigned to one of the three groups: control (CON), resistance trained (RT) and aerobic exercise trained (AT). Resistance trained rats were placed in a rodent squat apparatus and performed three sets of 10 repetitions at 75% of their one repetition maximum 3 days week-1 for 12 weeks. Aerobic exercise training consisted of running the rats 3 days week-1 for 45 min over a 12-week period on a motor-driven treadmill (32 m min-1, 15% grade). Following the training period, all animals were subjected to hind limb perfusion in the presence of 500 microU mL-1 insulin. Hind limb glucose uptake was similar in the RT (9.91 +/- 0.7 micromol g-1 h-1) and AT (10.23 +/- 1.0 micromol g-1 h-1) animals and significantly greater than control (CON) (6.40 +/- 0.6 micromol g-1 h-1). Rates of 3-O-methyl-d-glucose transport in the RT animals were elevated in the muscles utilized for RT while in the AT animals rates of 3-O-methyl-d-glucose transport were increased in those muscles recruited for running. The increased rates of 3-O-methyl-d-glucose transport in the skeletal muscles of the resistance trained and aerobic exercise trained animals appeared to be, in part, because of an increased GLUT4 protein concentration. These findings suggest that both resistance or aerobic training exercise can improve insulin-stimulated skeletal muscle glucose uptake and transport, but the training adaptations are restricted to the muscles recruited for the exercise performance.

Prior exercise increases net hepatic glucose uptake during a glucose load.

The aim of these studies was to determine whether prior exercise enhances net hepatic glucose uptake (NHGU) during a glucose load. Sampling catheters (carotid artery, portal, hepatic, and iliac veins), infusion catheters (portal vein and vena cava), and Doppler flow probes (portal vein, hepatic and iliac arteries) were implanted. Exercise (150 min; n = 6) or rest (n = 6) was followed by a 30-min control period and a 100-min experimental period (3.5 mg. kg-1. min-1 of glucose in portal vein and as needed in vena cava to clamp arterial blood glucose at approximately 130 mg/dl). Somatostatin was infused, and insulin and glucagon were replaced intraportally at fourfold basal and basal rates, respectively. During experimental period the arterial-portal venous (a-pv) glucose gradient (mg/dl) was -18 +/- 1 in sedentary and -19 +/- 1 in exercised dogs. Arterial insulin and glucagon were similar in the two groups. Net hepatic glucose balance (mg. kg-1. min-1) shifted from 1.9 +/- 0.2 in control period to -1.8 +/- 0.2 (negative rates represent net uptake) during experimental period in sedentary dogs (Delta3.7 +/- 0.5); with prior exercise it shifted from 4.1 +/- 0.3 (P < 0.01 vs. sedentary) in control period to -3.2 +/- 0.4 (P < 0.05 vs. sedentary) during experimental period (Delta7.3 +/- 0.7, P < 0.01 vs. sedentary). Net hindlimb glucose uptake (mg/min) was 4 +/- 1 in sedentary animals in control period and 13 +/- 2 during experimental period; in exercised animals it was 7 +/- 1 in control period (P < 0. 01 vs. sedentary) and 32 +/- 4 (P < 0.01 vs. sedentary) during experimental period. As the total glucose infusion rate (mg. kg-1. min-1) was 7 +/- 1 in sedentary and 11 +/- 1 in exercised dogs, approximately 30% of the added glucose infusion due to prior exercise could be accounted for by the greater NHGU. In conclusion, when determinants of hepatic glucose uptake (insulin, glucagon, a-pv glucose gradient, glycemia) are controlled, prior exercise increases NHGU during a glucose load due to an effect that is intrinsic to the liver. Increased glucose disposal in the postexercise state is therefore due to an improved ability of both liver and muscle to take up glucose.

Acute vasoconstriction-induced insulin resistance in rat muscle in vivo.

Insulin-mediated changes in blood flow are associated with altered blood flow distribution and increased capillary recruitment in skeletal muscle. Studies in perfused rat hindlimb have shown that muscle metabolism can be regulated by vasoactive agents that control blood flow distribution within the hindlimb. In the present study, the effects of a vasoconstrictive agent that has no direct effect on skeletal muscle metabolism but that alters perfusion distribution in rat hindlimb was investigated in vivo to determine its effects on insulin-mediated vascular action and glucose uptake. We measured the effects of alpha-methylserotonin (alpha-met5HT) on mean arterial blood pressure, heart rate, femoral blood flow, hindlimb vascular resistance, and glucose uptake in control and euglycemic insulin-clamped (10 mU x min(-1) x kg(-1)) anesthetized rats. Blood flow distribution within the hindlimb muscles was assessed by measuring the metabolism of 1-methylxanthine (1-MX), an exogenously added substrate for capillary xanthine oxidase. Alpha-met5HT (20 microg x min(-1) x kg(-1)) infusion alone increased mean arterial blood pressure by 25% and increased hindlimb vascular resistance but caused no change in femoral blood flow. These changes were associated with decreased hindlimb 1-MX metabolism indicating less capillary flow. Insulin infusion caused decreased hindlimb vascular resistance that was associated with increased femoral blood flow and 1-MX metabolism. Treatment with alpha-met5HT infusion commenced before insulin infusion prevented the increase in femoral blood flow and inhibited the stimulation of 1-MX metabolism. Alpha-met5HT infusion had no effect on hindlimb glucose uptake but markedly inhibited the insulin stimulation of glucose uptake (P < 0.05) and was associated with decreased glucose infusion rates to maintain euglycemia (P < 0.05). A significant correlation (P < 0.05) was observed between 1-MX metabolism and hindlimb glucose uptake but not between femoral blood flow and glucose uptake. The results indicate that in vivo, certain types of vasoconstriction in muscle such as elicited by 5HT2 agonists, which prevent normal insulin recruitment of capillary flow, cause impaired muscle glucose uptake. Moreover, if vasoconstriction of this kind results from stress-induced increase in sympathetic outflow, then this may provide a clue as to the link between hypertension and insulin resistance that is often observed in humans.

Effect of fast duration on disposition of an intraduodenal glucose load in the conscious dog.

The effects of prior fast duration (18 h, n = 8; 42 h, n = 8) on the glycemic and tissue-specific responses to an intraduodenal glucose load were studied in chronically catheterized conscious dogs. [3-3H]glucose was infused throughout the study. After basal measurements, glucose spiked with [U-14C]glucose was infused for 150 min intraduodenally. Arterial insulin and glucagon were similar in the two groups. Arterial glucose (mg/dl) rose approximately 70% more during glucose infusion after 42 h than after an 18-h fast. The net hepatic glucose balance (mg. kg-1. min-1) was similar in the two groups (basal: 1.8 +/- 0.2 and 2.0 +/- 0.3; glucose infusion: -2.2 +/- 0.5 and -2.2 +/- 0.7). The intrahepatic fate of glucose was 79% glycogen, 13% oxidized, and 8% lactate release after a 42-h fast; it was 23% glycogen, 21% oxidized, and 56% lactate release after an 18-h fast. Net hindlimb glucose uptake was similar between groups. The appearance of intraduodenal glucose during glucose infusion (mg/kg) was 900 +/- 50 and 1,120 +/- 40 after 18- and 42-h fasts (P < 0.01). glucose administration after prolonged fasting induces higher circulating glucose than a shorter fast (increased appearance of intraduodenal glucose); liver and hindlimb glucose uptakes and the hormonal response, however, are unchanged; finally, an intrahepatic redistribution of carbons favors glycogen deposition.