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Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and a β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (ECs). EC itgβ1 is necessary for the formation of capillary networks during embryonic development, and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study, we investigated the contribution of EC itgβ1 in microcirculatory function and glucose uptake, with an emphasis on skeletal muscle. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating "delivery"-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid the lethality of embryonic gene knockout and the deteriorating health resulting from early postnatal inducible gene deletion. We found that mice with ( itgβ1 fl/fl SCLcre) and without ( itgβ1 fl/fl ) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1 fl/fl SCLcre mice compared with itgβ1 fl/fl littermates have 1 ) deficits in capillary flow rate, flow heterogeneity, and capillary density; 2 ) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and 3 ) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation. NEW & NOTEWORTHY The microvasculature is an important site of resistance to muscle glucose uptake. We show that microvasculature integrins determine the exchange of glucose between the circulation and muscle. Specifically, a 30% reduction in the expression of endothelial integrin β1 subunit is sufficient to cause microcirculatory dysfunction and lead to insulin resistance. This emphasizes the importance of endothelial integrins in microcirculatory function and the importance of microcirculatory function for the ability of muscle to consume glucose.

Regular exercise elicits adaptations in glucose and lipid metabolism that allow the body to meet energy demands of subsequent exercise bouts more effectively and mitigate metabolic diseases including fatty liver. Energy discharged during the acute exercise bouts that comprise exercise training may be a catalyst for liver adaptations. During acute exercise, liver glycogenolysis and gluconeogenesis are accelerated to supply glucose to working muscle. Lower liver energy state imposed by gluconeogenesis and related pathways activates AMP-activated protein kinase (AMPK), which conserves ATP partly by promoting lipid oxidation. This study tested the hypothesis that AMPK is necessary for liver glucose and lipid adaptations to training. Liver-specific AMPKα1α2 knockout ( AMPKα1α2 fl/fl +AlbCre ) mice and littermate controls ( AMPKα1α2 fl/fl ) completed sedentary and exercise training protocols. Liver nutrient fluxes were quantified at rest or during acute exercise following training. Liver metabolites and molecular regulators of metabolism were assessed. Training increased liver glycogen in AMPKα1α2 fl/fl mice, but not in AMPKα1α2 fl/fl +AlbCre mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained AMPKα1α2 fl/fl +AlbCre mice. Deletion of AMPKα1α2 attenuated training-induced declines in liver diacylglycerides. In particular, training lowered the concentration of unsaturated and elongated fatty acids comprising diacylglycerides in AMPKα1α2 fl/fl mice, but not in AMPKα1α2 fl/fl +AlbCre mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of AMPKα1α2 fl/fl +AlbCre mice. These lipid responses were independent of differences in tricarboxylic acid cycle fluxes. In conclusion, AMPK is required for liver training adaptations that are critical to glucose and lipid metabolism. NEW & NOTEWORTHY This study shows that the energy sensor and transducer, AMP-activated protein kinase (AMPK), is necessary for an exercise training-induced: 1 ) increase in liver glycogen that is necessary for accelerated glycogenolysis during exercise, 2 ) decrease in liver glycerolipids independent of tricarboxylic acid (TCA) cycle flux, and 3 ) decline in the desaturation and elongation of fatty acids comprising liver diacylglycerides. The mechanisms defined in these studies have implications for use of regular exercise or AMPK-activators in patients with fatty liver.

These studies test, using intravital microscopy (IVM), the hypotheses that perfusion effects on insulin-stimulated muscle glucose uptake (MGU) are 1 ) capillary recruitment independent and 2 ) mediated through the dispersion of glucose rather than insulin. For experiment 1 , capillary perfusion was visualized before and after intravenous insulin. No capillary recruitment was observed. For experiment 2 , mice were treated with vasoactive compounds (sodium nitroprusside, hyaluronidase, and lipopolysaccharide), and dispersion of fluorophores approximating insulin size (10-kDa dextran) and glucose (2-NBDG) was measured using IVM. Subsequently, insulin and 2[ 14 C]deoxyglucose were injected and muscle phospho-2[ 14 C]deoxyglucose (2[C 14 ]DG) accumulation was used as an index of MGU. Flow velocity and 2-NBDG dispersion, but not perfused surface area or 10-kDa dextran dispersion, predicted phospho-2[ 14 C]DG accumulation. For experiment 3 , microspheres of the same size and number as are used for contrast-enhanced ultrasound (CEU) studies of capillary recruitment were visualized using IVM. Due to their low concentration, microspheres were present in only a small fraction of blood-perfused capillaries. Microsphere-perfused blood volume correlated to flow velocity. These findings suggest that 1 ) flow velocity rather than capillary recruitment controls microvascular contributions to MGU, 2 ) glucose dispersion is more predictive of MGU than dispersion of insulin-sized molecules, and 3 ) CEU measures regional flow velocity rather than capillary recruitment.

Extracellular matrix hyaluronan is increased in skeletal muscle of high-fat-fed insulin-resistant mice, and reduction of hyaluronan by PEGPH20 hyaluronidase ameliorates diet-induced insulin resistance (IR). CD44, the main hyaluronan receptor, is positively correlated with type 2 diabetes. This study determines the role of CD44 in skeletal muscle IR. Global CD44-deficient ( cd44 -/- ) mice and wild-type littermates ( cd44 +/+ ) were fed a chow diet or 60% high-fat diet for 16 wk. High-fat-fed cd44 -/- mice were also treated with PEGPH20 to evaluate its CD44-dependent action. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (ICv). High-fat feeding increased muscle CD44 protein expression. In the absence of differences in body weight and composition, despite lower clamp insulin during ICv, the cd44 -/- mice had sustained glucose infusion rate (GIR) regardless of diet. High-fat diet-induced muscle IR as evidenced by decreased muscle glucose uptake (Rg) was exhibited in cd44 +/+ mice but absent in cd44 -/- mice. Moreover, gastrocnemius Rg remained unchanged between genotypes on chow diet but was increased in high-fat-fed cd44 -/- compared with cd44 +/+ when normalized to clamp insulin concentrations. Ameliorated muscle IR in high-fat-fed cd44 -/- mice was associated with increased vascularization. In contrast to previously observed increases in wild-type mice, PEGPH20 treatment in high-fat-fed cd44 -/- mice did not change GIR or muscle Rg during ICv, suggesting a CD44-dependent action. In conclusion, genetic CD44 deletion improves muscle IR, and the beneficial effects of PEGPH20 are CD44-dependent. These results suggest a critical role of CD44 in promoting hyaluronan-mediated muscle IR, therefore representing a potential therapeutic target for diabetes.

Sepsis costs the healthcare system $23 billion annually and has a mortality rate between 10 and 40%. An early indication of sepsis is the onset of hyperglycemia, which is the result of sepsis-induced insulin resistance in skeletal muscle. Previous investigations have focused on events in the myocyte (e.g., insulin signaling and glucose transport and subsequent metabolism) as the causes for this insulin-resistant state. However, the delivery of insulin to the skeletal muscle is also an important determinant of insulin action. Skeletal muscle microvascular blood flow, which delivers the insulin to the muscle, is known to be decreased during sepsis. Here we test whether the reduced capillary blood flow to skeletal muscle belies the sepsis-induced insulin resistance by reducing insulin delivery to the myocyte. We hypothesize that decreased capillary flow and consequent decrease in insulin delivery is an early event that precedes gross cardiovascular alterations seen with sepsis. This hypothesis was examined in mice treated with either lipopolysaccharide (LPS) or polymicrobial sepsis followed by intravital microscopy of the skeletal muscle microcirculation. We calculated insulin delivery to the myocyte using two independent methods and found that LPS and sepsis rapidly reduce insulin delivery to the skeletal muscle by ~50%; this was driven by decreases in capillary flow velocity and the number of perfused capillaries. Furthermore, the changes in skeletal muscle microcirculation occur before changes in both cardiac output and arterial blood pressure. These data suggest that a rapid reduction in skeletal muscle insulin delivery contributes to the induction of insulin resistance during sepsis.

Integrin-linked kinase (ILK) is a critical intracellular signaling node for integrin receptors. Its role in liver development is complex, as ILK deletion at E10.5 (before hepatocyte differentiation) results in biochemical and morphological differences that resolve as mice age. Nevertheless, mice with ILK depleted specifically in hepatocytes are protected from the hepatic insulin resistance during obesity. Despite the potential importance of hepatocyte ILK to metabolic health, it is unknown how ILK controls hepatic metabolism or glucoregulation. The present study tested the role of ILK in hepatic metabolism and glucoregulation by deleting it specifically in hepatocytes, using a cre-lox system that begins expression at E15.5 (after initiation of hepatocyte differentiation). These mice develop the most severe morphological and glucoregulatory abnormalities at 6 wk, but these gradually resolve with age. After identifying when the deletion of ILK caused a severe metabolic phenotype, in depth studies were performed at this time point to define the metabolic programs that coordinate control of glucoregulation that are regulated by ILK. We show that 6-wk-old ILK-deficient mice have higher glucose tolerance and decreased net glycogen synthesis. Additionally, ILK was shown to be necessary for transcription of mitochondrial-related genes, oxidative metabolism, and maintenance of cellular energy status. Thus, ILK is required for maintaining hepatic transcriptional and metabolic programs that sustain oxidative metabolism, which are required for hepatic maintenance of glucose homeostasis.

Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations., (Copyright © 2015 the American Physiological Society.)

Intense interest has been focused on cell-based therapy for the infarcted heart given that stem cells have exhibited the ability to reduce infarct size and mitigate cardiac dysfunction. Despite this, it is unknown whether mesenchymal stem cell (MSC) therapy can prevent metabolic remodeling following a myocardial infarction (MI). This study examines the ability of MSCs to rescue the infarcted heart from perturbed substrate uptake in vivo. C57BL/6 mice underwent chronic ligation of the left anterior descending coronary artery to induce a MI. Echocardiography was performed on conscious mice at baseline as well as 7 and 23 days post-MI. Twenty-eight days following the ligation procedure, hyperinsulinemic euglycemic clamps assessed in vivo insulin sensitivity. Isotopic tracer administration evaluated whole body, peripheral tissue, and cardiac-specific glucose and fatty acid utilization. To gain insight into the mechanisms by which MSCs modulate metabolism, mitochondrial function was assessed by high-resolution respirometry using permeabilized cardiac fibers. Data show that MSC transplantation preserves insulin-stimulated fatty acid uptake in the peri-infarct region (4.25 ± 0.64 vs. 2.57 ± 0.34 vs. 3.89 ± 0.54 μmol·100 g(-1)·min(-1), SHAM vs. MI + PBS vs. MI + MSC; P < 0.05) and prevents increases in glucose uptake in the remote left ventricle (3.11 ± 0.43 vs. 3.81 ± 0.79 vs. 6.36 ± 1.08 μmol·100 g(-1)·min(-1), SHAM vs. MI + PBS vs. MI + MSC; P < 0.05). This was associated with an enhanced efficiency of mitochondrial oxidative phosphorylation with a respiratory control ratio of 3.36 ± 0.18 in MSC-treated cardiac fibers vs. 2.57 ± 0.14 in the infarct-only fibers (P < 0.05). In conclusion, MSC therapy exhibits the potential to rescue the heart from metabolic aberrations following a MI. Restoration of metabolic flexibility is important given the metabolic demands of the heart and the role of energetics in the progression to heart failure.

Hepatic glucagon action increases in response to accelerated metabolic demands and is associated with increased whole body substrate availability, including circulating lipids. The hypothesis that increases in hepatic glucagon action stimulate AMP-activated protein kinase (AMPK) signaling and peroxisome proliferator-activated receptor-α (PPARα) and fibroblast growth factor 21 (FGF21) expression in a manner modulated by fatty acids was tested in vivo. Wild-type (gcgr(+/+)) and glucagon receptor-null (gcgr(-/-)) littermate mice were studied using an 18-h fast, exercise, and hyperglucagonemic-euglycemic clamps plus or minus increased circulating lipids. Fasting and exercise in gcgr(+/+), but not gcgr(-/-) mice, increased hepatic phosphorylated AMPKα at threonine 172 (p-AMPK(Thr(172))) and PPARα and FGF21 mRNA. Clamp results in gcgr(+/+) mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, p-AMPK(Thr(172)), or PPARα and FGF21 mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPARα and FGF21 expression. All effects were absent in gcgr(-/-) mice. These findings demonstrate that glucagon exerts a critical regulatory role in liver to stimulate pathways linked to lipid metabolism in vivo and shows for the first time that effects of glucagon on PPARα and FGF21 expression are amplified by a physiological increase in circulating lipids.

This article addresses two topics. We provide an overview of the National Institutes of Health Mouse Metabolic Phenotyping Center (MMPC) Program. We then discuss some observations we have made during the first eight years of the Vanderbilt MMPC regarding common phenotyping practices. We include specific recommendations to improve phenotyping practices for tests of glucose tolerance and insulin action. We recommend that methods for experiments in vivo be described in manuscripts. We make specific recommendations for data presentation, interpretation, and experimental design for each test. To facilitate and maximize the exchange of scientific information, we suggest that guidelines be developed for methods used to assess glucose tolerance and insulin action in vivo.

Four grams of glucose circulates in the blood of a person weighing 70 kg. This glucose is critical for normal function in many cell types. In accordance with the importance of these 4 g of glucose, a sophisticated control system is in place to maintain blood glucose constant. Our focus has been on the mechanisms by which the flux of glucose from liver to blood and from blood to skeletal muscle is regulated. The body has a remarkable capacity to satisfy the nutritional need for glucose, while still maintaining blood glucose homeostasis. The essential role of glucagon and insulin and the importance of distributed control of glucose fluxes are highlighted in this review. With regard to the latter, studies are presented that show how regulation of muscle glucose uptake is regulated by glucose delivery to muscle, glucose transport into muscle, and glucose phosphorylation within muscle.

The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.

Islets in most species respond to increased glucose with biphasic insulin secretion, marked by a sharp first-phase peak and a slowly rising second phase. Mouse islets in vitro, however, lack a robust second phase. To date, this observation has not been extended in vivo. We thus compared insulin secretion from conscious mice with isolated mouse islets in vitro. The arterial plasma insulin response to a hyperglycemic clamp was measured in conscious mice 1 wk after surgical implantation of carotid artery and jugular vein catheters. Mice were transfused using clamps with blood from a donor mouse to maintain blood volume, allowing frequent arterial sampling. When plasma glucose in vivo was raised from approximately 5 to approximately 13 mM, insulin rose to a first-phase peak of 403+/-73% above basal secretion (n=5), followed by a rising second phase of mean 289+/- 41%. In contrast, perifused mouse islets ( approximately 75 islets/trial) responded with a similar first phase of 508+/- 94% (n=4) but a smaller and virtually flat second phase of 169+/- 9% (n=4, P<0.05). Furthermore, the slope of the second-phase response differed significantly from zero in mice (2.63+/-0.39%/min, P<0.01), in contrast to perifused islets (0.18+/- 0.14%/min, P>0.30). Mice also displayed pulsatile patterns in insulin concentration (period: 4.2+/- 0.4 min, n=8). Conscious mice thus responded to increased glucose with biphasic and pulsatile insulin secretion, as in other species. The robust second phase observed in vivo suggests that the processes needed to generate second-phase insulin secretion may be abrogated by islet isolation.

A portal venous 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion that results in hepatic 5-aminoimidazole-4-carboxamide-1-beta-D-ribosyl-5-monophosphate (ZMP) concentrations of approximately 4 micromol/g liver increases hepatic glycogenolysis and glucose output. ZMP is an AMP analog that mimics the regulatory actions of this nucleotide. The aim of this study was to measure hepatic AMP concentrations in response to increasing energy requirements to test the hypothesis that AMP achieves concentrations during exercise, consistent with a role in stimulation of hepatic glucose metabolism. Male C57BL/6J mice (27.4+/- 0.4 g) were subjected to 35 min of rest [sedentary (SED), n=8], underwent short-term (ST, 35 min) moderate (20 m/min, 5% grade) exercise (n=8), or underwent treadmill exercise under similar conditions but until exhaustion (EXH, n=8). Hepatic AMP concentrations were 0.82+/- 0.05, 1.17+/- 0.11, and 2.52+/- 0.16 micromol/g liver in SED, ST, and EXH mice, respectively (P< 0.05). Hepatic energy charge was 0.66+/- 0.01, 0.58+/- 0.02, and 0.33+/- 0.22 in SED, ST, and EXH mice, respectively (P< 0.05). Hepatic glycogen was 11.6+/- 1.0, 8.8+/- 2.2, and 0.0+/- 0.1 mg/g liver in SED, ST, and EXH mice, respectively (P< 0.05). Hepatic AMPK (Thr(172)) phosphorylation was 1.00+/- 0.14, 1.96+/- 0.16, and 7.44+/- 0.63 arbitrary units in SED, ST, and EXH mice, respectively (P< 0.05). Thus exercise increases hepatic AMP concentrations. These data suggest that the liver is highly sensitive to metabolic demands, as evidenced by dramatic changes in cellular energy indicators (AMP) and sensors thereof (AMP-activated protein kinase). In conclusion, AMP is sensitively regulated, consistent with it having an important role in hepatic metabolism.

Increased plasminogen activator inhibitor-1 (PAI-1) is linked to obesity and insulin resistance. However, the functional role of PAI-1 in adipocytes is unknown. This study was designed to investigate effects and underlying mechanisms of PAI-1 on glucose uptake in adipocytes and on adipocyte differentiation. Using primary cultured adipocytes from PAI-1(+/+) and PAI-1(-/-) mice, we found that PAI-1 deficiency promoted adipocyte differentiation, enhanced basal and insulin-stimulated glucose uptake, and protected against tumor necrosis factor-alpha-induced adipocyte dedifferentiation and insulin resistance. These beneficial effects were associated with upregulated glucose transporter 4 at basal and insulin-stimulated states and upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) and adiponectin along with downregulated resistin mRNA in differentiated PAI-1(-/-) vs. PAI-1(+/+) adipocytes. Similarly, inhibition of PAI-1 with a neutralizing anti-PAI-1 antibody in differentiated 3T3-L1 adipocytes further promoted adipocyte differentiation and glucose uptake, which was associated with increased expression of transcription factors PPARgamma, CCAAT enhancer-binding protein-alpha (C/EBPalpha), and the adipocyte-selective fatty acid-binding protein aP2, thus mimicking the phenotype in PAI-1(-/-) primary adipocytes. Conversely, overexpression of PAI-1 by adenovirus-mediated gene transfer in 3T3-L1 adipocytes inhibited differentiation and reduced PPARgamma, C/EBPalpha, and aP2 expression. This was also associated with a decrease in urokinase-type plasminogen activator mRNA expression, decreased plasmin activity, and increased collagen I mRNA expression. Collectively, these results indicate that absence or inhibition of PAI-1 in adipocytes protects against insulin resistance by promoting glucose uptake and adipocyte differentiation via increased PPARgamma expression. We postulate that these PAI-1 effects on adipocytes may, at least in part, be mediated via modulation of plasmin activity and extracellular matrix components.

This study aimed to test whether stimulation of net hepatic glucose output (NHGO) by increased concentrations of the AMP analog, 5-aminoimidazole-4-carboxamide-1-beta-d-ribosyl-5-monophosphate, can be suppressed by pharmacological insulin levels. Dogs had sampling (artery, portal vein, hepatic vein) and infusion (vena cava, portal vein) catheters and flow probes (hepatic artery, portal vein) implanted >16 days before study. Protocols consisted of equilibration (-130 to -30 min), basal (-30 to 0 min), and hyperinsulinemic-euglycemic (0-150 min) periods. At time (t) = 0 min, somatostatin was infused, and basal glucagon was replaced via the portal vein. Insulin was infused in the portal vein at either 2 (INS2) or 5 (INS5) mU.kg(-1).min(-1). At t = 60 min, 1 mg.kg(-1).min(-1) portal venous 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) infusion was initiated. Arterial insulin rose approximately 9- and approximately 27-fold in INS2 and INS5, respectively. Glucagon, catecholamines, and cortisol did not change throughout the study. NHGO was completely suppressed before t = 60 min. Intraportal AICAR stimulated NHGO by 1.9 +/- 0.5 and 2.0 +/- 0.5 mg.kg(-1).min(-1) in INS2 and INS5, respectively. AICAR stimulated tracer-determined endogenous glucose production similarly in both groups. Intraportal AICAR infusion significantly increased hepatic acetyl-CoA carboxylase (ACC, Ser(79)) phosphorylation in INS2. Hepatic ACC (Ser(79)) phosphorylation, however, was not increased in INS5. Thus intraportal AICAR infusion renders hepatic glucose output insensitive to pharmacological insulin. The effectiveness of AICAR in countering the suppressive effect of pharmacological insulin on NHGO occurs even though AICAR-stimulated ACC phosphorylation is completely blocked.

The role of heart-type cytosolic fatty acid-binding protein (H-FABP) in mediating whole body and muscle-specific long-chain fatty acid (LCFA) and glucose utilization was examined using exercise as a phenotyping tool. Catheters were chronically implanted in a carotid artery and jugular vein of wild-type (WT, n = 8), heterozygous (H-FABP(+/-), n = 8), and null (H-FABP(-/-), n = 7) chow-fed C57BL/6J mice, and mice were allowed to recover for 7 days. After a 5-h fast, conscious, unrestrained mice were studied during 30 min of treadmill exercise (0.6 mph). A bolus of [(125)I]-15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid and 2-deoxy-[(3)H]glucose was administered to obtain rates of whole body metabolic clearance (MCR) and indexes of muscle LCFA (R(f)) and glucose (R(g)) utilization. Fasting, nonesterified fatty acids (mM) were elevated in H-FABP(-/-) mice (2.2 +/- 0.9 vs. 1.3 +/- 0.1 and 1.3 +/- 0.2 for WT and H-FABP(+/-)). During exercise, blood glucose (mM) increased in WT (11.7 +/- 0.8) and H-FABP(+/-) (12.6 +/- 0.9) mice, whereas H-FABP(-/-) mice developed overt hypoglycemia (4.8 +/- 0.8). Examination of tissue-specific and whole body glucose and LCFA utilization demonstrated a dependency on H-FABP with exercise in all tissues examined. Reductions in H-FABP led to decreasing exercise-stimulated R(f) and increasing R(g) with the most pronounced effects in heart and soleus muscle. Similar results were seen for MCR with decreasing LCFA and increasing glucose clearance with declining levels of H-FABP. These results show that, in vivo, H-FABP has reciprocal effects on glucose and LCFA utilization and whole body fuel homeostasis when metabolic demands are elevated by exercise.

AMP-activated protein kinase (AMPK) independently increases glucose and long-chain fatty acid (LCFA) utilization in isolated cardiac muscle preparations. Recent studies indicate this may be due to AMPK-induced phosphorylation and activation of nitric oxide synthase (NOS). Given this, the aim of the present study was to assess the effects of AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 10 mg.kg(-1).min(-1)) on glucose and LCFA utilization in cardiac muscle and to determine the NOS dependence of any observed effects. Catheters were chronically implanted in a carotid artery and jugular vein of Sprague-Dawley rats. After 4 days of recovery, conscious, unrestrained rats were given either water or water containing 1 mg/ml nitro-L-arginine methyl ester (L-NAME) for 2.5 days. After an overnight fast, rats underwent one of four protocols: saline, AICAR, AICAR + L-NAME, or AICAR + Intralipid (20%, 0.02 ml.kg(-1).min(-1)). Glucose was clamped at approximately 6.5 mM in all groups, and an intravenous bolus of 2-deoxy-[(3)H]glucose and [(125)I]-15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid was administered to obtain indexes of glucose and LCFA uptake and clearance. Despite AMPK activation, as evidenced by acetyl-CoA carboxylase (Ser(221)) and AMPK phosphorylation (Thr(172)), AICAR increased cardiac LCFA but not glucose clearance. L-NAME + AICAR established that this effect was not due to NOS activation, and AICAR + Intralipid showed that increased cardiac LCFA clearance was not LCFA-concentration dependent. These results demonstrate that, in vivo, AMPK stimulation increases LCFA but not glucose clearance by a NOS-independent mechanism.

The purpose of this study was to determine whether the sedentary dog is able to autoregulate glucose production (R(a)) in response to non-insulin-induced changes (<20 mg/dl) in arterial glucose. Dogs had catheters implanted >16 days before study. Protocols consisted of basal (-30 to 0 min) and bilateral renal arterial phloridzin infusion (0-180 min) periods. Somatostatin was infused, and glucagon and insulin were replaced to basal levels. In one protocol (Phl +/- Glc), glucose was allowed to fall from t = 0-90 min. This was followed by a period when glucose was infused to restore euglycemia (90-150 min) and a period when glucose was allowed to fall again (150-180 min). In a second protocol (EC), glucose was infused to compensate for the renal glucose loss due to phloridzin and maintain euglycemia from t = 0-180 min. Arterial insulin, glucagon, cortisol, and catecholamines remained at basal in both protocols. In Phl +/- Glc, glucose fell by approximately 20 mg/dl by t = 90 min with phloridzin infusion. R(a) did not change from basal in Phl +/- Glc despite the fall in glucose for the first 90 min. R(a) was significantly suppressed with restoration of euglycemia from t = 90-150 min (P < 0.05) and returned to basal when glucose was allowed to fall from t = 150-180 min. R(a) did not change from basal in EC. In conclusion, the liver autoregulates R(a) in response to small changes in glucose independently of changes in pancreatic hormones at rest. However, the liver of the resting dog is more sensitive to a small increment, rather than decrement, in arterial glucose.

A marked sexual dimorphism exists in healthy individuals in the pattern of blunted neuroendocrine and metabolic responses following antecedent stress. It is unknown whether significant sex-related counterregulatory differences occur during prolonged moderate exercise after antecedent hypoglycemia in type 1 diabetes mellitus (T1DM). Fourteen patients with T1DM (7 women and 7 men) were studied during 90 min of euglycemic exercise at 50% maximal O(2) consumption after two 2-h episodes of previous-day euglycemia (5.0 mmol/l) or hypoglycemia of 2.9 mmol/l. Men and women were matched for age, glycemic control, duration of diabetes, and exercise fitness and had no history or evidence of autonomic neuropathy. Exercise was performed during constant "basal" intravenous infusion of regular insulin (1 U/h) and a 20% dextrose infusion, as needed to maintain euglycemia. Plasma glucose and insulin levels were equivalent in men and women during all exercise and glucose clamp studies. Antecedent hypoglycemia produced a relatively greater (P < 0.05) reduction of glucagon, epinephrine, norepinephrine, growth hormone, and metabolic (glucose kinetics) responses in men compared with women during next-day exercise. After antecedent hypoglycemia, endogenous glucose production (EGP) was significantly reduced in men only, paralleling a reduction in the glucagon-to-insulin ratio and catecholamine responses. In conclusion, a marked sexual dimorphism exists in a wide spectrum of blunted counterregulatory responses to exercise in T1DM after prior hypoglycemia. Key neuroendocrine (glucagon, catecholamines) and metabolic (EGP) homeostatic responses were better preserved during exercise in T1DM women after antecedent hypoglycemia. Preserved counterregulatory responses during exercise in T1DM women may confer greater protection against hypoglycemia than in men with T1DM.

Muscle glucose uptake (MGU) is determined by glucose delivery, transport, and phosphorylation. C57Bl/6J mice overexpressing GLUT4, hexokinase II (HK II), or both were used to determine the barriers to MGU. A carotid artery and jugular vein were catheterized for arterial blood sampling and venous infusions. Experiments were conducted in conscious mice approximately 7 days after surgery. 2-Deoxy-[3H]glucose was administered during rest or treadmill exercise to calculate glucose concentration-dependent (Rg) and -independent (Kg) indexes of MGU. Compared with wild-type controls, GLUT4-overexpressing mice had lowered fasting glycemia (165 +/- 6 vs. 115 +/- 6 mg/dl) and increased Rg by 230 and 166% in the gastrocnemius and superficial vastus lateralis (SVL) muscles under sedentary conditions. GLUT4 overexpression was not able to augment exercise-stimulated Rg or Kg. Whereas HK II overexpression had no effect on fasting glycemia (170 +/- 6 mg/dl) or sedentary Rg, it increased exercise-stimulated Rg by 82, 60, and 169% in soleus, gastrocnemius, and SVL muscles, respectively. Combined GLUT4 and HK II overexpression lowered fasting glycemia (106 +/- 6 mg/dl), increased nonesterified fatty acids, and increased sedentary Rg. Combined GLUT4 and HK II overexpression did not enhance exercise-stimulated Rg compared with HK II-overexpressing mice because of the reduced glucose concentration. GLUT4 combined with HK II overexpression resulted in a marked increase in exercise-stimulated Kg. In conclusion, control of MGU shifts from membrane transport at rest to phosphorylation during exercise. Glucose transport is not normally a significant barrier during exercise. However, when the phosphorylation barrier is lowered by HK II overexpression, glucose transport becomes a key site of control for regulating MGU during exercise.

Muscle glucose uptake (MGU) is distributively controlled by three serial steps: delivery of glucose to the muscle membrane, transport across the muscle membrane, and intracellular phosphorylation to glucose 6-phosphate by hexokinase (HK). During states of high glucose fluxes such as moderate exercise, the HK activity is of increased importance, since augmented muscle perfusion increases glucose delivery, and increased GLUT4 at the cell membrane increases glucose transport. Because HK II overexpression augments exercise-stimulated MGU, it was hypothesized that a reduction in HK II activity would impair exercise-stimulated MGU and that the magnitude of this impairment would be greatest in tissues with the largest glucose requirement. To this end, mice with a HK II partial knockout (HK+/-) were compared with their wild-type control (WT) littermates during either sedentary or moderate exercise periods. Rg, an index of glucose metabolism, was measured using 2-deoxy-[3H]glucose. No differences in glucose metabolism were detected between sedentary groups. The increase in Rg due to exercise was impaired in the highly oxidative heart and soleus muscles of HK+/- compared with WT mice (7 +/- 10 vs. 29 +/- 9 and 8 +/- 3 vs. 25 +/- 7 micromol. 100 g-1. min-1, respectively). However, the increase in Rg due to exercise was not altered in gastrocnemius and superficial vastus lateralis muscles in HK+/- and WT mice (8 +/- 2 vs. 12 +/- 3 and 5 +/- 2 vs. 8 +/- 2 micromol. 100 g-1. min-1, respectively). In conclusion, MGU is impaired by reductions in HK activity during exercise, a physiological condition characterized by high glucose flux. This impairment is critically dependent on the tissue's glucose metabolic rate and correlates with tissue oxidative capacity.

The aim of this study was to determine barriers limiting muscle glucose uptake (MGU) during increased glucose flux created by raising blood glucose in the presence of fixed insulin. The determinants of the maximal velocity (V(max)) of MGU in muscles of different fiber types were defined. Conscious rats were studied during a 4 mU x kg(-1) x min(-1) insulin clamp with plasma glucose at 2.5, 5.5, and 8.5 mM. [U-(14)C]mannitol and 3-O-methyl-[(3)H]glucose ([(3)H]MG) were infused to steady-state levels (t = -180 to 0 min). These isotope infusions were continued from 0 to 40 min with the addition of a 2-deoxy-[(3)H]glucose ([(3)H]DG) infusion. Muscles were excised at t = 40 min. Glucose metabolic index (R(g)) was calculated from muscle-phosphorylated [(3)H]DG. [U-(14)C]mannitol was used to determine extracellular (EC) H(2)O. Glucose at the outer ([G](om)) and inner ([G](im)) sarcolemmal surfaces was determined by the ratio of [(3)H]MG in intracellular to EC H(2)O and muscle glucose. R(g) was comparable at the two higher glucose concentrations, suggesting that rates of uptake near V(max) were reached. In summary, by defining the relationship of arterial glucose to [G](om) and [G](im) in the presence of fixed hyperinsulinemia, it is concluded that 1) V(max) for MGU is limited by extracellular and intracellular barriers in type I fibers, as the sarcolemma is freely permeable to glucose; 2) V(max) is limited in muscles with predominantly type IIb fibers by extracellular resistance and transport resistance; and 3) limits to R(g) are determined by resistance at multiple steps and are better defined by distributed control rather than by a single rate-limiting step.

Isotopic techniques were used to test the hypothesis that exercise and nitric oxide synthase (NOS) inhibition have distinct effects on tissue-specific fatty acid and glucose uptakes in a conscious, chronically catheterized mouse model. Uptakes were measured using the radioactive tracers (125)I-labeled beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) and deoxy-[2-(3)H]glucose (DG) during treadmill exercise with and without inhibition of NOS. [(125)I]BMIPP uptake at rest differed substantially among tissues with the highest levels in heart. With exercise, [(125)I]BMIPP uptake increased in both heart and skeletal muscles. In sedentary mice, NOS inhibition induced by nitro-L-arginine methyl ester (L-NAME) feeding increased heart and soleus [(125)I]BMIPP uptake. In contrast, exercise, but not L-NAME feeding, resulted in increased heart and skeletal muscle [2-(3)H]DG uptake. Significant interactions were not observed in the effects of combined exercise and L-NAME feeding on [(125)I]BMIPP and [2-(3)H]DG uptakes. In the conscious mouse, exercise and NOS inhibition produce distinct patterns of tissue-specific fatty acid and glucose uptake; NOS is not required for important components of exercise-associated metabolic signaling, or other mechanisms compensate for the absence of this regulatory mechanism.

To test whether hepatic insulin action and the response to an insulin-induced decrement in blood glucose are enhanced in the immediate postexercise state as they are during exercise, dogs had sampling (artery, portal vein, and hepatic vein) catheters and flow probes (portal vein and hepatic artery) implanted 16 days before a study. After 150 min of moderate treadmill exercise or rest, dogs were studied during a 150-min hyperinsulinemic (1 mU.kg(-1).min(-1)) euglycemic (n = 5 exercised and n = 9 sedentary) or hypoglycemic (65 mg/dl; n = 8 exercised and n = 9 sedentary) clamp. Net hepatic glucose output (NHGO) and endogenous glucose appearance (R(a)) and utilization (R(d)) were assessed with arteriovenous and isotopic ([3-(3)H]glucose) methods. Results show that, immediately after prolonged, moderate exercise, in relation to sedentary controls: 1) the glucose infusion rate required to maintain euglycemia, but not hypoglycemia, was higher; 2) R(d) was greater under euglycemic, but not hypoglycemic conditions; 3) NHGO, but not R(a), was suppressed more by a hyperinsulinemic euglycemic clamp, suggesting that hepatic glucose uptake was increased; 4) a decrement in glucose completely reversed the enhanced suppression of NHGO by insulin that followed exercise; and 5) arterial glucagon and cortisol were transiently higher in the presence of a decrement in glucose. In summary, an increase in insulin action that was readily evident under euglycemic conditions after exercise was abolished by moderate hypoglycemia. The means by which the glucoregulatory system is able to overcome the increase in insulin action during moderate hypoglycemia is related not to an increase in R(a) but to a reduction in insulin-stimulated R(d). The primary site of this reduction is the liver.