Search

The Mouse Metabolic Phenotyping Center (MMPC)Live Program was established in 2023 by the National Institute for Diabetes, Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH) to advance biomedical research by providing the scientific community with standardized, high quality phenotyping services for mouse models of diabetes and obesity. Emerging as the next iteration of the MMPC Program which served the biomedical research community for 20 years (2001–2021), MMPCLive is designed as an outwardly-facing consortium of service cores that collaborate to provide reduced-cost consultation and metabolic, physiologic, and behavioral phenotyping tests on live mice for U.S. biomedical researchers. Four MMPCLive Centers located at universities around the country perform complex and often unique procedures in vivo on a fee for service basis, typically on mice shipped from the client or directly from a repository or vendor. Current areas of expertise include energy balance and body composition, insulin action and secretion, whole body carbohydrate and lipid metabolism, cardiovascular and renal function, food intake and behavior, microbiome and xenometabolism, and metabolic pathway kinetics. Additionally, an opportunity arose to reduce barriers to access and expand the diversity of the biomedical research workforce by establishing the VIBRANT Program. Directed at researchers historically underrepresented in the biomedical sciences, VIBRANT-eligible investigators have access to testing services, travel and career development awards, expert advice and experimental design consultation, and short internships to learn test technologies. Data derived from experiments run by the Centers belongs to the researchers submitting mice for testing which can be made publicly available and accessible from the MMPCLive database following publication. In addition to services, MMPCLive staff provide expertise and advice to researchers, develop and refine test protocols, engage in outreach activities, publish scientific and technical papers, and conduct educational workshops and training sessions to aid researchers in unraveling the heterogeneity of diabetes and obesity. [ABSTRACT FROM AUTHOR]

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 (itgβ1fl/flSCLcre) and without (itgβ1fl/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 itgβ1fl/flSCLcre mice compared with itgβ1fl/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. [ABSTRACT FROM AUTHOR]

Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET)-producing enzyme, promotes insulin sensitivity, and Cyp2c44 −/− mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SCD)-fed male Cyp2c44 −/− mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared with wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol (DAG) levels. Cyp2c44 −/− mice had impaired glucose tolerance and increased hepatic plasma membrane–associated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SCD and high-fat diet (HFD)-fed Cyp2c44 −/− mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SCD-fed Cyp2c44 −/− mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44 −/− hepatocytes. Finally, Cyp2c44 −/− HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, DAG accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling. Article Highlights: Loss of epoxygenase Cyp2c44 leads to glucose intolerance, characterized by an increase in hepatic lipids and hyperlipidemia. Cyp2c44 participates in the regulation of hepatic fatty acid accumulation by limiting the plasma membrane localization of FATP2 and, in turn, intracellular levels of diacylglycerol. Cyp2c44-mediated downregulation of intracellular diacylglycerol levels results in decreased plasma membrane–associated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling. [ABSTRACT FROM AUTHOR]

Obesity-associated complications are causing increasing morbidity and mortality worldwide. Expansion of adipose tissue in obesity leads to a state of low-grade chronic inflammation and dysregulated metabolism, resulting in insulin resistance and metabolic syndrome. Adipose tissue macrophages (ATMs) accumulate in obesity and are a source of proinflammatory cytokines that further aggravate adipocyte dysfunction. Macrophages are rich sources of cyclooxygenase (COX), the rate limiting enzyme for prostaglandin E2 (PGE2) production. When mice were fed a high-fat diet (HFD), ATMs increased expression of COX-2. Selective myeloid cell COX-2 deletion resulted in increased monocyte recruitment and proliferation of ATMs, leading to increased proinflammatory ATMs with decreased phagocytic ability. There were increased weight gain and adiposity, decreased peripheral insulin sensitivity and glucose utilization, increased adipose tissue inflammation and fibrosis, and abnormal adipose tissue angiogenesis. HFD pair-feeding led to similar increases in body weight, but mice with selective myeloid cell COX-2 still exhibited decreased peripheral insulin sensitivity and glucose utilization. Selective myeloid deletion of the macrophage PGE2 receptor subtype, EP4, produced a similar phenotype, and a selective EP4 agonist ameliorated the metabolic abnormalities seen with ATM COX-2 deletion. Therefore, these studies demonstrated that an ATM COX-2/PGE2/ EP4 axis plays an important role in inhibiting adipose tissue dysfunction., Introduction There are currently about 2 billion people globally who are obese or overweight, leading to health complications, type 2 diabetes, cardiovascular disease, and liver disease. The expansion of adipose [...]

Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin's access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs., Introduction Interorgan communication is essential for maintaining physiological homeostasis. One way organs communicate with each other is by sending molecular signals through the circulation. Before these signals can stimulate biological [...]

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 (AMPKα1α2fl/fl+AlbCre) mice and littermate controls (AMPKα1α2fl/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 AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained AMPKα1α2fl/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 AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of AMPKα1α2fl/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. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) is among the most common and costly disorders worldwide (1). The goal of current medical management for T2D is to transiently ameliorate hyperglycemia through daily dosing of one or more antidiabetic drugs. Hypoglycemia and weight gain are common side effects of therapy, and sustained disease remission is not obtainable with nonsurgical approaches. On the basis of the potent glucose-lowering response elicited by activation of brain fibroblast growth factor (FGF) receptors (2-4), we explored the antidiabetic efficacy of centrally administered FGF1, which, unlike other FGF peptides, activates all FGF receptor subtypes (5). We report that a single intracerebroventricular injection of FGF1 at a dose one-tenth of that needed for antidiabetic efficacy following peripheral injection induces sustained diabetes remission in both mouse and rat models of T2D. This antidiabetic effect is not secondary to weight loss, does not increase the risk of hypoglycemia, and involves a novel and incompletely understood mechanism for increasing glucose clearance from the bloodstream. We conclude that the brain has an inherent potential to induce diabetes remission and that brain FGF receptors are potential pharmacological targets for achieving this goal., Growing evidence points to the brain as a potential target for the treatment of T2D (6,7). In rodent models of T2D, hyperglycemia can be ameliorated transiently by either systemic or [...]

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-[alpha] (PPAR[alpha] and fibroblast growth factor 21 (FGF21) expression in a manner modulated by fatty acids was tested in vivo. Wild-type ([gcgr.sup.+/+]) and glucagon receptor-null ([gcgr.sup.-/-]) 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.sup.+/+], but not [gcgr.sup.-/-] mice, increased hepatic phosphorylated AMPK[alpha] at threonine 172 ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) and PPAR[alpha] and FGF21 mRNA. Clamp results in [gcgr.sup.+/+] mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], or PPAR[alpha] and FGF21 mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPAR[alpha] and FGF21 expression. All effects were absent in [gcgr.sup.-/-] 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 PPARa and FGF21 expression are amplified by a physiological increase in circulating lipids. adenosine 5'-monophosphate-activated protein kinase; peroxisome proliferator-activated receptor-[alpha]; fibroblast growth factor 21 doi: 10.1152/ajpendo.00263.2010.

Endothelial nitric oxide synthase (eNOS) is associated with a number of physiological functions involved in the regulation of metabolism; however, the functional role of eNOS is poorly understood. We tested the hypothesis that eNOS is critical to muscle cell signaling and fuel usage during exercise in vivo, using 16-wk-old catheterized (carotid artery and jugular vein) C57BL/6J mice with wild-type (WT), partial (+/-), or no expression (-/-) of eNOS. Quantitative reductions in eNOS expression (~40%) elicited many of the phenotypic effects observed in [enos.sup.-/-] mice under fasted, sedentary conditions, with expression of oxidative phosphorylation complexes I to V and ATP levels being decreased, and total NOS activity and [Ca.sup.2+]/CaM kinase II [Thr.sup.286] phosphorylation being increased in skeletal muscle. Despite these alterations, exercise tolerance was markedly impaired in [enos.sup.-/-] mice during an acute 30-min bout of exercise. An eNOS-dependent effect was observed with regard to AMP-activated protein kinase signaling and muscle perfusion. Muscle glucose and long-chain fatty acid uptake, and hepatic and skeletal muscle glycogenolysis during the exercise bout was markedly accelerated in [enos.sup.-/-] mice compared with [enos.sup.+/-] and WT mice. Correspondingly, [enos.sup.-/-] mice exhibited hypoglycemia during exercise. Thus, the ablation of eNOS alters a number of physiological processes that result in impaired exercise capacity in vivo. The finding that a partial reduction in eNOS expression is sufficient to induce many of the changes associated with ablation of eNOS has implications for chronic metabolic diseases, such as obesity and insulin resistance, which are associated with reduced eNOS expression. mitochondria; oxidative phosphorylation; blood flow doi: 10.1152/ajpregu.00004.2009.

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. insulin resistance; insulin clamp; diabetes doi: 10.1152/ajpendo.90996.2008.

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor--null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism., Introduction The energy state in the cell is defined by adenine nucleotide levels and is critically coupled to nearly all metabolic processes (1). In the cell, the adenine nucleotides ATP, [...]

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the [H.sub.2][O.sub.2]-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial [H.sub.2][O.sub.2] emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial [H.sub.2][O.sub.2] emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity., Introduction Obesity has become a worldwide epidemic, the consequences of which represent a major health care challenge in the 21st century. A decrease in the sensitivity of skeletal muscle to [...]

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. insulin; mice; rat; dog; glycogen; epinephrine; hexokinase; glucose transport; glucose delivery

The present studies examined the relationship between fasting blood glucose and Hb [A.sub.1c] in C57BL/6J, DBA/2J, and KK/H1J 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.sub.1c], as determined with a hand-held automated device using an immunodetection method. [R.sup.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.sub.1c] than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb [A.sub.1c], we also measured total glycosylated hemoglobin using boronate HPLC. Hb [A.sub.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.sub.1c] than overnight-fasted blood glucose in these strains of mice. glycosylated hemoglobin; methodology; 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 ~5 to ~13 mM, insulin rose to a first-phase peak of 403 [+ or -] 73% above basal secretion (n = 5), followed by a rising second phase of mean 289 [+ or -] 41%. In contrast, perifused mouse islets (~75 islets/trial) responded with a similar first phase of 508 [+ or -] 94% (n = 4) but a smaller and virtually fiat second phase of 169 [+ or -] 9% (n = 4, P < 0.05). Furthermore, the slope of the second-phase response differed significantly from zero in mice (2.63 [+ or -] 0.39%/min, P < 0.01), in contrast to perifused islets (0.18 [+ or -] 0.14%/min, P > 0.30). Mice also displayed pulsatile patterns in insulin concentration (period: 4.2 [+ or -] 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. insulin; secretion; first phase; second phase; biphasic; mouse: in vivo; pulses; pulsatile

A portal venous 5-aminoimidazole-4-carboxamide-1-[beta]-D-fibofuranoside infusion that results in hepatic 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribosyl-5-monophosphate (ZMP) concentrations of ~4 [micro]mol/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 [+ or -] 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 [+ or -] 0.05, 1.17 [+ or -] 0.11, and 2.52 [+ or -] 0.16 [micro]mol/g liver in SED, ST, and EXH mice, respectively (P < 0.05). Hepatic energy charge was 0.66 [+ or -] 0.01, 0.58 [+ or -] 0.02, and 0.33 [+ or -] 0.22 in SED, ST, and EXH mice, respectively (P < 0.05). Hepatic glycogen was 11.6 [+ or -] 1.0, 8.8 [+ or -] 2.2, and 0.0 [+ or -] 0.1 mg/g liver in SED, ST, and EXH mice, respectively (P < 0.05). Hepatic AMPK ([Thr.sup.172]) phosphorylation was 1.00 [+ or -] 0.14, 1.96 [+ or -] 0.16, and 7.44 [+ or -] 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. adenosine 5'-monophosphate; adenosine 5'-monophosphate-activated protein kinase; glycogen

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.sup.+/+] and [PAI-1.sup.-/-] 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] (PPAR[gamma]) and adiponectin along with downregulated resistin mRNA in differentiated [PAI-1.sup.-/-] vs. [PAI-1.sup.+/+] 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 PPAR[gamma], CCAAT enhancer-binding protein-[alpha] (C/EBP[alpha]), and the adipocyte-selective fatty acid-binding protein aP2, thus mimicking the phenotype in [PAI-1.sup.-/-] primary adipocytes. Conversely, overexpression of PAI-1 by adenovirus-mediated gene transfer in 3T3-L1 adipocytes inhibited differentiation and reduced PPAR[gamma], C/EBP[alpha], and aP2 expression. This was also associated with a decrease in urokinasetype 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 PPAR[gamma] 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. plasminogen activator inhibitor-l; peroxisome proliferator-activated receptor-[gamma]; adipocyte differentiation; glucose uptake; insulin resistance

This study aimed to test whether stimulation of net hepatic glucose output (NHGO) by increased concentrations of the AMP analog, 5-aminoimidazole-4-carboxamide-l-[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.sup.-1]*[min.sup.-1]. At t = 60 min, 1 mg*[kg.sup.-1]*[min.sup.-1] portal venous 5-aminoimidazole-4-carbox-amide-l-[beta]-D-ribofuranoside (AICAR) infusion was initiated. Arterial insulin rose ~9- and ~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 [+ or -] 0.5 and 2.0 [+ or -] 0.5 mg*[kg.sup.-1]*[min.sup.-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.sup.79]) phosphorylation in INS2. Hepatic ACC ([Ser.sup.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. hepatic glucose production; glycogenolysis; AMP-activated protein kinase; adenosine 5'-monophosphate; 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribosyl-5-monophosphate

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-FAB[P.sup.+/-], n = 8), and null (H-FAB[P.sup.-/-], 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 [[sup.125]I]-15-(p-iodophenyl)-3-R, S-methylpentadecanoic acid and 2-deoxy-[[sup.3]H]glucose was administered to obtain rates of whole body metabolic clearance (MCR) and indexes of muscle LCFA ([R.sub.f]) and glucose ([R.sub.g]) utilization. Fasting, nonesterified fatty acids (mM) were elevated in H-FAB[P.sup.-/-] mice (2.2 [+ or -] 0.9 vs. 1.3 [+ or -] 0.1 and 1.3 [+ or -] 0.2 for WT and H-FAB[P.sup.+/-]). During exercise, blood glucose (mM) increased in WT (11.7 [+ or -] 0.8) and H-FAB[P.sup.+/-] (12.6 [+ or -] 0.9) mice, whereas H-FAB[P.sup.-/-] mice developed overt hypoglycemia (4.8 [+ or -] 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.sub.f] and increasing [R.sub.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. skeletal muscle; metabolism; substrate balance; 2-deoxyglucose; 15-(p-iodophenyl)-3-R, S-methylpentadecanoic acid

AMPK stimulation increases LCFA but not glucose clearance in cardiac muscle in vivo. Am J Physiol Endocrinol Metab 287: E871-E877. 2004. First published July 20, 2004; doi:10.1152/ajpendo.00125. 2004.--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.sup.-1]*[min.sup.-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. Alter 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.sup.-1]*[min.sup.-1]). Glucose was clamped at ~6.5 mM in all groups, and an intravenous bolus of 2-deoxy-[[sup.3]H]glucose and [[sup.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.sup.221]) and AMPK phosphorylation ([Thr.sup.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. substrate; lipid; carbohydrate; heart; interaction; AMP-activated protein kinase; long-chain fatty acid