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Obesity increases deposition of extracellular matrix (ECM) components of cardiac tissue. Since obesity aggregates with insulin resistance and heart disease, it is imperative to determine whether the increased ECM deposition contributes to this disease cluster. The hypotheses tested in this study were that in cardiac tissue of obese mice i) increased deposition of ECM components (collagens and hyaluronan) contributes to cardiac insulin resistance and that a reduction in these components improves cardiac insulin action and ii) reducing excess collagens and hyaluronan mitigates obesity-associated cardiac dysfunction. Genetic and pharmacological approaches that manipulated collagen and hyaluronan contents were employed in obese C57BL/6 mice fed a high fat (HF) diet. Cardiac insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and cardiac function was measured by pressure-volume loop analysis in vivo. We demonstrated a tight association between increased ECM deposition with cardiac insulin resistance. Increased collagen deposition by genetic deletion of matrix metalloproteinase 9 (MMP9) exacerbated cardiac insulin resistance and pirfenidone, a clinically available anti-fibrotic medication which inhibits collagen expression, improved cardiac insulin resistance in obese mice. Furthermore, decreased hyaluronan deposition by treatment with PEGylated human recombinant hyaluronidase PH20 (PEGPH20) improved cardiac insulin resistance in obese mice. These relationships corresponded to functional changes in the heart. Both PEGPH20 and pirfenidone treatment in obese mice ameliorated HF diet-induced abnormal myocardial remodelling. Our results provide important new insights into the role of ECM deposition in the pathogenesis of cardiac insulin resistance and associated dysfunction in obesity of distinct mouse models. These findings support the novel therapeutic potential of targeting early cardiac ECM abnormalities in the prevention and treatment of obesity-related cardiovascular complications. • Obesity increases deposition of ECM collogens and hyaluronan in cardiac tissue. • Increased collagen deposition exacerbates cardiac insulin resistance in obesity. • Pirfenidone, which inhibits collagen expression, improves cardiac insulin resistance. • Decreased hyaluronan deposition by PEGPH20 improves cardiac insulin resistance. • Both pirfenidone and PEGPH20 ameliorates obesity-associated cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28 °C) and room (laboratory) temperature (RT, ∼22 °C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue-specific glucose metabolic index were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole-body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis. • EE and insulin-mediated glucose fluxes are reduced in thermoneutral(TN)-adapted vs room 'laboratory' temperature (RT) mice. • Reduced insulin sensitivity manifests in TN mice whether they are TN-adapted or short-term transitioned from RT-adapted to TN. • TN-adapted mice are resistant to RT-induced increase in insulin sensitivity even though metabolic rate is increased. • TN-adapted mice switched to RT meets increased thermogenic needs by partitioning glucose from glycogen storage to glycolysis. • Brown fat glucose uptake sensitively increases with RT and decreases with TN by an insulin-independent mechanism. [ABSTRACT FROM AUTHOR]

Abstract Objective The loss of liver glycine N-methyltransferase (GNMT) promotes liver steatosis and the transition to hepatocellular carcinoma (HCC). Previous work showed endogenous glucose production is reduced in GNMT-null mice with gluconeogenic precursors being used in alternative biosynthetic pathways that utilize methyl donors and are linked to tumorigenesis. This metabolic programming occurs before the appearance of HCC in GNMT-null mice. The metabolic physiology that sustains liver tumor formation in GNMT-null mice is unknown. The studies presented here tested the hypothesis that nutrient flux pivots from glucose production to pathways that incorporate and metabolize methyl groups in GNMT-null mice with HCC. Methods 2H/13C metabolic flux analysis was performed in conscious, unrestrained mice lacking GNMT to quantify glucose formation and associated nutrient fluxes. Molecular analyses of livers from mice lacking GNMT including metabolomic, immunoblotting, and immunochemistry were completed to fully interpret the nutrient fluxes. Results GNMT knockout (KO) mice showed lower blood glucose that was accompanied by a reduction in liver glycogenolysis and gluconeogenesis. NAD+ was lower and the NAD(P)H-to-NAD(P)+ ratio was higher in livers of KO mice. Indices of NAD+ synthesis and catabolism, pentose phosphate pathway flux, and glutathione synthesis were dysregulated in KO mice. Conclusion Glucose precursor flux away from glucose formation towards pathways that regulate redox status increase in the liver. Moreover, synthesis and scavenging of NAD+ are both impaired resulting in reduced concentrations. This metabolic program blunts an increase in methyl donor availability, however, biosynthetic pathways underlying HCC are activated. Highlights • Loss of glycine N-methyltransferase results in hepatocellular carcinoma. • Metabolic reprogramming ensues to attenuate the increased S-adenosylmethionine. • The metabolic changes include dysregulated liver NAD+ homeostasis and redox state. • Liver glucose formation is reduced and precursors directed to biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Objective Hepatocyte deletion of estrogen receptor alpha (LKO-ERα) worsens fatty liver, dyslipidemia, and insulin resistance in high-fat diet fed female mice. However, whether or not hepatocyte ERα regulates reverse cholesterol transport (RCT) in mice has not yet been reported. Methods and results Using LKO-ERα mice and wild-type (WT) littermates fed a Western-type diet, we found that deletion of hepatocyte ERα impaired in vivo RCT measured by the removal of 3 H-cholesterol from macrophages to the liver, and subsequently to feces, in female mice but not in male mice. Deletion of hepatocyte ERα decreased the capacity of isolated HDL to efflux cholesterol from macrophages and reduced the ability of isolated hepatocytes to accept cholesterol from HDL ex vivo in both sexes. However, only in female mice, LKO-ERα increased serum cholesterol levels and increased HDL particle sizes. Deletion of hepatocyte ERα increased adiposity and worsened insulin resistance to a greater degree in female than male mice. All of the changes lead to a 5.6-fold increase in the size of early atherosclerotic lesions in female LKO-ERα mice compared to WT controls. Conclusions Estrogen signaling through hepatocyte ERα plays an important role in RCT and is protective against lipid retention in the artery wall during early stages of atherosclerosis in female mice fed a Western-type diet. [ABSTRACT FROM AUTHOR]

Summary Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway. [ABSTRACT FROM AUTHOR]

Summary: AS160 has emerged as a key player in insulin-mediated glucose transport through controlling GLUT4 trafficking, which is thought to be regulated by insulin-stimulated phosphorylation of sites including the 14-3-3 binding phospho-Thr649 (equivalent to Thr642 in human AS160). To define physiological roles of AS160-Thr649 phosphorylation and 14-3-3 binding in glucose homeostasis, we substituted this residue by a nonphosphorylatable alanine by knockin mutation in mice. The mutant protein was expressed at normal levels, while insulin-stimulated AS160 binding to 14-3-3s was abolished in homozygous knockin mice. These animals displayed impaired glucose disposal and insulin sensitivity, which were associated with decreased glucose uptake in vivo. Insulin-stimulated glucose transport and cell surface GLUT4 content were reduced in isolated muscles, but not in adipocytes. These results provide genetic evidence that insulin-induced AS160-Thr649 phosphorylation and/or its binding to 14-3-3 play an important role in regulating whole-body glucose homeostasis, at least in part through regulating GLUT4 trafficking in muscle. [ABSTRACT FROM AUTHOR]

We determined whether intraportal caffeine infusion, at rates designed to create concentrations similar to that seen with normal dietary intake, would enhance net hepatic glucose uptake (NHGU) during a glucose load. Dogs (n = 15) were implanted with sampling and infusion catheters as well as flow probes >16 d before the studies. After a basal sampling period, dogs were administered a somatostatin infusion (0-150 min) as well as intraportal infusions of glucose [18 micromol/(kg . min)], basal glucagon [0.5 ng/(kg . min)], and insulin [8.3 pmol/(kg . min)] to establish mild hyperinsulinemia. Arterial glucose was clamped at 10 mmol/L with a peripheral glucose infusion. At 80 min, either saline (Control; n = 7) or caffeine [1.5 micromol/(kg . min); n = 8] was infused into the portal vein. Arterial insulin, glucagon, norepinephrine, and glucose did not differ between groups. In dogs infused with caffeine, NHGU was significantly higher than in controls [21.2 +/- 4.3 vs. 11.2 +/- 1.6 micromol/(kg . min)]. Caffeine increased net hepatic lactate output compared with controls [12.5 +/- 3.8 vs. 5.5 +/- 1.5 micromol/(kg . min)]. These findings indicate that physiologic circulating levels of caffeine can enhance NHGU during a glucose load, and the added glucose consumed by the liver is in part converted to lactate. [ABSTRACT FROM AUTHOR]

Consumption of large amounts of coffee has been shown to decrease the incidence of type 2 diabetes. However, the specific compounds and mechanisms responsible for this effect are not known. The aim of this study was to determine the effects of a decaffeinated coffee extract and a synthetic quinide, representative of those found in roasted coffee, 3,4-diferuloyl-1,5-quinolactone, on insulin-stimulated glucose disposal and muscle glucose uptake. Experiments were performed on conscious rats during hyperinsulinemic, euglycemic clamps receiving gastric infusions of saline, a decaffeinated coffee extract (DECAF) (220 mg/kg), or 3,4-diferuloyl-1,5-quinide (DIFEQ) (110 mg/kg). Following treatment, rats received an intravenous bolus of deoxy-[2-3H] glucose to assess muscle glucose uptake (Rg, micromol x 100 g(-1) x min(-1)). Glucose infusions [mg/(kg x min)] required to maintain euglycemia during the tracer period were higher with DIFEQ (14.6 +/- 0.7) than with saline (10.8 +/- 0.7) and DECAF (11.5 +/- 1.1). Despite increased glucose requirements, Rg in skeletal (soleus, gastrocnemius, superficial vastus lateralis) and cardiac muscle were unchanged. DECAF or DIFEQ did not affect heart rate, blood pressure, plasma nonesterified fatty acids or liver aminotransferase activity. These results demonstrate that DIFEQ increases whole-body glucose disposal independently of skeletal muscle Rg. [ABSTRACT FROM AUTHOR]

To determine the contributions of transporter-mediated and passive absorption during an intraduodenal glucose infusion in a large animal model, six mongrel dogs had sampling catheters (portal vein, femoral artery, duodenum), infusion catheters (vena cava, duodenum) and a portal vein flow probe implanted 17 d before an experiment. Protocols consisted of a basal (-30 to 0 min) and an experimental (0-90 min) period. An intraduodenal glucose infusion of 44 micromol/(kg. min) was initiated at t = 0 min. At t = 20 and 80 min, 3-O-[3H]methylglucose and L-[14C]glucose (L-Glc) were injected intraduodenally. Phloridzin, an inhibitor of the Na+/K+ ATP-dependent transporter (SGLT1), was infused from t = 60 to 90 min in the presence of a peripheral isoglycemic clamp. Net gut glucose output was 21.1 +/- 3.0 micromol/(kg. min) from t = 0 to 60 min. Transporter-mediated glucose absorption was calculated using three approaches, which involved either direct measurements or indirect estimates of duodenal glucose analog radioactivities, to account for the assumptions and difficulties inherent to duodenal sampling. Values were essentially the same regardless of calculations used because transporter-mediated absorption was 89 +/- 1%, 90 +/- 2% and 91 +/- 2% of net gut glucose output. Phloridzin-induced inhibition of transporter-mediated absorption completely abolished passive absorption of L-Glc. We conclude that in dogs, transporter-mediated glucose absorption constitutes the vast majority of glucose absorbed from the gut and is required for passive glucose absorption. The method described here is applicable to investigation of the mechanisms of gut glucose absorption under a variety of nutritional, physiologic and pathophysiologic conditions. [ABSTRACT FROM AUTHOR]

Increased deposition of the extracellular matrix (ECM) in adipose tissue (AT) during obesity contributes to insulin resistance. The integrin receptors transmit changes in the extracellular environment causing corresponding intracellular adaptations. Integrin-linked kinase (ILK), an adaptor protein, is a central hub for intracellular signaling of integrins. This study determined the role of ILK in adipose function and insulin resistance. The pathogenic role of ILK in obesity and insulin resistance was studied in human adipose tissue and adipocyte-specific ILK-deficient mice (ILKlox/lox AdCre). ILKlox/lox AdCre mice together with wild-type littermates (ILKlox/lox) were fed a chow diet or 60% high-fat (HF) diet for 16 weeks. In vivo insulin sensitivity was determined by hyperinsulinemic-euglycemic clamps. AT ILK expression was increased by HF diet feeding in mice and increased in visceral fat of morbidly obese humans. The HF-fed ILKlox/lox AdCre mice displayed reduced fat mass and improved glucose tolerance relative to the HF-fed ILKlox/lox mice. During a hyperinsulinemic-euglycemic clamp, the HF-fed ILKlox/lox AdCre mice exhibited partially improved insulin resistance in AT. Lipolysis was suppressed to a greater extent by insulin and glucose uptake in brown AT (BAT) increased. Increased inhibition of lipolysis may have been attributed to increased vascularization in white AT, while increased glucose uptake in BAT was associated with increased Akt phosphorylation and P38/JNK dephosphorylation. Notably, AT insulin sensitivity in lean mice was not affected by ILK deletion. Moreover, reduced fat mass in the HF-fed ILKlox/lox AdCre mice may have been attributed to decreased free fatty acid uptake into adipocytes via the downregulation of CD36 gene expression. Consistent with the results in the mice, knockdown and knockout of ILK in 3T3-L1 cells decreased lipid accumulation and CD36 gene expression during adipogenesis. These data show that adipocyte ILK is important for regulating HF diet-mediated insulin resistance in AT in a manner consistent with AT function. • ILK protein increased in visceral adipose tissue of obese humans and mice. • Mice lacking adipocyte ILK had less fat and improved glucose tolerance in obesity. • Adipocyte ILK deletion improved anti-lipolytic action of insulin in obese mice. • Adipocyte ILK deletion stimulated brown adipose tissue glucose uptake in obese mice. [ABSTRACT FROM AUTHOR]

This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux. • Mitochondria lacking CrAT and Sirt3 are susceptible to extreme protein acetylation • Hyperacetylation is accompanied by disturbances in redox balance and insulin action • Hyperacetylation does not affect mitochondrial respiration and enhances fat oxidation • Sirt3 flux and acetyl-lysine turnover promote a fuel switch from fat to glucose Williams et al. show that double knockout (DKO) mice harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation are susceptible to extreme mitochondrial hyperacetylation and insulin resistance. However, DKO mitochondria have normal respiratory function and increased fat oxidation. The findings suggest that acetyl-lysine turnover, not stoichiometry, regulates mitochondrial fuel use. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is a highly-dynamic compartment that undergoes remodeling as a result of injury and repair. Over the past decade, mounting evidence in humans and rodents suggests that ECM remodeling is associated with diet-induced insulin resistance in several metabolic tissues. In addition, integrin receptors for the ECM have also been implicated in the regulation of insulin action. This review addresses what is currently known about the ECM, integrins, and insulin action in the muscle, liver, and adipose tissue. Understanding how ECM remodeling and integrin signaling regulate insulin action may aid in the development of new therapeutic targets for the treatment of insulin resistance and type 2 diabetes (T2D). [ABSTRACT FROM AUTHOR]

Abstract: This review summarizes the emerging role of AMP-activated protein kinase (AMPK) in mediating endocrine regulation of metabolic fluxes in the liver. There are a number of hormones which, when acting on the liver, alter AMPK activation. Here we describe those hormones associated with activation and de-activation of AMPK and the potential mechanisms for changes in AMPK activation state. The actions of these hormones, in many cases, are consistent with downstream effects of AMPK signaling thus strengthening the circumstantial case for AMPK-mediated hormone action. In recent years, genetic mouse models have also been used in an attempt to establish the role of AMPK in hormone-stimulated metabolism in the liver. Few experiments have, however, firmly established a causal relationship between hormone action at the liver and AMPK signaling. [Copyright &y& Elsevier]

Purpose: Diabetes mellitus is a delayed effect of radiation exposure in human and nonhuman primates. Diabetes mellitus is characterized by peripheral tissue insulin resistance, and as a result, irradiation exposure may cause important changes in insulin-sensitive tissues such as muscle and adipose.Methods and Materials: We prospectively investigated changes in response to irradiation (4 Gy whole body exposure) in 16 male rhesus macaques. We evaluated changes in body composition and glycemic control for 2 years. Insulin responsiveness, lipolysis, inflammation, and fibrosis were evaluated at study end.Results: Irradiated animals accumulate less fat and significantly increased percent glycation of hemoglobin A1c over time, such that 40% of irradiated monkeys had values that define them as diabetic at 2 years. Subcutaneous (SQ) adipose tissue was insulin resistant, as evidenced by reduced phosphorylation of the insulin receptor substrate-1 in response to insulin challenge and had increased basal lipolysis despite comparable insulin exposures to control animals. Irradiated SQ adipose tissue had more macrophage infiltration and adipocytes were larger. The observed hypertrophy was associated with decreased glycemic control and macrophage infiltration correlated with decreased adiponectin, signifying that inflammation is associated with worsening health. No evidence of SQ adipose fibrosis was detected.Conclusions: Our study is the first to prospectively illustrate that sublethal irradiation exposures directly propagate metabolic disease in the absence of obesity in nonhuman primates and implicate SQ adipose dysfunction as a target tissue. [ABSTRACT FROM AUTHOR]

Glycine N-methyltransferase (GNMT) is the most abundant liver methyltransferase regulating the availability of the biological methyl donor, S-adenosylmethionine (SAM). Moreover, GNMT has been identified to be down-regulated in hepatocellular carcinoma (HCC). Despite its role in regulatingSAMlevels and association of its down-regulation with liver tumorigenesis, the impact of reduced GNMT on metabolic reprogramming before the manifestation of HCC has not been investigated in detail. Herein, we used ²H/13C metabolic flux analysis in conscious, unrestrained mice to test the hypothesis that the absence of GNMT causes metabolic reprogramming. GNMT-null (KO) mice displayed a reduction in blood glucose that was associated with a decline in both hepatic glycogenolysis and gluconeogenesis. The reduced gluconeogenesis was due to a decrease in liver gluconeogenic precursors, citric acid cycle fluxes, and anaplerosis and cataplerosis. A concurrent elevation in both hepatic SAMand metabolites of SAM utilization pathways was observed in the KO mice. Specifically, the increase in metabolites of SAM utilization pathways indicated that hepatic polyamine synthesis and catabolism, transsulfuration, and de novo lipogenesis pathways were increased in the KO mice. Of note, these pathways utilize substrates that could otherwise be used for gluconeogenesis. Also, this metabolic reprogramming occurs before the welldocumented appearance of HCC in GNMT-null mice. Together, these results indicate that GNMT deletion promotes a metabolic shift whereby nutrients are channeled away from glucose formation toward pathways that utilize the elevated SAM. [ABSTRACT FROM AUTHOR]

Pathologies including diabetes and conditions such as exercise place an unusual demand on liver energy metabolism, and this demand induces a state of energy discharge. Hepatic AMP-activated protein kinase (AMPK) has been proposed to inhibit anabolic processes such as gluconeogenesis in response to cellular energy stress. However, bothAMPKactivation and glucose release from the liver are increased during exercise. Here, we sought to test the role of hepatic AMPK in the regulation of in vivo glucose-producing and citric acid cycle-related fluxes during an acute bout of muscular work. We used ²H/13C metabolic flux analysis to quantify intermediary metabolism fluxes in both sedentary and treadmill-running mice. Additionally, liver-specific AMPK α1 and α2 subunit KO and WT mice were utilized. Exercise caused an increase in endogenous glucose production, glycogenolysis, and gluconeogenesis from phosphoenolpyruvate. Citric acid cycle fluxes, pyruvate cycling, anaplerosis, and cataplerosis were also elevated during this exercise. Sedentary nutrient fluxes in the postabsorptive state were comparable for the WT and KO mice. However, the increment in the endogenous rate of glucose appearance during exercise was blunted in the KO mice because of a diminished glycogenolytic flux. This lower rate of glycogenolysis was associated with lower hepatic glycogen content before the onset of exercise and prompted a reduction in arterial glucose during exercise. These results indicate that liver AMPKα1α2 is required for maintaining glucose homeostasis during an acute bout of exercise. [ABSTRACT FROM AUTHOR]

Metabolic stress, as well as several antidiabetic agents, increases hepatic nucleotide monophosphate (NMP) levels, activates AMP-activated protein kinase (AMPK), and suppresses glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR; 8 mg⋅kg -1 ⋅ min-1)-euglycemic clamps were performed to elicit an increase in NMP in wild type (α1 α2lox/lox) and liver-specific AMPK knock-out mice (α1 α2lox/lox + Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5-h fasted α1 α2lox/lox and α1 α2lox/lox + Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose disappearance. These results demonstrate that AMPK is unnecessary for normal 5-h fasting glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK energy sensor paradigm. AICAR reduced hepatic energy charge by ~20% in α1 α2lox/lox, which was exacerbated by ~2-fold in α1 α2lox/lox + Albcre. This corresponded to a ~6-fold rise in AMP/ATP in α1 α2lox/lox + Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ~30% lower in α1 α2lox/lox + Albcre than α1 α2lox/lox livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKα2 subunit (α2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in α2-KD mice; expression of neuronal NOS (NOSμ) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 ± 8 and 50 ± 7%, respectively, in skeletal muscle of α2-KD mice (p < 0,05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSμ expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle. [ABSTRACT FROM AUTHOR]

Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during exercise because GLUT4 overexpression does not increase exercise-stimulated MGU. Instead of glucose transport, glucose phosphorylation is a primary limitation of exercise-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair exercise-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity was increased. Thus, C57BL/6J mice with hexokinase II (HKII) overexpression (HKTg), a GLUT4 partial knock-out (G4+/-), or both (HKTg + G4+/-) and wild-type (WT) littermates were implanted with carotid artery and jugular vein catheters for sampling and infusions at 4 months of age. After a 7-day recovery, 5-h fasted mice remained sedentary or ran on a treadmill at 0.6 mph for 30 min (n = 9-12 per group) and received a bolus of 2-deoxy[³H]glucose to provide an index of MGU (Rg). Arterial blood glucose and plasma insulin concentrations were similar in WT, G4+/-, HKTg, and HKTg + G4+/- mice. Sedentary Rg values were the same in all genotypes in all muscles studied, confirming that glucose transport is a significant barrier to basal glucose uptake. Gastrocnemius and soleus Rg were greater in exercising compared with sedentary mice in all genotypes. During exercise, G4+/- mice had a marked increase in blood glucose that was corrected by the addition of HK II overexpression. Exercise Rg (µmol/100g/min) was not different between WT and G4+/- mice in the gastrocnemius (24 ± 5 versus 21 ± 2) or the soleus (54 ± 6 versus 70 ± 7). In contrast, the enhanced exercise Rg observed in HKTg mice compared with that in WT mice was absent in HKTg + G4+/- mice in both the gastrocnemius (39 ± 7 versus 22 ± 6) and the soleus (98 ± 13 versus 65 ± 13). Thus, glucose transport is not a significant barrier to exercise-stimulated MGU despite a 50% reduction in GLUT4 content when glucose phosphorylation capacity is normal. However, when glucose phosphorylation capacity is increased by HK II overexpression, GLUT4 availability becomes a marked limitation to exercise-stimulated MGU. [ABSTRACT FROM AUTHOR]

Measurement of fractional lipogenesis by condensation polymerization methods assumes constant enrichment of lipogenic acetyl-CoA in all hepatocytes, mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) represent such methods and are based on the combinatorial analyses of mass isotopomer distributions (MIDs) of fatty acids and sterols. We previously showed that the concentration and enrichment of [13C]acetate decrease markedly across the dog liver because of the simultaneous uptake and production of acetate. To test for zonation of the enrichment of lipogenic acetyl-CoA, conscious dogs, prefitted with transhepatic catheters, were infused with glucose and [1,2-13C2]acetate in a branch of the portal vein. Analyses of MIDs of fatty acids and sterols isolated from liver, bile, and plasma very low density lipoprotein by a variant of ISA designed to detect gradients in precursor enrichment revealed marked zonation of enrichment of lipogenic acetyl-CoA. As control experiments where no zonation of acetyl-CoA enrichment would be expected, isolated rat livers were perfused with 10 nM [1,213C2]acetate. The ISA analyses of MIDs of fatty acids and sterols from liver and bile still revealed a zonation of acetyl-CoA enrichment. We conclude that zonation of hepatic acetyl-CoA enrichment occurs under a variety of animal models and physiological conditions. Failure to consider gradients of precursor enrichment can lead to underestimations of fractional lipogenesis calculated from the mass isotopomer distributions. The degree of such underestimation was modeled in vitro, and the data are reported in the companion paper (Bederman, I. R., Kasumov, T., Reszko, A. E., David, F., Brunengraber, H., and Kelleher, J. K. (2004) J. Biol. Chem. 279, 43217-43226). [ABSTRACT FROM AUTHOR]