Translate text to:
In JoVE (2)
- Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice
- Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Other Publications (33)
- The Journal of Nutrition
- American Journal of Physiology. Endocrinology and Metabolism
- Journal of Applied Physiology (Bethesda, Md. : 1985)
- American Journal of Physiology. Endocrinology and Metabolism
- American Journal of Physiology. Endocrinology and Metabolism
- American Journal of Physiology. Endocrinology and Metabolism
- The Journal of Biological Chemistry
- American Journal of Physiology. Endocrinology and Metabolism
- The Journal of Physiology
- Clinical and Experimental Pharmacology & Physiology
- The Journal of Physiology
- Canadian Journal of Applied Physiology = Revue Canadienne De Physiologie AppliquÃ©e
- Journal of Applied Physiology (Bethesda, Md. : 1985)
- The Journal of Physiology
- The Journal of Biological Chemistry
- Molecular Endocrinology (Baltimore, Md.)
- Molecular and Cellular Biology
- Biochimica Et Biophysica Acta
- The Journal of Biological Chemistry
- The Journal of Experimental Biology
- Obesity (Silver Spring, Md.)
- Molecular Endocrinology (Baltimore, Md.)
Articles by Patrick T. Fueger in JoVE
Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice
Julio E. Ayala1, Deanna P. Bracy2,3, Carlo Malabanan3, Freyja D. James2,3, Tasneem Ansari3, Patrick T. Fueger4, Owen P. McGuinness2,3, David H. Wasserman2,3
1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine
Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Patrick T. Fueger1,2, Angelina M. Hernandez2, Yi-Chun Chen2, E. Scott Colvin1,2
1Department of Pediatrics, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine
Other articles by Patrick T. Fueger on PubMed
Transporter-mediated Absorption is the Primary Route of Entry and is Required for Passive Absorption of Intestinal Glucose into the Blood of Conscious Dogs
The Journal of Nutrition. Jul, 2002 | Pubmed ID: 12097672
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.
American Journal of Physiology. Endocrinology and Metabolism. Mar, 2003 | Pubmed ID: 12556351
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.
Journal of Applied Physiology (Bethesda, Md. : 1985). Sep, 2003 | Pubmed ID: 12740315
The purpose of this study was to assess whether a prior bout of exercise enhances passive gut glucose absorption. Mongrel dogs had sampling catheters, infusion catheters, and a portal vein flow probe implanted 17 days before an experiment. Protocols consisted of either 150 min of exercise (n = 8) or rest (n = 7) followed by basal (-30 to 0 min) and a primed (150 mg/kg) intraduodenal glucose infusion [8.0 mg x kg-1x min-1, time (t) = 0-90 min] periods. 3-O-[3H]methylglucose (absorbed actively, facilitatively, and passively) and l-[14C]glucose (absorbed passively) were injected into the duodenum at t = 20 and 80 min. Phloridzin, an inhibitor of the active sodium glucose cotransporter-1 (SGLT-1), was infused (0.1 mg x kg-1 x min-1) into the duodenum from t = 60-90 min with a peripheral venous isoglycemic clamp. Duodenal, arterial, and portal vein samples were taken every 10 min during the glucose infusion, as well as every minute after each tracer bolus injection. Net gut glucose output in exercised dogs increased compared with that in the sedentary group (5.34 +/- 0.47 and 4.02 +/- 0.53 mg x kg-1x min-1). Passive gut glucose absorption increased approximately 100% after exercise (0.93 +/- 0.06 and 0.45 +/- 0.07 mg x kg-1 x min-1). Transport-mediated glucose absorption increased by approximately 20%, but the change was not significant. The infusion of phloridzin eliminated the appearance of both glucose tracers in sedentary and exercised dogs, suggesting that passive transport required SGLT-1-mediated glucose uptake. This study shows 1). that prior exercise enhances passive absorption of intraduodenal glucose into the portal vein and 2). that basal and the added passive gut glucose absorption after exercise is dependent on initial transport of glucose via SGLT-1.
Hexokinase II Partial Knockout Impairs Exercise-stimulated Glucose Uptake in Oxidative Muscles of Mice
American Journal of Physiology. Endocrinology and Metabolism. Nov, 2003 | Pubmed ID: 12865258
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.
Diabetes. Aug, 2003 | Pubmed ID: 12882903
To determine if prior exercise enhances insulin-stimulated extraction of glucose by the liver, chronically catheterized dogs were submitted to 150 min of treadmill exercise or rest. After exercise or rest, dogs received portal glucose (18 micro mol x kg(-1) x min(-1)), peripheral somatostatin, and basal portal glucagon infusions from t = 0 to 150 min. A peripheral glucose infusion was used to clamp arterial blood glucose at 8.3 mmol/l. Insulin was infused into the portal vein to create either basal levels or mild hyperinsulinemia. Prior exercise did not increase whole-body glucose disposal in the presence of basal insulin (25.5 +/- 1.5 vs. 20.3 +/- 1.7 micro mol x kg(-1) x min(-1)), but resulted in a marked enhancement in the presence of elevated insulin (97.2 +/- 15.1 vs. 64.4 +/- 7.4 micro mol x kg(-1) x min(-1)). Prior exercise also increased net hepatic glucose uptake in the presence of both basal insulin (7.5 +/- 1.2 vs. 2.9 +/- 2.4 micro mol x kg(-1) x min(-1)) and elevated insulin (22.0 +/- 3.5 vs. 11.5 +/- 1.8 micro mol x kg(-1) x min(-1)). Likewise, net hepatic glucose fractional extraction was increased by prior exercise with both basal insulin (0.04 +/- 0.01 vs. 0.01 +/- 0.01 micro mol x kg(-1) x min(-1)) and elevated insulin (0.10 +/- 0.01 vs. 0.05 +/- 0.01). Hepatic glycogen synthesis was increased by elevated insulin, but was not enhanced by prior exercise. Although the increase in glucose extraction after exercise could be ascribed to increased insulin action, the increase in hepatic glycogen synthesis was independent of it.
Distributed Control of Glucose Uptake by Working Muscles of Conscious Mice: Roles of Transport and Phosphorylation
American Journal of Physiology. Endocrinology and Metabolism. Jan, 2004 | Pubmed ID: 13129858
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.
Hexokinase II Overexpression Improves Exercise-stimulated but Not Insulin-stimulated Muscle Glucose Uptake in High-fat-fed C57BL/6J Mice
Diabetes. Feb, 2004 | Pubmed ID: 14747279
The aim of the present study was to determine the specific sites of impairment to muscle glucose uptake (MGU) in the insulin-resistant high-fat-fed, conscious C57BL/6J mouse. Wild type (WT) and hexokinase II overexpressing (HK(Tg)) mice were fed either a standard diet or high-fat diet and studied at 4 months of age. A carotid artery and jugular veins had catheters chronically implanted for sampling and infusions, respectively, and mice were allowed to recovery for at least 5 days. Mice were fasted for 5 h and underwent a hyperinsulinemic-euglycemic clamp or saline infusion for 120 min. Separate groups of mice were studied during 30-min sedentary or treadmill exercise periods. A bolus of 2-deoxy[(3)H]glucose was administered 25 min before the end of each study for determination of R(g), an index of tissue-specific glucose uptake. Fasting blood glucose was increased in high-fat compared with standard diet-fed WT (194 +/- 4 vs. 171 +/- 4 mg/dl) but not HK(Tg) (179 +/- 5 vs. 171 +/- 3 mg/dl) mice. High-fat feeding created hyperinsulinemia in both WT and HK(Tg) mice (58 +/- 8 and 77 +/- 15 micro U/ml) compared with standard diet-fed mice (21 +/- 2 and 20 +/- 1 micro U/ml). R(g) was not affected by genotype or diet during either saline infusion or sedentary conditions. HK II overexpression augmented insulin-stimulated R(g) in standard diet-fed but not high-fat-fed mice. Exercise-stimulated R(g) was impaired by high-fat feeding in WT mice, but this impairment was largely rectified in HK(Tg) mice. In conclusion, high-fat feeding impairs both insulin- and exercise-stimulated MGU, but only exercise-stimulated MGU was corrected by HK II overexpression.
AMP Kinase-induced Skeletal Muscle Glucose but Not Long-chain Fatty Acid Uptake is Dependent on Nitric Oxide
Diabetes. Jun, 2004 | Pubmed ID: 15161745
The purpose of this study was to examine the effects of AMP kinase (AMPK) activation on in vivo glucose and long-chain fatty acid (LCFA) uptake in skeletal muscle and to examine the nitric oxide (NO) dependence of any putative effects. Catheters were chronically implanted in the carotid artery and jugular vein of male Sprague-Dawley rats. After 4 days of recovery, rats were given either water or water containing 1 mg/ml nitro-l-arginine methylester (l-NAME) for 2.5 days. After an overnight fast, rats underwent one of five protocols: saline, 5-aminoimidazole-4-carboxamide-1-B-d-ribofuranoside (AICAR) (10 mg. kg(-1). min(-1)), l-NAME, AICAR + l-NAME, or AICAR + Intralipid (20%, 0.02 ml. kg(-1). min(-1)). Glucose was clamped at approximately 6.5 mmol/l 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 (K(g)) and LCFA (K(f)) uptake and clearance. At 150 min, soleus, gastrocnemius, and superficial vastus lateralis were excised for tracer determination. Both K(g) and K(f) increased with AICAR in all muscles studied. K(g) decreased with increasing muscle composition of type 1 slow-twitch fibers, whereas K(f) increased. In addition, AICAR-induced increases in K(g) but not K(f) were abolished by l-NAME in the majority of muscles examined. This shows that the mechanisms by which AMPK stimulates glucose and LCFA uptake are distinct.
American Journal of Physiology. Endocrinology and Metabolism. Nov, 2004 | Pubmed ID: 15265760
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.
Regulation of Insulin-stimulated Muscle Glucose Uptake in the Conscious Mouse: Role of Glucose Transport is Dependent on Glucose Phosphorylation Capacity
Endocrinology. Nov, 2004 | Pubmed ID: 15284204
Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during hyperinsulinemia, because glucose phosphorylation is the more formidable barrier to insulin-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair insulin-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity is increased. Thus, chow-fed C57BL/6J mice with a GLUT4 partial knockout (GLUT4(+/-)), hexokinase II overexpression (HK(Tg)), or both (HK(Tg) + GLUT4(+/-)) and wild-type littermates were studied. Carotid artery and jugular vein catheters were implanted for sampling and infusions at 4 months of age. After a 5-d recovery, 5-h fasted mice (n = 8-11/group) underwent a 120-min saline infusion or insulin clamp (4 mU/kg.min insulin with glucose maintained at 165 mg/dl) and received a 2-deoxy[(3)H]glucose bolus to provide an index of MGU (R(g)) for the soleus, gastrocnemius, and superficial vastus lateralis. Basal R(g) from all muscles studied from saline-infused mice were not changed by any of the genetic modifications. HK(Tg) mice had augmented insulin-stimulated R(g) in all muscles studied compared with remaining genotypes. Insulin-stimulated R(g) was not impaired in any of the muscles studied from GLUT4(+/-) mice. However, the enhanced insulin-stimulated R(g) created by HK overexpression was ablated in HK(Tg) + GLUT4(+/-) mice. Thus, a 50% reduction of normal GLUT4 content in the presence of normal HK activity does not impair insulin-stimulated MGU. However, when the glucose phosphorylation barrier is lowered by HK overexpression, GLUT4 availability becomes a limitation to insulin-stimulated MGU.
Control of Exercise-stimulated Muscle Glucose Uptake by GLUT4 is Dependent on Glucose Phosphorylation Capacity in the Conscious Mouse
The Journal of Biological Chemistry. Dec, 2004 | Pubmed ID: 15456776
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 (HK(Tg)), a GLUT4 partial knock-out (G4(+/-)), or both (HK(Tg) + 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 (micromol/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.
Exercise-induced Changes in Insulin and Glucagon Are Not Required for Enhanced Hepatic Glucose Uptake After Exercise but Influence the Fate of Glucose Within the Liver
Diabetes. Dec, 2004 | Pubmed ID: 15561932
To test whether pancreatic hormonal changes that occur during exercise are necessary for the postexercise enhancement of insulin-stimulated net hepatic glucose uptake, chronically catheterized dogs were exercised on a treadmill or rested for 150 min. At the onset of exercise, somatostatin was infused into a peripheral vein, and insulin and glucagon were infused in the portal vein to maintain basal levels (EX-Basal) or simulate the response to exercise (EX-Sim). Glucose was infused as needed to maintain euglycemia during exercise. After exercise or rest, somatostatin infusion was continued in exercised dogs and initiated in dogs that had remained sedentary. In addition, basal glucagon, glucose, and insulin were infused in the portal vein for 150 min to create a hyperinsulinemic-hyperglycemic clamp in EX-Basal, EX-Sim, and sedentary dogs. Steady-state measurements were made during the final 50 min of the clamp. During exercise, net hepatic glucose output (mg x kg(-1) x min(-1)) rose in EX-Sim (7.6 +/- 2.8) but not EX-Basal (1.9 +/- 0.3) dogs. During the hyperinsulinemic-hyperglycemic clamp that followed either exercise or rest, net hepatic glucose uptake (mg x kg(-1) x min(-1)) was elevated in both EX-Basal (4.0 +/- 0.7) and EX-Sim (4.6 +/- 0.5) dogs compared with sedentary dogs (2.0 +/- 0.3). Despite this elevation in net hepatic glucose uptake after exercise, glucose incorporation into hepatic glycogen, determined using [3-3H]glucose, was not different in EX-Basal and sedentary dogs, but was 50 +/- 30% greater in EX-Sim dogs. Exercise-induced changes in insulin and glucagon, and consequent glycogen depletion, are not required for the increase in net hepatic glucose uptake after exercise but result in a greater fraction of the glucose consumed by the liver being directed to glycogen.
Heart-type Fatty Acid-binding Protein Reciprocally Regulates Glucose and Fatty Acid Utilization During Exercise
American Journal of Physiology. Endocrinology and Metabolism. Feb, 2005 | Pubmed ID: 15454399
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.
Control of Muscle Glucose Uptake: Test of the Rate-limiting Step Paradigm in Conscious, Unrestrained Mice
The Journal of Physiology. Feb, 2005 | Pubmed ID: 15576451
The aim of this study was to test whether in fact glucose transport is rate-limiting in control of muscle glucose uptake (MGU) under physiological hyperinsulinaemic conditions in the conscious, unrestrained mouse. C57Bl/6J mice overexpressing GLUT4 (GLUT4(Tg)), hexokinase II (HK(Tg)), or both (GLUT4(Tg) + HK(Tg)), were compared to wild-type (WT) littermates. Catheters were implanted into a carotid artery and jugular vein for sampling and infusions at 4 month of age. After a 5-day recovery period, conscious mice underwent one of two protocols (n = 8-14/group) after a 5-h fast. Saline or insulin (4 mU kg(-1) min(-1)) was infused for 120 min. All mice received a bolus of 2-deoxy[(3)H]glucose (2-(3)HDG) at 95 min. Glucose was clamped at approximately 165 mg dl(-1) during insulin infusion and insulin levels reached approximately 80 microU ml(-1). The rate of disappearance of 2-(3)HDG from the blood provided an index of whole body glucose clearance. Gastrocnemius, superficial vastus lateralis and soleus muscles were excised at 120 min to determine 2-(3)HDG-6-phosphate levels and calculate an index of MGU (R(g)). Results show that whole body and tissue-specific indices of glucose utilization were: (1) augmented by GLUT4 overexpression, but not HKII overexpression, in the basal state; (2) enhanced by HKII overexpression in the presence of physiological hyperinsulinaemia; and (3) largely unaffected by GLUT4 overexpression during insulin clamps whether alone or combined with HKII overexpression. Therefore, while glucose transport is the primary barrier to MGU under basal conditions, glucose phosphorylation becomes a more important barrier during physiological hyperinsulinaemia in all muscles. The control of MGU is distributed rather than confined to a single rate-limiting step such as glucose transport as glucose transport and phosphorylation can both become barriers to skeletal muscle glucose influx.
5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside Causes Acute Hepatic Insulin Resistance in Vivo
Diabetes. Feb, 2005 | Pubmed ID: 15677492
The infusion of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) causes a rise in tissue concentrations of the AMP analog 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranotide (ZMP), which mimics an elevation of cellular AMP levels. The purpose of this work was to determine the effect of raising hepatic ZMP levels on hepatic insulin action in vivo. Dogs had sampling and infusion catheters as well as flow probes implanted 16 days before an experiment. After an 18-h fast, blood glucose was 82 +/- 1 mg/dl and basal net hepatic glucose output 1.5 +/- 0.2 mg . kg(-1) . min(-1). Dogs received portal venous glucose (3.2 mg . kg(-1) . min(-1)), peripheral venous somatostatin, and basal portal venous glucagon infusions from -90 to 60 min. Physiological hyperinsulinemia was established with a portal insulin infusion (1.2 mU . kg(-1) . min(-1)). Peripheral venous glucose infusion was used to clamp arterial blood glucose at 150 mg/dl. Starting at t = 0 min, dogs received portal venous AICAR infusions of 0, 1, or 2 mg . kg(-1) . min(-1). Net hepatic glucose uptake was 2.4 +/- 0.5 mg . kg(-1) . min(-1) (mean of all groups) before t = 0 min. In the absence of AICAR, net hepatic glucose uptake was 1.9 +/- 0.4 mg . kg(-1) . min(-1) at t = 60 min. The lower-dose AICAR infusion caused a complete suppression of net hepatic glucose uptake (-1.0 +/- 1.7 mg . kg(-1) . min(-1) at t = 60 min). The higher AICAR dose resulted in a profound shift in hepatic glucose balance from net uptake to a marked net output (-6.1 +/- 1.9 mg . kg(-1) . min(-1) at t = 60 min), even in the face of hyperglycemia and hyperinsulinemia. These data show that elevations in hepatic ZMP concentrations, induced by portal venous AICAR infusion, cause acute hepatic insulin resistance. These findings have important implications for the targeting of AMP kinase for the treatment of insulin resistance, using AMP analogs.
Clinical and Experimental Pharmacology & Physiology. Apr, 2005 | Pubmed ID: 15810998
1. Glucose phosphorylation is the first irreversible step of the muscle glucose uptake pathway and is catalysed by a hexokinase isozyme. 2. While glucose transport is the primary barrier to muscle glucose uptake during basal conditions, glucose phosphorylation becomes an important barrier to muscle glucose uptake during stimulated conditions such as hyperinsulinaemia or exercise. 3. High fat feeding markedly impairs insulin- and exercise-stimulated muscle glucose uptake. As hexokinase II overexpression corrects this dietary-induced deficit during exercise, glucose phosphorylation is a site of impairment following high fat feeding. 4. Exercise is an important tool for diagnosing deficits in glucose phosphorylation.
The Journal of Physiology. Jul, 2005 | Pubmed ID: 15878951
Hexokinase (HK) II content is elevated in fatigue resistant muscle fibres and exercise trained muscle. The aim of this study was to determine if exercise capacity is dependent on muscle HK protein content. C57Bl/6 mice with a 50% HK knockout (HK+/-), no genetic manipulation (wild-type, WT) and an approximately 3-fold HK overexpression (HKTg) were tested. Mice (n = 12/group) completed both a maximal oxygen consumption test(VO2max) test and an endurance capacity test (run at approximately 75% VO2max) on an enclosed treadmill equipped to measure gas exchange. Arterial and venous catheters were surgically implanted into separate groups of mice (n = 9-11/group) in order to measure an index of muscle glucose uptake Rg during 30 min of treadmill exercise. Maximum work rate (0.95 +/- 0.05, 1.00 +/- 0.04 and 1.06 +/- 0.07 kg m min-1), (137 +/- 3, 141 +/- 4 and 141 +/- 5 ml kg-1 min-1) and maximal respiratory exchange ratio (1.04 +/- 0.02, 1.00 +/- 0.03 and 1.04 +/- 0.04) were similar in HK+/-, WT and HKTg, respectively. Exercise endurance capacity (measured as time to exhaustion) increased as HK content increased (55 +/- 11, 77 +/- 5 and 98 +/- 9 min) and this was related to Rg measured in mice during 30 min of exercise (13 +/- 2, 24 +/- 5 and 42 +/- 5 micromol (100 g)-1 min-1). Muscle glycogen in sedentary HK+/-mice and HK+/- mice following 30 min of exercise were significantly lower than in HKTg and WT mice. However, the net exercise-induced muscle glycogen breakdown was equal in the three genotypes. In summary, HK protein content within the range studied (a) was not associated with a difference in the capacity to perform maximal intensity exercise, (b) was a powerful determinant of the ability to sustain moderate intensity exercise, as reducing HK content impaired endurance and increasing HK content enhanced endurance, and (c) although directly related to exercise endurance, was not a determinant of net muscle glycogen usage during exercise. In conclusion, adaptations that increase HK protein content and/or functional activity such as regular exercise contribute to increased muscular endurance.
Canadian Journal of Applied Physiology = Revue Canadienne De Physiologie AppliquÃ©e. Jun, 2005 | Pubmed ID: 16129894
The liver is anatomically well situated to regulate blood glucose. It is positioned downstream from the pancreas, which releases the key regulatory hormones glucagon and insulin. It is also just downstream from the gut, permitting efficient extraction of ingested glucose and preventing large excursions in systemic glucose after a glucose-rich meal. The position of the liver is not as well situated from the standpoint of experimentation and clinical assessment, as its primary blood supply is impossible to access in conscious human subjects. Over the last 20 years, to study hepatic glucose metabolism during and after exercise, we have utilized a conscious dog model which permits sampling of the blood that perfuses (portal vein, artery) and drains (hepatic vein) the liver. Our work has demonstrated the key role of exercise-induced changes in glucagon and insulin in stimulating hepatic glycogenolysis and gluconeogenesis during exercise. Recently we showed that portal venous infusion of the pharmacological agent 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside leads to a marked increase in hepatic glucose production. Based on this, we propose that the concentration of AMP may be a component of a physiological pathway for stimulating hepatic glucose production during exercise. Insulin-stimulated hepatic glucose uptake is increased following exercise by an undefined mechanism that is independent of liver glycogen content. The fate of glucose taken up by the liver is critically dependent on hepatic glycogen stores, however, as glycogen deposition is greatly facilitated by prior glycogen depletion.
Partial Gene Deletion of Heart-type Fatty Acid-binding Protein Limits the Severity of Dietary-induced Insulin Resistance
Diabetes. Nov, 2005 | Pubmed ID: 16249436
The aim of this study was to determine the contribution of heart-type fatty acid-binding protein (H-FABP) to glucose and long-chain fatty acid (LCFA) utilization in dietary-induced insulin resistance. We tested the hypothesis that H-FABP facilitates increases in LCFA flux present in glucose-intolerant states and that a partial reduction in the amount of this protein would compensate for all or part of the impairment. Transgenic H-FABP heterozygotes (HET) and wild-type (WT) littermates were studied following chow diet (CHD) or high-fat diet (HFD) for 12 weeks. Catheters were surgically implanted in the carotid artery and jugular vein for sampling and infusions, respectively. Following 5 days of recovery, mice received either a saline infusion or underwent a euglycemic insulin clamp (4 mU x kg(-1) x min(-1)) for 120 min. At 90 min, a bolus of 2-deoxyglucose and [125I]-15-(rho-iodophenyl)-3-R,S-methylpentadecanoic acid were administered to obtain indexes of glucose and LCFA utilization. At 120 min, skeletal muscles were excised for tracer determination. All HFD mice were obese and hyperinsulinemic; however, only HFD-WT mice were hyperglycemic. Glucose infusion rates during insulin clamps were 49 +/- 4, 59 +/- 4, 16 +/- 4, and 33 +/- 4 mg x kg(-1) x min(-1) for CHD-WT, CHD-HET, HFD-WT, and HFD-HET mice, respectively, showing that HET limited the severity of whole-body insulin resistance with HFD. Insulin-stimulated muscle glucose utilization was attenuated in HFD-WT but unaffected in HFD-HET mice. Conversely, rates of LCFA clearance were increased with HFD feeding in HFD-WT but not in HFD-HET mice. In conclusion, a partial reduction in H-FABP protein normalizes fasting glucose levels and improves whole-body insulin sensitivity in HFD-fed mice despite obesity.
Pro- and Antiapoptotic Proteins Regulate Apoptosis but Do Not Protect Against Cytokine-mediated Cytotoxicity in Rat Islets and Beta-cell Lines
Diabetes. May, 2006 | Pubmed ID: 16644697
Type 1 diabetes results from islet beta-cell death and dysfunction induced by an autoimmune mechanism. Proinflammatory cytokines such as interleukin-1beta and gamma-interferon are mediators of this beta-cell cytotoxicity, but the mechanism by which damage occurs is not well understood. In the current study, we present multiple lines of evidence supporting the conclusion that cytokine-induced killing of rat beta-cells occurs predominantly by a nonapoptotic mechanism, including the following: 1) A rat beta-cell line selected for resistance to cytokine-induced cytotoxicity (833/15) is equally sensitive to killing by the apoptosis-inducing agents camptothecin and etoposide as a cytokine-sensitive cell line (832/13). 2) Overexpression of a constitutively active form of the antiapoptotic protein kinase Akt1 in 832/13 cells provides significant protection against cell killing induced by camptothecin and etoposide but no protection against cytokine-mediated damage. 3) Small interfering RNA-mediated suppression of the proapoptotic protein Bax enhances viability of 832/13 cells upon exposure to the known apoptosis-inducing drugs but not the inflammatory cytokines. 4) Exposure of primary rat islets or 832/13 cells to the inflammatory cytokines causes cell death as evidenced by the release of adenylate kinase activity into the cell medium, with no attendant increase in caspase 3 activation or annexin V staining. In contrast, camptothecin- and etoposide-induced killing is associated with robust increases in caspase 3 activation and annexin V staining. 5) Camptothecin increases cellular ATP levels, whereas inflammatory cytokines lower ATP levels in both beta-cell lines and primary islets. We conclude that proinflammatory cytokines cause beta-cell cytotoxicity primarily through a nonapoptotic mechanism linked to a decline in ATP levels.
Point-Counterpoint: Glucose Phosphorylation Is/is Not a Significant Barrier to Muscle Glucose Uptake by the Working Muscle
Journal of Applied Physiology (Bethesda, Md. : 1985). Dec, 2006 | Pubmed ID: 17106068
Chronic Treatment with Sildenafil Improves Energy Balance and Insulin Action in High Fat-fed Conscious Mice
Diabetes. Apr, 2007 | Pubmed ID: 17229936
Stimulation of nitric oxide-cGMP signaling results in vascular relaxation and increased muscle glucose uptake. We show that chronically inhibiting cGMP hydrolysis with the phosphodiesterase-5 inhibitor sildenafil improves energy balance and enhances in vivo insulin action in a mouse model of diet-induced insulin resistance. High-fat-fed mice treated with sildenafil plus L-arginine or sildenafil alone for 12 weeks had reduced weight and fat mass due to increased energy expenditure. However, uncoupling protein-1 levels were not increased in sildenafil-treated mice. Chronic treatment with sildenafil plus L-arginine or sildenafil alone increased arterial cGMP levels but did not adversely affect blood pressure or cardiac morphology. Sildenafil treatment, with or without l-arginine, resulted in lower fasting insulin and glucose levels and enhanced rates of glucose infusion, disappearance, and muscle glucose uptake during a hyperinsulinemic (4 mU x kg(-1) x min(-1))-euglycemic clamp in conscious mice. These effects occurred without an increase in activation of muscle insulin signaling. An acute treatment of high fat-fed mice with sildenafil plus l-arginine did not improve insulin action. These results show that phosphodiesterase-5 is a potential target for therapies aimed at preventing diet-induced energy imbalance and insulin resistance.
The Journal of Physiology. Jul, 2007 | Pubmed ID: 17495042
The absence of GLUT4 severely impairs basal glucose uptake in vivo, but does not alter glucose homeostasis or circulating insulin. Glucose uptake in isolated contracting skeletal muscle (MGU) is also impaired by the absence of GLUT4, and onset of muscle fatigue is hastened. Whether the body can compensate and preserve glucose homeostasis during exercise, as it does in the basal state, is unknown. One aim was to test the effectiveness of glucoregulatory compensation for the absence of GLUT4 in vivo. The absence of GLUT4 was also used to further define the role of hexokinase (HK) II, which catalyses glucose phosphorylation after it is transported in the cell. HK II increases MGU during exercise, as well as exercise endurance. In the absence of GLUT4, HK II expression will not affect MGU. A second aim was to test whether, in the absence of GLUT4, HK II retains its ability to increase exercise endurance. Wild-type (WT), GLUT4 null (GLUT4(-/-)), and GLUT4 null overexpressing HK II (GLUT4(-/-)HK(Tg)) mice were studied using a catheterized mouse model that allows blood sampling and isotope infusions during treadmill exercise. The impaired capacity of working muscle to take up glucose in GLUT4(-/-) is partially offset by an exaggerated increase in the glucagon: insulin ratio, increased liver glucose production, hyperglycaemia, and a greater capillary density in order to increase the delivery of glucose to the exercising muscle of GLUT4(-/-). Hearts of GLUT4(-/-) also exhibited a compensatory increase in HK II expression and a paradoxical increase in glucose uptake. Exercise tolerance was reduced in GLUT4(-/-) compared to WT. As expected, MGU in GLUT4(-/-)HK(Tg) was the same as in GLUT4(-/-). However, HK II overexpression retained its ability to increase exercise endurance. In conclusion, unlike the basal state where glucose homeostasis is preserved, hyperglycaemia results during exercise in GLUT4(-/-) due to a robust stimulation of liver glucose release in the face of severe impairments in MGU. Finally, studies in GLUT4(-/-)HK(Tg) show that HK II improves exercise tolerance, independent of its effects on MGU.
Phosphorylation Barriers to Skeletal and Cardiac Muscle Glucose Uptakes in High-fat Fed Mice: Studies in Mice with a 50% Reduction of Hexokinase II
Diabetes. Oct, 2007 | Pubmed ID: 17639019
Muscle glucose uptake (MGU) is regulated by glucose delivery to, transport into, and phosphorylation within muscle. The aim of this study was to determine the role of limitations in glucose phosphorylation in the control of MGU during either physiological insulin stimulation (4 mU x kg(-1) x min(-1)) or exercise with chow or high-fat feeding.
The Journal of Biological Chemistry. Feb, 2008 | Pubmed ID: 18096703
Relatively little is known about the in vivo functions of the alpha subunit of the heterotrimeric G protein Gz (Galphaz). Clues to one potential function recently emerged with the finding that activation of Galphaz inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708-31713). To extend this study in vivo, a Galphaz knock-out mouse model was utilized to determine whether Galphaz function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between Galphaz-null and wild-type mice. There was also no difference between the insulin sensitivity of Galphaz-null mice and wild-type controls, as measured by insulin tolerance tests. Galphaz-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in Galphaz-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from Galphaz-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in Galphaz-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Galphaz-null islets in the absence of exogenous stimulation. These findings indicate that Galphaz may be a potential new target for therapeutics aimed at ameliorating beta-cell dysfunction in Type 2 diabetes.
Trefoil Factor 3 Stimulates Human and Rodent Pancreatic Islet Beta-cell Replication with Retention of Function
Molecular Endocrinology (Baltimore, Md.). May, 2008 | Pubmed ID: 18258687
Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.
Stimulation of Human and Rat Islet Beta-cell Proliferation with Retention of Function by the Homeodomain Transcription Factor Nkx6.1
Molecular and Cellular Biology. May, 2008 | Pubmed ID: 18347054
The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet beta-cell development, but its effects on adult beta-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-(3)H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-betaGAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are beta cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [(3)H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating beta-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of beta-cell mass in treatment of both major forms of diabetes.
Diabetes. Jul, 2008 | Pubmed ID: 18398139
To characterize differences in whole-body glucose metabolism between commonly used inbred mouse strains.
Metabolic Implications of Reduced Heart-type Fatty Acid Binding Protein in Insulin Resistant Cardiac Muscle
Biochimica Et Biophysica Acta. Oct, 2008 | Pubmed ID: 18692568
Insulin resistance is characterized by elevated rates of cardiac fatty acid utilization resulting in reduced efficiency and cardiomyopathy. One potential therapeutic approach is to limit the uptake and oxidation of fatty acids. The aims of this study were to determine whether a quantitative reduction in heart-type fatty acid binding protein (FABP3) normalizes cardiac substrate utilization without altering cardiac function. Transgenic (FABP3(+/-)) and wild-type (WT) littermates were studied following low fat (LF) or high fat (HF) diets, with HF resulting in obese, insulin-resistant mice. Cardiovascular function (systolic blood pressure, % fractional shortening) and heart dimension were measured at weaning and every month afterward for 3 mo. During this period cardiovascular function was the same independent of genotype and diet. Catheters were surgically implanted in the carotid artery and jugular vein for sampling and infusions in mice at 4 mo of age. Following 5 d recovery, mice underwent either a saline infusion or a hyperinsulinemic-euglycemic clamp (4 mU kg(-1) min(-1)). Indices of long chain fatty acid and glucose utilization (R(f), R(g); mumol g wet weight(-1) min(-1)) were obtained using 2-deoxy[(3)H]glucose and [(125)I]-15-rho-iodophenyl)-3-R,S-methylpentadecanoic acid. FABP3(+/-) had enhanced cardiac R(g) compared with WT during saline infusion in both LF and HF. FABP3(+/-) abrogated the HF-induced decrement in insulin-stimulated cardiac R(g). On a HF diet, FABP(+/-) but not WT had an increased reliance on fatty acids (R(f)) during insulin stimulation. In conclusion, cardiac insulin resistance and glucose uptake is largely corrected by a reduction in FABP3 in vivo without contemporaneous deleterious effects on cardiac function.
Rap1 Promotes Multiple Pancreatic Islet Cell Functions and Signals Through Mammalian Target of Rapamycin Complex 1 to Enhance Proliferation
The Journal of Biological Chemistry. May, 2010 | Pubmed ID: 20339002
Recent studies have implicated Epac2, a guanine-nucleotide exchange factor for the Rap subfamily of monomeric G proteins, as an important regulator of insulin secretion from pancreatic beta-cells. Although the Epac proteins were originally identified as cAMP-responsive activators of Rap1 GTPases, the role of Rap1 in beta-cell biology has not yet been defined. In this study, we examined the direct effects of Rap1 signaling on beta-cell biology. Using the Ins-1 rat insulinoma line, we demonstrate that activated Rap1A, but not related monomeric G proteins, promotes ribosomal protein S6 phosphorylation. Using isolated rat islets, we show that this signaling event is rapamycin-sensitive, indicating that it is mediated by the mammalian target of rapamycin complex 1-p70 S6 kinase pathway, a known growth regulatory pathway. This newly defined beta-cell signaling pathway acts downstream of cAMP, in parallel with the stimulation of cAMP-dependent protein kinase, to drive ribosomal protein S6 phosphorylation. Activated Rap1A promotes glucose-stimulated insulin secretion, islet cell hypertrophy, and islet cell proliferation, the latter exclusively through mammalian target of rapamycin complex 1, suggesting that Rap1 is an important regulator of beta-cell function. This newly defined signaling pathway may yield unique targets for the treatment of beta-cell dysfunction in diabetes.
The Journal of Experimental Biology. Jan, 2011 | Pubmed ID: 21177945
Skeletal muscle glucose uptake increases dramatically in response to physical exercise. Moreover, skeletal muscle comprises the vast majority of insulin-sensitive tissue and is a site of dysregulation in the insulin-resistant state. The biochemical and histological composition of the muscle is well defined in a variety of species. However, the functional consequences of muscle biochemical and histological adaptations to physiological and pathophysiological conditions are not well understood. The physiological regulation of muscle glucose uptake is complex. Sites involved in the regulation of muscle glucose uptake are defined by a three-step process consisting of: (1) delivery of glucose to muscle, (2) transport of glucose into the muscle by GLUT4 and (3) phosphorylation of glucose within the muscle by a hexokinase (HK). Muscle blood flow, capillary recruitment and extracellular matrix characteristics determine glucose movement from the blood to the interstitium. Plasma membrane GLUT4 content determines glucose transport into the cell. Muscle HK activity, cellular HK compartmentalization and the concentration of the HK inhibitor glucose 6-phosphate determine the capacity to phosphorylate glucose. Phosphorylation of glucose is irreversible in muscle; therefore, with this reaction, glucose is trapped and the uptake process is complete. Emphasis has been placed on the role of the glucose transport step for glucose influx into muscle with the past assertion that membrane transport is rate limiting. More recent research definitively shows that the distributed control paradigm more accurately defines the regulation of muscle glucose uptake as each of the three steps that define this process are important sites of flux control.
Cafeteria Diet is a Robust Model of Human Metabolic Syndrome with Liver and Adipose Inflammation: Comparison to High-fat Diet
Obesity (Silver Spring, Md.). Jun, 2011 | Pubmed ID: 21331068
Obesity has reached epidemic proportions worldwide and reports estimate that American children consume up to 25% of calories from snacks. Several animal models of obesity exist, but studies are lacking that compare high-fat diets (HFD) traditionally used in rodent models of diet-induced obesity (DIO) to diets consisting of food regularly consumed by humans, including high-salt, high-fat, low-fiber, energy dense foods such as cookies, chips, and processed meats. To investigate the obesogenic and inflammatory consequences of a cafeteria diet (CAF) compared to a lard-based 45% HFD in rodent models, male Wistar rats were fed HFD, CAF or chow control diets for 15 weeks. Body weight increased dramatically and remained significantly elevated in CAF-fed rats compared to all other diets. Glucose- and insulin-tolerance tests revealed that hyperinsulinemia, hyperglycemia, and glucose intolerance were exaggerated in the CAF-fed rats compared to controls and HFD-fed rats. It is well-established that macrophages infiltrate metabolic tissues at the onset of weight gain and directly contribute to inflammation, insulin resistance, and obesity. Although both high fat diets resulted in increased adiposity and hepatosteatosis, CAF-fed rats displayed remarkable inflammation in white fat, brown fat and liver compared to HFD and controls. In sum, the CAF provided a robust model of human metabolic syndrome compared to traditional lard-based HFD, creating a phenotype of exaggerated obesity with glucose intolerance and inflammation. This model provides a unique platform to study the biochemical, genomic and physiological mechanisms of obesity and obesity-related disease states that are pandemic in western civilization today.
PPAR-Î³ Activation Restores Pancreatic Islet SERCA2 Levels and Prevents Î²-Cell Dysfunction Under Conditions of Hyperglycemic and Cytokine Stress
Molecular Endocrinology (Baltimore, Md.). Feb, 2012 | Pubmed ID: 22240811
The maintenance of intracellular Ca(2+) homeostasis in the pancreatic Î²-cell is closely regulated by activity of the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) pump. Our data demonstrate a loss of Î²-cell SERCA2b expression in several models of type 2 diabetes including islets from db/db mice and cadaveric diabetic human islets. Treatment of 832/13 rat INS-1-derived cells with 25 mm glucose and the proinflammatory cytokine IL-1Î² led to a similar loss of SERCA2b expression, which was prevented by treatment with the peroxisome proliferator-activated receptor (PPAR)-Î³ agonist, pioglitazone. Pioglitazone was able to also protect against hyperglycemia and cytokine-induced elevations in cytosolic Ca(2+) levels, insulin-secretory defects, and cell death. To determine whether PPAR-Î³ was a direct transcriptional regulator of the SERCA2 gene, luciferase assays were performed and showed that a -259 bp region is sufficient to confer PPAR-Î³ transactivation; EMSA and chromatin immunoprecipitation experiments confirmed that PPAR-Î³ directly binds a PPAR response element in this proximal region. We next sought to characterize the mechanisms by which SERCA2b was down-regulated. INS-1 cells were exposed to high glucose and IL-1Î² in time course experiments. Within 2 h of exposure, activation of cyclin-dependent kinase 5 (CDK5) was observed and correlated with increased serine-273 phosphorylation of PPAR-Î³ and loss of SERCA2 protein expression, findings that were prevented by pioglitazone and roscovitine, a pharmacological inhibitor of CDK5. We conclude that pioglitazone modulates SERCA2b expression through direct transcriptional regulation of the gene and indirectly through prevention of CDK5-induced phosphorylation of PPAR-Î³.