Changes in microvascular density differentiate metabolic health outcomes in monkeys with prior radiation exposure and subsequent skeletal muscle ECM remodeling.

Radiation exposure accelerates the onset of age-related diseases such as diabetes, cardiovascular disease, and neoplasia and, thus, lends insight into in vivo mechanisms common to these disorders. Fibrosis and extracellular matrix (ECM) remodeling, which occur with aging and overnutrition and following irradiation, are risk factors for development of type 2 diabetes mellitus. We previously demonstrated an increased incidence of skeletal muscle insulin resistance and type 2 diabetes mellitus in monkeys that had been exposed to whole body irradiation 5-9 yr prior. We hypothesized that irradiation-induced fibrosis alters muscle architecture, predisposing irradiated animals to insulin resistance and overt diabetes. Rhesus macaques (Macaca mulatta, n = 7-8/group) grouped as nonirradiated age-matched controls (Non-Rad-CTL), irradiated nondiabetic monkeys (Rad-CTL), and irradiated monkeys that subsequently developed diabetes (Rad-DM) were compared. Prior radiation exposure resulted in persistent skeletal muscle ECM changes, including a relative overabundance of collagen IV and a trend toward increased transforming growth factor-ß1. Preservation of microvascular markers differentiated the irradiated diabetic and nondiabetic groups. Microvascular density and plasma nitrate and heat shock protein 90 levels were lower in Rad-DM than Rad-CTL. These results are consistent with a protective effect of abundant microvasculature in maintaining glycemic control within radiation-induced fibrotic muscle.

AMP-activated protein kinase (AMPK)α2 plays a role in determining the cellular fate of glucose in insulin-resistant mouse skeletal muscle.

AIMS/HYPOTHESIS: We determined whether: (1) an acute lipid infusion impairs skeletal muscle AMP-activated protein kinase (AMPK)α2 activity, increases inducible nitric oxide synthase (iNOS) and causes peripheral insulin resistance in conscious, unstressed, lean mice; and (2) restoration of AMPKα2 activity during the lipid infusion attenuates the increase in iNOS and reverses the defect in insulin sensitivity in vivo. METHODS: Chow-fed, 18-week-old C57BL/6J male mice were surgically catheterised. After 5 days they received: (1) a 5 h infusion of 5 ml kg(-1) h(-1) Intralipid + 6 U/h heparin (Lipid treatment) or saline (Control); (2) Lipid treatment or Control, followed by a 2 h hyperinsulinaemic-euglycaemic clamp (insulin clamp; 4 mU kg(-1) min(-1)); and (3) infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) (1 mg kg(-1) min(-1)), or saline during Lipid treatment, followed by a 2 h insulin clamp. In a separate protocol, mice producing a muscle-specific kinase-dead AMPKα2 subunit (α2-KD) underwent an insulin clamp to determine the role of AMPKα2 in insulin-mediated muscle glucose metabolism. RESULTS: Lipid treatment decreased AMPKα2 activity, increased iNOS abundance/activation and reduced whole-body insulin sensitivity in vivo. AICAR increased AMPKα2 activity twofold; this did not suppress iNOS or improve whole-body or tissue-specific rates of glucose uptake during Lipid treatment. AICAR caused a marked increase in insulin-mediated glycogen synthesis in skeletal muscle. Consistent with this latter result, lean α2-KD mice exhibited impaired insulin-stimulated glycogen synthesis even though muscle glucose uptake was not affected. CONCLUSIONS/INTERPRETATION: Acute induction of insulin resistance via lipid infusion in healthy mice impairs AMPKα2, increases iNOS and causes insulin resistance in vivo. However, these changes do not appear to be interrelated. Rather, a functionally active AMPKα2 subunit is required for insulin-stimulated muscle glycogen synthesis.

Regulation of glucose kinetics during exercise by the glucagon-like peptide-1 receptor.

In response to oral glucose, glucagon-like peptide-1 receptor (Glp1r) knockout (Glp1r−/−) mice become hyperglycaemic due to impaired insulin secretion. Exercise also induces hyperglycaemia in Glp1r−/− mice. In contrast to oral glucose, exercise decreases insulin secretion. This implies that exercise-induced hyperglycaemia in Glp1r−/− mice results from the loss of a non-insulinotropic effect mediated by the Glp1r. Muscle glucose uptake (MGU) is normal in exercising Glp1r−/− mice. Thus, we hypothesize that exercise-induced hyperglycaemia in Glp1r−/− mice is due to excessive hepatic glucose production (HGP). Wild-type (Glp1r+/+) and Glp1r−/− mice implanted with venous and arterial catheters underwent treadmill exercise or remained sedentary for 30 min. [3-3H]glucose was used to estimate rates of glucose appearance (Ra), an index of HGP, and disappearance (Rd). 2[14C]deoxyglucose was used to assess MGU. Glp1r−/− mice displayed exercise-induced hyperglycaemia due to an excessive increase in Ra but normal Rd and MGU. Exercise-induced glucagon levels were ∼2-fold higher in Glp1r−/− mice, resulting in a ∼2-fold higher glucagon:insulin ratio. Since inhibition of the central Glp1r stimulates HGP, we tested whether intracerebroventricular (ICV) infusion of the Glp1r antagonist exendin(9­39) (Ex9) in Glp1r+/+ mice would result in exercise-induced hyperglycaemia. ICV Ex9 did not enhance glucose levels or HGP during exercise, suggesting that glucoregulatory effects of Glp1 during exercise are mediated via the pancreatic Glp1r. In conclusion, functional disruption of the Glp1r results in exercise-induced hyperglycaemia associated with an excessive increase in glucagon secretion and HGP. These results suggest an essential role for basal Glp1r signalling in the suppression of alpha cell secretion during exercise.

Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets.

AIMS/HYPOTHESIS: Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. METHODS: We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2](-/-)) and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As (-/-) mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. RESULTS: Fasting glucose concentrations were reduced in As (-/-) mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As (-/-) than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As (-/-) mice even during high sodium intake. There was no difference in insulin sensitivity between As (-/-) and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose- and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. CONCLUSIONS/INTERPRETATION: We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess.

Obesity impairs skeletal muscle AMPK signaling during exercise: role of AMPKα2 in the regulation of exercise capacity in vivo.

OBJECTIVE: Skeletal muscle AMP-activated protein kinase (AMPK)α2 activity is impaired in obese, insulin-resistant individuals during exercise. We determined whether this defect contributes to the metabolic dysregulation and reduced exercise capacity observed in the obese state. DESIGN: C57BL/6J wild-type (WT) mice and/or mice expressing a kinase dead AMPKα2 subunit in skeletal muscle (α2-KD) were fed chow or high-fat (HF) diets from 3 to 16 weeks of age. At 15 weeks, mice performed an exercise stress test to determine exercise capacity. In WT mice, muscle glucose uptake and skeletal muscle AMPKα2 activity was assessed in chronically catheterized mice (carotid artery/jugular vein) at 16 weeks. In a separate study, HF-fed WT and α2-KD mice performed 5 weeks of exercise training (from 15 to 20 weeks of age) to test whether AMPKα2 is necessary to restore work tolerance. RESULTS: HF-fed WT mice had reduced exercise tolerance during an exercise stress test, and an attenuation in muscle glucose uptake and AMPKα2 activity during a single bout of exercise (P<0.05 versus chow). In chow-fed α2-KD mice, running speed and time were impaired ∼45 and ∼55%, respectively (P<0.05 versus WT chow); HF feeding further reduced running time ∼25% (P<0.05 versus α2-KD chow). In response to 5 weeks of exercise training, HF-fed WT and α2-KD mice increased maximum running speed ∼35% (P<0.05 versus pre-training) and maintained body weight at pre-training levels, whereas body weight increased in untrained HF WT and α2-KD mice. Exercise training restored running speed to levels seen in healthy, chow-fed mice. CONCLUSION: HF feeding impairs AMPKα2 activity in skeletal muscle during exercise in vivo. Although this defect directly contributes to reduced exercise capacity, findings in HF-fed α2-KD mice show that AMPKα2-independent mechanisms are also involved. Importantly, α2-KD mice on a HF-fed diet adapt to regular exercise by increasing exercise tolerance, demonstrating that this adaptation is independent of skeletal muscle AMPKα2 activity.

Impact of macrophage toll-like receptor 4 deficiency on macrophage infiltration into adipose tissue and the artery wall in mice.

AIMS/HYPOTHESIS: Toll-like receptor 4 (TLR4) is a receptor for saturated fatty acids (SFAs), global deficiency of which has been shown to protect against inflammation, insulin resistance and atherosclerotic lesion formation. Because macrophages express Tlr4 and are important in insulin resistance and atherosclerotic lesion formation due to their infiltration of white adipose tissue (WAT) and the artery wall, respectively, we hypothesised that deficiency of macrophage TLR4 could protect against these disorders. METHODS: Bone marrow transplantation of agouti, LDL-receptor deficient (A(y)/a; Ldlr (-/-)) mice with marrow from either C57BL/6 or Tlr4 (-/-) mice was performed. Recipient mice with Tlr4 (+/+) marrow (MthetaTLR4(+/+)) or with Tlr4 (-/-) marrow (MthetaTLR4(-/-)) were then placed on one of four diets: (1) low fat; (2) high fat; (3) high fat rich in SFAs (HF(SFA)); and (4) HF(SFA) supplemented with fish oil. RESULTS: There were no differences in body composition or plasma lipids between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice on any of the diets. However, we observed a decrease in some macrophage and inflammatory markers in WAT of female low fat-fed MthetaTLR4(-/-) mice compared with MthetaTLR4(+/+) mice. MthetaTLR4(-/-) mice fed low-fat diet also displayed decreased atherosclerotic lesion area. There were no differences in macrophage accrual in WAT or atherosclerosis between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed any of the high-fat diets. Finally, no difference was seen in insulin sensitivity between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed the HF(SFA) diet. CONCLUSIONS/INTERPRETATION: These data suggest that under certain dietary conditions, macrophage expression of Tlr4 can be an important mediator of macrophage accumulation in WAT and the artery wall.

Metabolomic profiling of dietary-induced insulin resistance in the high fat-fed C57BL/6J mouse.

The predictive ability of metabolic profiling to detect obesity-induced perturbations in metabolism has not been clearly established. Complex aetiologies interacting with environmental factors highlight the need to understand how specific manipulations alter metabolite profiles in this state. The aim of this study was to determine if targeted metabolomic profiling could be employed as a reliable tool to detect dietary-induced insulin resistance in a small subset of experimental animals (n = 10/treatment). Following weaning, male C57BL/6J littermates were randomly divided into two dietary groups: chow and high fat. Following 12 weeks of dietary manipulation, mice were fasted for 5 h prior to serum collection. The resultant high fat-fed animals were obese and insulin resistant as shown by a euglycaemic-hyperinsulinaemic clamp. Sera were analysed by proton nuclear magnetic resonance spectroscopy, and 46 known compounds were identified and quantified. Multivariate analysis by orthogonal partial least squares discriminant analysis, a projection method for class separation, was then used to establish models of each treatment. Models were able to predict class separation between diets with 90% accuracy. Variable importance plots revealed the most important metabolites in this discrimination to include lysine, glycine, citrate, leucine, suberate and acetate. These metabolites are involved in energy metabolism and may be representative of the perturbations taking place with insulin resistance. Results show metabolomics to reliably describe the metabolic effects of insulin resistance in a small subset of samples and are an initial step in establishing metabolomics as a tool to understand the biochemical signature of insulin resistance.

Effects of chronic coffee consumption on glucose kinetics in the conscious rat.

Epidemiological studies indicate that regular coffee consumption reduces the risk of developing type 2 diabetes. Despite these findings, the biological mechanisms by which coffee consumption exerts these effects are unknown. The aim of this study was twofold: to develop a rat model that would further delineate the effects of regular coffee consumption on glucose kinetics, and to determine whether coffee, with or without caffeine, alters the actions of insulin on glucose kinetics in vivo. Male Sprague-Dawley rats were fed a high-fat diet for 4 weeks in combination with one of the following: (i) drinking water as placebo (PL), (ii) decaffeinated coffee (2 g/100 mL) (DC), or (iii) alkaloid caffeine (20 mg/100 mL) added to decaffeinated coffee (2 g/100 mL) (CAF). Catheters were chronically implanted in a carotid artery and jugular vein for sampling and infusions, respectively. Recovered animals (5 days postoperative) were fasted for 5 h before hyperinsulinemic-euglycemic clamps (2 mU x kg(-1) x min(-1)). Glucose was clamped at 6 mmol/L and isotopes (2-deoxy-[(14)C]glucose and [3-(3)H]glucose) were administered to obtain indices of whole-body and tissue-specific glucose kinetics. Glucose infusion rates and measures of whole-body metabolic clearance were greater in DC than in PL or CAF, indicating increased whole-body insulin sensitivity. As the only difference between DC and CAF was the addition of alkaloid caffeine, it can be concluded that caffeine antagonizes the beneficial effects of DC. Given these findings, decaffeinated coffee may represent a nutritional means of combating insulin resistance.

Glycogen synthase kinase 3 inhibition improves insulin-stimulated glucose metabolism but not hypertension in high-fat-fed C57BL/6J mice.

AIMS/HYPOTHESIS: In the current study, the effect of a highly specific peptide inhibitor of glycogen synthase kinase 3 (GSK3) (L803-mts) on glucose metabolism and BP was examined in a high-fat (HF) fed mouse model of diabetes. METHODS: C57/BL6J mice were placed on an HF diet for 3 months and treated with L803-mts for 20 days, following which glucose metabolism was examined by euglycaemic-hyperinsulinaemic clamp studies. BP and heart rate were measured by radio-telemetry. RESULTS: The HF mice were obese, with impaired glucose tolerance and high plasma insulin and leptin levels. L803-mts treatment significantly reduced the insulin levels and doubled the glucose infusion rate required to maintain a euglycaemic condition in the HF+L803-mts group compared with the HF group. Insulin failed to suppress the endogenous glucose production rate in the HF group while decreasing it by 75% in the HF+L803-mts group, accompanied by increased liver glycogen synthase activity and net hepatic glycogen synthesis. GSK3 inhibition also reduced peripheral insulin resistance. Plasma glucose disappearance rate increased by 60% in the HF+L803-mts group compared with the HF group. In addition, glucose uptake in heart and gastrocnemius muscle was markedly improved. Although mean arterial pressure increased following the HF diet, it did not change significantly during the 12 days of L803-mts treatment. CONCLUSIONS/INTERPRETATION: These studies demonstrate that GSK3 inhibition improved hepatic and peripheral insulin resistance in a mouse model of HF-induced diabetes, but it failed to have an effect on BP. GSK3 may represent an important therapeutic target for insulin resistance.

Role of carotid bodies in control of the neuroendocrine response to exercise.

This study was aimed at assessing the role of carotid body function in neuroendocrine and glucoregulatory responses to exercise. The carotid bodies and associated nerves were removed (CBR, n = 6) or left intact (Sham, n = 6) in anesthetized dogs >16 days before experiments, and infusion and sampling catheters were implanted. Conscious dogs were studied at rest and during 150 min of exercise. Isotopic dilution was used to assess glucose production (R(a)) and disappearance (R(d)). Arterial glucagon was reduced in CBR compared with Sham at rest (29 +/- 3 vs. 47 +/- 3 pg/ml). During exercise, glucagon increased more in Sham than in CBR (47 +/- 9 vs. 15 +/- 2 pg/ml). Cortisol and epinephrine levels were similar in the two groups at rest and during exercise. Basal norepinephrine was similar in CBR and Sham. During exercise, norepinephrine increased by 432 +/- 124 pg/ml in Sham, but by only 201 +/- 28 pg/ml in CBR. Basal arterial plasma glucose was 108 +/- 2 and 105 +/- 2 mg/dl in CBR and Sham, respectively. Arterial glucose dropped by 10 +/- 3 mg/dl at onset of exercise in CBR (P < 0.01) but was unchanged in Sham (decrease of 3 +/- 2 mg/dl, not significant). Basal glucose kinetics were equal in Sham and CBR. At onset of exercise, R(a) and R(d) were transiently uncoupled in CBR (i.e., R(d) > R(a)) but were closely matched in Sham. In steady-state exercise, R(a) and R(d) were closely matched in both groups. Insulin was equal in the basal period and decreased similarly during exercise. These studies suggest that input from the carotid bodies, or receptors anatomically close to them, 1) is important in control of basal glucagon and the exercise-induced increment in glucagon, 2) is involved in the sympathetic response to exercise, and 3) participates in the non-steady-state coupling of R(a) to R(d), but 4) is not essential to glucoregulation during sustained exercise.

Sexual dimorphism in counterregulatory responses to hypoglycemia after antecedent exercise.

After antecedent hypoglycemia, counterregulatory responses to subsequent hypoglycemia exhibit greater blunting in men than in women. Because physical exercise and hypoglycemia share multiple counterregulatory mechanisms, we hypothesized that prior exercise may also result in gender-specific blunting of counterregulatory responses to subsequent hypoglycemia. Thirty healthy subjects (15 women and 15 men; age, 28 +/- 3 yr; body mass index, 23 +/- 1 kg/m2) were studied during 2-d experiments. Day 1 consisted of either identical 90-min morning and afternoon cycle exercise at 50% maximum oxygen expenditure or two 2-h episodes of hyperinsulinemic euglycemia. Day 2 consisted of a 2-h morning hyperinsulinemic-hypoglycemic clamp. Endogenous glucose production was measured using [3-(3)H]glucose. Muscle sympathetic nerve activity was measured using microneurography. Day 2 insulin (540 +/- 36 pmol/liter) and plasma glucose (2.9 +/- 0.06 pmol/liter) levels were similar in men and women during the last 30 min of hypoglycemia. Compared with antecedent euglycemia, d 1 exercise produced significant blunting of d 2 counterregulatory responses to hypoglycemia. Several key d 2 counterregulatory responses were blunted to a greater extent in men than in women: glucagon (men, -105 +/- 14; women, -25 +/- 7 ng/liter; P < 0.0001), epinephrine (men, -2625 +/- 257 pmol/liter; women, -212 +/- 573; P < 0.001), norepinephrine (men, -0.50 +/- 0.12 nmol/liter; women, -0 +/- 0.11; P < 0.001), and muscle sympathetic nerve activity (men, -13 +/- 4; women, -4 +/- 4 bursts/min; P < 0.01). Cardiovascular responses (heart rate and systolic and mean arterial blood pressures) were also more blunted by antecedent exercise in men than in women. After d 1 exercise, the amount of glucose infused during d 2 hypoglycemia in men was increased 6-fold compared with that after d 1 euglycemia. This amount was significantly increased (P < 0.01) compared with the 2-fold (P < 0.01) increment in glucose infusion that was required in women after d 1 exercise. Lipolysis was unaffected by d 1 exercise in women, but was significantly blunted during d 2 hypoglycemia in men. In summary, two bouts of prolonged, moderate exercise (90 min at 50% maximum oxygen expenditure) induced a marked sexual dimorphism in key neuroendocrine (glucagon, catecholamines, and muscle sympathetic nerve activity) and metabolic (glucose kinetic, lipolysis) responses to next day hypoglycemia.

Effect of morning exercise on counterregulatory responses to subsequent, afternoon exercise.

The aim of this study was to determine whether a bout of morning exercise (EXE(1)) can alter neuroendocrine and metabolic responses to subsequent afternoon exercise (EXE(2)) and whether these changes follow a gender-specific pattern. Sixteen healthy volunteers (8 men and 8 women, age 27 +/- 1 yr, body mass index 23 +/- 1 kg/m(2), maximal O(2) uptake 31 +/- 2 ml x kg(-1) x min(-1)) were studied after an overnight fast. EXE(1) and EXE(2) each consisted of 90 min of cycling on a stationary bike at 48 +/- 2% of maximal O(2) uptake separated by 3 h. To avoid the confounding effects of hypoglycemia and glycogen depletion, carbohydrate (1.5 g/kg body wt po) was given after EXE(1), and plasma glucose was maintained at euglycemia during both episodes of exercise by a modification of the glucose-clamp technique. Basal insulin levels (7 +/- 1 microU/ml) and exercise-induced insulin decreases (-3 microU/ml) were similar during EXE(1) and EXE(2). Plasma glucose was 5.2 +/- 0.1 and 5.2 +/- 0.1 mmol/l during EXE(1) and EXE(2), respectively. The glucose infusion rate needed to maintain euglycemia during the last 30 min of exercise was increased during EXE(2) compared with EXE(1) (32 +/- 4 vs. 7 +/- 2 micromol x kg(-1) x min(-1)). Although this increased need for exogenous glucose was similar in men and women, gender differences in counterregulatory responses were significant. Compared with EXE(1), epinephrine, norepinephrine, growth hormone, pancreatic polypeptide, and cortisol responses were blunted during EXE(2) in men, but neuroendocrine responses were preserved or increased in women. In summary, morning exercise significantly impaired the body's ability to maintain euglycemia during later exercise of similar intensity and duration. We conclude that antecedent exercise can significantly modify, in a gender-specific fashion, metabolic and neuroendocrine responses to subsequent exercise.

Effects of antecedent prolonged exercise on subsequent counterregulatory responses to hypoglycemia.

In the present study the hypothesis tested was that prior exercise may blunt counterregulatory responses to subsequent hypoglycemia. Healthy subjects [15 females (f)/15 males (m), age 27 +/- 1 yr, body mass index 22 +/- 1 kg/m(2), hemoglobin A(Ic) 5.6 +/- 0.5%] were studied during 2-day experiments. Day 1 involved either 90-min morning and afternoon cycle exercise at 50% maximal O2 uptake (VO2(max)) (priorEXE, n = 16, 8 m/8 f) or equivalent rest periods (priorREST, n = 14, 7 m/7 f). Day 2 consisted of a 2-h hypoglycemic clamp in all subjects. Endogenous glucose production (EGP) was measured using [3-3H]glucose. Muscle sympathetic nerve activity (MSNA) was measured using microneurography. Day 2 insulin (87 +/- 6 microU/ml) and plasma glucose levels (54 +/- 2 mg/dl) were equivalent after priorEXE and priorREST. Significant blunting (P < 0.01) of day 2 norepinephrine (-30 +/- 4%), epinephrine (-37 +/- 6%), glucagon (-60 +/- 4%), growth hormone (-61 +/- 5%), pancreatic polypeptide (-47 +/- 4%), and MSNA (-90 +/- 8%) responses to hypoglycemia occurred after priorEXE vs. priorREST. EGP during day 2 hypoglycemia was also suppressed significantly (P < 0.01) after priorEXE compared with priorREST. In summary, two bouts of exercise (90 min at 50% VO2(max)) significantly reduced glucagon, catecholamines, growth hormone, pancreatic polypeptide, and EGP responses to subsequent hypoglycemia. We conclude that, in normal humans, antecedent prolonged moderate exercise blunts neuroendocrine and metabolic counterregulatory responses to subsequent hypoglycemia.

Stimulation of splanchnic glucose production during exercise in humans contains a glucagon-independent component.

To determine the importance of basal glucagon to the stimulation of net splanchnic glucose output (NSGO) during exercise, seven healthy males performed cycle exercise during a pancreatic islet cell clamp. In one group (BG), glucagon was replaced at basal levels and insulin was adjusted to achieve euglycemia. In another group (GD), only insulin was replaced at the identical rate used in BG, and basal glucagon was not replaced. Exogenous glucose infusion was necessary to maintain euglycemia during exercise in BG and during rest and exercise in GD. Arterial glucagon was at least twofold greater in BG than in GD throughout the pancreatic islet cell clamp. Although basal NSGO remained stable in BG (2.5 +/- 0.5 mg x kg(-1) x min(-1)), basal NSGO dropped by 70% in GD (0.7 +/- 0.3 mg. kg(-1) x min(-1)). NSGO was also greater in BG than in GD at 10 min of moderate exercise, most likely due to the residual effect of basal glucagon replacement. However, NSGO increased slightly and remained similar throughout the remainder of moderate and heavy exercise in BG and GD. Therefore, a mechanism independent of changes in pancreatic hormones and/or the level of glycemia contributes toward modest stimulation of NSGO during moderate and heavy exercise.

Functional limitations to glucose uptake in muscles comprised of different fiber types.

Skeletal muscle glucose uptake requires delivery of glucose to the sarcolemma, transport across the sarcolemma, and the irreversible phosphorylation of glucose by hexokinase (HK) inside the cell. Here, a novel method was used in the conscious rat to address the roles of these three steps in controlling the rate of glucose uptake in soleus, a muscle comprised of type I fibers, and two muscles comprised of type II fibers. Experiments were performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed, constant infusions of 3-O-methyl-[3H]glucose (3-O-MG) and [1-14C]mannitol. Total muscle glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-MG concentration, which distributes based on the transsarcolemmal glucose gradient (TSGG), were used to calculate glucose concentrations at the inner and outer sarcolemmal surfaces ([G](im) and [G](om), respectively) in muscle. Muscle glucose uptake was much lower in muscle comprised of type II fibers than in soleus under both basal and insulin-stimulated conditions. Under all conditions, the TSGG in type II muscle exceeded that in soleus, indicating that glucose transport plays a more important role to limit glucose uptake in type II muscle. Although hyperinsulinemia increased [G](im) in soleus, indicating that phosphorylation was a limiting factor, type II muscle was limited primarily by glucose delivery and glucose transport. In conclusion, the relative importance of glucose delivery, transport, and phosphorylation in controlling the rate of insulin-stimulated muscle glucose uptake varies between muscle fiber types, with glucose delivery and transport being the primary limiting factors in type II muscle.

Limitations to basal and insulin-stimulated skeletal muscle glucose uptake in the high-fat-fed rat.

Rats fed a high-fat diet display blunted insulin-stimulated skeletal muscle glucose uptake. It is not clear whether this is due solely to a defect in glucose transport, or if glucose delivery and phosphorylation are also impaired. To determine this, rats were fed standard chow (control rats) or a high-fat diet (HF rats) for 4 wk. Experiments were then performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed constant infusions of 3-O-methyl-[(3)H]glucose (3-O-MG) and [1-(14)C]mannitol. Total muscle glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-MG concentration [which distributes based on the transsarcolemmal glucose gradient (TSGG)] were used to calculate glucose concentrations at the inner and outer sarcolemmal surfaces ([G](im) and [G](om), respectively) in soleus. Total muscle glucose was also measured in two fast-twitch muscles. Muscle glucose uptake was markedly decreased in HF rats. In control rats, hyperinsulinemia resulted in a decrease in soleus TSGG compared with basal, due to increased [G](im). In HF rats during hyperinsulinemia, [G](im) also exceeded zero. Hyperinsulinemia also decreased muscle glucose in HF rats, implicating impaired glucose delivery. In conclusion, defects in extracellular and intracellular components of muscle glucose uptake are of major functional significance in this model of insulin resistance.

Evidence that carotid bodies play an important role in glucoregulation in vivo.

The carotid bodies are sensitive to glucose in vitro and can be stimulated to cause hyperglycemia in vivo. The aim of this study was to determine if the carotid bodies are involved in basal glucoregulation or the counterregulatory response to an insulin-induced decrement in arterial glucose in vivo. Dogs were surgically prepared >16 days before the experiment. The carotid bodies and their associated nerves were removed (carotid body resected [CBR]) or left intact (Sham), and infusion and sampling catheters were implanted. Removal of carotid bodies was verified by the absence of a ventilatory response to NaCN. Experiments were performed in 18-h fasted conscious dogs and consisted of a tracer ([3-3H]glucose) equilibration period (-120 to -40 min), a basal period (-40 to 0 min), and an insulin infusion (1 mU x kg(-1) x min(-1)) period (0-150 min) during which glucose was infused as needed to clamp at mildly hypoglycemic (65 mg/dl) or euglycemic (105 mg/dl) levels. Basal (8 microU/ml) and clamp (40 microU/ml) insulin levels were similar in both groups. Basal arterial glucagon was reduced in CBR compared with Sham (30 + 2 vs. 40 +/- 2 pg/ml) and remained reduced in CBR during hypoglycemia (peak levels of 36 +/- 3 vs. 52 +/- 7 pg/ml). Cortisol levels were not significantly different between the 2 groups in the basal state, but were reduced during the hypoglycemic clamp in CBR. Catecholamine levels were not significantly different between the 2 groups in the basal and hypoglycemic periods. The glucose infusion rate required to clamp glucose at 65 mg/dl was 2.5-fold greater in CBR compared with Sham (4.0 +/- 0.4 vs. 1.6 +/- 0.4 mg x kg(-1) x min(-1)). Basal endogenous glucose appearance (R(a)) was equal in CBR and Sham (2.5 +/- 0.1 vs. 2.5 +/- 0.2 mg x kg(-1) x min(-1)). During the hypoglycemic clamp, insulin suppressed R(a) in CBR but not Sham (1.1 +/- 0.2 vs. 2.5 +/- 0.2 mg x kg(-1) x min(-1) during the last 30 min of the clamp), reflecting impaired counterregulation. Glucose disappearance (R(d)) in the basal state was similar in CBR and Sham, whereas it was elevated in CBR during the hypoglycemic clamp (4.8 +/- 0.1 vs. 3.9 +/- 0.1 mg x kg(-1) x min(-1) during the last 30 min of the clamp). R(d) was also elevated in euglycemic clamp studies, indicating an effect of carotid body resection independent of hypoglycemia. There were no other measured systematic endocrine or metabolic effects of carotid body resection during euglycemic clamps. In conclusion, we found that the carotid bodies (or receptors anatomically close by) play an important role in the insulin-induced counterregulatory response to mild hypoglycemia.

Glucagon response to exercise is critical for accelerated hepatic glutamine metabolism and nitrogen disposal.

The aim of this study was to determine the role of glucagon in hepatic glutamine (Gln) metabolism during exercise. Sampling (artery, portal vein, and hepatic vein) and infusion (vena cava) catheters and flow probes (portal vein, hepatic artery) were implanted in anesthetized dogs. At least 16 days after surgery, an experiment, consisting of a 120-min equilibration period, a 30-min basal sampling period, and a 150-min exercise period, was performed in these animals. [5-(15)N]Gln was infused throughout experiments to measure gut and liver Gln kinetics and the incorporation of Gln amide nitrogen into urea. Somatostatin was infused throughout the study. Glucagon was infused at a basal rate until the beginning of exercise, when the rate was either 1) gradually increased to simulate the glucagon response to exercise (n = 5) or 2) unchanged to maintain basal glucagon (n = 5). Insulin was infused during the equilibration and basal periods at rates designed to achieve stable euglycemia. The insulin infusion was reduced in both protocols to simulate the exercise-induced insulin decrement. These studies show that the exercise-induced increase in glucagon is 1) essential for the increase in hepatic Gln uptake and fractional extraction, 2) required for the full increment in ureagenesis, 3) required for the specific transfer of the Gln amide nitrogen to urea, and 4) unrelated to the increase in gut fractional Gln extraction. These data show, by use of the physiological perturbation of exercise, that glucagon is a physiological regulator of hepatic Gln metabolism in vivo.

Hepatic alpha- and beta-adrenergic receptors are not essential for the increase in R(a) during exercise in diabetes.

The purpose of this study was to determine the role of direct hepatic adrenergic stimulation in the control of endogenous glucose production (R(a)) during moderate exercise in poorly controlled alloxan-diabetic dogs. Chronically catheterized and instrumented (flow probes on hepatic artery and portal vein) dogs were made diabetic by administration of alloxan. Each study consisted of a 120-min equilibration, 30-min basal, 150-min moderate exercise, 30-min recovery, and 30-min blockade test period. Either vehicle (control; n = 6) or alpha (phentolamine)- and beta (propranolol)-adrenergic blockers (HAB; n = 6) were infused in the portal vein. In both groups, epinephrine (Epi) and norepinephrine (NE) were infused in the portal vein during the blockade test period to create suprapharmacological levels at the liver. Isotopic ([3-(3)H]glucose, [U-(14)C]alanine) and arteriovenous difference methods were used to assess hepatic function. Arterial plasma glucose was similar in controls (345 +/- 24 mg/dl) and HAB (336 +/- 23 mg/dl) and was unchanged by exercise. Basal arterial insulin was 5 +/- 1 mU/ml in controls and 4 +/- 1 mU/ml in HAB and fell by approximately 50% during exercise in both groups. Basal arterial glucagon was similar in controls (56 +/- 10 pg/ml) and HAB (55 +/- 7 pg/ml) and rose similarly, by approximately 1.4-fold, with exercise in both groups. Despite greater arterial Epi and NE levels in HAB compared with controls during the basal and exercise periods, exercise-induced increases in catecholamines from basal were similar in both groups. Gluconeogenic conversion from alanine and lactate and the intrahepatic efficiency of this process were increased by twofold during exercise in both groups. R(a) rose similarly by 2.9 +/- 0.7 and 2.7 +/- 1.0 mg. kg(-1). min(-1) at time = 150 min during exercise in controls and HAB. During the blockade test period, arterial plasma glucose and R(a) rose to 454 +/- 43 mg/dl and 11.3 mg. kg(-1). min(-1) in controls, respectively, but were essentially unchanged in HAB. The attenuated response to the blockade test in HAB substantiates the effectiveness of the hepatic adrenergic blockade. In conclusion, these results demonstrate that direct hepatic adrenergic stimulation does not play a role in the stimulation of R(a) during exercise in poorly controlled diabetes.

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.