In JoVE (1)

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Articles by Owen P. McGuinness in JoVE

 JoVE Medicine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

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


JoVE 3188

Other articles by Owen P. McGuinness on PubMed

Physiological Consequences of Phasic Insulin Release in the Normal Animal

Diabetes. Feb, 2002  |  Pubmed ID: 11815467

The dose-response relationship between the hepatic sinusoidal insulin level and glucose production by the liver is such that a half-maximally effective concentration is at or slightly below the hormone levels seen basally after an overnight fast. In the normal individual, the direct effect of the hormone on the hepatocyte is far more important in restraining glucose production than its indirect effect mediated via a suppression of lipolysis. Because insulin regulates the liver in a direct fashion, its effect occurs within several minutes. Thus, the speed with which insulin works and the sensitivity of the liver to it predict that first-phase insulin release should have a significant effect in quickly suppressing hepatic glucose production. On the other hand, nonhepatic tissues are much less sensitive to insulin and respond slowly as a result of the need for insulin to cross the endothelial barrier. As a result, first-phase insulin is unlikely to significantly alter peripheral glucose disposal. Simulation studies in humans and dogs in which the effects of first-phase insulin were simulated confirmed the aforementioned predictions. In addition, they confirmed the ability of second-phase insulin release to have significant effects on both glucose production and utilization.

Impact of Intraportal N(omega)-nitro-L-arginine Infusion on Hepatic Glucose Metabolism in Total Parenteral Nutrition-adapted Dogs: Interaction with Infection

Metabolism: Clinical and Experimental. Mar, 2002  |  Pubmed ID: 11887160

During chronic total parenteral nutrition (TPN), liver glucose uptake and lactate release are markedly elevated. However, in the presence of an infection, hepatic glucose uptake and lactate release are reduced. Glucose delivery (the product of liver blood flow and inflowing glucose concentration) is a major determinant of liver glucose uptake. Hepatic blood flow is increased during infection, and increased nitric oxide (NO) biosynthesis is thought to contribute to the increase. Our aim was to determine if the increase in liver blood flow served to limit the infection-induced decrease in hepatic glucose uptake and metabolism. Chronically catheterized conscious dogs received TPN for 5 days at a rate designed to match daily basal energy requirements. On the third day of TPN administration, a sterile (SHAM) or Escherichia coli (E. coli)-containing (INF) fibrin clot was implanted in the peritoneal cavity. Forty-two hours later, somatostatin was infused with intraportal replacement of insulin (10 +/- 2 v 23 +/- 2 microU/mL, SHAM v INF, respectively) and glucagon (22 +/- 4 v 90 +/- 8 pg/mL) to match concentrations observed in sham and infected animals. Tracer and arteriovenous difference techniques were used to assess hepatic glucose metabolism. Following a 120-minute basal sampling period, sham and infected animals received either intraportal saline or N(omega)-nitro-L-arginine (L-NNA; 37 microg x kg(-1) x min(-1)) infusion for 180 minutes. Isoglycemia (120 mg/dL) was maintained with a variable glucose infusion. In the infected group L-NNA infusion decreased hepatic arterial blood flow (23.3 +/- 0.7 to 8.6 +/- 0.5 mL x kg(-1) x min(-1)), but not portal vein blood flow. Neither portal vein nor hepatic artery blood flow were altered by L-NNA infusion in the sham group. Hepatic glucose uptake and lactate metabolism were not altered by L-NNA infusion in either group. In summary, during infection, an increase in NO biosynthesis contributes to the increase in hepatic arterial blood flow, while it exerts no effect on hepatic glucose metabolism.

Impact of Chronic Fructose Infusion on Hepatic Metabolism During TPN Administration

American Journal of Physiology. Endocrinology and Metabolism. Dec, 2002  |  Pubmed ID: 12424103

During chronic total parenteral nutrition (TPN), net hepatic glucose uptake (NHGU) is markedly elevated. However, NHGU is reduced by the presence of an infection. We recently demonstrated that a small, acute (3-h) intraportal fructose infusion can correct the infection-induced impairment in NHGU. The aim of this study was to determine whether the addition of fructose to the TPN persistently enhances NHGU in the presence of an infection. TPN was infused continuously into the inferior vena cava of chronically catheterized dogs for 5 days. On day 3, a bacterial clot was implanted in the peritoneal cavity, and either saline (CON, n = 5) or fructose (+FRUC, 1.0 mg. kg(-1). min(-1), n = 6) infusion was included with the TPN. Forty-two hours after the infection was induced, hepatic glucose metabolism was assessed in conscious dogs with arteriovenous and tracer methods. Arterial plasma glucose concentration was lower with chronic fructose infusion (120 +/- 4 vs. 131 +/- 3 mg/dl, +FRUC vs. CON, P < 0.05); however, NHGU was not enhanced (2.2 +/- 0.5 vs. 2.8 +/- 0.4 mg. kg(-1). min(-1)). Acute removal of the fructose infusion dramatically decreased NHGU (2.2 +/- 0.5 to -0.2 +/- 0.5 mg. kg(-1). min(-1)), and net hepatic lactate release also fell (1.6 +/- 0.3 to 0.5 +/- 0.3 mg. kg(-1). min(-1)). This led to an increase in the arterial plasma glucose (Delta13 +/- 3 mg/dl, P < 0.05) and insulin (Delta5 +/- 2 micro U/ml) concentrations and to a decrease in glucagon (Delta-11 +/- 3 pg/ml) concentration. In conclusion, the addition of chronic fructose infusion to TPN during infection does not lead to a persistent augmentation of NHGU.

Infection Impairs Insulin-dependent Hepatic Glucose Uptake During Total Parenteral Nutrition

American Journal of Physiology. Endocrinology and Metabolism. Mar, 2003  |  Pubmed ID: 12441309

Total parenteral nutrition (TPN) markedly augments net hepatic glucose uptake (NHGU) and hepatic glycolysis in the presence of mild hyperglycemia and hyperinsulinemia. This increase is impaired by an infection. We determined whether the adaptation to TPN alters the responsiveness of the liver to insulin and whether infection impairs that response. Chronically catheterized dogs received TPN for 5 days. On day 3 of TPN, either a nonlethal hypermetabolic infection was induced (INF, n = 5) or a sham surgery was performed (SHAM, n = 5). Forty-two hours after clot implantation, somatostatin and glucagon (34 +/- 3 vs. 84 +/- 11 pg/ml in artery, SHAM vs. INF) were infused, and a three-step (120 min each) isoglycemic (approximately 120 mg/dl) hyperinsulinemic (approximately 12, 25, and 50 microU/ml) clamp was performed to simulate levels seen in normal, infected, and exogenous insulin treatment states. In SHAM, NHGU (3.5 +/- 0.2 to 4.2 +/- 0.4 to 4.6 +/- 0.5 mg x kg(-1) x min(-1)) modestly increased. In INF, NHGU was consistently lower at each insulin step (1.1 +/- 0.5 to 2.6 +/- 0.5 to 2.8 +/- 0.7 mg x kg(-1) x min(-1)). Although NHGU increased from the first to the second step in INF, it did not increase further with the highest dose of insulin. Despite increases in NHGU, net hepatic lactate release did not increase in SHAM and fell in INF. In summary, in the TPN-adapted state, liver glucose uptake is unresponsive to increases in insulin above the basal level. Although the infection-induced increase in insulin sustains NHGU, further increments in insulin enhance neither NHGU nor glycolysis.

Effects of Fructose on Hepatic Glucose Metabolism

Current Opinion in Clinical Nutrition and Metabolic Care. Jul, 2003  |  Pubmed ID: 12806219

The liver plays an important role in glucose tolerance. A number of studies have suggested fructose improves glucose tolerance especially in insulin resistant settings. This review summarizes the recent work suggesting that fructose enhances glucose tolerance by augmenting liver glucose uptake. This increase may be mediated by the translocation and activation of hepatic glucokinase.

Impact of Infection on Glucose-dependent Liver Glucose Uptake During TPN: Interaction with Insulin

American Journal of Physiology. Endocrinology and Metabolism. Feb, 2004  |  Pubmed ID: 14532169

Chronic total parenteral nutrition (TPN) markedly augments net hepatic glucose uptake (NHGU). This adaptive increase is impaired by an infection despite accompanying hyperinsulinemia. In the nonadapted state, NHGU is dependent on the prevailing glucose levels. Our aims were to determine whether the adaptation to TPN alters the glucose dependence of NHGU, whether infection impairs this dependence, and whether insulin modulates the glucose dependence of NHGU during infection. Chronically catheterized dogs received TPN for 5 days. On day 3 of TPN, dogs received either a bacterial fibrin clot to induce a nonlethal infection (INF, n = 9) or a sterile fibrin clot (Sham, n = 6). Forty-two hours after clot implantation, somatostatin was infused. In Sham, insulin and glucagon were infused to match the level seen in Sham (9 +/- 1 microU/ml and 23 +/- 4 pg/ml, respectively). In infected animals, either insulin and glucagon were infused to match the levels seen in infection (25 +/- 2 microU/ml and 101 +/- 15 pg/ml; INF-HI; n = 5) or insulin was replaced to match the lower levels seen in Sham (13 +/- 2 microU/ml), whereas glucagon was kept elevated (97 +/- 9 pg/ml; INF-LO; n = 4). Then a four-step (90 min each) hyperglycemic (120, 150, 200, or 250 mg/dl) clamp was performed. NHGU increased at each glucose step in Sham (from 3.6 +/- 0.6 to 5.4 +/- 0.7 to 8.9 +/- 0.9 to 12.1 +/- 1.1 mg.kg(-1).min(-1)); the slope of the relationship between glucose levels and NHGU (i.e., glucose dependence) was higher than that seen in nonadapted animals. Infection impaired glucose-dependent NHGU in both INF-HI (1.3 +/- 0.4 to 2.9 +/- 0.5 to 5.5 +/- 1.0 to 7.7 +/- 1.6 mg.kg(-1).min(-1)) and INF-LO (0.5 +/- 0.7 to 2.2 +/- 0.6 to 4.2 +/- 1.0 to 5.8 +/- 0.8 mg.kg(-1).min(-1)). In summary, TPN augments glucose-dependent NHGU, the presence of infection decreases glucose-dependent NHGU, and the accompanying hyperinsulinemia associated with infection does not sustain the glucose dependence of NHGU.

Prevention of Obesity and Insulin Resistance in Mice Lacking Plasminogen Activator Inhibitor 1

Diabetes. Feb, 2004  |  Pubmed ID: 14747283

Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment.

Microfluidic Glucose Stimulation Reveals Limited Coordination of Intracellular Ca2+ Activity Oscillations in Pancreatic Islets

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2004  |  Pubmed ID: 15317941

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet beta cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over approximately 7 mM generate synchronous oscillations in beta cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that beta cell [Ca2+]i oscillations occur only within regions stimulated with more than approximately 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems.

Time Course of the Hepatic Adaptation to TPN: Interaction with Glycogen Depletion

American Journal of Physiology. Endocrinology and Metabolism. Jan, 2005  |  Pubmed ID: 15339746

In response to chronic (5 days) TPN, the liver becomes a major site of glucose disposal, removing approximately 45% (4.5 mg.kg(-1).min(-1)) of exogenous glucose. Moreover, approximately 70% of glucose is not stored but released as lactate. We aimed to determine in chronically catheterized conscious dogs the time course of adaptation to TPN and the glycogen depletion impact on early time course. After an 18-h (n = 5) fast, TPN was infused into the inferior vena cava for 8 (n = 5) or 24 h (n = 6). A third group, of 42-h-fasted animals (n = 6), was infused with TPN for 8 h. TPN was infused at a rate designed to match the dog's calculated basal energy and nitrogen requirements. NHGU (-2.3 +/- 0.1 to 2.2 +/- 0.7 to 3.9 +/- 0.6 vs. -1.7 +/- 0.3 to 1.1 +/- 0.5 to 2.9 +/- 0.4 mg.kg(-1).min(-1), basal to 4 to 8 h, 18 vs. 42 h) and net hepatic lactate release (0.7 +/- 0.3 to 0.6 +/- 0.1 to 1.4 +/- 0.2 vs. -0.6 +/- 0.1 to 0.1 +/- 0.1 to 0.8 +/- 0.1 mg.kg(-1).min(-1), basal to 4 to 8 h) increased progressively. Net hepatic glycogen repletion and tracer determined that glycogen syntheses were similar. After 24 h of TPN, NHGU (5.4 +/- 0.6 mg.kg(-1).min(-1)) and net hepatic lactate release (2.6 +/- 0.4 mg.kg(-1).min(-1)) increased further. In summary, 1) most hepatic adaptation to TPN occurs within 24 h after initiation of TPN, and 2) prior glycogen depletion does not augment hepatic adaptation rate.

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.

Impact of Continuous and Pulsatile Insulin Delivery on Net Hepatic Glucose Uptake

American Journal of Physiology. Endocrinology and Metabolism. Aug, 2005  |  Pubmed ID: 15755768

The pancreas releases insulin in a pulsatile manner; however, studies assessing the liver's response to insulin have used constant infusion rates. Our aims were to determine whether the secretion pattern of insulin [continuous (CON) vs. pulsatile] in the presence of hyperglycemia 1) influences net hepatic glucose uptake (NHGU) and 2) entrains NHGU. Chronically catheterized conscious dogs fasted for 42 h received infusions including peripheral somatostatin, portal insulin (0.25 mU x kg(-1) x min(-1)), peripheral glucagon (0.9 ng x kg(-1) x min(-1)), and peripheral glucose at a rate double the glucose load to the liver. After the basal period, insulin was infused for 210 min at either four times the basal rate (1 mU x kg(-1) x min(-1)) or an identical amount in pulses of 1 and 4 min duration, followed by intervals of 11 and 8 min (CON, 1/11, and 4/8, respectively) in which insulin was not infused. A variable peripheral glucose infusion containing [3H]glucose clamped glucose levels at twice the basal level ( approximately 200 mg/dl) throughout each study. Hepatic metabolism was assessed by combining tracer and arteriovenous difference techniques. Arterial plasma insulin (microU/ml) either increased from basal levels of 6 +/- 1 to a constant level of 22 +/- 4 in CON or oscillated from 5 +/- 1 to 416 +/- 79 and from 6 +/- 1 to 123 +/- 43 in 1/11 and 4/8, respectively. NHGU (-0.8 +/- 0.3, 0.4 +/- 0.2, and -0.9 +/- 0.4 mg x kg(-1) x min(-1)) and net hepatic fractional extraction of glucose (0.04 +/- 0.01, 0.04 +/- 0.01, and 0.05 +/- 0.01 mg x kg(-1) x min(-1)) were similar during the experimental period. Spectral analysis was performed to assess whether a correlation existed between the insulin secretion pattern and NHGU. NHGU was not augmented by pulsatile insulin delivery, and there is no evidence of entrainment in hepatic glucose metabolism. Thus the loss of insulin pulsatility per se likely has little or no impact on the effectiveness of insulin in regulating liver glucose uptake.

Route-dependent Effect of Nutritional Support on Liver Glucose Uptake

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Nov, 2005  |  Pubmed ID: 15994371

The liver is a major site of glucose disposal during chronic (5 day) total parenteral (TPN) and enteral (TEN) nutrition. Net hepatic glucose uptake (NHGU) is dependent on the route of delivery when only glucose is delivered acutely; however, the hepatic response to chronic TPN and TEN is very similar. We aimed to determine whether the route of nutrient delivery altered the acute (first 8 h) response of the liver and whether chronic enteral delivery of glucose alone could augment the adaptive response to TPN. Chronically catheterized conscious dogs received either TPN or TEN containing glucose, Intralipid, and Travasol for either 8 h or 5 days. Another group received TPN for 5 days, but approximately 50% of the glucose in the nutrition was given via the enteral route (TPN+EG). Hepatic metabolism was assessed with tracer and arteriovenous difference techniques. In the presence of similar arterial plasma glucose levels (approximately 6 mM), NHGU and net hepatic lactate release increased approximately twofold between 8 h and 5 days in TPN and TEN. NHGU (26 +/- 1 vs. 23 +/- 3 micromol.kg(-1).min(-1)) and net hepatic lactate release (44 +/- 1 vs. 34 +/- 6 micromol.kg(-1).min(-1)) in TPN+EG were similar to results for TPN, despite lower insulin levels (96 +/- 6 vs. 58 +/- 16 pM, TPN vs. TPN+EG). TEN does not acutely enhance NHGU or disposition above that seen with TPN. However, partial delivery of enteral glucose is effective in decreasing the insulin requirement during chronic TPN.

Defective Glucose Homeostasis During Infection

Annual Review of Nutrition. 2005  |  Pubmed ID: 16011457

Infection leads to profound alterations in whole-body metabolism, which is characterized by marked acceleration of glucose, fat and protein, and amino acid flux. One of the complications of infection, especially in the nutritionally supported setting, is hyperglycemia. The hyperglycemia is caused by peripheral insulin resistance and alterations in hepatic glucose metabolism. The defects in hepatic glucose metabolism include overproduction of glucose and a failure of the liver to appropriately adapt when nutritional support is administered. Investigators have suggested that multiple factors contribute to the observed defects. In this review, I focus primarily on alterations in carbohydrate metabolism, examining both the metabolic response to infection and inflammatory stress, the role of the accompanying neuroendocrine and inflammatory responses in the metabolic response, and the interaction between the endocrine response to infection and nutritional support.

Suppression of Aging in Mice by the Hormone Klotho

Science (New York, N.Y.). Sep, 2005  |  Pubmed ID: 16123266

A defect in Klotho gene expression in mice accelerates the degeneration of multiple age-sensitive traits. Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.

Individual Mice Can Be Distinguished by the Period of Their Islet Calcium Oscillations: is There an Intrinsic Islet Period That is Imprinted in Vivo?

Diabetes. Dec, 2005  |  Pubmed ID: 16306370

Pulsatile insulin secretion in vivo is believed to be derived, in part, from the intrinsic glucose-dependent intracellular calcium concentration ([Ca2+]i) pulsatility of individual islets. In isolation, islets display fast, slow, or mixtures of fast and slow [Ca2+]i oscillations. We show that the period of islet [Ca2+]i oscillations is unique to each mouse, with the islets from an individual mouse demonstrating similar rhythms to one another. Based on their rhythmic period, mice were broadly classified as being either fast (0.65 +/- 0.1 min; n = 6 mice) or slow (4.7 +/- 0.2 min; n = 15 mice). To ensure this phenomenon was not an artifact of islet-to-islet communication, we confirmed that islets cultured in isolation (period: 2.9 +/- 0.1 min) were not statistically different from islets cultured together from the same mouse (3.1 +/- 0.1 min, P > 0.52, n = 5 mice). We also compared pulsatile insulin patterns measured in vivo with islet [Ca2+]i patterns measured in vitro from six mice. Mice with faster insulin pulse periods corresponded to faster islet [Ca2+]i patterns, whereas slower insulin patterns corresponded to slower [Ca2+]i patterns, suggesting that the insulin rhythm of each mouse is preserved to some degree by its islets in vitro. We propose that individual mice have characteristic oscillatory [Ca2+]i patterns, which are imprinted in vivo through an unknown mechanism.

Insulin Secretion in the Conscious Mouse is Biphasic and Pulsatile

American Journal of Physiology. Endocrinology and Metabolism. Mar, 2006  |  Pubmed ID: 16249252

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

Overexpression of the Insulin Receptor Inhibitor PC-1/ENPP1 Induces Insulin Resistance and Hyperglycemia

American Journal of Physiology. Endocrinology and Metabolism. Apr, 2006  |  Pubmed ID: 16278247

The ectoenzyme PC-1 is an insulin receptor inhibitor that is elevated in cells and tissues of humans with type 2 diabetes (T2D). We have recently shown that acute PC-1 overexpression in liver causes insulin resistance and glucose intolerance in mice (3), but the chronic effects of PC-1 overexpression on these functions are unknown. Herein we produced transgenic mice overexpressing the potent q allele of human PC-1 in muscle and liver. Compared with controls, these mice had 2- to 3-fold elevations of PC-1 content in liver and 5- to 10-fold elevations in muscle. In the fed state, the PC-1 animals had 100 mg/dl higher glucose levels and sixfold higher insulin levels compared with controls. During glucose tolerance tests, these PC-1 animals had peak glucose levels that were >150 mg/dl higher than controls. In vivo uptake of 2-deoxy-d-glucose in muscle during insulin infusion was decreased in the PC-1 animals. These in vivo data support the concept, therefore, that PC-1 plays a role in insulin resistance and hyperglycemia and suggest that animals with overexpression of human PC-1 in insulin-sensitive tissues may be important models to investigate insulin resistance.

Considerations in the Design of Hyperinsulinemic-euglycemic Clamps in the Conscious Mouse

Diabetes. Feb, 2006  |  Pubmed ID: 16443772

Despite increased use of the hyperinsulinemic-euglycemic clamp to study insulin action in mice, the effects of experimental parameters on the results obtained have not been addressed. In our studies, we determined the influences of sampling sites, fasting duration, and insulin delivery on results obtained from clamps in conscious mice. Carotid artery and jugular vein catheters were implanted in C57BL/6J mice (n = 6-10/group) fed a normal diet for sampling and infusions. After a 5-day recovery period, mice underwent a 120-min clamp (2.5-mU . kg(-1) . min(-1) insulin infusion; approximately 120-130 mg/dl glucose) while receiving [3-(3)H]glucose to determine glucose appearance (endoR(a)) and disappearance (R(d)). Sampling large volumes (approximately 100 mul) from the cut tail resulted in elevated catecholamines and basal glucose compared with artery sampling. Catecholamines were not elevated when taking small samples ( approximately 5 mul) from the cut tail. Overnight (18-h) fasting resulted in greater loss of total body, lean, and fat masses and hepatic glycogen but resulted in enhanced insulin sensitivity compared with 5-h fasting. Compared with a 16-mU/kg insulin prime, a 300-mU/kg prime resulted in hepatic insulin resistance and slower acquisition of steady-state glucose infusion rates (GIR) after a 5-h fast. The steady-state GIR was expedited after the 300-mU/kg prime in 18-h-fasted mice. The GIR and R(d) rose with increasing insulin infusions (0.8, 2.5, 4, and 20 mU . kg(-1) . min(-1)), but endoR(a) was fully suppressed with doses higher than 0.8 mU . kg(-1) . min(-1). Thus, common variations in experimental factors yield different results and should be considered in designing and interpreting clamps.

Glucocorticoid-deficient Corticotropin-releasing Hormone Knockout Mice Maintain Glucose Requirements but Not Autonomic Responses During Repeated Hypoglycemia

American Journal of Physiology. Endocrinology and Metabolism. Jul, 2006  |  Pubmed ID: 16449297

Glucocorticoids have been implicated in hypoglycemia-induced autonomic failure but also contribute to normal counterregulation. To determine the influence of normal and hypoglycemia-induced levels of glucocorticoids on counterregulatory responses to acute and repeated hypoglycemia, we compared plasma catecholamines, corticosterone, glucagon, and glucose requirements in male wild-type (WT) and glucocorticoid-deficient, corticotropin-releasing hormone knockout (CRH KO) mice. Conscious, chronically cannulated, unrestrained WT and CRH KO mice underwent a euglycemic (Prior Eu) or hypoglycemic clamp (Prior Hypo) on day 1 followed by a hypoglycemic clamp on day 2 (blood glucose both days, 65 +/- 1 mg/dl). Baseline epinephrine and glucagon were similar, and norepinephrine was elevated, in CRH KO vs. WT mice. CRH KO corticosterone was almost undetectable (<1.5 microg/dl) and unresponsive to hypoglycemia. CRH KO glucose requirements were significantly higher during day 1 hypoglycemia despite epinephrine and glucagon responses that were comparable to or greater than those in WT. Hyperinsulinemic euglycemia did not increase hormones or glucose requirements above baseline. On day 2, Prior Hypo WT had significantly higher glucose requirements and significantly lower corticosterone and glucagon responses. Prior Hypo and Prior Eu CRH KO mice had similar day 2 glucose requirements. However, Prior Hypo CRH KO mice had significantly lower day 2 epinephrine and norepinephrine vs. Prior Eu CRH KO and tended to have lower glucagon than on day 1. We conclude that glucocorticoid insufficiency in CRH KO mice correlates with 1) impaired counterregulation during acute hypoglycemia and 2) complex effects after repeated hypoglycemia, neither preventing decreased hormone responses nor worsening glucose requirements.

Counterregulatory Deficits Occur Within 24 H of a Single Hypoglycemic Episode in Conscious, Unrestrained, Chronically Cannulated Mice

American Journal of Physiology. Endocrinology and Metabolism. Apr, 2006  |  Pubmed ID: 16533951

Hypoglycemia-induced counterregulatory failure is a dangerous complication of insulin use in diabetes mellitus. Controlled hypoglycemia studies in gene knockout models, which require the use of mice, would aid in identifying causes of defective counterregulation. Because stress can influence counterregulatory hormones and glucose homeostasis, we developed glucose clamps with remote blood sampling in conscious, unrestrained mice. Male C57BL/6 mice implanted with indwelling carotid artery and jugular vein catheters were subjected to 2 h of hyperinsulinemic glucose clamps 24 h apart, with a 6-h fast before each clamp. On day 1, blood glucose was maintained (euglycemia, 178 +/- 4 mg/dl) or decreased to 62 +/- 1 mg/dl (hypoglycemia) by insulin (20 mU x kg(-1) x min(-1)) and variable glucose infusion. Donor blood was continuously infused to replace blood sample volume. Baseline plasma epinephrine (32 +/- 8 pg/ml), corticosterone (16.1 +/- 1.8 microg/dl), and glucagon (35 +/- 3 pg/ml) were unchanged during euglycemia but increased significantly during hypoglycemia, with a glycemic threshold of approximately 80 mg/dl. On day 2, all mice underwent a hypoglycemic clamp (blood glucose, 64 +/- 1 mg/dl). Compared with mice that were euglycemic on day 1, previously hypoglycemic mice had significantly higher glucose requirements and significantly lower plasma glucagon and corticosterone (n = 6/group) on day 2. Epinephrine tended to decrease, although not significantly, in repeatedly hypoglycemic mice. Pre- and post-clamp insulin levels were similar between groups. We conclude that counterregulatory responses to acute and repeated hypoglycemia in unrestrained, chronically cannulated mice reproduce aspects of counterregulation in humans, and that repeated hypoglycemia in mice is a useful model of counterregulatory failure.

Impact of Portal Glucose Delivery on Glucose Metabolism in Conscious, Unrestrained Mice

American Journal of Physiology. Endocrinology and Metabolism. Dec, 2006  |  Pubmed ID: 16822956

Previous studies in mice suggest that portal venous infusion of glucose at a low rate paradoxically causes hypoglycemia; this does not occur in dogs, rats, and humans. A possible explanation is that fasting status in the mouse studies may have altered the response. We sought to determine whether the response to portal glucose delivery in the mouse was similar to that seen in other species and whether it was dependent on fasting status. Studies were performed on chronically catheterized conscious mice. Catheters were placed into the portal and jugular veins and carotid artery 5 days before study. After a 5- or 16-h fast, glucose was infused into either the portal (PO) or the jugular vein (JU) for 6 h at 25 microg.g(-1).min(-1). [3-(3)H]glucose was infused into the JU to measure glucose turnover. In 5-h-fasted mice, PO and JU exhibited similar increases in arterial blood glucose from 155 +/- 11 to 173 +/- 19 and 147 +/- 8 to 173 +/- 10 mg/dl, respectively. Endogenous glucose production decreased and arterial insulin increased to the same extent in both PO and JU. A similar response was observed in 16-h-fasted mice; however, the proportion of hepatic glycogen synthesis occurring by the indirect pathway was increased by fasting. In summary, portal glucose delivery in the mouse did not cause hypoglycemia even when the duration of the fast was extended. The explanation of the differing response from previous reports in the mouse is unclear.

Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/glucose-6-phosphatase Catalytic Subunit-beta Results in Lowered Plasma Cholesterol and Elevated Glucagon

The Journal of Biological Chemistry. Dec, 2006  |  Pubmed ID: 17023421

In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type 1a, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-beta. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by approximately 50% in homogenates of UGRP(-/-) mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP(-/-) mice exhibit growth retardation as do G6Pase(-/-) mice and patients with GSD type 1a. However, in contrast to G6Pase(-/-) mice and patients with GSD type 1a, UGRP(-/-) mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP(-/-) mice are not hypoglycemic, female UGRP(-/-) mice have elevated ( approximately 60%) plasma glucagon and reduced ( approximately 20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP(-/-) mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis in vivo.

Monoclonal Antibody Antagonists of Hypothalamic FGFR1 Cause Potent but Reversible Hypophagia and Weight Loss in Rodents and Monkeys

American Journal of Physiology. Endocrinology and Metabolism. Mar, 2007  |  Pubmed ID: 17132826

We generated three fully human monoclonal antibody antagonists against fibroblast growth factor receptor-1 (FGFR1) that potently block FGF signaling. We found that antibodies targeting the c-splice form of the receptor (FGFR1c) were anorexigenic when administered intraperitoneally three times weekly to mice, resulting in rapid, dose-dependent weight loss that plateaued (for doses>4 mg/kg) at 35-40% in 2 wk. Animals appeared healthy during treatment and regained their normal body weights and growth trajectories upon clearance of the antibodies from the bloodstream. Measurements of food consumption and energy expenditure indicated that the rapid weight loss was induced primarily by decreased energy intake and not by increased energy expenditure or cachexia and was accompanied by a greater reduction in fat than lean body mass. Hypophagia was not caused through malaise or illness, as indicated by absence of conditioned taste aversion, pica behavior, and decreased need-induced salt intake in rats. In support of a hypothalamic site of action, we found that, after intraperitoneal injections, anti-FGFR1c (IMC-A1), but not a control antibody, accumulated in the median eminence and adjacent mediobasal hypothalamus and that FGFR1c is enriched in the hypothalamus of mice. Furthermore, a single intracerebroventricular administration of 3 microg of IMC-A1 via the 3rd ventricle to mice caused an approximately 36% reduction in food intake and an approximately 6% weight loss within the ensuing 24 h. Our data suggest that FGF signaling through FGFR1c may play a physiological role in hypothalamic feeding circuit and that blocking it leads to hypophagia and weight loss.

Glucagon Chronically Impairs Hepatic and Muscle Glucose Disposal

American Journal of Physiology. Endocrinology and Metabolism. Mar, 2007  |  Pubmed ID: 17132827

Defects in insulin secretion and/or action contribute to the hyperglycemia of stressed and diabetic patients, and we hypothesize that failure to suppress glucagon also plays a role. We examined the chronic impact of glucagon on glucose uptake in chronically catheterized conscious depancreatized dogs placed on 5 days of nutritional support (NS). For 3 days of NS, a variable intraportal infusion of insulin was given to maintain isoglycemia (approximately 120 mg/dl). On day 3 of NS, animals received a constant low infusion of insulin (0.4 mU.kg-1.min-1) and either no glucagon (CONT), basal glucagon (0.7 ng.kg-1.min-1; BasG), or elevated glucagon (2.4 ng.kg-1.min-1; HiG) for the remaining 2 days. Glucose in NS was varied to maintain isoglycemia. An additional group (HiG+I) received elevated insulin (1 mU.kg-1.min-1) to maintain glucose requirements in the presence of elevated glucagon. On day 5 of NS, hepatic substrate balance was assessed. Insulin and glucagon levels were 10+/-2, 9+/-1, 7+/-1, and 24+/-4 microU/ml, and 24+/-5, 39+/-3, 80+/-11, and 79+/-5 pg/ml, CONT, BasG, HiG, and HiG+I, respectively. Glucagon infusion decreased the glucose requirements (9.3+/-0.1, 4.6+/-1.2, 0.9+/-0.4, and 11.3+/-1.0 mg.kg-1.min-1). Glucose uptake by both hepatic (5.1+/-0.4, 1.7+/-0.9, -1.0+/-0.4, and 1.2+/-0.4 mg.kg-1.min-1) and nonhepatic (4.2+/-0.3, 2.9+/-0.7, 1.9+/-0.3, and 10.2+/-1.0 mg.kg-1.min-1) tissues decreased. Additional insulin augmented nonhepatic glucose uptake and only partially improved hepatic glucose uptake. Thus, glucagon impaired glucose uptake by hepatic and nonhepatic tissues. Compensatory hyperinsulinemia restored nonhepatic glucose uptake and partially corrected hepatic metabolism. Thus, persistent inappropriate secretion of glucagon likely contributes to the insulin resistance and glucose intolerance observed in obese and diabetic individuals.

Point-counterpoint: Interleukin-6 Does/does Not Have a Beneficial Role in Insulin Sensitivity and Glucose Homeostasis

Journal of Applied Physiology (Bethesda, Md. : 1985). Feb, 2007  |  Pubmed ID: 17323468

Macrophage TNF-alpha Contributes to Insulin Resistance and Hepatic Steatosis in Diet-induced Obesity

American Journal of Physiology. Endocrinology and Metabolism. Sep, 2007  |  Pubmed ID: 17578885

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-alpha. To test this hypothesis, we transplanted male wild-type (WT) and TNF-alpha deficient (KO) mice with either TNF-alpha-sufficient (TNF-alpha(+/+)) or TNF-alpha-deficient (TNF-alpha(-/-)) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-alpha, which was partially explained by reduced epididymal fat pad and liver weight. TNF-alpha(-/-) -->KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-alpha(+/+)-->KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-alpha(-/-) bone marrow in WT mice compared with TNF-alpha(+/+)-->WT. The preserved insulin sensitivity seen in TNF-alpha(-/-)-->KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-alpha contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-alpha.

Glucagon-mediated Impairments in Hepatic and Peripheral Tissue Nutrient Disposal Are Not Aggravated by Increased Lipid Availability

American Journal of Physiology. Endocrinology and Metabolism. May, 2009  |  Pubmed ID: 19208853

Glucose, fat, and glucagon availability are increased in diabetes. The normal response of the liver to chronic increases in glucose availability is to adapt to become a marked consumer of glucose. Yet this fails to occur in diabetes. The aim was to determine whether increased glucagon and lipid interact to impair the adaptation to increased glucose availability. Chronically catheterized well controlled depancreatized conscious dogs (n = 21) received 3 days of continuous parenteral nutrition (TPN) that was either high in glucose [C; 75% nonprotein calories (NPC)] or in lipid (HL; 75% NPC) in the presence or absence of a low dose (one-third basal) chronic intraportal infusion of glucagon (GN; 0.25 ng.kg(-1).min(-1)). During the 3 days of TPN, all groups received the same insulin algorithm; the total amount of glucose infused (GIR) was varied to maintain isoglycemia ( approximately 120 mg/dl). On day 3 of TPN, hepatic metabolism was assessed. Glucose and insulin levels were similar in all groups. GIR was decreased in HL and C + GN ( approximately 30%) and was further decreased in HL + GN (55%). Net hepatic glucose uptake was decreased approximately 15% in C + GN, and HL and was decreased approximately 50% in HL + GN. Lipid alone or combined with glucagon decreased glucose uptake by peripheral tissues. Despite impairing whole body glucose utilization, HL did not limit whole body energy disposal. In contrast, glucagon suppressed whole body energy disposal irrespective of the diet composition. In summary, failure to appropriately suppress glucagon secretion adds to the dietary fat-induced impairment in both hepatic and peripheral glucose disposal. In addition, unlike increasing the percentage of calories as fat, inappropriate glucagon secretion in the absence of compensatory hyperinsulinemia limits whole body nutrient disposition.

Insulin Signaling in Alpha Cells Modulates Glucagon Secretion in Vivo

Cell Metabolism. Apr, 2009  |  Pubmed ID: 19356716

Glucagon plays an important role in glucose homeostasis by regulating hepatic glucose output in both normo- and hypoglycemic conditions. In this study, we created and characterized alpha cell-specific insulin receptor knockout (alphaIRKO) mice to directly explore the role of insulin signaling in the regulation of glucagon secretion in vivo. Adult male alphaIRKO mice exhibited mild glucose intolerance, hyperglycemia, and hyperglucagonemia in the fed state and enhanced glucagon secretion in response to L-arginine stimulation. Hyperinsulinemic-hypoglycemic clamp studies revealed an enhanced glucagon secretory response and an abnormal norepinephrine response to hypoglycemia in alphaIRKO mice. The mutants also exhibited an age-dependent increase in beta cell mass. Furthermore, siRNA-mediated knockdown of insulin receptor in glucagon-secreting InR1G cells promoted enhanced glucagon secretion and complemented our in vivo findings. Together, these data indicate a significant role for intraislet insulin signaling in the regulation of alpha cell function in both normo- and hypoglycemic conditions.

Inactivation of NF-kappaB P50 Leads to Insulin Sensitization in Liver Through Post-translational Inhibition of P70S6K

The Journal of Biological Chemistry. Jul, 2009  |  Pubmed ID: 19433583

In this study, we investigated the metabolic phenotype of the NF-kappaB p50 knock-out (p50-KO) mice. Compared with wild type mice, the p50-KO mice had an increase in food intake, but a decrease in body fat content. On chow diet, their blood glucose dropped much more than the wild type (WT) mice in the insulin tolerance test. Their glucose infusion rate was 30% higher than that of the WT mice in the hyperinsulinemic-euglycemic clamp. Their hepatic glucose production was suppressed more actively by insulin, and their insulin-induced glucose uptake was not altered in skeletal muscle or adipose tissue. In the liver, their p70S6K (S6K1) protein was significantly lower, and tumor necrosis factor-alpha (TNF-alpha) expression was much higher. Their S6K1 protein was reduced by TNF-alpha treatment in the primary culture of hepatocytes. S6K1 reduction was blocked by the proteasome inhibitor MG132. In their livers, IKK2 (IKKbeta) activity was reduced together with IKKgamma. Their S6K1 degradation was dependent on IKK2 deficiency. Reconstitution of the S6K1 protein in their liver blocked the increase in insulin sensitivity. S6K1 degradation was not observed in hepatocytes of the WT mice. The data suggest that inactivation of NF-kappaB p50 leads to suppression of IKK2 activity in the liver. IKK2 deficiency leads to S6K1 inhibition through TNF-induced protein degradation. The S6K1 reduction may contribute to insulin sensitivity in p50-KO mice. This study suggests that hepatic S6K1 may be a drug target in the treatment of insulin resistance.

Deletion of the Mouse Slc30a8 Gene Encoding Zinc Transporter-8 Results in Impaired Insulin Secretion

The Biochemical Journal. Aug, 2009  |  Pubmed ID: 19450229

The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid residue 325 of human ZnT-8 are associated with altered susceptibility to Type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in Type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analysed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioural changes or differences in body weight were observed between wild-type and ZnT-8-/- mice, and ZnT-8-/- mouse islets were indistinguishable from wild-type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8-/- mouse islets, as evaluated both by Timm's histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16-week-old, 6 h fasted animals of either gender; however, plasma insulin concentrations were reduced in both female (approximately 31%) and male (approximately 47%) ZnT-8-/- mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8-/- mice, but glucose-stimulated insulin secretion from isolated islets was reduced approximately 33% relative to wild-type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

Skeletal Muscle AMP-activated Protein Kinase is Essential for the Metabolic Response to Exercise in Vivo

The Journal of Biological Chemistry. Sep, 2009  |  Pubmed ID: 19525228

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKalpha2 subunit (alpha2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in alpha2-KD mice; expression of neuronal NOS (NOSmicro) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 +/- 8 and 50 +/- 7%, respectively, in skeletal muscle of alpha2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSmicro expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.

NIH Experiment in Centralized Mouse Phenotyping: the Vanderbilt Experience and Recommendations for Evaluating Glucose Homeostasis in the Mouse

American Journal of Physiology. Endocrinology and Metabolism. Oct, 2009  |  Pubmed ID: 19638507

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

Lost in Translation

Diabetes. Sep, 2009  |  Pubmed ID: 19720822

Circadian Clock Gene Bmal1 is Not Essential; Functional Replacement with Its Paralog, Bmal2

Current Biology : CB. Feb, 2010  |  Pubmed ID: 20153195

Most of the central circadian clock genes in the mouse exist as paralog pairs (Per1 and Per2, Cry1 and Cry2, Clock and Npas2) in which each gene of the pair must be knocked out to confer arrhythmicity. The only exception to this pattern is Bmal1 (also known as Mop3), the single knockout of which confers arrhythmicity, despite the presence of its paralog, Bmal2 (also known as Mop9). The knockout of Bmal1 also has significant effects on longevity, metabolism, etc. These results have led to the conclusion that Bmal1 is a singularly essential clock gene and that Bmal2 has a minimal role in the clock system. In contrast, we find that expression of Bmal2 from a constitutively expressed promoter can rescue the clock and metabolic phenotypes of Bmal1-knockout mice, including rhythmic locomotor activity, rhythmic metabolism, low body weight, and enhanced fat deposition. Combined with the data of Bunger and colleagues, who reported that knockout of Bmal1 downregulates Bmal2, we conclude that Bmal1 and Bmal2 form a circadian paralog pair that is functionally redundant and that, in the mouse, Bmal2 is regulated by Bmal1 such that knockout of Bmal1 alone results in a functionally double Bmal1 and Bmal2 knockout. Therefore, the role(s) of Bmal2 may be more important than has been appreciated heretofore.

Impaired Glucose Tolerance and Predisposition to the Fasted State in Liver Glycogen Synthase Knock-out Mice

The Journal of Biological Chemistry. Apr, 2010  |  Pubmed ID: 20178984

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

Advantages of Dynamic "closed Loop" Stable Isotope Flux Phenotyping over Static "open Loop" Clamps in Detecting Silent Genetic and Dietary Phenotypes

Metabolomics : Official Journal of the Metabolomic Society. Jun, 2010  |  Pubmed ID: 20445758

In vivo insulin sensitivity can be assessed using "open loop" clamp or "closed loop" methods. Open loop clamp methods are static, and fix plasma glucose independently from plasma insulin. Closed loop methods are dynamic, and assess glucose disposal in response to a stable isotope labeled glucose tolerance test. Using PPARalpha(-/-) mice, open and closed loop assessments of insulin sensitivity/glucose disposal were compared. Indirect calorimetry done for the assessment of diurnal substrate utilization/metabolic flexibility showed that chow fed PPARalpha(-/-) mice had increased glucose utilization during the light (starved) cycle. Euglycemic clamps showed no differences in insulin stimulated glucose disposal, whether for chow or high fat diets, but did show differences in basal glucose clearance for chow fed PPARalpha(-/-) versus SV129J-wt mice. In contrast, the dynamic stable isotope labeled glucose tolerance tests reveal enhanced glucose disposal for PPARalpha(-/-) versus SV129J-wt, for chow and high fat diets. Area under the curve for plasma labeled and unlabeled glucose for PPARalpha(-/-) was approximately 1.7-fold lower, P < 0.01 during the stable isotope labeled glucose tolerance test for both diets. Area under the curve for plasma insulin was 5-fold less for the chow fed SV129J-wt (P < 0.01) but showed no difference on a high fat diet (0.30 +/- 0.1 for SV129J-wt vs. 0.13 +/- 0.10 for PPARalpha(-/-), P = 0.28). This study demonstrates that dynamic stable isotope labeled glucose tolerance test can assess "silent" metabolic phenotypes, not detectable by the static, "open loop", euglycemic or hyperglycemic clamps. Both open loop and closed loop methods may describe different aspects of metabolic inflexibility and insulin sensitivity.

Standard Operating Procedures for Describing and Performing Metabolic Tests of Glucose Homeostasis in Mice

Disease Models & Mechanisms. Sep-Oct, 2010  |  Pubmed ID: 20713647

The Mouse Metabolic Phenotyping Center (MMPC) Consortium was established to address the need to characterize the growing number of mouse models of metabolic diseases, particularly diabetes and obesity. A goal of the MMPC Consortium is to propose standard methods for assessing metabolic phenotypes in mice. In this article, we discuss issues pertaining to the design and performance of various tests of glucose metabolism. We also propose guidelines for the description of methods, presentation of data and interpretation of results. The recommendations presented in this article are based on the experience of the MMPC Consortium and other investigators.

Metabolic Response to Endotoxin in Vivo in the Conscious Mouse: Role of Interleukin-6

Metabolism: Clinical and Experimental. Jan, 2011  |  Pubmed ID: 20102773

Inflammation and insulin resistance are characteristics of endotoxemia. Although the role of interleukin (IL)-6 in insulin-resistant states has been characterized, little is known of its role in the metabolic response to inflammation. To study the role of IL-6, conscious chronically catheterized mice were used. Five days before being studied, catheters were implanted in the carotid artery and jugular vein. After a 5-hour fast, Escherichia coli (250 μg per mouse) lipopolysaccharide (LPS) was injected in IL-6⁻/⁻ (KO, n = 13) and IL-6+/+ (WT, n = 10) littermates. The IL-6 response to LPS was simulated in an additional group of KO mice (KO + IL-6, n = 10). Interleukin-6 increased in WT (15 ± 0.7 ng/mL) 4 hours after LPS and was undetectable in KO. Interleukin-6 replacement in the KO restored circulating IL-6 to levels observed in the WT group (14 ± 0.3 ng/mL). Tumor necrosis factor-α increased more rapidly in WT than in both KO and KO + IL-6 mice. The KO mice exhibited a more profound glucose excursion 30 minutes after LPS injection and no apparent hypoglycemia at 4 hours (95 ± 5 vs 70 ± 8 mg/dL, KO vs WT), despite having a blunted glucagon and epinephrine response. Glucose levels in KO + IL-6 mice, while decreased (93 ± 4 mg/dL) at 4 hours, remained higher than those in WT mice. In summary, the absence of IL-6 protected against LPS-induced hypoglycemia. Acute restoration of the IL-6 response to LPS did not potentiate hypoglycemia but partially restored the glucagon response. Thus, although IL-6 promotes glucose intolerance in insulin-resistant states, IL-6 promotes hypoglycemia during acute inflammation.

Continuous Low-dose Fructose Infusion Does Not Reverse Glucagon-mediated Decrease in Hepatic Glucose Utilization

Metabolism: Clinical and Experimental. Jun, 2011  |  Pubmed ID: 20940071

An adaptation to continuous total parenteral nutrition (TPN; 75% of nonprotein calories as glucose) is the liver becomes a major consumer of glucose with lactate release as a by-product. The liver is able to further increase liver glucose uptake when a small dose of fructose is acutely infused via the portal system. Glucagon, commonly elevated during inflammatory stress, is a potent inhibitor of glucose uptake by the liver during TPN. The aim was to determine if continuous fructose infusion could overcome the glucagon-mediated decrease in hepatic glucose uptake. Studies were performed in conscious, insulin-treated, chronically catheterized, pancreatectomized dogs that adapted to TPN for 33 hours. They were then assigned to 1 of 4 groups: TPN (C), TPN + fructose (4.4 μmol kg(-1) min(-1); F), TPN + glucagon (0.2 pmol kg(-1) min(-1); GGN), or TPN + fructose and glucagon (F + GGN) for an additional 63 hours (33-96 hours). Insulin, fructose, and glucagon were infused into the portal vein. During that period, all animals received a fixed insulin infusion of 0.4 mU·kg(-1)·min(-1) (33-96 hours); and the glucose infusion rates were adjusted to maintain euglycemia (6.6 mmol/L). Continuous fructose infusion was unable to further enhance net hepatic glucose uptake (in micromoles per kilogram per minute) (31.1 ± 2.8 vs 36.1 ± 5.0; C vs F), nor was it able to overcome glucagon-mediated decrease in net hepatic glucose uptake (10.0 ± 4.4 vs 12.2 ± 3.9; GGN vs F + GGN). In summary, continuous fructose infusion cannot augment liver glucose uptake during TPN; nor can it overcome the inhibitory effects of glucagon.

Assessment of Different Bariatric Surgeries in the Treatment of Obesity and Insulin Resistance in Mice

Annals of Surgery. Jul, 2011  |  Pubmed ID: 21522012

To assess the effects of different bariatric surgical procedures on the treatment of obesity and insulin resistance in high fat diet-induced obese (DIO) mice.

Ablation of Ghrelin Receptor Reduces Adiposity and Improves Insulin Sensitivity During Aging by Regulating Fat Metabolism in White and Brown Adipose Tissues

Aging Cell. Dec, 2011  |  Pubmed ID: 21895961

Aging is associated with increased adiposity in white adipose tissues and impaired thermogenesis in brown adipose tissues; both contribute to increased incidences of obesity and type 2 diabetes. Ghrelin is the only known circulating orexigenic hormone that promotes adiposity. In this study, we show that ablation of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) improves insulin sensitivity during aging. Compared to wild-type (WT) mice, old Ghsr(-/-) mice have reduced fat and preserve a healthier lipid profile. Old Ghsr(-/-) mice also exhibit elevated energy expenditure and resting metabolic rate, yet have similar food intake and locomotor activity. While GHS-R expression in white and brown adipose tissues was below the detectable level in the young mice, GHS-R expression was readily detectable in visceral white fat and interscapular brown fat of the old mice. Gene expression profiles reveal that Ghsr ablation reduced glucose/lipid uptake and lipogenesis in white adipose tissues but increased thermogenic capacity in brown adipose tissues. Ghsr ablation prevents age-associated decline in thermogenic gene expression of uncoupling protein 1 (UCP1). Cell culture studies in brown adipocytes further demonstrate that ghrelin suppresses the expression of adipogenic and thermogenic genes, while GHS-R antagonist abolishes ghrelin's effects and increases UCP1 expression. Hence, GHS-R plays an important role in thermogenic impairment during aging. Ghsr ablation improves aging-associated obesity and insulin resistance by reducing adiposity and increasing thermogenesis. Growth hormone secretagogue receptor antagonists may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.

Disassociation of Muscle Insulin Signaling and Insulin-Stimulated Glucose Uptake During Endotoxemia

PloS One. 2012  |  Pubmed ID: 22276152

Lipopolysaccharide (LPS) elicits a strong immune response, which leads to the release of inflammatory cytokines. Increased cytokine production has been shown to impair insulin-mediated glucose disposal. LPS can alter other factors, such as muscle blood flow and insulin signaling in the myocyte,that can influence glucose disposal. We hypothesize that LPS induced impairments in cardiovascular function contribute to the associated impairments in insulin action in vivo. Male wild-type C57BL/6J mice had a catheter implanted in the jugular vein for infusions and the carotid artery for sampling 5 days prior to the hyperinsulinemic-euglycemic clamp. Mice were treated with vehicle, low-(1 ug/gBW) or high-dose (10 ug/gBW) LPS 4 hours prior to the clamp. Muscle glucose uptake (MGU) was assessed using [2-(14)C] deoxyglucose. While both low- and high-dose LPS inhibited insulin-stimulated MGU compared to vehicle-treated mice, the impairment was more significant with the high-dose treatment (∼25% in soleus and ∼70% in both gastrocnemius and vastus lateralis). Interestingly, insulin signaling through the PI3-kinase pathway in the muscle was not affected by this treatment suggesting that the decrease in MGU is not directly due to impairments in muscle insulin action. Echocardiography demonstrated that high-dose LPS treatment significantly decreased stroke volume (∼30%), heart rate (∼35%), and cardiac output (∼50%). These observations were not seen with vehicle or low-dose LPS treatment. High-dose LPS treatment also significantly decreased muscle blood flow (∼70%) and whole body oxygen consumption (∼50%). Thus, in vivo acute endotoxemia does not impair insulin signaling through the PI3-kinase pathway in skeletal muscle and decreased tissue blood flow likely plays a central role in the impairment of glucose uptake in the muscle.

Adropin Deficiency is Associated with Increased Adiposity and Insulin Resistance

Obesity (Silver Spring, Md.). Feb, 2012  |  Pubmed ID: 22318315

Adropin is a secreted peptide that improves hepatic steatosis and glucose homeostasis when administered to diet-induced obese mice. It is not clear if adropin is a peptide hormone regulated by signals of metabolic state. Moreover, the significance of a decline in adropin expression with obesity with respect to metabolic disease is also not clear. We investigated the regulation of serum adropin by metabolic status and diet. Serum adropin levels were high in chow-fed conditions and were suppressed by fasting and diet-induced obesity. High adropin levels were observed in mice fed a high-fat low carbohydrate diet, while lower levels were observed in mice fed a low fat high carbohydrate diet. To investigate the role of adropin deficiency in metabolic homeostasis, we generated adropin knockout mice (AdrKO) on the C57BL/6J background. AdrKO displayed a 50%-increase in increase in adiposity, although food intake and energy expenditure were normal. AdrKO also exhibited dyslipidemia and impaired suppression of endogenous glucose production in hyperinsulinemic-euglycemic clamp conditions, suggesting insulin resistance. While homo- and heterozygous carriers of the null adropin allele exhibited normal diet-induced obesity relative to controls, impaired glucose tolerance associated with weight gain was more severe in both groups. In summary, adropin is a peptide hormone regulated by fasting and feeding. In fed conditions, adropin levels are regulated dietary macronutrients, and increase with dietary fat content. Adropin is not required for regulating food intake, however it's functions impact on adiposity and are involved in preventing insulin resistance, dyslipidemia and impaired glucose tolerance.

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