Deletion of the gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantigen results in a mild metabolic phenotype.

AIMS/HYPOTHESIS: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences. METHODS: To address this concern and assess the physiological function of G6PC2, we generated G6pc2-null mice and performed a phenotypic analysis focusing principally on energy metabolism. RESULTS: No differences in body weight were observed and no gross anatomical or behavioural changes were evident. In 16-week-old animals, following a 6-h fast, a small but significant decrease in blood glucose was observed in both male (-14%) and female (-11%) G6pc2 (-/-) mice, while female G6pc2 (-/-) mice also exhibited a 12% decrease in plasma triacylglycerol. Plasma cholesterol, glycerol, insulin and glucagon concentrations were unchanged. CONCLUSIONS/INTERPRETATION: These results argue against the possibility of G6PC2 playing a major role in pancreatic islet stimulus secretion coupling or energy homeostasis under physiological conditions imposed by conventional animal housing. This indicates that manipulating the expression of G6PC2 for therapeutic ends may be feasible.

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.

Fructose augments infection-impaired net hepatic glucose uptake during TPN administration.

During chronic total parenteral nutrition (TPN), net hepatic glucose uptake (NHGU) and net hepatic lactate release (NHLR) are markedly reduced (downward arrow approximately 45 and approximately 65%, respectively) with infection. Because small quantities of fructose are known to augment hepatic glucose uptake and lactate release in normal fasted animals, the aim of this work was to determine whether acute fructose infusion with TPN could correct the impairments in NHGU and NHLR during infection. Chronically catheterized conscious dogs received TPN for 5 days via the inferior vena cava at a rate designed to match daily basal energy requirements. On the third day of TPN administration, a sterile (SHAM, n = 12) or Escherichia coli-containing (INF, n = 11) fibrin clot was implanted in the peritoneal cavity. Forty-two hours later, somatostatin was infused with intraportal replacement of insulin (12 +/- 2 vs. 24 +/- 2 microU/ml, SHAM vs. INF, respectively) and glucagon (24 +/- 4 vs. 92 +/- 5 pg/ml) to match concentrations previously observed in sham and infected animals. After a 120-min basal period, animals received either saline (Sham+S, n = 6; Inf+S, n = 6) or intraportal fructose (0.7 mg x kg(-1) x min(-1); Sham+F, n = 6; Inf+F, n = 5) infusion for 180 min. Isoglycemia of 120 mg/dl was maintained with a variable glucose infusion. Combined tracer and arteriovenous difference techniques were used to assess hepatic glucose metabolism. Acute fructose infusion with TPN augmented NHGU by 2.9 +/- 0.4 and 2.5 +/- 0.3 mg x kg(-1) x min(-1) in Sham+F and Inf+F, respectively. The majority of liver glucose uptake was stored as glycogen, and NHLR did not increase substantially. Therefore, despite an infection-induced impairment in NHGU and different hormonal environments, small amounts of fructose enhanced NHGU similarly in sham and infected animals. Glycogen storage, not lactate release, was the preferential fate of the fructose-induced increase in hepatic glucose disposal in animals adapted to TPN.

Impact of enteral and parenteral nutrition on hepatic and muscle glucose metabolism.

Liver and muscle metabolism were assessed in dogs adapted to long-term total parenteral (TPN) and enteral (TEN) nutrition. Studies were done in 13 conscious long-term catheterized dogs in which sampling (artery, portal and hepatic vein, and iliac vein), infusion catheters (inferior vena cava, duodenum), and transonic flow probes (hepatic artery, portal vein, and iliac artery) were implanted. Fourteen days after surgery dogs were grouped to receive TPN or TEN. After 5 days of TPN/TEN, substrate balances across the liver and limb were assessed. The liver was a marked net consumer of glucose in both groups (23.6 +/- 3.3 vs 22.6 +/- 2.8 micromol x kg(-1) x min(-1), TPN vs TEN) despite near normoglycemia (6.5 +/- 0.3 vs 6.7 +/- 0.2 mmol/L). Arterial insulin levels were higher during TEN (96 +/- 6 vs 144 +/- 30 pmol/L; p < .05). The majority (79 +/- 13 vs 76% +/- 7%) of the glucose taken up by the liver was released as lactate. Despite higher insulin levels during TEN the nonsplanchnic tissues consumed a lessor quantity of glucose (25.9 +/- 3.3 vs 16.1 +/- 3.9 micro x mol x kg(-1) x min(-1)). In summary, the liver undergoes a profound adaptation to TPN and TEN making it a major site of glucose uptake and conversion to lactate irrespective of the route of nutrient delivery. However, the insulin requirements are higher with TEN possibly secondary to impaired peripheral glucose removal.

Regulation of fertilization-induced Ca(2+)spiking in the mouse zygote.

Fertilization-induced Ca(2+)spiking in mouse zygotes ceases at the end of pre-G1 as pronuclei (PN) form. In the present studies we found that there was no consistent temporal relationship between PN formation and cessation of spiking. We also show that nucleate and anucleate fragments of zygotes, obtained by bisection of fertilized eggs prior to PN formation, both ceased spiking at times that did not depend on the presence of the PN. We, therefore, concluded that formation of the PN does not cause spiking cessation. The possibility that cessation of the fertilization-induced Ca(2+)spiking may be mediated by a redox sensitive mechanism affecting the sensitivity of Ca(2+)release from internal stores is proposed. At first mitosis, a small proportion of zygotes show low amplitude calcium spikes prior to pronuclear envelope breakdown (PNEBD), whereas all zygotes spiked at this time in the presence of high extracellular Ca(2+)and dithiothreitol. Nucleated zygotic fragments also spiked before PNEBD whereas anucleated ones rarely did. Exit from G2 was required for this spiking to be observed in nucleated zygotes or fragments. Arrest in M-phase resulted in the appearance of a prolonged series of small amplitude spikes. It is concluded that the spiking at mitosis is cell cycle regulated and may differ qualitatively in its control from that at fertilization.

Hepatic glucose metabolism during intraduodenal glucose infusion: impact of infection.

We previously reported that infection decreases hepatic glucose uptake when glucose is given as a constant peripheral glucose infusion (8 mg. kg(-1) x min(-1)). This impairment persisted despite greater hyperinsulinemia in the infected group. In a normal setting, hepatic glucose uptake can be further enhanced if glucose is given gastrointestinally. Thus the aim of this study was to determine whether hepatic glucose uptake is impaired during an infection when glucose is given gastrointestinally. Thirty-six hours before study, a sham (SH, n = 7) or Escherichia coli-containing (2 x 10(9) organisms/kg; INF; n = 7) fibrin clot was placed in the peritoneal cavity of chronically catheterized dogs. After the 36 h, a glucose bolus (150 mg/kg) followed by a continuous infusion (8 mg. kg(-1). min(-1)) of glucose was given intraduodenally to conscious dogs for 240 min. Tracer ([3-(3)H]glucose and [U-(14)C]glucose) and arterial-venous difference techniques were used to assess hepatic and intestinal glucose metabolism. Infection increased hepatic blood flow (35 +/- 5 vs. 47+/-3 ml x g(-1) x min(-1); SH vs. INF) and basal glucose rate of appearance (2.1+/-0.2 vs. 3.3+/-0.1 mg x kg(-1) x min(-1)). Arterial insulin concentrations increased similarly in SH and INF during the last hour of glucose infusion (38+/-8 vs. 46+/-20 microU/ml), and arterial glucagon concentrations fell (62+/-14 to 30+/-3 vs. 624+/-191 to 208+/-97 pg/ml). Net intestinal glucose absorption was decreased in INF, attenuating the increase in blood glucose caused by the glucose load. Despite this, net hepatic glucose uptake (1.6+/-0.8 vs. 2.4+/- 0.9 mg x kg(-1) x min(-1); SH vs. INF) and consequently tracer-determined glycogen synthesis (1.3+/-0.3 vs. 1.0+/-0.3 mg. kg(-1) x min(-1)) were similar between groups. In summary, infection impairs net glucose absorption, but not net hepatic glucose uptake or glycogen deposition, when glucose is given intraduodenally.

Hyperinsulinemia compensates for infection-induced impairment in net hepatic glucose uptake during TPN.

In animals receiving total parenteral nutrition (TPN), infection impairs net hepatic glucose uptake (NHGU) by 40% and induces mild hyperinsulinemia. In the normal animal, the majority of the glucose taken up by the liver is diverted to lactate, but in the infected state, lactate release is curtailed. Because of the hyperinsulinemia and reduced NHGU, more glucose is utilized by peripheral tissues. Our aims were to determine the role of infection-induced hyperinsulinemia in 1) limiting the fall in NHGU and hepatic lactate release and 2) increasing the proportion of glucose disposed of by peripheral tissues. Chronically catheterized dogs received TPN for 5 days via the inferior vena cava. On day 3, a fibrin clot with a nonlethal dose of E. coli was placed into the peritoneal cavity; sham dogs received a sterile clot. On day 5, somatostatin was infused to prevent endogenous pancreatic hormone secretion, and insulin and glucagon were replaced at rates matching incoming hormone concentrations observed previously in sham or infected dogs. The TPN-derived glucose infusion was adjusted to maintain a constant arterial plasma glucose level of approximately 120 mg/dl. after a basal blood sampling period, the insulin infusion rate was either maintained constant (infected time control, Hi-Ins, n = 6; sham time control, Sham, n = 6) or decreased (infected + reduced insulin, Lo-Ins; n = 6) for 180 min to levels seen in noninfected dogs (from 23 +/- 2 to 12 +/- 1 microU/ml). Reduction of insulin to noninfected levels decreased NHGU by 1.4 +/- 0.5 mg x kg(-1) x min(-1) (P < 0.05) and nonhepatic glucose utilization by 4.8 +/- 0.8 mg x kg(-1) x min(-1) (P < 0.01). The fall in NHGU was caused by a decline in HGU (Delta-0.6 +/- 0.4 mg x kg(-1) x min(-1)) and a concomitant increase in hepatic glucose production (HGP, Delta0.8 +/- 0.5 mg x kg(-1) x min(-1)); net hepatic lactate release was not altered. Hyperinsulinemia that accompanies infection 1) primarily diverts glucose carbon to peripheral tissues, 2) limits the fall in NHGU by enhancing HGU and suppressing HGP, and 3) does not enhance hepatic lactate release, thus favoring hepatic glucose storage. Compensatory hyperinsulinemia plays a critical role in facilitating hepatic and peripheral glucose disposal during an infection.

Impact of acute epinephrine removal on hepatic glucose metabolism during stress hormone infusion.

We examined the effect of acute discontinuation of an epinephrine (EPI) infusion on hepatic glucose metabolism during stress hormone infusion (SHI). Glucose metabolism was assessed in 11 conscious, 20-hour fasted dogs using tracer and arteriovenous techniques after a 3-day exposure to SHI. SHI increased EPI, norepinephrine, cortisol, and glucagon levels (approximately sixfold to 10-fold), which led to marked hyperglycemia, hyperinsulinemia, and accelerated glucose metabolism. On day 3, EPI infusion was acutely discontinued for 180 minutes in five dogs while infusion of the other hormones was continued (SHI - EPI). In the remaining six dogs, all hormones were continued for the duration of the study (SHI + EPI). In SHI - EPI, EPI levels decreased from 1,678+/-191 to 161+/-47 pg/mL. Isoglycemia (183+/-10 to 185+/-15 mg/dL) was maintained with an exogenous glucose infusion. Arterial insulin levels increased from 41+/-8 to 64+/-8 microU/mL. Whole-body glucose utilization increased from 3.5+/-0.5 to 9.4+/-1.9 mg/kg/min. Nonesterified fatty acids ([NEFAs] 763+/-292 to 147+/-32 micromol/L) decreased. Net hepatic glucose output decreased (2.6+/-0.6 to 0.1+/-0.3 mg/kg/min). In SHI + EPI, hepatic glucose metabolism remained unaltered. In summary, EPI plays a pivotal role during SHI by stimulating glucose production and inhibiting glucose utilization. In part, these effects are mediated by restraining pancreatic insulin secretion.

Hepatic production and intestinal uptake of IGF-I: response to infection.

The role of the liver and gut in contributing to the infection-induced fall in circulating insulin-like growth factor I (IGF-I) was examined in chronically catheterized conscious dogs. Two weeks before study, catheters and Doppler flow probes were implanted to assess hepatic and gut balance of IGF-I. To control nutrient intake, dogs were placed on total parenteral nutrition (TPN) as their sole caloric source. After dogs received TPN for 5 days, net hepatic and intestine IGF-I balances were assessed. A hypermetabolic infected state was then induced by the intraperitoneal implantation of a fibrin clot containing Escherichia coli. TPN was continued, and organ IGF-I balance was assessed 24 and 48 h after induction of infection. Arterial IGF-I levels were significantly decreased following infection (111 +/- 18, 62 +/- 10, and 63 +/- 8 ng/ml before and 24 and 48 h after, respectively). Net hepatic IGF-I output decreased markedly (221 +/- 73, to 73 +/- 41 and 41 +/- 17 ng. kg-1. min-1 before and 24 and 48 h after, respectively). The infection-induced decrease in hepatic IGF-I output could not be explained by concomitant alterations in plasma cortisol or insulin levels. The gut demonstrated a net uptake of IGF-I before infection (178 +/- 29 ng. kg-1. min-1). However, after infection, intestinal IGF-I uptake was completely suppressed (-10 +/- 15 and -8 +/- 36 ng. kg-1. min-1). In summary, infection decreases net hepatic IGF-I release 65-80% and completely suppresses net IGF-I uptake by the intestine. As a consequence of these reciprocal changes in IGF-I balance across the liver and intestine, splanchnic production of IGF-I was unchanged by infection. These data suggest that changes in the clearance and/or production of IGF-I by extrasplanchnic tissues contribute to the infection-induced decrease in circulating IGF-I levels.

Hepatic and muscle glucose metabolism during total parenteral nutrition: impact of infection.

We examined the impact of infection on hepatic and muscle glucose metabolism in dogs adapted to chronic total parenteral nutrition (TPN). Studies were done in five conscious chronically catheterized dogs, in which sampling (artery, portal and hepatic vein, and iliac vein), infusion catheters (inferior vena cava), and Transonic flow probes (hepatic artery, portal vein, and iliac artery) were implanted. Fourteen days after surgery, dogs were placed on TPN. After 5 days of TPN, an infection was induced, and the TPN was continued. The balance of substrates across the liver and limb was assessed on the day before infection (day 0) and 18 (day 1) and 42 h (day 2) after infection. On day 0, the liver was a marked net consumer of glucose (4.3 +/- 0.6 mg. kg-1. min-1) despite near normoglycemia (117 +/- 5 mg/dl) and only mild hyperinsulinemia (16 +/- 2 microU/ml). In addition, the majority (79 +/- 13%) of the glucose taken up by the liver was released as lactate (34 +/- 6 micromol. kg-1. min-1). After infection, net hepatic glucose uptake decreased markedly on day 1 (1.6 +/- 0.9 mg. kg-1. min-1) and remained suppressed on day 2 (2.4 +/- 0.5 mg. kg-1. min-1). Net hepatic lactate output also decreased on days 1 and 2 (15 +/- 5 and 12 +/- 3 micromol. kg-1. min-1, respectively). This occurred despite increases in arterial plasma glucose on days 1 and 2 (135 +/- 9 and 144 +/- 9 mg/dl, respectively) and insulin levels on days 1 and 2 (57 +/- 14 and 34 +/- 9 microU/ml, respectively). In summary, the liver undergoes a profound adaptation to TPN, making it a major site of glucose disposal and conversion to lactate. Infection impairs hepatic glucose uptake, forcing TPN-derived glucose to be removed by peripheral tissues.

Regulation of glucose production by NEFA and gluconeogenic precursors during chronic glucagon infusion.

We previously reported that simulation of the chronic hyperglucagonemia seen during infection was unable to recreate the infection-induced increase in hepatic glucose production. However, chronic hyperglucagonemia was accompanied by a fall in the arterial levels of gluconeogenic precursors as opposed to a rise as is seen during infection. Thus our aim was to determine whether an infusion of gluconeogenic precursors could increase hepatic glucose production in a setting of hyperglucagonemia. Studies were done in 11 conscious chronically catheterized dogs in which sampling (artery and portal and hepatic veins) and infusion catheters (splenic vein) were implanted 17 days before study. Forty-eight hours before infusion of gluconeogenic (GNG) precursors, a sterile fibrinogen clot was placed into the peritoneal cavity. Glucagon was infused over the subsequent 48-h period to simulate the increased glucagon levels (approximately 500 pg/ml) seen during infection. On the day of the experiment, somatostatin was infused peripherally, and basal insulin and simulated glucagon were infused intraportally. After a basal period, a two-step increase in lactate and alanine was initiated (120 min/step; n = 5). Lactate (Delta479 +/- 25 and Delta1, 780 +/- 85 microM; expressed as change from basal in periods I and II, respectively) and alanine (Delta94 +/- 13 and Delta287 +/- 44 microM) levels were increased. Despite increases in net hepatic GNG precursor uptake (Delta0.7 +/- 0.3 and Delta1.1 +/- 0.4 mg glucose . kg-1 . min-1), net hepatic glucose output did not increase. Because nonesterified fatty acid (NEFA) levels fell, in a second series of studies, the fall in NEFA was eliminated. Intralipid and heparin were infused during the two-step substrate infusion to maintain the NEFA levels constant in period I and increase NEFA availability in period II (Delta -29 +/- 29 and Delta689 +/- 186 microM; n = 6). In the presence of similar increases in net hepatic GNG precursor uptake and despite increases in arterial glucose levels (Delta17 +/- 5 and Delta38 +/- 12 mg/dl), net hepatic glucose output increased (Delta0.6 +/- 0.1 and Delta0.7 +/- 0.2 mg . kg-1 . min-1). In summary, a chronic increase in glucagon, when combined with an acute increase in gluconeogenic precursor and maintenance of NEFA supply, increases hepatic glucose output as is seen during infection.

Alterations in hepatic gluconeogenic amino acid uptake and gluconeogenesis in the endotoxin treated conscious dog.

We examined the effect of a 240 min intraportal infusion of a nonlethal dose of Escherichia coli endotoxin (.21 g x kg(-1) x min[-1]) on hepatic amino acid and glucose metabolism in chronically catheterized 42 h fasted conscious dogs (n = 8). Hepatic metabolism was assessed using tracer (3-[3H]glucose [U-14C]alanine) and arteriovenous difference techniques. After endotoxin administration net hepatic glucose output increased twofold. Arterial plasma insulin levels decreased by 25%, whereas arterial plasma glucagon and cortisol levels increased 10- and 6-fold, respectively. Arterial lactate levels increased 6.4-fold, whereas net hepatic lactate uptake was not increased. Arterial alanine levels (1.6-fold) and net hepatic alanine uptake (1.3-fold) increased, whereas net hepatic alanine fractional extraction was unaltered. In contrast, the arterial levels of the other gluconeogenic amino acids (glutamine, glycine, serine, and threonine) decreased. Despite this decrease, net uptake of these amino acids by the liver did not decrease, because net hepatic amino acid fractional extraction increased. Total net hepatic gluconeogenic precursor uptake was unaltered (1.1 +/- .1 to 1.3 +/- .3 mg x kg(-1) x min(-1) expressed in glucose equivalents). In summary, gluconeogenesis does not increase after endotoxin administration. Thus, an increase in net hepatic glycogenolysis accounts for the majority of the increase in hepatic glucose production. The lack of an increase in alanine fractional extraction, despite hyperglucagonemia and a rise in the fractional extraction of other gluconeogenic amino acids, suggests that endotoxin specifically impairs hepatic alanine entry in vivo.

Role of epinephrine and norepinephrine in the metabolic response to stress hormone infusion in the conscious dog.

The role of epinephrine and norepinephrine in contributing to the alterations in hepatic glucose metabolism during a 70-h stress hormone infusion (SHI) was investigated in four groups of chronically catheterized (20-h-fasted) conscious dogs. SHI increased glucagon (approximately 5-fold), epinephrine (approximately 10-fold), norepinephrine (approximately 10-fold), and cortisol (approximately 6-fold) levels. Dogs received either all the hormones (SHI; n = 5), all the hormones except epinephrine (SHI-Epi; n = 6), or all the hormones except norepinephrine (SHI-NE; n = 6). In addition, six dogs received saline only (Sal). Glucose production (Ra) and gluconeogenesis were assessed after a 70-h hormone or saline infusion with the use of tracer ([3-(3)H]glucose and [U-(14)C]alanine) and arteriovenous difference techniques. SHI increased glucose levels (108 +/- 2 vs. 189 +/- 10 mg/dl) and Ra (2.6 +/- 0.2 vs. 4.1 +/- 0.3 mg x kg(-1) x min(-1)) compared with Sal. The absence of an increase in epinephrine markedly attenuated these changes (glucose and Ra were 140 +/- 6 mg/dl and 2.7 +/- 0.4 mg x kg(-1) x min(-1), respectively). Only 25% of the blunted rise in Ra could be accounted for by an attenuation of the rise in net hepatic gluconeogenic precursor uptake (0.9 +/- 0.1, 1.5 +/- 0.1, and 1.1 +/- 0.2 mg x kg(-1) x min(-1) for Sal, SHI, and SHI-Epi, respectively). The absence of an increase in norepinephrine did not blunt the rise in arterial glucose levels, Ra, or net hepatic gluconeogenic precursor uptake (they rose to 195 +/- 21 mg/dl, 3.7 +/- 0.5 mg x kg(-1) x min(-1), and 1.7 +/- 0.2 mg x kg(-1) min(-1), respectively). In summary, during chronic SHI, the rise in epinephrine exerts potent stimulatory effects on glucose production principally by enhancing hepatic glycogenolysis, although the rise in circulating norepinephrine has minimal effects.

Assessment of insulin action on glucose uptake and production during a euglycemic-hyperinsulinemic clamp in dog: a new kinetic analysis.

We evaluated the validity of the traditional method of assessment of the speed of insulin action during a euglycemic-hyperinsulinemic clamp. We first estimated the error of Steele's model on glucose uptake in these experimental conditions. We tested the appropriateness of estimating the half-time of insulin action by expressing the glucose flux changes as a percent of the maximal change (normalization on a 0% to 100% scale). For this purpose, we performed a 390-minute euglycemic-hyperinsulinemic (2 mU.min-1.kg-1) clamp in five chronically catheterized conscious dogs. We used [3-3H]glucose to assess glucose kinetics. We used a novel analysis based on a circulatory model, which allowed us to overcome the limitations of compartmental analysis. We found that the primary effect of insulin (increased from 12.3 +/- 1.6 to 104 +/- 15 microU/mL) was to increase the whole-body fractional extraction of glucose (3.0% +/- 0.3% to 18% +/- 2%). Insulin did not alter the mean whole-body artery-vein transit time (3.1 +/- 0.2 v 2.9 +/- 0.4 minutes). In contrast to the assumptions of the Steele model, which assumes that glucose uptake and rate of appearance (Ra) are equal during the clamp, during the initial 30 minutes of the clamp the increase in glucose uptake preceded (by approximately 4 minutes) the increase in Ra. Thus, during this period uptake exceeded Ra by about 15%. The maximal difference between Ra and uptake (1 to 1.5 mg.min-1.kg-1) occurred approximately 15 minutes after insulin infusion. Finally, to estimate the half-time of the insulin signal that controls glucose uptake and production, we accounted for the nonlinear relationship between insulin concentration and glucose uptake and production. We found that the traditional normalization of the glucose flux changes on a 0% to 100% scale underestimated the half-time of onset of the insulin signal that controls glucose uptake (half-time, 20 v 54 minutes) and glucose production (half-time, 25 v 39 minutes). Accounting for the nonlinearity of the dose-response curves may thus be of crucial importance in the evaluation of the onset and offset of insulin action.

A direct measurement of increased divalent cation influx in fertilised mouse oocytes.

On fertilisation of mouse oocytes, the fusing spermatozoon triggers a series of repetitive calcium (Ca2+) spikes. The Ca2+ spikes seem to be necessary for successful progression through the cell cycle and are regulated in a cell-cycle-dependent manner. The spikes appear to require the linkage of continuous Ca2+ influx to the periodic release of Ca2+ from intracellular stores by a process of Ca(2+)-induced Ca2+ release. The precise role of Ca2+ influx was explored using the manganese (Mn2+)-quench technique to monitor unidirectional cation influx into single mouse oocytes. There was a marked stimulation of cation influx associated closely with the upsweep of the first and subsequent fertilisation Ca2+ spikes. A smaller but significant increase in the rate of cation influx persisted in the interspike period in fertilised oocytes. Spike-associated entry was not as apparent in oocytes stimulated to spike repetitively by thimerosal or acetylcholine application. Instead, there was a continuous increase in cation influx underlying Ca2+ spiking which commenced with the onset of the first spike. Using the specific microsomal inhibitor thapsigargin and the Ca2+ ionophore ionomycin, we found evidence for a capacitative entry mechanism in mouse oocytes. We propose that the persistent influx of Ca2+ observed in response to all stimuli examined is controlled by a capacitative mechanism and sets the frequency of spiking by determining the time taken to refill the internal stores to a point where they are again sensitive enough to initiate the next spike.

Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+ spiking.

This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLC beta 1, PLC beta 3 and PLC gamma isoenzymes. Furthermore, immunodetection of PLC gamma 1 using monoclonal antibodies reveals that PLC gamma 1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLC beta protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 microM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 microM), herbimycin (10 microM) and geldanamycin (0.6 microM) which could affect signalling through PLC gamma hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.

Hepatic release of tumor necrosis factor in the endotoxin-treated conscious dog.

The effects of a 4 h intraportal infusion of Escherichia coli lipopolysaccharide (LPS, .21 mu g/kg/min) on the release of tumor necrosis factor (TNF) by hepatic and nonhepatic splanchnic tissues was assessed in the chronically catheterized conscious dog (n = 7) using arteriovenous difference techniques. TNF levels were measured using both a WEHI-164 cytotoxicity assay (WEHI) and a h-TNF-alpha EIA kit (ELISA; Biosource, Camarillo, CA). Using WEHI, arterial TNF levels increased from 10 + or - 6 pg/mL to a peak of 4667 + or - 1442 pg/mL 100 min after LPS and fell to 443 + or - 199 pg/mL by 240 min. Using ELISA, arterial TNF levels increased from 5 + or - 5 pg/mL to a peak of 12,234 + or - 2046 pg/mL at 100 min and fell to 3511 + or - 991 pg/mL by 240 min. WEHI could not be used to assess organ TNF release due to excessive assay variability. Based upon ELISA, net hepatic TNF output increased from undetectable release at basal to 23.0 + or - 10.7 ng/kg/min at 60 min and returned toward basal by 240 min (4.7 + or - 3.8 ng/kg/min). Net release of TNF by the nonhepatic splanchnic bed was not observed. One compartment analysis of the arterial TNF response indicated that net release of TNF by the liver accounted for the majority of the increase in the arterial TNF levels. In summary, after intraportal LPS infusion, it was determined that 1) both assays predict similar qualitative TNF response, while the quantitative response differs, 2) the liver is the major site of TNF production, and 3) the nonhepatic splanchnic bed is not a net producer of TNF.

Juvenile Krabbe's Leukodystrophy precipitated by influenza A infection.

Juvenile Krabbe's disease, a neurometabolic disorder, presented as an acute demyelinating episode characterised by an abnormal gait. The neurological presentation was preceded by influenza A infection.

Role of cortisol in the metabolic response to stress hormone infusion in the conscious dog.

The role of cortisol in directing the metabolic response to a combined infusion of glucagon, epinephrine, norepinephrine, and cortisol (stress hormones) was investigated. Chronically catheterized, conscious fasted dogs were studied before hormone infusion and after a 70-hour stress hormone infusion containing glucagon, epinephrine, norepinephrine, and cortisol (n = 11) or containing all these hormones except cortisol (n = 5). Combined stress hormone infusion increased arterial plasma glucagon, cortisol, epinephrine, and norepinephrine approximately sixfold. Whole-body glucose production (Ra), glycogenolysis, and gluconeogenesis were assessed using tracer and arteriovenous-difference techniques. The absence of an increase in cortisol during stress hormone infusion attenuated the increase in arterial plasma glucose concentration and Ra (delta 81 +/- 16 v 24 +/- 3 mg/dL and 1.7 +/- 0.3 v 0.8 +/- 0.4 mg/ kg/min, respectively). However, it did not alter the increase in net hepatic glucose output (delta 0.7 +/- 0.3 v 0.8 +/- 0.4 mg/kg/min). When the increase in cortisol was absent, the increase in net hepatic gluconeogenic precursor uptake was attenuated (delta 0.7 +/- 0.3 v 0.1 +/- 0.3 mg glucose/kg/min) due to a decrease in gluconeogenic precursor levels. The efficiency of gluconeogenesis increased to a greater extent (delta 0.19 +/- 0.07 v 0.31 +/- 0.11) when cortisol was not infused. The absence of an increase in cortisol also led to marked glycogen depletion in the liver (10 +/- 4 v 55 +/- 10 mg/g liver). Cortisol thus plays a pivotal role in the metabolic response to stress hormone infusion by sustaining gluconeogenesis through a stimulatory effect on hepatic gluconeogenic precursor supply and by maintaining hepatic glycogen availability.

Hyperglucagonemia and hepatic glucose metabolism during infection in the conscious dog.

The chronic and acute roles of hyperglucagonemia in sustaining the increased glucose production observed in the conscious infected dog were examined. Three groups of dogs were studied: a sham group (SHAM; n = 10), an infected group (INFXN; n = 11), and a sham group in which the chronic (42-h) increase in glucagon observed in INFXN was simulated (SimGGN; n = 5). INFXN and SimGGN were studied in the presence of hyperglucagonemia. In addition, glucagon was selectively decreased for 180 min in INFXN by use of somatostatin with basal intraportal insulin replacement and in SimGGN by discontinuing the exogenous glucagon infusion. Tracer and arteriovenous difference techniques were used to assess hepatic glucose metabolism and gluconeogenesis. Whereas the rate of glucose appearance (Ra) was increased by 30% (3.3 +/- 0.1 vs. 2.5 +/- 0.1 mg.kg-1.min-1) in INFXN vs. SHAM, Ra did not increase in SimGGN (2.4 +/- 0.2 mg.kg-1.min-1). In addition, the 30% increase in net hepatic gluconeogenic precursor uptake seen in INFXN did not occur in SimGGN despite an augmented net hepatic alanine fractional extraction (0.62 +/- 0.03 vs. 0.47 +/- 0.05, SimGGN vs. INFXN). With acute removal of hyperglucagonemia, endogenous Ra decreased in SimGGN and INFXN by 1.0 +/- 0.2 and 1.4 +/- 0.3 mg.kg-1.min-1, respectively. Net hepatic alanine fractional extraction in INFXN, leading to a greater rise in arterial blood alanine levels. In summary, chronic hyperglucagonemia alone cannot explain the increase in Ra observed during an infection. The marked hyperglucagonemia seen during infection plays an essential role in sustaining normal net hepatic fractional alanine extraction to compensate for an impairment in glucagon-stimulated hepatic amino acid transport activation.