Phillip E. Williams

Role of Brain in Counterregulation of Insulin-Induced Hypoglycemia in Dogs

The role of the brain in directing counterregulation during hypoglycemia induced by insulin infusion was assessed in overnight-fasted conscious dogs. Concomitant brain and peripheral hypoglycemia was induced in one group of dogs (n = 5) by infusing insulin peripherally at a rate of 3.5 mU · kg−1 · min−1. In another group (n = 4), insulin was infused as described above to induce peripheral hypoglycemia, and brain hypoglycemia was minimized by infusing glucose bilaterally into the carotid and vertebral arteries to maintain the brain glucose level at a calculated concentration of 85 mg/dl. Glucose was also infused peripherally as needed so that the peripheral glucose levels in both of the protocols were similar (45 ± 2 mg/dl with and 48 ± 3 mg/dl without brain glucose infusion, both P < .05). The responses (in terms of change of area under the curve) of epinephrine, norepinephrine, cortisol, and pancreatic polypeptide when brain glycemia was controlled during insulin infusion were only 14 ± 6, 39 ± 12, 17 ± 8, and 9 ± 4%, respectively, of those present during insulin infusion without concomitant brain glucose infusion (all P < .05). Of particular interest was the glucagon response that occurred when head hypoglycemia was minimized; the glucagon level was only 21 ± 8% of that present when marked brain hypoglycemia accompanied insulin infusion (P < .05). During hypoglycemia resulting from insulin infusion, endogenous glucose production (EGP), as assessed with [3-3H]glucose, rose from 2.6 ± 0.1 to 4.4 ± 0.5 mg · kg−1 min−1 (P < .05). In contrast, EGP decreased from 2.7 ± 0.2 to 2.0 ± 0.3 mg · kg−1 · min−1 When brain hypoglycemia was minimized. In an additional set of studies, when insulin was infused at 3.5 mU · kg−1 · min−1 and glucose was infused peripherally to maintain both the head and peripheral glucose concentrations at 88 ± 6 mg/dl, EGP decreased from 2.6 ± 0.1 to 1.2 ± 0.2 mg · kg−1 · min−1. These results suggest that under marked hyperinsulinemic conditions the brain is the primary director of glucagon release and that it is responsible for ∼ 75% of the life-sustaining glucose production.

Glucose disposal during insulinopenia in somatostatin-treated dogs. The roles of glucose and glucagon.

The first aim of this study was to determine whether the plasma glucose level can regulate hepatic glucose balance in vivo independent of its effects on insulin and glucagon secretion. To accomplish this, glucose was infused into conscious dogs whose basal insulin and glucagon secretion had been replaced by exogenous intraportal insulin and glucagon infusion after somatostatin inhibition of endogenous pancreatic hormone release. The acute induction of hyperglycemia (mean increment of 121 mg/dl) in the presence of basal levels of insulin (7+/-1 muU/ml) and glucagon (76+/-3 pg/ml) resulted in a 56% decrease in net hepatic glucose production but did not cause net hepatic glucose uptake. The second aim of the study was to determine whether a decrease in the plasma glucagon level would modify the effect of glucose on the liver. The above protocol was repeated with the exception that glucagon was withdrawn (83% decrease in plasma glucagon) coincident with the induction of hyperglycemia. Under this circumstance, with the insulin level basal (7+/-1 muU/ml) and the glucagon levels reduced (16+/-2 pg/ml), hyperglycemia (mean increment of 130 mg/dl) promoted marked net hepatic glucose uptake (1.5+/-0.2 mg/kg per min) and glycogen deposition. In conclusion, (a) physiological increments in the plasma glucose concentration, independent of their effects on insulin and glucagon secretion, can significantly reduce net hepatic glucose production in vivo but at levels as high as 230 mg/dl cannot induce net hepatic glucose storage and (b) in the presence of basal insulin the ability of hyperglycemia to stimulate net hepatic glucose storage is influenced by the plasma glucagon concentration.

Role of Gluconeogenesis in Sustaining Glucose Production During Hypoglycemia Caused by Continuous Insulin Infusion in Conscious Dogs

The roles of glycogenolysis and gluconeogenesis in sustaining glucose production during insulin-induced hypoglycemia were assessed in overnight-fasted conscious dogs. Insulin was infused intraportally for 3 h at 5 mU · kg−1 · min−1 in five animals, and glycogenolysis and gluconeogenesis were measured by using a combination of tracer [(3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous difference techniques. In response to the elevated insulin level (263 ± 39 μU/ml), plasma glucose level fell (41 ± 3 mg/dl), and levels of the counterregulatory hormones glucagon, epinephrine, norepinephrine, and cortisol increased (91 ± 29 to 271 ± 55 pg/ml, 83 ± 26 to 2356 ± 632 pg/ml, 128 ± 31 to 596 ± 81 pg/ml, and 1.5 ± 0.4 to 11.1 ± 1.0 μg/dl, respectively; for all, P < .05). Glucose production fell initially and then doubled (3.1 ± 0.3 to 6.1 ± 0.5 mg · kg−1 · min−1; P < .05) by 60 min. Net hepatic gluconeogenic precursor uptake increased ∼ eightfold by the end of the hypoglycemic period. By the same time, the efficiency with which the liver converted the gluconeogenic precursors to glucose rose twofold. Five control experiments in which euglycemia was maintained by glucose infusion during insulin administration (5.0 mU · kg−1 · min−1) provided baseline data. Glycogenolysis accounted for 69–88% of glucose production during the 1st h of hypoglycemia, whereas gluconeogenesis accounted for 48–88% of glucose production during the 3rd h of hypoglycemia. These data suggest that gluconeogenesis is the key process for the normal counterregulatory response to prolonged and marked hypoglycemia.

Bile diversion to the distal small intestine has comparable metabolic benefits to bariatric surgery

Roux-en-Y gastric bypass (RYGB) is highly effective in reversing obesity and associated diabetes. Recent observations in humans suggest a contributing role of increased circulating bile acids in mediating such effects. Here we use a diet-induced obesity (DIO) mouse model and compare metabolic remission when bile flow is diverted through a gallbladder anastomosis to jejunum, ileum or duodenum (sham control). We find that only bile diversion to the ileum results in physiologic changes similar to RYGB, including sustained improvements in weight, glucose tolerance and hepatic steatosis despite differential effects on hepatic gene expression. Circulating free fatty acids and triglycerides decrease while bile acids increase, particularly conjugated tauro-β-muricholic acid, an FXR antagonist. Activity of the hepatic FXR/FGF15 signalling axis is reduced and associated with altered gut microbiota. Thus bile diversion, independent of surgical rearrangement of the gastrointestinal tract, imparts significant weight loss accompanied by improved glucose and lipid homeostasis that are hallmarks of RYGB.

The Relative Importance of First- and Second-Phase Insulin Secretion in Countering the Action of Glucagon on Glucose Turnover in the Conscious Dog

While it is known that glucagon induces a biphasic release of insulin when infused into a normal animal, it is not known whether the resultant pattern of insulin secretion has important metabolic consequences. To ascertain whether such is the case, glucagon was elevated fourfold in the presence of first-, second-, or combined first- and second-phase insulin release to determine ability of the latter hormone to antagonize the effect of glucagon on glucose turnover in the conscious dog. To separate the effects of the different phases of insulin release the “pancreatic clamp” technique, in which somatostatin is given to inhibit the endocrine pancreas and replacement amounts of insulin and glucagon are given intraportally, was used. In this way, a rise in glucagon (∼220 pg/ml) was brought about in the presence of simulated first-phase (peak IRI 25 μU/ml at 5 min; basal by 30 min), secondphase (peak IRI 19 μU/ml at 30 min and sustained elevation thereafter), or first- plus second-phase (peak IRI 33 μU/ml at 5 min; 17 μU/ml at 30 min and sustained elevation thereafter) insulin release. Optimal glycemic control required both first- and second-phase insulin release. A selective deficiency of first-phase release resulted in a transient (2 h) worsening of the glucagon-induced hyperglycemia (twofold the normal increment). This defect was attributable to a larger initial rise in glucose production (3.6 ± 0.6 mg/kg · min) than that observed when both phases of insulin release were present (0.9 ± 0.4 mg/kg · min). First-phase insulin release had no significant effect on glucose clearance. A selective deficiency of second-phase release resulted in marked (sixfold) and prolonged worsening of the glucagon-induced hyperglycemia. In this case, however, the hyperglycemia was primarily the result of a defect in glucose clearance. Glucose clearance fell by 29 ± 7% instead of rising by 30 ± 4% as it did when both firstand second-phase release were present. Glucose production was mildly elevated between 15 and 75 min when second-phase insulin release was deficient relative to that apparent when both the first- and secondphases of release were present; this also contributed to the abnormal hyperglycemia. We conclude that both phases of insulin release are vital to full counterregulation of the action of glucagon on glucose metabolism. First-phase insulin release is important to counter the quick effect of glucagon on glucose production, while second-phase insulin release is important to sustain that inhibition and to augment glucose utilization. Absence of first-phase release results in a transient (2 h) and moderate (20–30 mg/dl) glycemic defect while an absence of second-phase release results in a prolonged and dramatic (70–100 mg/dl) defect.

Effects of Insulin on the Metabolic Control of Hepatic Gluconeogenesis In Vivo

OBJECTIVE Insulin represses the expression of gluconeogenic genes at the mRNA level, but the hormone appears to have only weak inhibitory effects in vivo. The aims of this study were 1) to determine the maximal physiologic effect of insulin, 2) to determine the relative importance of its effects on gluconeogenic regulatory sites, and 3) to correlate those changes with alterations at the cellular level. RESEARCH DESIGN AND METHODS Conscious 60-h fasted canines were studied at three insulin levels (near basal, 4×, or 16×) during a 5-h euglycemic clamp. Pancreatic hormones were controlled using somatostatin with portal insulin and glucagon infusions. Glucose metabolism was assessed using the arteriovenous difference technique, and molecular signals were assessed. RESULTS Insulin reduced gluconeogenic flux to glucose-6-phosphate (G6P) but only at the near-maximal physiological level (16× basal). The effect was modest compared with its inhibitory effect on net hepatic glycogenolysis, occurred within 30 min, and was associated with a marked decrease in hepatic fat oxidation, increased liver fructose 2,6-bisphosphate level, and reductions in lactate, glycerol, and amino acid extraction. No further diminution in gluconeogenic flux to G6P occurred over the remaining 4.5 h of the study, despite a marked decrease in PEPCK content, suggesting poor control strength for this enzyme in gluconeogenic regulation in canines. CONCLUSIONS Gluconeogenic flux can be rapidly inhibited by high insulin levels in canines. Initially decreased hepatic lactate extraction is important, and later reduced gluconeogenic precursor availability plays a role. Changes in PEPCK appear to have little or no acute effect on gluconeogenic flux.

Acute lung injury after hepatic cryoablation: Correlation with NF-κB activation and cytokine production

Background: Previous clinical reports have documented multisystem organ injury after hepatic cryoablation. We hypothesized that hepatic cryosurgery, but not partial hepatectomy, induces a systemic inflammatory response characterized by distant organ injury and overproduction of nuclear factor κB (NF-κB)–dependent, proinflammatory cytokines. Methods: In this study, rats underwent either cryoablation of 35% of liver parenchyma or a similar resection of left hepatic tissue. Serum tumor necrosis factor-α and macrophage inflammatory protein-2 levels and NF-κB activation were assessed by electrophoretic mobility shift assay at 30 minutes 1, 2, 6, and 24 hours after either procedure. Results: Cryoablation of 35% of liver (n = 22 rats) resulted in lung injury and a 45% mortality rate within 24 hours of surgery, whereas 7% treated with 35% hepatectomy (n = 15 rats) died during the 24 hours after surgery (P < .05, cryoablation vs hepatectomy). Serum tumor necrosis factor-α and macrophage inflammatory protein-2 levels were markedly increased in rats (n = 10 rats) 1 hour after hepatic cryoablation compared with rats that underwent partial hepatectomy (P < .005). We evaluated NF-κB activation by electrophoretic mobility shift assay in nuclear extracts of liver and lung after cryosurgery and found that NF-κB activation was strikingly increased in the liver but not the lung at 30 minutes and in both organs 1 hour after cryosurgery, and returned to baseline in both organs by 2 hours. In rats undergoing 35% hepatectomy, no increase in NF-κB activation was detected in nuclear extracts of either liver or lung at any time point. Conclusions: These data show that hepatic cryosurgery results in systemic inflammation with activation of NF-κB and increased production of NF-κB–dependent cytokines. Our data suggest that lung injury and death in this animal model is mediated by an exaggerated inflammatory response to cryosurgery. (Surgery 1999:126:518-26.)

Exercise-Induced Fall in Insulin and Increase in Fat Metabolism During Prolonged Muscular Work

The role of the exercise-induced fall in insulin in fat metabolism was studied in dogs during 150 min of treadmill exercise alone (controls) or with insulin clamped at basal levels by an intraportal infusion to prevent the normal fall in insulin concentration (ICs). To counteract the suppressive effect of insulin on glucagon release, glucagon was supplemented by an intraportal infusion in ICs. In all dogs, catheters were placed in a carotid artery and in the portal and hepatic veins for sampling and in the vena cava and the splenic vein for infusion purposes. Glucose levels were clamped in ICs to recreate the glycemic response evident in controls. In controls, insulin fell by 7 ± 1 μxU/ml but was unchanged from basal levels in ICs (0 ± 2 μU/ml). Glucagon, norepinephrine, epinephrine, and cortisol rose similarly in controls and ICs. Arterial free-fatty acid (FFA) levels rose by 644 ± 126 μeq/L in controls but did not increase in ICs (−12 ± 148 μeq/L). Arterial glycerol levels rose by 337 ± 43 and 183 ± 19 (μM in controls and ICs. Hepatic FFA delivery and fractional extraction increased by 17 ± 3 and 0.06 ± 0.02 μmol · kg−1 · min−1, respectively, in controls. In ICs, hepatic FFA delivery increased by only 1 ± 2 (μmol · kg−1 · min−1, whereas hepatic fractional extraction fell slightly (−0.03 ± 0.03). Consequently, net hepatic FFA uptake rose by 4.8 ± 1.5 μxmol · kg−1 · min−1 in controls but decreased slightly in ICs (−0.5 ± 1.1 μmol · kg−1 · min−1). At least partly because of the differences in hepatic FFA uptake, arterial β-hydroxybutyrate (50 ± 14 vs. 23 ± 8 μM) and net hepatic β-hydroxybutyrate output (2.2 ± 0.7 vs. 0.4 ± 0.3 μmol · kg−1 · min−1) rose more in controls than in ICs. In summary, the exercise-induced fall in insulin 7) is essential to the increase in FFA levels during prolonged muscular work, 2) facilitates hepatic FFA uptake by enhancing the delivery of FFAs to the liver and their extraction by the liver, and 3) enhances the net β-hydroxybutyrate output by the liver, at least in part through the effects described in 1 and 2. Hence, the exercise-induced fall in insulin is essential for the transition to the increased rate of FFA metabolism that occurs as work duration progresses.

Effects of low- and high-intensity exercise on plasma and cerebrospinal fluid levels of ir-β-endorphin, ACTH, cortisol, norepinephrine and glucose in the conscious dog

This study was designed to assess effects of exercise on plasma and cerebrospinal fluid (CSF) levels of immunoreactive (ir) beta-endorphin, ACTH, cortisol, norepinephrine, and glucose in the conscious dog. Dogs were exercised on a treadmill at low or high intensity (4.2 miles/h and a 6% or 20% incline) for 90 min, and were allowed to recover for 90 additional min. Neither intensity of exercise changed plasma glucose levels, but dose-related changes in glucose kinetics did occur. CSF glucose declined in both groups. During low intensity exercise, plasma levels of ir-beta-endorphin, ACTH, and cortisol increased with duration of exercise. During high intensity exercise, ACTH, ir-beta-endorphin and cortisol increased faster, and the integrated plasma response of these hormones was greater. Thus, peripheral release of ir-beta-endorphin, ACTH, and cortisol during exercise is dose-related with respect to time and intensity. CSF ir-beta-endorphin and ACTH both increased during low- but not high-intensity exercise. CSF cortisol rose markedly in both exercise groups. During high-intensity exercise there was a 50% increase in CSF norepinephrine, indicating that exercise induces alterations in central noradrenergic turnover. We conclude that exercise is a physiologic regulator of both peripheral and central neuroendocrine systems.

Parenteral glutamine infusion alters insulin-mediated glucose metabolism.

BACKGROUND Glutamine is a conditionally essential amino acid that is critical for many basic cellular processes. Its supplementation has been found to be beneficial during several critical illnesses. This study examines the effects of increased glutamine availability on insulin-mediated glucose homeostasis in vivo in multicatheterized conscious canines (n = 5). METHODS Two weeks before the study, catheters were placed in the femoral artery and the portal, hepatic, femoral, and renal veins for blood sampling and in the splenic vein for intraportal infusion of insulin and glucagon. Doppler probes were placed to measure blood flow. The metabolic study consisted of equilibration, basal, and experimental periods during which [3-3H]glucose was infused to measure glucose kinetics. During the 5-hour experimental period, a hyperinsulinemic-euglycemic clamp was performed by infusing somatostatin, basal glucagon, fivefold basal insulin, and glucose to maintain euglycemia. The experimental period was divided evenly into two subperiods performed in random order: (1) i.v. glutamine infusion (0.72 mmol kg(-1) h(-1)) and (2) i.v. saline infusion. RESULTS With glutamine, the glucose required to maintain euglycemia was increased 46% over saline (6.8 +/- 1.0 to 9.9 +/- 1.7 mg kg(-1) min(-1). In addition, whole-body glucose production and utilization were increased by 1.4 and 4.6 mg kg(-1) min(-1), respectively. Finally, the increase in whole-body glucose utilization was manifested by increased hepatic and hindlimb glucose utilization. CONCLUSIONS Increased glutamine availability blunted insulins action on glucose production and enhanced insulin-mediated glucose utilization with the changes in utilization being threefold greater than the changes in production. Thus parenteral glutamine has potential benefit as a nutrient adjuvant during clinical situations associated with insulin resistance.