Alan D. Cherrington

Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.

Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxPtechnology in combination with gene targeting to perform global, β cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. β cell- and hepatocyte-specific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a β cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in β cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the β cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both β cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Glucagonocentric restructuring of diabetes: A pathophysiologic and therapeutic makeover

The hormone glucagon has long been dismissed as a minor contributor to metabolic disease. Here we propose that glucagon excess, rather than insulin deficiency, is the sine qua non of diabetes. We base this on the following evidence: (a) glucagon increases hepatic glucose and ketone production, catabolic features present in insulin deficiency; (b) hyperglucagonemia is present in every form of poorly controlled diabetes; (c) the glucagon suppressors leptin and somatostatin suppress all catabolic manifestations of diabetes during total insulin deficiency; (d) total β cell destruction in glucagon receptor-null mice does not cause diabetes; and (e) perfusion of normal pancreas with anti-insulin serum causes marked hyperglucagonemia. From this and other evidence, we conclude that glucose-responsive β cells normally regulate juxtaposed α cells and that without intraislet insulin, unregulated α cells hypersecrete glucagon, which directly causes the symptoms of diabetes. This indicates that glucagon suppression or inactivation may provide therapeutic advantages over insulin monotherapy.

The role of insulin and glucagon in the regulation of basal glucose production in the postabsorptive dog.

The aim of the present experiments was to determine the role of insulin and glucagon in the regulation of basal glucose production in dogs fasted overnight. A deficiency of either or both pancreatic hormones was achieved by infusin somatostatin (1 mug/kg per min), a potent inhibitor of both insulin and glucagon secretion, alone or in combination with intraportal replacement infusions of either pancreatic hormone. Infusion of somatostatin alone caused the arterial levels of insulin and glucagon to drop rapidly by 72+/-6 and 81+/-8%, respectively. Intraportal infusion of insulin and glucagon at rates of 400 muU/kg per min and 1 ng/kg per min, respectively, resulted in the maintenance of the basal levels of each hormone. Glucose production was measured using tracer (primed constant infusion of [3-3H]glucose) and arteriovenous difference techniques. Isolated glucagon deficiency resulted in a 35+/-5% (P less than 0.05) rapid and sustained decrease in glucose production which was abolished upon restoration of the plasma glucagon level. Isolated insulin deficiency resulted in a 52+/-16% (P less than 0.01) increase in the rate of glucose production which was abolished when the insulin level was restored. Somatostatin had no effect on glucose production when the changes in the pancreatic hormone levels which it normally induces were prevented by simultaneous intraportal infusion of both insulin and glucagon. In conclusion, in the anesthetized dog fasted overnight; (a) basal glucagon is responsible for at least one-third of basal glucose production, (b) basal insulin prevents the increased glucose production which would result from the unrestrained action of glucagon, and (c) somatostatin has no acute effects on glucose turnover other than those it induces through perturbation of pancreatic hormone secretion. This study indicates that the opposing actions of the two pancreatic hormones are important in the regulation of basal glucose production in the postabsorptive state.

Effect of glucagon on glucose production during insulin deficiency in the dog.

The aim of the present experiments was to determine the effects of basal glucagon on glucose production after induction of prolonged insulin lack in normal conscious dogs fasted overnight. A selective deficiency of insulin or a combined deficiency of both pancreatic hormones was created by infusing somatostatin alone or in combination with an intraportal replacement infusion of glucagon. Glucose production (GP) was measured by a primed constant infusion of [3H-3]glucose, and gluconeogenesis (GNG) was assessed by determining the conversion rate of circulating [14C]alanine and [14C]lactate into [14C]glucose. When insulin deficiency was induced in the presence of basal glucagon the latter hormone caused GP to double and then to decline so that after 4 h it had returned to the conrol rate. The conversion of alanine and lactate into glucose, on the other hand, increased throughout the period of insulin lack. Withdrawal of glucagon after GP had normalized resulted in a 40% fall in GP, a 37% decrease in GNG, and a marked decrease in the plasma glucose concentration. Induction of insulin deficiency in the absence of basal glucagon resulted in an initial (30%) drop in GP followed by a restoration of normal GP after 2--3 h and moderately enhanced glucose formation from alanine and lactate. It can be concluded that (a) the effect of relative hyperglucagonemia on GP is short-lived; (b) the waning of the effect of glucagon is attributable solely to a diminution of glycogenolysis because GNG remains stimulated; (c) basal glucagon markedly enhances the GNG stimulation apparent after induction of insulin deficiency; and (d) basal glucagon worsens the hyperglycemia pursuant on the induction of insulin deficiency both by triggering an initial overproduction of glucose and by maintaining the basal production rate thereafter.

Importance of the route of intravenous glucose delivery to hepatic glucose balance in the conscious dog.

To assess the importance of the route of glucose delivery in determining net hepatic glucose balance (NHGB) eight conscious overnight-fasted dogs were given glucose via the portal or a peripheral vein. NHGB was measured using the arteriovenous difference technique during a control and two 90-min glucose infusion periods. The sequence of infusions was randomized. Insulin and glucagon were held at constant basal levels using somatostatin and intraportal insulin and glucagon infusions during the control, portal, and peripheral glucose infusion periods (7 +/- 1, 7 +/- 1, 7 +/- 1 microU/ml; 100 +/- 3, 101 +/- 6, 101 +/- 3 pg/ml, respectively). In the three periods the hepatic blood flow, glucose infusion rate, arterial glucose level, hepatic glucose load, arterial-portal glucose difference and NHGB were 37 +/- 1, 34 +/- 1, 32 +/- 3 ml/kg per min; 0 +/- 0, 4.51 +/- 0.57, 4.23 +/- 0.34 mg/kg per min; 101 +/- 5, 200 +/- 15, 217 +/- 13 mg/dl; 28.5 +/- 3.5, 57.2 +/- 6.7, 54.0 +/- 6.4 mg/kg per min; +2 +/- 1, -22 +/- 3, +4 +/- 1 mg/dl; and 2.22 +/- 0.28, -1.41 +/- 0.31, and 0.08 +/- 0.23 mg/kg per min, respectively. Thus when glucose was delivered via a peripheral vein the liver did not take up glucose but when a similar glucose load was delivered intraportally the liver took up 32% (P less than 0.01) of it. In conclusion portal glucose delivery provides a signal important for the normal hepatic-peripheral distribution of a glucose load.

Effects of Increased Glucokinase Gene Copy Number on Glucose Homeostasis and Hepatic Glucose Metabolism

The relationship between glucokinase (GK) gene copy number and glucose homeostasis was studied in transgenic mice with additional copies of the entire GK gene locus (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem.272, 22564–22569). The plasma glucose concentration was reduced by 25 ± 3% and 37 ± 4% in mice with one or two extra copies of the gene locus, respectively. The basis for the hypoglycemic phenotype was determined using metabolic tracer techniques in chronically cannulated, conscious mice with one extra GK gene copy. Under basal conditions (6-h fasted) transgenic mice had a lower blood glucose concentration (−12 ± 1%) and a slightly higher glucose turnover rate (+8 ± 3%), resulting in a significantly higher glucose clearance rate (+21 ± 2%). Plasma insulin levels were not different, suggesting that increased glucose clearance was due to augmented hepatic, not islet, GK gene expression. Under hyperglycemic clamp conditions the transgenic mice had glucose turnover and clearance rates similar to the controls, but showed a lower plasma insulin response (−48 ± 5%). Net hepatic glycogen synthesis was markedly elevated (+360%), whereas skeletal muscle glycogen synthesis was decreased (−40%). These results indicate that increased GK gene dosage leads to increased hepatic glucose metabolism and, consequently, a lower plasma glucose concentration. Increased insulin secretion was not observed, even though the transgene is expressed in islets, because hypoglycemia causes a down-regulation in islet GK content (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, in press).

Evidence for an important role of glucagon in the regulation of hepatic glucose production in normal man.

To investigate the role of glucagon in regulating hepatic glucose production in man, selective glucagon deficiency was produced in four normal men by infusing somatostatin (0.9 mg/h) and regular pork insulin (150-muU/kg per min) for 2 h. Exogenous glucose was infused to maintain euglycemia. Arterial plasma glucagon levels fell by greater than 50% whereas plasma insulin levels were maintained in the range of 10-14 muU/ml. In response to these hormonal changes, net splanchnic glucose production (NSGP) fell by 75% and remained suppressed for the duration of the study. In contrast, when somatostatin alone was administered to normal men, resulting in combined insulin and glucagon deficiency (euglycemia again maintained), NSGP fell markedly but only transiently, reaching its nadir at 15 min. Thereafter, NSGP rose progressively, reaching the basal rate at 105 min. These data indicate that the induction of selective glucagon deficiency in man (with basal insulin levels maintained) is associated with a marked and sustained fall in hepatic glucose production. We conclude, therefore, that basal glucagon plays an important role in the maintenance of basal hepatic glucose production in normal man.

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.

The Role of Fatty Acids in Mediating the Effects of Peripheral Insulin on Hepatic Glucose Production in the Conscious Dog

We investigated the mechanism by which a selective increase in arterial insulin can suppress hepatic glucose production in vivo. Isotopic (3-3H-glucose) and arteriovenous difference methods were used in overnight-fasted, conscious dogs. A pancreatic clamp (somatostatin, basal portal insulin, and glucagon infusions) was used to control the endocrine pancreas. Equilibration (100 min) and basal (40 min) periods were followed by a 180-min test period. In control dogs (n = 5), basal insulin delivery was continued throughout the study. In the other two groups, peripheral insulin was selectively increased at the beginning of the test period by stopping the portal insulin infusion and infusing insulin peripherally at twice the basal portal rate. One group (INS + FAT; n = 6) received an infusion of 20% intralipid + heparin (0.5 U · kg−1 · min−1) to clamp the nonesterified fatty acid (NEFA) levels during hyperinsulinemia; the other group (INS; n = 7) received only saline during the experimental period. In the INS group, a selective increase in peripheral insulin of 84 pmol/l was achieved (36 ± 6 to 120 ± 24 pmol/l, last 30 min) while portal insulin was unaltered (84 ± 18 pmol/l). In the INS + FAT group, a similar increase in peripheral insulin was achieved (36 ± 6 to 114 ± 6 pmol/l, last 30 min); again, portal insulin was unaltered (96 ± 12 pmol/l). In the control group, basal insulin did not change. Glucagon and glucose remained near basal values in all protocols. In the INS group, NEFA levels dropped from 700 ± 90 (basal) to 230 ± 65 μmol/l (last 30 min; P > 0.05), but in the INS + FAT group changed minimally (723 ± 115 [basal] to 782 ± 125 μmol/l [last 30 min]). In the INS group, net hepatic glucose output dropped by 6.7 μmol · kg−1 · min−1 (P < 0.05), whereas in the INS + FAT group it dropped by 3.9 μmol · kg−1 · min−1 (P < 0.05). When insulin levels were not increased (i.e., in the control group), net hepatic glucose output dropped 1.7 μmol · kg−1 · min−1 (P , 0.05). In all groups, the net hepatic glucose output data were confirmed by the tracer-determined glucose production data. In the INS group, net hepatic gluconeogenic substrate uptake (alanine, glutamine, glutamate, glycerol, glycine, lactate, threonine, and serine) fell slightly (10.4 ± 1.3 [basal] to 7.2 ± 1.3 μmol · kg−1 · min−1 [last 30 min]), whereas in the INS + FAT group it did not change (7.3 ±1.5 [basal] to 7.4 ± 0.6 μmol · kg−1 · min−1 [last 30 min]), and in the control group it increased slightly (9.6 ± 1.3 [basal] to 10.3 ± 1.4 μmol · kg−1 · min−1 [last 30 min]). These results indicate that peripheral insulins ability to regulate hepatic glucose production is partially linked to its inhibition of lipolysis. When plasma NEFA levels were prevented from falling during a selective arterial hyperinsulinemia, ∼55% of insulin%s inhibition of net hepatic glucose output (NHGO) was eliminated. The fall in NEFA levels brings about a redirection of glycogenolytically derived carbon within the hepatocyte such that there is an increase in lactate efflux and a corresponding decrease in NHGO.

Differential Time Course of Glucagon's Effect on Glycogenolysis and Gluconeogenesis in the Conscious Dog

The evanescence of glucagons effect on glucose production (GP) is well documented, but it is unclear (1) whether this response involves both glycogenolysis and gluconeogenesis and (2) whether the liver becomes dependent on the increased glucagon level for the maintenance of a basal supply of glucose. To answer these questions, conscious overnight-fasted dogs were given somatostatin (0.8 μg/kg · min) plus basal intraportal replacement amounts of insulin (273 μU/kg min) and glucagon (0.65 ng/kg · min) for 2 h, after which the rate of glucagon infusion was increased fourfold for 3 h and then returned to basal for 1.5 h. GP was determined using a primed infusion of [3-3H[ glucose, and gluconeogenesis (GNG) was estimatedby determining the conversion rate of alanine and lactate to glucose. An increase in the plasma glucagon level from 55 to 206 pg/ml resulted in an initial 180% increase in GP, followed by a decline such that after 3 h of hyperglucagonemia GP was increased by only 41%. Contrary to overall GP, gluconeogenesis increased progressively throughout the hyperglucagonemic period, eventually reaching a rate 3 times basal. Restoration of the basal glucagon level (63 pg/ml) caused a marked decline in GP and GNG. In fact, GP fell to a level 29% below the initial control rate and consequently the plasma glucose level fell rapidly. The data suggest that (1) the downregulation of glucagonstimulated GP is attributable to a decline in glycogenolysis and not gluconeogenesis, and (2) following adaptation to the hormone, the liver becomes dependent on the elevated glucagon concentration for the maintenance of basal glucose production.