G. K. Hendrick

Glucagonlike Peptide I (7–37) Actions on Endocrine Pancreas

Glucagonlike peptide I (7–37) [GLP-I-(7–37)], encoded with glucagon and glucagonlike peptide II and intervening peptide II in the rat and human glucagon gene, is processed from proglucagon in both pancreas and intestine and is a potent stimulator of insulin secretion. Unequivocal insulin release from the isolated perfused rat pancreas is elicited by a 10−11 M concentration of this peptide, and a weak response is found at 10−12 M. We found that GLP-I-(7–37) is ∼100 times more potent than glucagon in the stimulation of insulin secretion. Insulin release in response to GLP-I-(7–37) is highly dependent on the ambient glucose concentration; no response is detectable at a glucose concentration of 2.8 mM, and at 6.6 and 16.7 mM, insulin release is augmented by 4.7 and 22.8 ng/ml, respectively. The pattern of insulin secretion stimulated by GLP-I-(7–37) is biphasic, with an initial spike followed by a plateau of sustained release. The effects on insulin release of GLP-I-(7–36) amide, a GLP-I analogue, and GLP-I-(7–37) at concentrations of 10−11 M were indistinguishable. We also found that GLP-I-(7–37) at 10−9 M does not influence glucagon secretion and that glucagonlike peptide II and the intervening peptide II, two other peptides encoded by the glucagon gene, have no detectable effects on insulin secretion.

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.

Similar Dose Responsiveness of Hepatic Glycogenolysis and Gluconeogenesis to Glucagon In Vivo

This study was undertaken to determine whether the dose-dependent effect of glucagon on gluconeogenesis parallels its effect on hepatic glycogenolysis in conscious overnight-fasted dogs. Endogenous insulin and glucagon secretion were inhibited by somatostatin (0.8 μg · kg−1 · min−1), and intraportal replacement infusions of insulin (213 ± 28 (αU kg−1 · min−1) and glucagon (0.65 ng · kg−1 · min−1) were given to maintain basal hormone concentrations for 2 h (12 ± 2 and 108 ± 23 pg/ml, respectively). The glucagon infusion was then increased 2-, 4-, 8-, or 12-fold for 3 h, whereas the rate of insulin infusion was left unchanged. Glucose production (GP) was determined with 3-[3H]glucose, and gluconeogenesis (GNG) was assessed with tracer (U-[14C]alanine conversion to [14C]glucose) and arteriovenous difference (hepatic fractional extraction of alanine, FEA) techniques. Increases in plasma glucagon of 53 ± 8, 199 ± 48, 402 ± 28, and 697 ±149 pg/ml resulted in initial (15- 30 min) increases in GP of 1.1 ± 0.4 (N = 4), 4.9 ± 0.5 (N = 4), 6.5 ± 0.6 (N = 6), and 7.7 ± 1.4 (N = 4) mg kg−1 · min−1, respectively; increases in GNG (∼3 h) of 48 ± 19, 151 ± 50, 161 ± 25, and 157 ± 7%, respectively; and increases in FEA (3 h) of 0.14 ± 0.07, 0.37 ± 0.05, 0.42 ± 0.04, and 0.40 ± 0.17, respectively. In conclusion, GNG and glycogenolysis were similarly sensitive to stimulation by glucagon in vivo, and the dose-response curves were markedly parallel.

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.

Interaction Between Insulin and Glucose-Delivery Route in Regulation of Net Hepatic Glucose Uptake in Conscious Dogs

In the presence of fixed basal levels of insulin, the route of intravenous glucose delivery (portal vs. peripheral) determines whether net hepatic glucose uptake (NHGU) occurs. Our aims were to determine if the route of intravenous glucose delivery also plays a role in regulating NHGU in the presence of hyperinsulinemia and to determine if length of fast (18 vs. 36 h) influences regulation of NHGU. Five conscious dogs fasted 18 h were given somatostatin and replacement insulin (245 ± 34 μU · kg−1 · min−1) and glucagon (0.65 ng · kg−1 · min−1) infusions intraportally. After a 40-min control period, the insulin infusion rate was increased fourfold, and glucose was infused for 3 h. Glucose was given either through a peripheral vein or the portal vein for 90 min to double the glucose load reaching the liver. The order of infusions was randomized. NHGU was measured with the arterial – venous difference technique. Insulin and glucagon levels were 12 ± 2, 35 ± 6, and 36 ± 5 μU/ml and 55 ± 12, 61 ± 13, and 59 ± 7 pg/ml during the control, peripheral, and portal infusions, respectively. The glucose infusion rate, the load of glucose reaching the liver, and the arterial-portal plasma glucose gradient were 0, 9.58 ± 2.28, and 10.44 ± 2.94 mg · kg−1 · min−1; 29.4 ± 3.6, 56.8 ± 3.4, and 56.8 ± 2.8 mg · kg−1 · min−1; and 2 ± 1, 5 ± 1, and −51 ± 15 mg/dl during the same periods. The liver switched from net glucose output (2.5 ± 0.4 mg · kg−1 · min−1) to uptake of 1.4 ± 0.7 and 3.5 ± 0.8 mg · kg−1 · min−1 during peripheral and portal glucose delivery, respectively. Despite a similar hormonal milieu and indistinguishable glucose loads, there was significantly (P < 0.01) more NHGU when glucose was infused intraportally. Identical studies in five conscious dogs fasted 36 h gave results similar to those of dogs fasted 18 h. After 36 h of fasting, NHGU was 1.6 ± 0.4 and 4.0 ± 0.4 mg · kg−1 · min−1 during the peripheral and portal glucose infusions, respectively (P < 0.005). In conclusion, the route of intravenous glucose administration plays an important role in regulating NHGU even in the presence of hyperinsulinemia in conscious dogs fasted 18 and 36 h.

Glucagon-like peptide-I-(7–37) suppresses hyperglycemia in rats☆☆☆

Glucagon-like peptide-(GLP) I-(7-37) is an endogenous hormone that has recently been demonstrated to be a potent insulin secretagogue. In these studies, GLP was administered during oral and intravenous (IV) glucose tolerance tests (OGTT and IVGTT, respectively) to determine whether this peptide could enhance postprandial insulin levels and thus reduce glycemic excursions. Surprisingly, during OGTT, GLP administration did not augment insulin secretion; however, GLP administration resulted in significantly lower glycemic excursions. In fasted rats, glycemic excursions were significantly reduced 10 and 20 minutes after receiving GLP (P < .001). Fed rats that received GLP had virtually no initial increase in plasma glucose level after administration of oral glucose. During IVGTT, glucose alone increased insulin levels eightfold, while administration of both glucose and GLP resulted in a 15-fold increase (P < .001). These IVGTT data support previous studies that show GLP to be a potent and glucose-dependent insulin secretagogue. Furthermore, all of these studies suggest that GLP reduces postprandial glycemic excursion and thus may be useful in the treatment of non-insulin-dependent diabetes mellitus.

Stimulation of glucose production through hormone secretion and other mechanisms during insulin-induced hypoglycemia

To assess the role of counterregulatory hormones per se in the response to continuous insulin infusion, overnight-fasted dogs were given 5 mil · kg−1 · min−1 insulin intraportally either alone (INS, n = 5), with glucose to maintain euglycemia (INS + GLU, n = 5), or with glucose and hormone replacement [i.e., glucagon, epinephrine, norepinephrine, and cortisol infusions (INS + GLU + HR, n = 6)]. The increases in counterregulatory hormones that occurred during insulin-induced hypoglycemia were simulated in the latter group. In this way, it was possible to separate the effects of hypoglycemia per se from those due to the associated counterregulatory hormone response. Glycogenolysis and gluconeogenesis were measured with a combination of tracer ([3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous (AV) difference techniques during a 40-min control and a 180-min experimental period. Insulin levels increased similarly in all groups (to ≃250 μU/ml), whereas plasma glucose levels decreased in INS (115 ± 3 to 41 ± 3 mg/dl; P < .05) and rose slightly in both INS + GLU (108 ± 2 to 115 ± 4 mg/dl; P < .05) and INS + GLU + HR (111 ± 3 to 120 ± 3 mg/dl; P < .05) due to glucose infusion. Glucagon, epinephrine, norepinephrine, and cortisol were replaced in INS + GLU + HR so that the increments in their levels were 102 ± 6, 106 ± 14, 117 ± 9, and 124 ± 37%, respectively, of their increments in INS. At no time was there a significant difference between the hormone levels in INS and INS + GLU + HR. The rise in the counterregulatory hormones per se accounted for only half (53 ± 9% by the AV difference method and 54 ± 10% by tracer method) of the glucose production associated with hypoglycemia resulting from insulin infusion. The rate and efficiency of alanine conversioto glucose in the hormone-replacement studies were only 29 ± 10 and 50 ± 27% of what occurred during hypoglycemia induced by insulin infusion. In conclusion, the counterregulatory hormones alone (i.e., without accompanying hypoglycemia) can account for only 50% of the glucose production that is present during insulin-induced hypoglycemia. The remaining 50%, therefore, must result from effects of hypoglycemia other than its ability to trigger hormone release.

Oxidation of rat insulin II, but not I, leads to anomalous elution profiles upon HPLC analysis of insulin-related peptides

Rat insulin II, unlike rat insulin I and other non‐rodent insulins, contains a unique methionine residue at position B29. Reversed‐phase HPLC allows for separation of the two rat insulins, with insulin I typically eluting faster than insulin II. An anomalous peak of insulin immunoreactive material was found eluting even faster than insulin I following acid extraction of rat insulin‐producing cells. This early peak co‐eluted with [Met‐O B29]insulin II suggesting that during cell extraction and subsequent purification steps, rat insulin II is subject to selective oxidation at MetB29. Such oxidation of rat proinsulin II affords improved separation from rat proinsulin I compared to the native form.