Endocrinology 98: 819, 1976 EFFECTS OF NEUROTENSIN AND SUBSTANCE P ON PLASMA INSULIN, GLUCAGON AND GLUCOSE LEVELS. Marvin Brown and Wylie Vale The Salk Institute for Biological Studies, La Jolla, California,
92037
ABSTRACT: Neurotensin and substance P given iv 5, 10, 20 and 30 minutes prior to blood collection produce hypoinsulinemia, hyperglucagonemia and hyperglycemia in the rat. Glucagon similarly produces hyperglycemia in rats but results in hyperinsulinemia. On a molar basis neurotensin is _ca. 10 and 30 times more active in inducing hyperglycemia than substance P and glucagon, respectively. The enhanced glucogenic effects of neurotensin and substance P over glucagon may well result from their inhibition of insulin release. Neurotensin and substance P may be important in glucose homeostasis.
Neurotensin, a tridecapeptide isolated and characterized from extracts of bovine hypothalamus, produces hypotension, gut contraction and hyperglycemia in the rat (1,2,3). Thishyperglycemia is associated with a decrease in liver glycogen content and an increase in liver glycogenphosphorylase A activity. The mechanism of these events has not been explained (3), Substance P, an undecapeptide isolated and characterized from extracts of bovine hypothalamus (4,5) and equine intestine (6), also produces hypotension and gut contraction but its effects on glucoregulation have not been reported (4). In these studies, we describe the effects of neurotensin and substance P on plasma insulin, glucagon and glucose levels in the rat. MATERIALS AND METHODS Eat bioassay: Fed male Sprague-Dawley rats weighing 200-250 g were used in all experiments. All animals were fed tap water and Purina Rat Chow ad_ libitum and housed in temperature and humidity controlled quarters with 14 h of light (0700 - 2100) and 10 h of dark. All experiments were performed between 1400 and 1600 h. Administration of peptides or drugs was made in a volume of 0.2 ml via the external jugular vein in ether anesthetized animals. Saline control groups were included in all experiments. Each Received November 11, 1975
point in the figures represents six rats sacrificed by decapitation at the times indicated in the text. Hormone determinations: Blood samples were collected into chilled tubes containing 10 ng EDTA and 50 yl of 2 M benzamidine per ml of blood. Insulin was determined by the method of Herbert _et _al. (7) utilizing porcine insulin antiserum obtained from Miles Laboratories, and [12->I] iodinated insulin from New England Nuclear Corp. Rat insulin standard was a gift from Eli Lilly Co. Plasma glucagon was determined by the method of Faloona and Unger (8) utilizing glucagon antiserum 30K (pool 1, lot 25) generously supplied by Dr. Roger Unger, Dallas, Texas. [•*• ^1]iodinated glucagon was obtained from Nuclear Medical Laboratories, Dallas, Texas, and glucagon for standard was a gift from Eli Lilly Co. Glucose was determined by the glucose oxidase method, utilizing a Beckman Glucose Analyzer. Drugs and pep.tides: Substance P and neurotensin were synthesized by Jean Rivier using solid phase methodology as previously described (9,10). Bovine glucagon was obtained from CalBiochem, La Jolla, Cal. Bradykinin was obtained from Beckman Instruments, Palo Alto Cal. Statistical analysis: All experiments were carried out in a randomized block design. Following analysis of variance,
819
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Endo • 1976 Vol 98 • No 3
RAPID COMMUNICATIONS
820
differences between treatments were determined by the multiple range tests of Dunnett and Duncan. All calculations were carried out by the UCLA IBM 360 Model 95 computer with the program EXBIOL and HUBA RESULTS AND DISCUSSION Figure 1 shows elevations of plasma glucose 30 minutes following iv administration of graded doses of neurotensin, substance P, glucagon and bradykinin. As determined graphically neurotensin is £a_. 10 and 30 times more active on a molar basis for inducing hyperglycemia than substance P and glucagon, respectively. Bradykinin, which produces hypotension over the same dose range as neurotensin and substance P (11), did not elevate plasma glucose levels, thus suggesting that neurotensin and substance P elevate plasma glucose levels independent of their hypotensive actions.
o en
z 200
o u
Administration of 2 yg neurotensin (Fig. 2) or 6 yg substance P (Fig.3) results in the following: 1) elevation of plasma glucose levels at 10 minutes which remain elevated throughout the 30 min time course; 2) an initial suppression of plasma insulin which returns to control levels at 10 min, appears suppressed at 20 min and is significantly elevated at 30 min; 3) an initial elevation of plasma glucagon (5 min) which returns to control values within 10 min. Plasma insulin levels are suppressed or inappropriately low for the degree of hyperglycemia up to 20 min. Although the plasma glucagon values return to control range, they are elevated considering the level of hyperglycemia. Thus, there is an absolute or relative hyperglucagonemia and hypoinsulinemia up to 20 rain throughout the time course. This absolute or relative hyperglucagonemia may be secondary to the low insulin levels observed throughout the time course, or may be secondary to a stimulating effect of neurotensin or substance P. It is improbable that the hypoinsulinemia is secondary to an elevation in plasma glucagon, as exogenous glucagon (10 yg) administration is associated with stimulation, not suppression, of insulin release (Fig. A ) .
150
BRADYKININ
-4
100 0.1
0.3
J 10
LOG DOSE PEPTIDE (/ig/IOOg BW)
Fig. Z* Effects oi. graded doses of neurotensin, substance P, glucagon and bradykinin on plasma glucose levels. All peptides were given iv 30 minutes prior to blood collection. Each point represents six animals. **p
92037
ABSTRACT: Neurotensin and substance P given iv 5, 10, 20 and 30 minutes prior to blood collection produce hypoinsulinemia, hyperglucagonemia and hyperglycemia in the rat. Glucagon similarly produces hyperglycemia in rats but results in hyperinsulinemia. On a molar basis neurotensin is _ca. 10 and 30 times more active in inducing hyperglycemia than substance P and glucagon, respectively. The enhanced glucogenic effects of neurotensin and substance P over glucagon may well result from their inhibition of insulin release. Neurotensin and substance P may be important in glucose homeostasis.
Neurotensin, a tridecapeptide isolated and characterized from extracts of bovine hypothalamus, produces hypotension, gut contraction and hyperglycemia in the rat (1,2,3). Thishyperglycemia is associated with a decrease in liver glycogen content and an increase in liver glycogenphosphorylase A activity. The mechanism of these events has not been explained (3), Substance P, an undecapeptide isolated and characterized from extracts of bovine hypothalamus (4,5) and equine intestine (6), also produces hypotension and gut contraction but its effects on glucoregulation have not been reported (4). In these studies, we describe the effects of neurotensin and substance P on plasma insulin, glucagon and glucose levels in the rat. MATERIALS AND METHODS Eat bioassay: Fed male Sprague-Dawley rats weighing 200-250 g were used in all experiments. All animals were fed tap water and Purina Rat Chow ad_ libitum and housed in temperature and humidity controlled quarters with 14 h of light (0700 - 2100) and 10 h of dark. All experiments were performed between 1400 and 1600 h. Administration of peptides or drugs was made in a volume of 0.2 ml via the external jugular vein in ether anesthetized animals. Saline control groups were included in all experiments. Each Received November 11, 1975
point in the figures represents six rats sacrificed by decapitation at the times indicated in the text. Hormone determinations: Blood samples were collected into chilled tubes containing 10 ng EDTA and 50 yl of 2 M benzamidine per ml of blood. Insulin was determined by the method of Herbert _et _al. (7) utilizing porcine insulin antiserum obtained from Miles Laboratories, and [12->I] iodinated insulin from New England Nuclear Corp. Rat insulin standard was a gift from Eli Lilly Co. Plasma glucagon was determined by the method of Faloona and Unger (8) utilizing glucagon antiserum 30K (pool 1, lot 25) generously supplied by Dr. Roger Unger, Dallas, Texas. [•*• ^1]iodinated glucagon was obtained from Nuclear Medical Laboratories, Dallas, Texas, and glucagon for standard was a gift from Eli Lilly Co. Glucose was determined by the glucose oxidase method, utilizing a Beckman Glucose Analyzer. Drugs and pep.tides: Substance P and neurotensin were synthesized by Jean Rivier using solid phase methodology as previously described (9,10). Bovine glucagon was obtained from CalBiochem, La Jolla, Cal. Bradykinin was obtained from Beckman Instruments, Palo Alto Cal. Statistical analysis: All experiments were carried out in a randomized block design. Following analysis of variance,
819
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 06 July 2015. at 06:33 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1976 Vol 98 • No 3
RAPID COMMUNICATIONS
820
differences between treatments were determined by the multiple range tests of Dunnett and Duncan. All calculations were carried out by the UCLA IBM 360 Model 95 computer with the program EXBIOL and HUBA RESULTS AND DISCUSSION Figure 1 shows elevations of plasma glucose 30 minutes following iv administration of graded doses of neurotensin, substance P, glucagon and bradykinin. As determined graphically neurotensin is £a_. 10 and 30 times more active on a molar basis for inducing hyperglycemia than substance P and glucagon, respectively. Bradykinin, which produces hypotension over the same dose range as neurotensin and substance P (11), did not elevate plasma glucose levels, thus suggesting that neurotensin and substance P elevate plasma glucose levels independent of their hypotensive actions.
o en
z 200
o u
Administration of 2 yg neurotensin (Fig. 2) or 6 yg substance P (Fig.3) results in the following: 1) elevation of plasma glucose levels at 10 minutes which remain elevated throughout the 30 min time course; 2) an initial suppression of plasma insulin which returns to control levels at 10 min, appears suppressed at 20 min and is significantly elevated at 30 min; 3) an initial elevation of plasma glucagon (5 min) which returns to control values within 10 min. Plasma insulin levels are suppressed or inappropriately low for the degree of hyperglycemia up to 20 min. Although the plasma glucagon values return to control range, they are elevated considering the level of hyperglycemia. Thus, there is an absolute or relative hyperglucagonemia and hypoinsulinemia up to 20 rain throughout the time course. This absolute or relative hyperglucagonemia may be secondary to the low insulin levels observed throughout the time course, or may be secondary to a stimulating effect of neurotensin or substance P. It is improbable that the hypoinsulinemia is secondary to an elevation in plasma glucagon, as exogenous glucagon (10 yg) administration is associated with stimulation, not suppression, of insulin release (Fig. A ) .
150
BRADYKININ
-4
100 0.1
0.3
J 10
LOG DOSE PEPTIDE (/ig/IOOg BW)
Fig. Z* Effects oi. graded doses of neurotensin, substance P, glucagon and bradykinin on plasma glucose levels. All peptides were given iv 30 minutes prior to blood collection. Each point represents six animals. **p
