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Endocrinology Vol. 139, No. 11 4540-4546
Copyright © 1998 by The Endocrine Society

ARTICLES

Insulin, But Not Glucose Lowering Corrects the Hyperglucagonemia and Increased Proglucagon Messenger Ribonucleic Acid Levels Observed in Insulinopenic Diabetes1

Eric Dumonteil2, Christophe Magnan2, Beate Ritz-Laser, Paolo Meda, Philippe Dussoix, Marc Gilbert, Alain Ktorza and Jacques Philippe

Unité de Diabétologie Clinique, Department of Medicine (E.D., B.R.-L., P.D., J.P.), and the Department of Morphology (P.M.), Centre Médical Universitaire, 1211 Geneva 4, Switzerland; and Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique, URA 307, Université Paris 7-Denis Diderot (C.M., M.G., A.K.), 75005 Paris, France

Address all correspondence and requests for reprints to: Jacques Philippe, M.D., Unité de Diabétologie Clinique, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland. E-mail: philippe{at}cmu.unige.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The factors that regulate glucagon biosynthesis and proglucagon gene expression are poorly defined. We previously reported that insulin inhibits proglucagon gene expression in vitro. In vivo, however, the effects of insulin on the regulation of the proglucagon gene have been controversial. Furthermore, whether glucose plays any role alone or in conjunction with insulin on proglucagon gene expression is unknown. We investigated the consequences of insulinopenic diabetes on glucagon gene expression in the endocrine pancreas and intestine and whether insulin and/or glucose could correct the observed abnormalities. We show here that in the first 3 days after induction of hyperglycemia by streptozotocin, rats have levels of plasma glucagon and proglucagon messenger RNA comparable to those of normoglycemic controls despite hyperglycemia. With more prolonged diabetes, plasma glucagon and proglucagon messenger RNA levels increase; this increase is corrected by insulin treatment, but not by phloridzin despite normalization of the glycemia by both treatments. Proglucagon gene expression exhibits the same regulatory response to glucose and insulin in both pancreas and ileum. We conclude that insulin tonically inhibits proglucagon gene expression in the pancreas and ileum and that glucose plays a minor, if any, role in this regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ENDOCRINE pancreas plays a central role in the control of glucose homeostasis. Insulin and glucagon are the two major hormones that antagonistically control the balance between glucose storage and consumption and maintain plasma glucose levels within a very narrow range. Plasma insulin and glucagon levels are inversely correlated; insulin is secreted in response to nutrients to promote energy storage, whereas glucagon secretion is increased in the fasting state to activate glycogenolysis and gluconeogenesis.

The proglucagon gene is expressed in pancreatic {alpha}-cells, intestinal L cells, and neurons of hypothalamus and medulla oblongata (1). Glucagon is derived from a larger precursor, proglucagon, which also contains in tandem two glucagon-like peptides, GLP-1 and GLP-2. Posttranslational processing of proglucagon is tissue specific, inasmuch as glucagon is the primary peptide synthesized in the pancreas, whereas glicentin, oxyntomodulin, GLP-1 and GLP-2 are released from the intestine (1).

The fact that hyperglycemia is a hallmark of both insulin-dependent (IDDM) and noninsulin-dependent diabetes mellitus stimulated many studies on the role of the different factors regulating glucagon secretion and biosynthesis (2, 3). However, the precise roles of these factors in pancreatic {alpha}-cell function are still poorly understood. Control of glucagon secretion is probably mediated by multiple factors, including circulating intermediary metabolites, neurotransmitters, and hormones. However, the plasma glucose concentration, the insulin level, and the activity of the autonomic nervous system appear to be the major determinants (2). Physiologically, glucagon secretion is suppressed by hyperglycemia; this suppression is lost in insulinopenic diabetes, inasmuch as hyperglycemia is accompanied by hyperglucagonemia, which, in turn, perpetuates hyperglycemia by stimulating hepatic glucose output (3). The mechanisms responsible for glucose suppression of glucagon secretion and for abnormal regulation of {alpha}-cells in insulinopenic diabetes are still debated. It has been proposed that {alpha}-cell suppression could be secondary to glucose, insulin, glucose together with intraislet insulin, or somatostatin (3). These different hypotheses are not exclusive. Recent studies suggest that both insulin and glucose are necessary to inhibit glucagon secretion (4, 5, 6, 7). The respective roles of insulin and glucose on glucagon biosynthesis and gene expression are unknown. Although insulin has been suggested to regulate pancreatic proglucagon gene expression in vitro (8, 9), conflicting results have appeared in vivo. In the insulinopenic, streptozotocin-induced diabetic rat model, proglucagon messenger RNA (mRNA) levels have been reported to be either elevated (10) or comparable to those in nondiabetic rats (7, 11).

To gain more insight into the roles of insulin and glucose in proglucagon gene expression, we performed in vivo experiments with streptozotocin-induced diabetic rats that were either left untreated or given insulin or phloridzin to achieve euglycemia. We report here that in vivo, severe insulinopenia induced by streptozotocin is accompanied by hyperglucagonemia and an increase in proglucagon mRNA levels in the pancreas, which are both suppressed by insulin, but not by phloridzin, treatment. In addition, proglucagon gene expression in the ileum exhibits the same regulatory response to glucose and insulin. We conclude that proglucagon gene expression is tonically inhibited by insulin regardless of the glucose concentration; derepressed glucagon gene expression occurs in insulin deficiency and can only be corrected by insulin treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and in vivo studies
Three-month-old female Wistar rats, weighing 220–240 g, that had free access to water and standard laboratory chow pellets (UAR 113, Usine d’Alimentation Rationnelle, Villemoisson sur Orge, France) were used. For the in vivo studies, the rats were randomly allocated into control and diabetic groups. Diabetes was induced with 65 mg/kg streptozotocin (Sigma Chemical Co., St. Louis, MO) injected into the saphenous vein. After streptozotocin injection, there was a significant loss of weight during the first 48 h (from 232 ± 5 to 206 ± 4 g; P < 0.001) for both experiments; weight then remained lower than that in controls throughout the study. In experiments designed to normalize glycemia in diabetic rats, insulin or phloridzin was infused during 3 days through a catheter implanted under ketamine anesthesia (125 mg/kg, ip; Imalgene, Mérieux, France) in the right atrium via the jugular vein, and animals were allowed to recover from the surgery for 3 days before starting two series of experiments (12, 13). In the first series, the infusion period started 3 days after streptozotocin injection and lasted for 2 days. In the second series, the infusion period started 10 days after streptozotocin injection and lasted for 3 days. Each series of animals was divided into four groups: 1) control rats infused with 0.9% saline, 2) rats made diabetic by streptozotocin injection, 3) diabetic rats infused with 1.5 µmol/liter (at a rate of 30 pmol/min) insulin (Actrapid Novo, Copenhagen, Denmark), and 4) diabetic rats infused with 1 mg/min·kg (at a rate of 26 µl/min) phloridzin diluted in dimethylsulfoxide. Dimethylsulfoxide at the amount used does not affect blood glucose or insulin levels (14, 15). Before and throughout the infusion period, plasma glucose, glucagon, and insulin concentrations were measured once daily (100 µl blood/assay).

Northern blot and ribonuclease (RNase) protection analyses
Rat pancreas and ileum were removed, immediately frozen, and stored at -80 C until RNA isolation by the guanidine isothiocyanate method followed by centrifugation through a cesium chloride gradient (16). Insulin mRNA levels were determined by Northern blot analyses. One to 2 µg total RNA were size-fractionated on a 1% agarose gel, transferred onto a nylon membrane (Nytran, Schleicher & Schuell, Inc., Keene, NH), and UV cross-linked. Blots were sequentially hybridized with a random primed complementary DNA (cDNA) probe for rat insulin I (17) and an 18S ribosomal RNA oligonucleotide probe. Quantification of RNA signals was performed using a PhosporImager (Molecular Dynamics, Inc., Sunnyvale, CA), and results were expressed as the insulin mRNA/18S ribosomal RNA ratio.

RNase protection analyses were performed for the measurement of proglucagon and ß-actin mRNA levels. Uniformly labeled RNA probes were synthesized by in vitro transcription according to standard protocols (16) using plasmid pGem3-Gluc containing a 1.1-kb SacI/PstI fragment of the rat proglucagon cDNA (18). As an internal control for RNA quantity, a riboprobe was generated complementary to codons 220–303 of the mouse ß-actin cDNA (Ambion, Inc., Lugano, Switzerland). RNase protection analyses were performed following standard protocols (16) with 50 µg (pancreas) and 30 µg (ileum) total RNA; the relative intensities of protected fragments were quantified using a PhosporImager, and results were expressed as the proglucagon/ß-actin ratio.

Analytical methods
Plasma glucose was determined by the glucose oxidase technique using a glucose analyzer (YSI, Inc., Yellow Springs, OH). Plasma immunoreactive insulin and glucagon were determined by RIAs using kits from CEA (Gif-sur-Yvette, France) and Biodata (Rome, Italy), respectively (19). The lower limit of the assay was 15 pmol/liter (4 µU/ml) for insulin and 14.5 ng/liter for glucagon, and the coefficient of variation within and between assays was 6% for the insulin and glucagon kits.

Presentation of the results and data analysis
Results are presented as the mean ± SEM. Statistical analysis of differences between groups was performed using ANOVA, followed, when significant, by a post-hoc test (Scheffe’s test) using the ANOVA output.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin, but not glucose, regulates proglucagon gene expression in vivo
To test the effects of insulin and/or glucose on proglucagon gene expression in vivo, we chose the streptozotocin-induced insulinopenic rat model. Chronic hyperglycemia may indeed render {alpha}-cells insensitive to the prevailing glucose concentration, whereas insulin may affect proglucagon gene expression through a tonic inhibition (1, 2, 3, 20, 21). To test these hypotheses we designed a two-phase model. In the first phase model, a state of insulinopenic hyperglycemia was obtained in the presence of normal plasma glucagon levels, as observed within the first few days after the induction of diabetes (22), to assess the effects of the low intraislet insulin levels (23) on proglucagon gene expression. In the second phase model, experiments were performed 10 days after the induction of diabetes, at a time when intraislet insulin levels are barely detectable (23) and plasma glucagon levels are high (22), to investigate the consequences of minimal intraislet insulin levels on proglucagon gene expression and the possible regulatory changes induced by insulin and phloridzin treatment. Phloridzin was used to inhibit renal tubular reabsorption of glucose to obtain a euglycemic state independently of insulin. Both phases of the experiments were conducted on a group of control rats and three groups of streptozotocin-treated animals. In the first phase model, one group of diabetic rats remained untreated, whereas 3 days after streptozotocin administration, the other groups received either insulin or phloridzin for an additional 2-day period. On day 5, rats were killed, and ileum and pancreas were removed and stored at -70 C.

Figure 1Go illustrates plasma glucose, insulin, and glucagon levels in the four groups of rats. As expected, glucose levels increased within 24 h up to 350–400 mg/dl in the streptozotocin-treated rats and returned to near-normal levels when the animals received either insulin or phloridzin. Accordingly, plasma insulin levels fell in the diabetic rats within 24 h to low, but still detectable, levels. In these rats, insulinemia was not altered by phloridzin treatment, whereas it increased to more than 3-fold compared with controls in rats treated with insulin. Glucagonemia, by contrast, was not significantly different among the four groups of rats, except on days 4 and 5, when it was slightly, but significantly, lower in the diabetic rats treated with phloridzin. This decrease is intriguing and could be due either to phloridzin itself or to an indirect effect through the correction of hyperglycemia, changes in the plasma concentration of other nutrients, such as amino acids or hormones, or even the local release of neuromediators. Phloridzin is unlikely to be directly involved, as it is associated with hyperglucagonemia; we thus favor an indirect inhibiting effect that may be due to the correction of hyperglycemia. We have indeed observed that glucagon-producing cells exposed chronically to high glucose decrease their glucagon release when glucose is lowered to normal levels (Dumonteil, E., manuscript in preparation). The fact that we do not observe the same phenomenon with insulin treatment might be explained by the concomitant activation of the sympathetic nervous system by insulin, resulting in an activation of glucagon release.



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  		Figure 1. Plasma glucose, insulin, and glucagon levels in the first phase experimental model of insulinopenic streptozotocin-induced diabetes. A, Plasma glucose (milligrams per dl) was determined by the glucose oxidase technique. B and C, Plasma immunoreactive insulin (181 U/ml) and glucagon (pg/ml) were measured by RIAs. {square}, Control rats (n = 6); {blacksquare}, streptozotocin-injected rats (STZ; n = 6); {circ}, diabetic rats treated with insulin (STZi; n = 6); •, diabetic rats treated with phloridzin (STZp; n = 6). **, P < 0.01; ***, P < 0.001 (significantly different from control rats). Shown are the mean ± SEM of six rats per group.

 
To investigate the effects of glucose and insulin on proglucagon gene expression, we isolated total RNA from the pancreas and ileum of control and diabetic rats and quantified proglucagon mRNA levels by Northern analyses. As shown in Fig. 2AGo, we found that the levels of pancreatic insulin mRNA were reduced by 95% in the streptozotocin-treated animals, suggesting that most of the ß-cells had been destroyed. Pancreatic proglucagon mRNA levels were also assessed by Northern analyses; however, as the signals were not sufficiently reproducible, we measured proglucagon mRNA levels by RNase protection assays. Figure 2BGo shows that pancreatic proglucagon transcripts were similar to control levels in both treated and untreated rats. These results indicate that despite markedly decreased peripheral insulin and low intraislet insulin levels (as suggested by the low pancreatic insulin mRNA levels), glucagon biosynthesis and proglucagon gene expression, as reflected by similar peripheral glucagonemia and pancreatic proglucagon mRNA levels, respectively, were not altered. We can conclude from this experimental model, that high glucose levels maintained during 5 days in the presence of low peripheral insulin levels do not influence proglucagon gene expression in the pancreas.



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  		Figure 2. Proinsulin and proglucagon mRNA levels in control, untreated, and treated diabetic rats during the first phase experimental model. Pancreas and ileum were removed on day 5 and frozen. Total RNA was extracted. A, Proinsulin mRNA levels were determined by Northern blots using the rat insulin I cDNA probe, labeled by random priming with [32P]deoxy-CTP. B, Proglucagon mRNA levels were measured by RNase protection analyses with uniformly labeled RNA probes using plasmids containing either 1.1 kb of the rat proglucagon cDNA (16 ) or 252 bp of the mouse ß-actin cDNA used as an internal control. Results are expressed as percentages of the values obtained in control rats and are corrected for the ß-actin mRNA levels. *, P < 0.05; +, P < 0.001.

 
The proglucagon gene is expressed not only in the pancreas but also in the intestine (1). We thus assessed the consequences of diabetes on proglucagon mRNA levels in the terminal ileum. In untreated diabetic rats, proglucagon mRNA levels increased by 2.3-fold over control values; treatment with insulin reduced these levels to values comparable to those measured in control rats, whereas phloridzin treatment had no effect (Fig. 2BGo). These results suggest that, in contrast to the data obtained in the pancreas, proglucagon gene expression in the ileum is activated by the state of insulinopenic diabetes and that this activation is corrected by insulin treatment, but not by euglycemia.

The different regulation of the proglucagon gene in the pancreas and intestine in response to diabetes could be due to the residual intraislet insulin concentrations, which even when low could still be sufficient to exert a tonic inhibitory effect on pancreatic glucagon biosynthesis and proglucagon gene expression. The increased proglucagon mRNA levels found in the intestine would then result from the markedly decreased peripheral plasma insulin levels. In favor of this hypothesis, plasma glucagon concentrations in diabetic rats were comparable to those in nondiabetic controls (Fig. 1CGo), suggesting that glucagon biosynthesis, which is mainly contributed by the pancreas, was similar in the two groups of rats.

To test whether a progressive decrease in intraislet insulin concentrations could result in an increase in proglucagon gene expression in the pancreas, we waited 10 days after streptozotocin injection to obtain elevated plasma glucagon levels (22). Insulin and phloridzin treatment were then started and continued for 3 days. The batch of streptozotocin used for the second phase experiments was probably more active than the first batch, inasmuch as for the same injected dose, more rats died after receiving streptozotocin, glycemia in diabetic rats was slightly higher, and plasma glucagon levels had increased by 48 h. As shown in Fig. 3AGo, glycemia in the diabetic animals ranged from 400–500 mg/dl and promptly returned to basal values with insulin or phloridzin treatment. Insulinemia was very low in diabetic rats and increased with insulin treatment to about 3-fold over control values, whereas no change was noted with phloridzin treatment (Fig. 3BGo).



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  		Figure 3. Plasma glucose, insulin, and glucagon levels in the second phase experimental model of streptozotocin-induced diabetes. Plasma glucose (milligrams per dl; A), insulin (microunits per ml; B), and glucagon (picograms per ml; C) levels were determined as described in Fig. 1Go. {square}, Control rats (n = 4); {blacksquare}, streptozotocin-injected rats (STZ; n = 4); {circ}, diabetic rats treated with insulin (STZi; n = 4); •, diabetic rats treated with phloridzin (STZp; n = 4). ***, P < 0.001 (significantly different from control rats). Shown are the mean ± SEM.

 
Plasma glucagon levels slightly increased 48 h after streptozotocin administration and plateaued on day 6 at 400–600% of basal values. Insulin treatment rapidly normalized these levels, whereas phloridzin had no effect (Fig. 3CGo). Under these conditions, proglucagon mRNA levels were elevated 2-fold (Fig. 4Go). Insulin decreased proglucagon mRNA levels to values comparable to those found in control rats, whereas phloridzin had no effect. Similar results were obtained in the ileum (Fig. 4Go). These results indicate that in severe insulinopenic diabetes, there is a marked increase in peripheral plasma glucagon levels, as previously reported (3), accompanied by increased proglucagon mRNA levels in both pancreas and intestine. This increase in proglucagon gene expression is largely due to a lack of insulin, inasmuch as insulin treatment corrects both the increased proglucagon mRNA and the peripheral glucagon levels, whereas normalization of the plasma glucose concentrations has no effect. We thus conclude from our studies that insulin tonically inhibits glucagon biosynthesis and glucagon gene expression in both pancreas and intestine, whereas glucose alone has no major effect.



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  		Figure 4. Proglugagon mRNA levels in control, untreated, and treated diabetic rats of the second phase experimental model of streptozotocin-induced diabetes. Proglucagon and ß-actin mRNA levels were determined in both pancreas and ileum by RNase protection analyses as described in Materials and Methods. Results are expressed as percentages of the values obtained in control rats and are corrected for the ß-actin mRNA levels. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies indicate that proglucagon gene expression is tonically inhibited by insulin; thus, 5 days after the induction of diabetes, plasma glucagon and proglucagon mRNA levels were comparable in diabetic and control rats, indicating that proglucagon gene expression is sensitive to relatively low concentrations of intraislet insulin. Similar observations had been noted previously for glucagon release in vitro (24). In addition, the concept of the tonic inhibition of glucagon by insulin has been substantiated over the last 30 yr by the elegant studies of Samols et al. (21). It is unlikely that under our experimental conditions, hyperglycemia had a suppressive effect on glucagon production and release, inasmuch as the return to euglycemia by phloridzin treatment was accompanied by a slight decrease, rather than an increase, in plasma glucagon levels. By contrast, our observations in the second phase experiment show that, as previously reported (10), proglucagon gene expression is increased and accompanied by hyperglucagonemia, a likely consequence of the progressive decrease in intraislet insulin levels that occurs after streptozotocin treatment. We thus conclude that the chronic hyperglucagonemia characteristic of untreated insulinopenic diabetes is due to increased proglucagon gene expression. Our data suggest, however, that derepressed proglucagon gene expression may not be the sole mechanism explaining the high plasma glucagon levels. Proglucagon mRNA levels are indeed only increased 2-fold in insulinopenic diabetes, whereas plasma glucagon levels are increased 4- to 6-fold compared with control values. It is thus likely that hyperglucagonemia reflects not only increased gene expression, but also increased glucagon biosynthesis, possibly due to enhanced mRNA translation. Hence, insulin could affect glucagon biosynthesis at multiple levels. Our conclusion is in disagreement with previous results reported using a different experimental model (11). In these studies and despite insulin treatment, hyperglycemia persisted. Proglucagon mRNA levels were comparable in diabetic and nondiabetic rats, suggesting an effect of insulin under hyperglycemic conditions. In our studies, insulin treatment was sufficient to normalize not only hyperglucagonemia, but also the elevated proglucagon mRNA levels. By contrast, normalizing plasma glucose alone by phloridzin treatment had no impact on either plasma glucagon or proglucagon mRNA levels, indicating that hyperglycemia alone cannot suppress proglucagon gene expression. However, some modulation of the effects of insulin by changing glucose concentrations cannot be excluded by our studies, because the expression of the proglucagon gene in diabetic rats treated with insulin was only assessed in a state of hyperglycemia. The results obtained by Brubaker et al. do not favor this possibility however (11).

The regulation of proglucagon gene expression appears, therefore, to differ from that of glucagon secretion, which is affected by both hypo- and hyperglycemia. For instance, in severe insulin-deficient diabetes, chronic hyperglycemia has been proposed to mediate at least in part the glucose insensitivity of {alpha}-cells that is characteristic of diabetes (3, 4, 25, 26). In addition, impaired glucagon responsiveness to hypoglycemia is partially improved by an insulin-independent correction of hyperglycemia, again stressing the role of normoglycemia in maintaining the glucose sensitivity of the {alpha}-cells (7).

Several studies suggest, nevertheless, that insulin deficiency is a critical factor in the sensitivity of {alpha}-cells to glucose through the loss of either a suppressive effect of local insulin or a permissive effect of insulin on the ability of glucose to suppress {alpha}-cells (6, 27, 28, 29). The response of glucagon secretion to changing glucose concentrations is thus likely to involve both insulin- and glucose-dependent mechanisms.

In contrast, the suppressive effect of insulin on the high glucagon levels observed in insulinopenic diabetes may not be dependent on glucose. Plasma glucagon levels in insulin-deprived diabetic dogs decline during insulin infusion at the same rate whether the animals are hyperglycemic or are made normoglycemic by phloridzin treatment, indicating that there is no relationship between the ambient glucose concentration and the magnitude of the insulin-mediated suppression of glucagon (5). These results are consistent with our conclusions on the regulation of proglucagon gene expression.

An additional important finding of this study is the differential regulation of pancreatic and ileal proglucagon gene expression. This expression is down-regulated by insulin in both the ileum and pancreas, but increases only in the ileum of diabetic rats, which have normal plasma glucagon and pancreatic proglucagon mRNA levels. This difference may be due to the relatively higher intraislet insulin concentrations, which are then sufficient to inhibit pancreatic proglucagon gene expression and glucagon release, compared with peripheral plasma insulin levels, which may fall below the threshold level for inhibiting ileal proglucagon gene expression.

The treatment of IDDM is hampered by symptomatic hypoglycemia related to an impaired glucagon response (30), perhaps due to intensive insulin therapy (31). On the other hand, pancreatic {alpha}-cell dysfunction contributes to the deterioration of glycemic control in diabetes mellitus. Our results suggest that insulin plays a major role in suppressing {alpha}-cell function and, notably, glucagon release and proglucagon gene expression; therefore, high insulin levels resulting from insulin therapy in IDDM and basal hyperinsulinemia in obese noninsulin-dependent diabetic patients may be the main factors responsible for impaired glucagon response in diabetes. Although glucose may also be critical for {alpha}-cell functions, particularly for glucagon secretion, we propose that it has little impact, if any, on proglucagon gene expression. Finally, proglucagon gene expression in the pancreas and ileum is similarly regulated by insulin, suggesting that the same insulin regulatory DNA elements in the proglucagon gene promoter (9) may be operative in both organs.


   Acknowledgments
 
We thank F. Kaempfen for typing the manuscript.


   Footnotes
 
1 This work was supported by the Swiss National Fund (Grant 32–46816.96 to J.P. and Grant 32–34086.95 to P.M.), the Institute for Human Genetics and Biochemistry, the Nägeli Wolfermann Foundation, the Horten Foundation, and the Juvenile Diabetes Foundation International (Grant 197124). Back

2 E.D. and C.M. contributed equally to this work. Back

Received March 31, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  7. Shi ZQ, Rastogi KS, Lekas M, Efendic S, Drucker DJ, Vranic M 1996 Glucagon response to hypoglycemia is improved by insulin-independent restoration of normoglycemia in diabetic rats. Endocrinology 137:3193–3199[Abstract]
  8. Philippe J 1989 Glucagon gene transcription is negatively regulated by insulin in a hamster islet cell line. J Clin Invest 84:672–677
  9. Philippe J 1991 Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element. Proc Natl Acad Sci USA 88:7224–7227[Abstract/Free Full Text]
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E. Dumonteil, B. Ritz-Laser, C. Magnan, I. Grigorescu, A. Ktorza, and J. Philippe
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