Abstract
To determine unambiguously if suppression of glucagon action will eliminate manifestations of diabetes, we expressed glucagon receptors in livers of glucagon receptor-null (GcgR−/−) mice before and after β-cell destruction by high-dose streptozotocin. Wild type (WT) mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR−/− mice with similar β-cell destruction remained clinically normal without hyperglycemia, impaired glucose tolerance, or hepatic glycogen depletion. Restoration of receptor expression using adenovirus containing the GcgR cDNA restored hepatic GcgR, phospho-cAMP response element binding protein (P-CREB), and phosphoenol pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycemia appeared. When GcgR mRNA spontaneously disappeared 7 d later, P-CREB declined and hyperglycemia disappeared. In conclusion, the metabolic manifestations of diabetes cannot occur without glucagon action and, once present, disappear promptly when glucagon action is abolished. Glucagon suppression should be a major therapeutic goal in diabetes.
Keywords: glucagon receptor knockout, glucose turnover, type 1 diabetes
So momentous was the discovery of insulin (1) that a companion hormone, subsequently named “glucagon” (2), was ignored. Initially considered a contaminant of the extraction process, glucagon gained hormonal status only after five decades of biochemical, physiological, and morphological research. The discoveries that glucagon is localized in pancreatic (3) and gastric α-cells (4), that it activates hepatic glycogenolysis and gluconeogenesis (5–7) via cAMP (7), that its secretion is stimulated by need for fuels (8, 9), and that it is suppressed by insulin (10), provided compelling hormonal credentials. Subsequent recognition of similarity between the effects of glucagon on the liver (5, 6) and the hepatic abnormalities of untreated insulin deficiency raised the possibility of a role for glucagon in the pathogenesis of diabetes. This idea gained further impetus from the finding that every form of diabetes in man and animal is accompanied by absolute or relative hyperglucagonemia (11, 12). This finding includes total pancreatectomy (4), in which gastric α-cells produce the glucagon, and congenital lipodystrophy (13). This finding further led to the characterization of diabetes as a bihormonal disease (14), in which hepatic overproduction of fuels is caused by glucagon excess, rather than directly by insulin deficiency (15).
Experimental and clinical studies since the mid-1970s (15–17) have demonstrated that glucagon suppression and blockade of its action (18) correct the hyperglycemia of insulin deficiency, consistent with an essential role in the pathogenesis of diabetes (19). However, every glucagon-suppressing hormone also has antidiabetic actions unrelated to glucagon. For example, somatostatin suppresses growth hormone (20) and the exocrine secretions of the pancreas (21). Leptin reduces food intake (22), increases fatty acid oxidation, and enhances insulin sensitivity (23). Glucagon-like peptide-1 reduces food intake, slows gastric emptying, and stimulates insulin secretion by β-cells that might have escaped destruction (24, 25). Even the recent demonstration in glucagon receptor-null (GcgR−/−) mice that diabetes does not develop following β-cell destruction (26) has been attributed to putative hepatic developmental flaws rather than to lack of glucagon action. This finding may account for the skepticism that still prevails (27), and for failure to mount a major clinical effort to evaluate glucagon suppression as a valid therapeutic strategy in type 1 diabetes (T1DM).
To determine whether elimination of glucagon action reverses the metabolic consequences of complete insulin deficiency, we transiently restore GcgR expression via adenoviral delivery and observe whether or not this produces a transient facsimile of the streptozotocin (STZ)-induced diabetes of the WT mice. The ability to turn glucagon action on and off without administering hormones with multiple actions makes possible a reliable assessment of the therapeutic value of abolishing of glucagon action in diabetes.
Results
Completeness of STZ-Induced β-Cell Destruction.
In an effort to eliminate completely the source of insulin, we treated 10- to 12-wk-old C57BL/6J GcgR−/− mice (26) and WT controls with STZ given intravenously 1 wk apart in two or three doses of 75–100 mg/kg of body weight. To assess the completeness of β-cell destruction, each mouse was killed after its experiments, and the pancreas was processed for morphometric quantification of immunocytochemically positive insulin-containing cells. After STZ treatment, residual β-cell area averaged 715 ± 75 μm2 in the WT mice compared with 801 ± 86 μm2 in the GcgR−/− mice (not significant). The normal β-cell area averages 23,279 ± 5,614 μm2. In addition, no mouse was included in the study if it exhibited a rise in plasma C-peptide levels in response to glucose.
Metabolic Effects of β-Cell Destruction in GcgR−/− and WT Mice.
In their intact pretreatment state the 10:00 AM blood glucose levels (4 h after feeding) in WT averaged 134 ± 7 mg/dL and in GcgR−/− averaged 122 ± 4 mg/dL (not significant). Adenovirus GcgR (Adv-GcgR) treatment did not alter blood glucose levels significantly in either group (134 ± 14 mg/dL and 126 ± 4 mg/dL; not significant) (Fig. 1A). Food intake and body weight were constant in the groups.
Fig. 1.
Comparison of postprandial glycemia, glucose turnover, oral glucose tolerance, and C-peptide responses of normal wild-type mice and GcgR−/− mice before and after β-cell destruction (A) Postprandial (10:00 AM) blood glucose levels (mean ± SEM) in intact WT (open column) and in GcgR−/− mice (solid column) before and after treatment with high-dose STZ. (B) Glucose turnover in 5-h-fasted mice implanted with jugular catheters 5 d prior and infused with [3-3H] glucose for 90 min to achieve steady-state (n = 6–7 per group). Blood samples were taken from the cut tail at t = 90, 110, and 120 min to calculate glucose turnover. *P < 0.05 vs. vehicle-treated GcgR+/+ mice; †P < 0.05 vs. STZ-treated GcgR+/+ mice; and #P < 0.05 compared with vehicle-treated GcgR−/− mice. (C) OGTT in GcgR−/− mice before (○) and after (■) STZ treatment. (D) The peak level of C-peptide rise during an oral glucose challenge before and after high-dose STZ before and after STZ (*P < 0.01).
STZ-induced β-cell destruction caused severe hyperglycemia in WT mice (>400 mg/dL by 7 d), but a similar degree of β-cell destruction in GcgR−/− mice did not cause hyperglycemia throughout the 4-wk period of observation (121 ± 6.8 mg/dL). In confirmation of our earlier report (26), β-cell destruction in the GcgR−/− mice did not alter the postprandial glucose levels (Fig. 1A). These data are consistent with elevated glucose turnover in STZ-treated WT mice. In addition, glucose turnover in STZ-treated GcgR−/− mice is lowered compared with the other groups (Fig. 1B). Also in confirmation of our earlier report, β-cell destruction in GcgR−/− mice did not negatively impact glucose tolerance tested during an oral glucose tolerance test (OGTT) (Fig. 1C). Neither value differed significantly from the pre-STZ values, despite the absence of a C-peptide response after STZ (Fig. 1D). The lack of GcgR did not alter plasma triglyceride (TG) levels, although liver TG was significantly lower. Plasma cholesterol levels were significantly higher in GcgR−/− mice. Adenovirus delivery of GcgR did not correct the differences. β-Cell destruction did not result in any important changes in lipid content or in the expression of lipogenic transcription factors and their target enzymes (Table S1).
Turning Glucagon Action On and Off; Molecular and Metabolic Consequences.
To provide glucagon action in the liver of GcgR−/− mice, we intravenously injected 1 × 109 pfu of adenovirus containing the GcgR cDNA (Adv-GcgR). Hepatic glucagon receptor mRNA, which had been undetectable by qRT-PCR, rose to a peak at 3 d after injection and declined thereafter (Fig. 2A).
Fig. 2.
The effects of transient transgenic expression of GcgR in the liver of insulin-deficient GcgR-null mice upon markers of glucagon action and upon blood glucose levels. (A) Pattern of liver GcgR mRNA expression before and after injecting Adv-GcgR into insulin-deficient GcgR−/− mice. (B) Densitometric measurements of CREB, a transducer of glucagon action, in GcgR−/− mice before, during, and after expression of adenovirally delivered GcgR cDNA (P < 0.01). (See Fig. S1 for a representative immunoblot). (C) PEPCK mRNA in intact WT (open column) and in streptozotocinized GcgR−/− mice (solid column) before and at 5 and 10 d after the administration of adenovirus containing the GcgR cDNA (*P < 0.01; **P < 0.001). (D) Blood glucose levels (mean ± SEM) in GcgR−/− mice injected adv-β-gal (○) and Adv-GcgR (■) after destruction of β-cells by high-dose STZ (**P < 0.001).
Based on the fact that GcgR−/− mice have marked hyperglucagonemia (28), we assumed that restoration of a functioning glucagon receptor expressed in the liver of such mice would quickly activate molecular markers of glucagon action. Therefore, to test the functionality of the GcgR transgene, we measured hepatic phospho-cAMP response element binding protein (P-CREB), a major component of glucagon’s signal transduction pathway (29), and the mRNA of phosphoenol pyruvate carboxykinase (PEPCK), a glucagon-responsive gluconeogenic target enzyme (30). Before the administration of Adv-GcgR, hepatic P-CREB protein and PEPCK mRNA in GcgR−/− mice were significantly below WT controls (Fig. 2 B and C). At 5 d after injection, when GcgR expression was high, both P-CREB protein and PEPCK mRNA were significantly increased. However, 10 d after Adv-GcgR injection, when GcgR mRNA had declined, P-CREB and PEPCK mRNA had returned to preinjection levels. These findings provide strong evidence that glucagon action had been restored by the adenoviral delivery of the GcgR transgene.
Glucose levels appeared to reflect GcgR expression levels and the resulting glucagon activity. Glucose averaged 121 ± 9 mg/dL before injection of Adv-GcgR but had risen by the third day, reaching a peak of 470 ± 21 mg/dL at 8 d after injection (Fig. 2D). Thus, the presence of GcgR in the livers of insulin-deficient GcgR−/− mice was required to produce the same diabetic phenotype that in WT mice occurred with STZ treatment alone. The results exclude the possibility that the congenital absence of glucagon receptors had somehow impaired the development of normal hepatic responses to insulin deficiency independently of the loss of glucagon action.
To obtain evidence that turning off glucagon action would, by itself, reverse the diabetes without confounding actions of hormonal suppressors, we waited for the transiently expressed GcgR mRNA to disappear spontaneously. At 7 d postinjection, GcgR had declined to 1.5% of the peak value. To assess glucagon activity we again measured P-CREB protein and PEPCK mRNA. By10 d postinjection, both P-CREB and PEPCK mRNA had returned to similar levels as those before GcgR restoration, indicating that glucagon action had subsided. At 10 d postinjection, when GcgR mRNA, P-CREB, and PEPCK mRNA were virtually undetectable, the high glucose levels had declined to 129 ± 17 mg/dL, not different from pre-STZ levels of intact WT controls (Fig. 2D). Thus, the elimination of glucagon action completely abolished the hyperglycemia of insulin deficiency.
Finally, to exclude the unlikely possibility that the virus itself was diabetogenic, we injected 1 × 109 pfu of adenovirus containing the β-galactosidase cDNA into the insulin-deficient GcgR−/− mice. Blood glucose levels remained around 100 mg/dL throughout the weeks, evidence that the GcgR, not the virus, was diabetogenic.
Glycogen, Insulin, and the Glucagon Receptor.
To compare tissue glycogen content of the groups after glucose loading, mice were killed immediately after the final OGTT blood sample had been obtained. Glycogen content of liver and skeletal muscle was measured by the method of Hachey et al. (31). In GcgR−/− mice after β-cell destruction, liver glycogen averaged 145 ± 15 mg/g of tissue, 2.7-times their content before STZ treatment, and 7.6-times that of intact WT mice (Fig. 3A). Skeletal muscle glycogen content was markedly reduced in insulin-deficient GcgR−/− mice (Fig. 3B).
Fig. 3.
Glycogen content of liver and skeletal muscle in normal mice and in GcgR−/− mice with and without insulin deficiency. Comparison of liver (A) and skeletal muscle (B) glycogen content in intact WT mice (white column) and in GcgR−/− mice before (black column) and after (grey column) treatment with high-dose STZ (*P < 0.02; **P < 0.001; ***P < 0.0001).
The mechanism of the increase in liver glycogen in the GcgR−/− mice in the absence of insulin action is unclear. We suspect that this increase may reflect an expanded glucose pool with very low glucose turnover, with accumulation of glucose in those tissues in which insulin is not required for transport into cells. Thus, glucose entry into liver cells would be unimpeded, and hepatic glycogenesis would be unimpeded by glucagon action.
Suppression of Hyperglucagonemia by Adv-GcgR.
Before the injection of Adv-GcgR, marked hyperglucagonemia exceeding 2,000 pg/mL was present, confirming the report of Gelling et al. (28); fell to 132 ± 11 pg/mL in the insulin-deficient GcgR−/− mice 5 d after the injection.
Discussion
These results provide unique and unambiguous evidence that elimination of glucagon action by itself eliminates diabetic hyperglycemia. All previous studies of glucagon suppression used hormones with multiple antidiabetic actions. In this model of total insulin deficiency, glucagon action was turned on and off without the use of exogenous hormones or drugs. The results clearly indicate that the glycemic abnormalities of insulin deficiency do not occur in the absence of glucagon action and, once present, can be restored to normal by eliminating glucagon action. These abnormalities include fasting hyperglycemia, glucose intolerance, and depletion of liver glycogen. Their absence in insulin-deficient GcgR−/− mice and their appearance after restoration of GcgR expression provides evidence of the essential pathogenic role of glucagon. The return of normoglycemia after disappearance of the receptor mRNA argues for the therapeutic potential of glucagon suppression/inactivation in diabetes. If, as these results suggest, glucagon is the sine qua non of diabetic hyperglycemia, they should apply to type 2 diabetes as well (32, 33). Although favorable results in animals do not guarantee similar therapeutic efficacy in humans, previous interspecies comparisons of glucagon suppression have shown no species differences (15–17).
The fact that glucose intolerance cannot occur without glucagon receptors implies that the frequent hyperglycemic spikes might be eliminated by glucagon suppression (34, 35, 36). Clearly, current insulin monotherapy cannot duplicate the around-the-clock control of α-cell secretion provided by the normal paracrine action of endogenous insulin secreted from juxtaposed nondiabetic β-cells (37, 38).
The use of an α-cell suppressor without insulin-like actions on peripheral glucose disposition should eliminate the peaks of hyperglycemia, while reducing the insulin dose should eliminate or reduce the nadirs of glycemic volatility, as shown in preclinical studies in mice with T1DM (35). If this process can be successfully translated to humans with T1DM, it should also eliminate the need for frequent glucose monitoring and insulin injections. The quality and quantity of life for patients with T1DM would thereby be enhanced. However, risk of hypoglycemia induced, not by insulin but by fasting and exercise, would probably be increased, so patients should be alerted to avoid these challenges.
The elegant studies of Jamison et al. (39) provide strong tangible support for this theory. As recently reviewed, the α-cell defect in that disorder consists of failure to suppress glucagon during influx of nutrients because of defective paracrine signaling from β-cells (38).
Methods
Animals.
C57BL/6J GcgR−/− mice were housed in individual cages with constant temperature and 12 h of light and alternating darkness, and fed Teklad 6% fat (g/g) mouse/rat diet (Teklad) with free access to water. Animals were killed at various time points before and the end of experimental periods, and tissues were freeze-clamped before death and immediately placed in liquid nitrogen. Tissues were stored at −80 °C until assay. The protocol was approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center and the Institutional Animal Care and Use Committee of the North Texas Veterans Administration Medical Center, Dallas, TX.
Chemical Destruction of β-Cells.
β-Cells were destroyed in 10- to 12-wk-old GcgR−/− mice and WT controls with intravenous injections of STZ (100 mg/kg body weight) separated by 7 d. Food intake, body weight, and blood glucose were measured at weekly intervals.
Adv-GcgR Administration.
Adv-GcgR was purchased from Vector Biolabs. Adv-GcgR or β-galactosidase was administered intravenously (1 × 109 pfu/10 g mice).
Plasma Measurements.
Blood specimens were collected at 0, 3, 5, 8, and 10 d after adenovirus injection and blood glucose levels were measured with glucose meter (Bayer Healthcare). Plasma C-peptide levels were measured using a mouse C-peptide ELISA kit (ALPCO). Plasma TG were measured using a glycerol phosphate oxidase-Trinder triglyceride kit (Sigma-Aldrich).
TG Content of Tissues.
Total lipids from liver were extracted and dried under N2 gas. TG content was assed as previously described (40).
Immunoblotting.
Liver CREB and P-CREB were measured by Western blotting (Cell Signaling Technology). γ-Tubulin (Sigma-Aldrich) was used as a loading control.
Real-Time Quantitative PCR.
Total RNA was extracted from the liver by TRIsol isolation method (Life Technologies). All PCR reactions were performed in triplicate as previously described (26). Primer sequences are shown in Table 1.
Table 1.
Primer sequences used in real-time quantitative PCR in WT and GcgR−/− mice
| Genes | Forward primer | Reverse primer |
| 36B4 | 5′-GGACCCGAGAAGACCTCCTT-3′ | 5′-GGTGCCTCTGGAGATTTTCG-3′ |
| PEPCK | 5′-CGCAAGCTGAAGAAAATATGACAA-3′ | 5′-TCGATCCTGGCCACATCTC-3′ |
| GcgR | 5′-ATTGGCGATGACCTCAGTGTGA-3′ | 5′-GCAATAGTTGGCTATGATGCCG-3′ |
| ACC-1 | 5′-CCCAGCAGAATAAAGCTACTTTGG-3′ | 5′-TCCTTTTGTGCAACTAGGAACGT-3′ |
| ACO | 5′-GGCCAACTATGGTGGACATCA-3′ | 5′-ACCAATCTGGCTGCACGAA-3 |
| CPT-1 | 5′-ACCACTGGCCGAATGTCAAG-3′ | 5′-AGCGAGTAGCGCATGGTCAT-3′ |
| GPAT | 5′-ATCTTCAGAACAGCAAAATCGAAA-3′ | 5′-CAGCGGAAAACTCCAAATCC-3′ |
| FAS | 5′-CCTGGATAGCATTCCGAACCT-3′ | 5′-AGCACATCTCGAAGGCTACACA-3′ |
| DGAT2 | 5′-CCGCAAAGGCTTTGTGAA-3′ | 5′-GGAATAAGTGGGAACCAGATCAG-3′ |
| SREBP1-C | 5′-GGCACTAAGTGCCCTCAACCT-3′ | 5′-GCCACATAGATCTCTGCCAGTGT-3′ |
| SREBP2 | 5′-CAGACAGCCGCCCTTCAAGT-3′ | 5′-GCTGTTCATTGACCTTCTCCCG-3′ |
ACC-1, acetyl CoA carboxylase-1; ACO, acetyl CoA oxidase; CPT-1, carnithine palmityol transferase; DGAT2, diacyl glycerol Transferase 2; FAS, fatty acid synthase; GcgR, glucagon receptor; GPAT, glycerol phosphate acyl transferase; PEPCK, phosphoenol pyruvate carboxy kinase; SREBP1-C, sterol regulatory element binding protein 1-C; SREBP2, sterol regulatory element binding protein 2.
Glycogen Measurement in Liver and Muscle.
Tissue glycogen was measured in mice killed immediately after the final OGTT blood sample (2 mg/kg glucose; 10% (g/vol) of glucose was [U-13C] glucose) using previously described gas chromatography/mass spectrometry techniques (31).
Measurement of Glucose Turnover.
Mice were implanted with jugular catheters and infused with [3-3H] glucose, as previously described (41).
Statistical Analysis.
Results are reported as mean ± SEM. Statistical analysis of the data was performed with a Student t test.
Supplementary Material
Acknowledgments
We thank S. Kay McCorkle for editorial and technical contributions and Dr. J. Goldstein for critical review. This work was supported by private donations, a Veterans Administration Merit Review, and Amylin Pharmaceuticals.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1205983109/-/DCSupplemental.
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