Delayed onset of hyperglycaemia in a mouse model with impaired glucagon secretion demonstrates that dysregulated glucagon secretion promotes hyperglycaemia and type 2 diabetes
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- Gustavsson, N., Seah, T., Lao, Y. et al. Diabetologia (2011) 54: 415. doi:10.1007/s00125-010-1950-2
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Type 2 diabetes is caused by relative deficiency of insulin secretion and is associated with dysregulation of glucagon secretion during the late stage of diabetes development. Like insulin secretion from beta cells, glucagon secretion is dependent on calcium signals and a calcium sensing protein, synaptotagmin-7. In this study, we tested the relative contribution of dysregulated glucagon secretion and reduced insulin release in the development of hyperglycaemia and type 2 diabetes by using synaptotagmin-7 knockout (KO) mice, which exhibit glucose intolerance, reduced insulin secretion and nearly abolished Ca2+-stimulated glucagon secretion.
We fed the synaptotagmin-7 KO and control mice with a high-fat diet (HFD) for 14 weeks, and compared their body weight, glucose levels, glucose and insulin tolerance, and insulin and glucagon secretion.
On the HFD, synaptotagmin-7 KO mice showed progressive impairment of glucose tolerance and insulin secretion, along with continued maintenance of a low glucagon level. The control mice were less affected in terms of glucose intolerance, and showed enhanced insulin secretion with a concurrent increase in glucagon levels. Unexpectedly, after 14 weeks of HFD feeding, only the control mice displayed resting hyperglycaemia, whereas in synaptotagmin-7 KO mice defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion.
Our data uncover a previously overlooked role of dysregulated glucagon secretion in promoting hyperglycaemia and the ensuing diabetes, and strongly suggest maintenance of adequate regulation of glucagon secretion as an important therapeutic target in addition to the preservation of beta cell function and mass in the prevention and treatment of diabetes.
KeywordsExocytosis Glucose tolerance test Hepatic glucose production Insulin resistance Insulin secretion Insulin tolerance test Knockout mice Pancreatic islets Pyruvate tolerance test
Glucose tolerance test
Insulin tolerance test
Glucose homeostasis is maintained by two counteracting hormones from the pancreas: insulin from beta cells and glucagon from alpha cells . Under normal conditions, insulin is released when blood glucose is high to stimulate glucose uptake and to inhibit glucose production, while low glucose stimulates glucagon secretion, which acts on the liver to promote glycogenolysis and gluconeogenesis [1, 2]. Insulin and glucagon levels are tightly regulated to achieve a proper balance for the maintenance of glucose homeostasis. When the balance of these two hormones is disrupted, proper glucose levels cannot be maintained.
Type 2 diabetes is the result of relative insulin deficiency, i.e. impaired insulin secretion due to beta cell dysfunction and/or reduced beta cell mass that fail to overcome peripheral insulin insensitivity [3, 4, 5]. Consequently, most previous studies on the pathophysiology of type 2 diabetes were mainly focused on the interplay between beta cell dysfunction and insulin resistance . Although dysregulated glucagon secretion and elevated glucagon levels are known to be associated with high blood glucose in type 2 diabetes [7, 8], these conditions are usually considered a consequence  instead of a cause  of diabetes, and it remains controversial whether glucagon, particularly dysregulated oversupply of glucagon, plays an active role in diabetes development .
Exocytosis of both insulin and glucagon granules is a calcium-dependent process, and is regulated by the same calcium sensor, synaptotagmin-7 [11, 12, 13, 14]. Deletion of synaptotagmin-7 results in glucose intolerance, and impaired insulin secretion . Synaptotagmin-7 knockout (KO) mice maintain normoglycaemia and exhibit normal insulin sensitivity, insulin synthesis, islet architecture and beta cell ultrastructural organisation . They also have lower body weight than their littermate controls . Furthermore, synaptotagmin-7 regulates insulin and glucagon secretion to different extents. While insulin secretion is partially impaired, glucagon secretion is nearly abolished in synaptotagmin-7 KO mice [12, 13].
In the present study we investigated the relative contributions of impaired insulin response and dysregulated glucagon secretion in the development of hyperglycaemia and diabetes. We challenged synaptotagmin-7 KO and control mice with a high-fat diet (HFD) to induce peripheral insulin insensitivity, and measured their insulin and glucagon responses, as well as blood glucose levels. Our results show that dysregulated glucagon secretion plays a previously underappreciated role in promoting hyperglycaemia and the ensuing diabetes.
All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of A*STAR Biomedical Sciences Institutes.
Synaptotagmin-7 KO mice
The synaptotagmin-7 KO mice were generated as previously described . All mice used in this study were bred and housed in our animal care facilities. At 12 weeks of age, synaptotagmin-7 KO and control male mice were each divided into two groups and were fed either an HFD or a low-fat diet (LFD, catalogue no. D12451 and D12450B, respectively; Research Diets, New Brunswick, NJ, USA) for 14 weeks. Body weight and resting glucose levels were measured every 1 or 2 weeks.
Blood glucose, insulin and glucagon measurements
Blood samples (4 μl) for both resting (non-fasted) synaptotagmin-7 KO and control mice were taken from the tail vein every 1 or 2 weeks. Blood glucose concentrations were determined by using an Accu-check advantage glucometer (Roche, Mannheim, Germany). Resting insulin and glucagon levels were measured at 4 and 14 weeks of HFD feeding after 2 h of fasting (to avoid postprandial insulin increase) by using mouse endocrine multiplex kits (Linco Research, St Charles, MO, USA). Fasting glucose and insulin levels at 4 and 14 weeks of HFD feeding were measured in mice that had been fasted overnight.
Glucose tolerance tests, insulin tolerance tests and in vivo insulin secretion measurements
Glucose tolerance tests (GTT), insulin tolerance tests (ITT) and in vivo insulin secretion measurements were performed on synaptotagmin-7 KO and control mice after 14 weeks of HFD or LFD feeding essentially as previously described . Plasma insulin concentrations were determined by Ultrasensitive Mouse Insulin enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden). The AUC was calculated by using Interactive Data Language (IDL) based on a five-point Newton–Cotes integration formula. For the ITT, data are presented as a percentage of the basal glucose level.
In vivo glucagon secretion measurements
Mice were fasted for 2 h with free access to water before they were weighed and injected i.p. with insulin (Actrapid) at 1 U/kg of body weight. Blood samples of ∼35 μl were collected from the tail vein for determination of blood glucose and plasma glucagon levels before and 20 and 40 min after the injection. Blood glucose was determined using an Accu-check advantage glucometer (Roche), and plasma glucagon using a glucagon RIA (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. AUC was calculated by following the trapezoid rule after baseline subtraction.
Islet isolation and glucagon secretion measurements
Islets were isolated from synaptotagmin-7 KO and control mice at the end of 14 weeks of HFD feeding by liberase (Roche) digestion, and cultured in RPMI Advanced (Invitrogen, Auckland, New Zealand) supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/l l-glutamine, 1% penicillin–streptomycin and 15 mmol/l HEPES. All subsequent handling was performed in Krebs–Ringer–HEPES (KRH) medium containing (in mmol/l): 130 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.56 CaCl2, 1 mg/ml BSA and 20 mmol/l HEPES (pH 7.4) supplemented with 10 mmol/l glucose. Similar-sized islets from a single mouse (ten islets per batch) were first incubated in 200 μl of KRH buffer containing 1 mmol/l glucose for 15 min at 37°C. The medium was collected and islets were further incubated in 200 μl of KRH containing 10 mmol/l glucose for 15 min. Glucagon concentrations in the two fractions were measured using a Glucagon RIA (Linco Research). Inhibition of glucagon secretion at 10 mmol/l glucose was expressed as the percentage of stimulated secretion at 1 mmol/l glucose.
Pyruvate tolerance tests
Pyruvate tolerance tests were performed in HFD-fed synaptotagmin-7 KO and control mice after 24 h of fasting. The mice were injected i.p. with pyruvate (2 g/kg body weight), and blood glucose levels were determined before and 30, 60, 90 and 120 min after injection .
Liver samples from fasted and non-fasted mice on the LFD and fasted mice on the HFD were snap-frozen in liquid nitrogen. Approximately 50 mg from each sample was boiled in 1 ml of 30% KON for 20 min and centrifuged at 16,000 g for 10 min. After protein separation, glycogen was precipitated by 100% ethanol, centrifuged at 16,000 g for 5 min, washed and dissolved in 100 μl of distilled water. Glycogen concentration was measured using a Glycogen Assay Kit (Biovision, Mountain View, CA, USA).
Data are presented as mean ± SEM. Statistical comparisons were made by two-tailed Student’s t test or by ANOVA followed by Tukey’s test. The significance limit was set at p < 0.05.
HFD-induced obesity and hyperglycaemia in control mice
Continued glucose intolerance in synaptotagmin-7 KO mice after HFD feeding
Synaptotagmin-7 KO mice on a normal chow diet exhibited normal fasting blood glucose levels, but glucose intolerance during the GTT . We first tested fasting glucose levels after 4 and 14 weeks of HFD feeding in synaptotagmin-7 KO and control mice. Feeding the HFD led to elevated fasting glucose levels in both genotypes when compared with age-matched mice that were fed the LFD (data not shown). However, fasting glucose levels were not different between synaptotagmin-7 KO and control mice after either 4 weeks (6.5 ± 0.4 vs 6.5 ± 0.2 mmol/l, synaptotagmin-7 KO vs control; n = 10 for each genotype, NS) or 14 weeks of HFD feeding (7.5 ± 0.5 vs 7.9 ± 0.7 mmol/l, synaptotagmin-7 KO vs control; n = 10 for each genotype, NS).
Impaired insulin sensitivity in HFD-fed synaptotagmin-7 KO and control mice
Compensatory enhancement of insulin secretion in HFD-fed synaptotagmin-7 KO and control mice
Lack of inhibition of glucagon secretion by high glucose in HFD-fed control mice
Increased hepatic glucose production in HFD-fed control mice
Type 2 diabetes develops when pancreatic beta cells fail to secrete sufficient insulin to meet peripheral insulin demands . Because of the beta cell- or insulin-centric view of the disease, overwhelmingly more efforts have been devoted to the study of beta cell functions and dysfunctions and of insulin secretion regulation and failure in normal physiology and during disease progression than to the study of pancreatic alpha cells and glucagon . Glucagon is closely involved in the control of glucose levels by regulating glucose production in the liver, and forms a yin–yang partnership with insulin in the regulation of glucose homeostasis. Although increased glucagon secretion is commonly described in diabetes, it is rarely mentioned in prediabetic conditions [1, 2, 8, 9]. Moreover, elevated glucagon secretion is usually referred to as a consequence of diabetes, and an active role of glucagon in promoting hyperglycaemia remains controversial or ignored [2, 9, 10].
An alternative to the insulin-centric view in diabetes development is the bi-hormonal abnormality hypothesis proposed by Unger and Orci in 1975 . This model states that the major consequence of absolute or relative insulin deficiency is glucose underutilisation, and that absolute or relative glucagon excess is the principal factor in the overproduction of glucose in diabetes . In support of the bi-hormonal model, a number of studies have demonstrated that pancreatic alpha cells are hyporesponsive to glucose-induced suppression and hyperresponsive to the stimulatory effects of amino acids in type 2 diabetes, although these defects in alpha cell function could also be considered as secondary to defective glucose sensing in beta cells (see review and references cited in ).
Synaptotagmin-7 KO mice show impaired insulin secretion and glucose intolerance , so we expected they would develop hyperglycaemia much sooner when combined with HFD-induced insulin insensitivity. Unexpectedly, however, defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion. The HFD-fed control mice, which showed an extent of reduced insulin sensitivity similar to that seen in the HFD-fed synaptotagmin-7 KO mice, but significantly elevated insulin secretion, developed resting hyperglycaemia, with a concurrent increase and dysregulation in glucagon secretion. HFD-fed mice of both genotypes showed delayed glucose clearance, indicating progressive beta cell dysfunction . Accordingly, both synaptotagmin-7 KO and control mice became more glucose-intolerant. Why did the control mice, but not the synaptotagmin-7 KO mice, develop hyperglycaemia even though their beta cell dysfunction and glucose intolerance were less severe than in synaptotagmin-7 KO mice? There are two likely possibilities: first, synaptotagmin-7 KO mice developed insulin resistance at a slower pace than the control mice, and thus the onset of hyperglycaemia was delayed. We evaluated insulin sensitivity by an ITT and found that synaptotagmin-7 KO mice had higher insulin sensitivity at the end of 4 weeks of HFD feeding (Fig. 3a), although the difference in insulin sensitivity disappeared at the end of 14 weeks of HFD feeding (Fig. 3b). The ITT is a commonly used method of assessing insulin sensitivity; however, the gold standard method is hyperinsulinaemic euglycaemic clamping. Future studies are needed to determine to what extent delayed insulin resistance contributes to protecting synaptotagmin-7 KO mice from developing hyperglycaemia. Second, synaptotagmin-7 KO mice continued to maintain their low glucagon level and glucose inhibition of glucagon secretion even after prolonged HFD feeding, while the HFD-fed control mice exhibited a significantly higher glucagon level and loss of glucose inhibition of glucagon secretion, and the elevated glucagon level led to increased hepatic glucose production (Fig. 6) and the consequent hyperglycaemia.
Glucagon secretion regulation is highly complex. Besides the intrinsic control mechanisms in the regulation of glucagon production, secretory granule biogenesis and glucagon granule exocytosis, external factors, such as paracrine factors from neighbouring beta and delta cells, and neuroendocrine factors also play major roles in the regulation of glucagon secretion [1, 18]. Insulin , along with substances released together with insulin, such as γ-aminobutyric acid [20, 21], islet amyloid polypeptide , Zn2+  and ATP , suppresses the function of alpha cells (for review, see [1, 25]). One hypothesis regarding glucose inhibition of glucagon secretion is that such action is mediated through insulin or its co-released factors [26, 27]. Interestingly, glucose-induced insulin secretion in synaptotagmin-7 KO mice is impaired , which should result in less pronounced inhibition of glucagon. Instead, these mice demonstrate adequate suppression of glucagon release in the presence of high glucose, indicating that inhibition of glucagon secretion by high glucose is also mediated by other factors. Further studies on the regulation of glucagon secretion in synaptotagmin-7 KO mice may aid in understanding how high glucose inhibits glucagon secretion, and in providing valuable insights into mechanisms of dysregulated glucagon secretion in diabetes.
Dysregulation of glucagon secretion is linked to dysfunction of beta cells, but the extent to which insulin secretion must be impaired to cause such an effect is unknown. Therefore, it is possible that dysregulation of glucagon secretion is present at even early stages of beta cell dysfunction, long before diabetes is diagnosed.
In summary, our data show that combined beta cell dysfunction and insulin insensitivity may not be sufficient to induce hyperglycaemia without dysregulated glucagon secretion, and thus reveal a previously underappreciated role of the regulation of glucagon secretion in the development of diabetes. Increased glucagon secretion does not merely aggravate hyperglycaemia at late stages of diabetes, but also actively promotes diabetes development at early stages of diabetes pathogenesis. Our study thus suggests that glucagon control may be an important therapeutic strategy for the management of hyperglycaemia and diabetes.
We thank C. Li for discussions and advice and Y. Liu, J. Kusunoki, G. Smith and P. Shepherd for help with hepatic glucose production measurements. This study was supported by intramural funding from A*STAR (Agency for Science, Technology and Research, Singapore, Republic of Singapore) Biomedical Research Council (W. Han).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.