882 Diabetes Volume 69, May 2020 The Limited Role of Glucagon for Ketogenesis During Fasting or in Response to SGLT2 Inhibition Megan E. Capozzi,1 Reilly W. Coch,1,2 Jepchumba Koech,1 Inna I. Astapova,1,2 Jacob B. Wait,1 Sara E. Encisco,1 Jonathan D. Douros,1 Kimberly El,1 Brian Finan,3 Kyle W. Sloop,4 Mark A. Herman,1,2,5 David A. D’Alessio,1,2 and Jonathan E. Campbell1,2,5 Diabetes 2020;69:882–892 | https://doi.org/10.2337/db19-1216 Glucagon is classically described as a counterregula- the oxidation of fatty acids, a shift in fuel utilization that tory hormone that plays an essential role in the pro- coordinates energy needs and glucose production (2). The tection against hypoglycemia. In addition to its role in actions of glucagon to increase lipid oxidation, including the regulation of glucose metabolism, glucagon has the production of ketone bodies that is a downstream end been described to promote ketosis in the fasted state. point of this process, have been defined by numerous – Sodium glucose cotransporter 2 inhibitors (SGLT2i) are experiments with cultured hepatocytes (3–5). Moreover, a new class of glucose-lowering drugs that act primarily the classic studies of Gerich et al. (6), using somatostatin to in the kidney, but some reports have described direct reduce circulating glucagon and mitigate diabetic ketoaci- effects of SGLT2i on a-cells to stimulate glucagon se- dosis (DKA), add to the now ingrained belief that glucagon cretion. Interestingly, SGLT2 inhibition also results in increased endogenous glucose production and ketone has both glucogenic and ketogenic activities. – production, features common to glucagon action. Here, Sodium glucose cotransporter 2 inhibitors (SGLT2i) we directly test the ketogenic role of glucagon in mice, are a new class of medications used in type 2 diabetes METABOLISM demonstrating that neither fasting- nor SGLT2i-induced (T2D) that have been implicated in causing ketosis. These ketosis is altered by interruption of glucagon signaling. drugs act by blocking reabsorption of glucose in the Moreover, any effect of glucagon to stimulate ketogen- proximal tubules of the kidneys, leading to substantial esis is severely limited by its insulinotropic actions. glucosuria (7). In addition to improved glycemia and Collectively, our data suggest that fasting-associated modest weight loss, reductions of adverse cardiovascular ketosis and the ketogenic effects of SGLT2 inhibitors and renal complications of T2D have been demonstrated occur almost entirely independent of glucagon. with use of SGLT2i in recent clinical trials (8). However, it is now clear that SGLT2i have consistent effects to raise circulating ketone bodies and, in uncommon instances, can The current view of glucagon physiology is that it acts as precipitate DKA, particularly among insulinopenic patients the catabolic counterbalance to insulin, maintaining ade- (9–16). While the mechanism by which SGLT2i causes quate amounts of blood glucose in states where it is ketosis has not been established, patients treated with threatened, such as starvation, exercise, and hypoglycemia these agents have elevations in circulating glucagon and (1,2). Glucagon is secreted into the hepato-portal circula- increased hepatic glucose production. Remarkably, there is tion and has well-described effects to promote hepatic some evidence that SGLT2 is expressed by the islet a-cells glucose production by increasing glycogenolysis and glu- that secrete glucagon, leading to the hypothesis that in- coneogenesis (1). Another important effect of glucagon in creased glucagon mediates SGLT2i effects in the liver the liver is to reduce glucose consumption by increasing (17–19). 1Duke Molecular Physiology Institute, Duke University, Durham, NC This article contains Supplementary Data online at https://diabetes.diabetesjournals 2Division of Endocrinology, Department of Medicine, Duke University, Durham, NC .org/lookup/suppl/doi:10.2337/db19-1216/-/DC1. 3 Novo Nordisk Research Center, Indianapolis, IN © 2020 by the American Diabetes Association. Readers may use this article as 4 Diabetes and Complications, Lilly Research Laboratories, Eli Lilly and Company, long as the work is properly cited, the use is educational and not for profit, and the Indianapolis, IN work is not altered. More information is available at https://www.diabetesjournals 5 Department of Pharmacology and Cancer Biology, Duke University, Durham, NC .org/content/license. Corresponding author: Jonathan E. Campbell, [email protected] Received 9 December 2019 and accepted 27 January 2020 diabetes.diabetesjournals.org Capozzi and Associates 883 Ketogenesis is considered to be controlled by the islet 8- to 20-week-old mice of the C57Bl6 background. Mice hormones,insulinandglucagon(20).Insulinstrongly were housed under a 12-h light/dark cycle and provided inhibits ketosis, predominantly by reducing lipolysis in free access to a normal chow diet. WT mice were either adipocytes and reducing the supply of free fatty acids, the bred in-house or purchased from The Jackson Laboratory. 2 2 substrate for ketone body production. In addition, insulin Gcg / mice, described previously (30), were generated by 1 2 1 1 may have direct effects at the level of the hepatocyte by breeding Gcg / mice. Cage-matched Gcg / mice served lowering intracellular cAMP (21). In contrast, glucagon as controls. potently increases cAMP in hepatocytes, a signal that has been tied to both lipid and glucose metabolism (22). Dapagliflozin Treatment Consequently, the plasma insulin-to-glucagon ratio has Mice were fasted for 3 h in the morning before receiv- long been purported to dictate the rates of ketogenesis ing orally administered dapagliflozin (10 mg/kg) in PBS. (23). For example, it has been proposed that DKA second- Plasma was collected 3 or 6 h post–dapagliflozin treat- ary to SGLT2 inhibition occurs through a drug-induced ment. Mice had ad libitum access to water throughout the increase in glucagon in an insulinopenic patient (10). experiments. For glucagon studies, glucagon (20 mg/kg) However, this explanation was recently put in doubt in was injected 3 h post–dapagliflozin treatment. For studies a study that implicated dehydration and insulinopenia as using Ab-4, Ab-4 (10 mg/kg) was administered 24 h prior necessary components of dapagliflozin-induced ketosis in to dapagliflozin treatment. a rat model (24). In this study, the authors demonstrated that glucagon was elevated in response to SGLT2i inde- Fast and Refeed pendent of hydration state, while ketosis in this study Food was removed from mice at the end of the light cycle. required dehydration in the setting of insulinopenia, rais- Plasma was collected at the beginning of the following light ing some doubt as to the contribution of glucagon to cycle after ;12–14 h of fasting. Food was then provided SGLT2i-induced ketosis. and plasma collected after 30–60 min of refeeding. For We recently challenged the primacy of a catabolic, studies using Ab-4, 10 mg/kg i.p. was administered 24 h counterregulatory role for glucagon by demonstrating that prior to fasting plasma collection. glucagon has a significant impact on glucose metabolism as an insulin secretagogue (25,26), an observation also noted S661 and STZ Treatment by other investigators (27,28). In the current study, we S661 was injected (20 nmol/mouse i.p.). For STZ treat- sought to directly investigate the importance of glucagon ment, mice were injected for two consecutive days with in the regulation of physiologically (fasting) or pharma- 120 mg/kg i.p. STZ in sodium citrate buffer. Control mice cologically (SGLT2i) induced ketosis. Herein, we demon- were injected with sodium citrate buffer. Mice received strate that loss of glucagon signaling does not modulate subsequent doses of STZ as needed until fed glycemia ketone production in response to fasting or SGLT2i. was $350 mg/dL. For experiments using STZ/S661, only Importantly, we demonstrate that glucagon is only capable mice with postfeeding blood glucose .400 mg/dL were of increasing ketone production in the context of complete used. For STZ/S661 plus dapagliflozin experiments, mice loss of insulin signaling. Moreover, we reveal that SGLT2i- were fasted for 3 h prior to dapagliflozin administration at induced ketosis occurs independently of the actions of t 5 0. S661 was administered 1 h before blood glucose insulin and glucagon. and ketone measurement at t 5 3 h. For STZ/S661 plus glucagon experiments, mice were fasted for 5 h and then RESEARCH DESIGN AND METHODS injected at t 5260 min with S661, and glucagon was Reagents injected 1 h later at t 5 0. Dapagliflozin was purchased from Advanced ChemBlocks Inc. and prepared fresh in PBS. Glucagon receptor–blocking Metabolic and Hormone Measurements antibody (Ab-4) was prepared in PBS and kindly provided by Blood glucose was measured from tail blood using a gluco- Eli Lilly and Company. Glucagon (Gcg) was purchased from meter (Contour). b-Hydroxybutyrate (bOHB) was mea- Sigma-Aldrich, and stocks were prepared in 0.3% acetic sured using a ketometer (Precision Xtra), which we validated acid. Epinephrine was purchased from Sigma-Aldrich and by the measurement of known concentrations of bOHB prepared fresh in PBS for each experiment. Streptozotocin spiked into either plasma from a fed mouse to achieve low [STZ] was purchased from Sigma-Aldrich and prepared in bOHB levels. Values from the ketometer averaged 614.25% sodium citrate buffer. S661, an insulin receptor antagonist of the expected concentrations across a range of bOHB (29), was prepared in PBS and was kindly provided by B.F. from 0 to 2 mmol/L. Assay variability was 12.9% when the from Novo Nordisk. same sample was read three times. Plasma nonesterified fatty acid (NEFA) concentrations were measured by enzy- Animals matic assay (Wako Diagnostics). Plasma insulin and glu- All mouse procedures were approved and performed in cagon were measured by ELISA (Mercodia). Insulin and accordance with the Duke University Institutional Animal glucagon levels that were undetectable were assigned the Care and Use Committee.