Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1-/- mouse model

Mangala M Soundarapandian; Christine A Juliana; Jinghua Chai; Patrick A Haslett; Kevin Fitzgerald; Diva D De León

doi:10.1371/journal.pone.0236892

. 2020 Jul 31;15(7):e0236892. doi: 10.1371/journal.pone.0236892

Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1^-/- mouse model

Mangala M Soundarapandian ^1,^*, Christine A Juliana ², Jinghua Chai ², Patrick A Haslett ¹, Kevin Fitzgerald ¹, Diva D De León ^2,^3,^*

Editor: Michael Bader⁴

PMCID: PMC7394442 PMID: 32735622

Abstract

There is a significant unmet need for a safe and effective therapy for the treatment of children with congenital hyperinsulinism. We hypothesized that amplification of the glucagon signaling pathway could ameliorate hyperinsulinism associated hypoglycemia. In order to test this we evaluated the effects of loss of Prkar1a, a negative regulator of Protein Kinase A in the context of hyperinsulinemic conditions. With reduction of Prkar1a expression, we observed a significant upregulation of hepatic gluconeogenic genes. In wild type mice receiving a continuous infusion of insulin by mini-osmotic pump, we observed a 2-fold increase in the level of circulating ketones and a more than 40-fold increase in Kiss1 expression with reduction of Prkar1a. Loss of Prkar1a in the Sur1^-/- mouse model of K_ATP hyperinsulinism significantly attenuated fasting induced hypoglycemia, decreased the insulin/glucose ratio, and also increased the hepatic expression of Kiss1 by more than 10-fold. Together these data demonstrate that amplification of the hepatic glucagon signaling pathway is able to rescue hypoglycemia caused by hyperinsulinism.

Introduction

Congenital Hyperinsulinism (HI) is a genetic disorder of the pancreatic β-cells that causes dysregulated insulin secretion and persistent hypoglycemia. There are at least nine different genetic subtypes of hyperinsulinism, but the most common and severe form is caused by inactivating mutations in ABCC8 or KCNJ11, the genes encoding the two component of the β-cell K_ATP channel [1]. HI is the most common cause of persistent hypoglycemia in neonates, infants and children and is associated with high risk for serious complications (seizures, intellectual deficiencies, brain damage, and coma) with the rate of neurodevelopmental deficits in these patients as high as 48% [2]. Only about 40% of patients respond to the limited number of existing therapies and this is not without significant limitations and side effects [3–5]. Thus, there is a serious and unmet need for development of safe and effective therapies for treatment of HI.

The liver offers several possible avenues for therapeutic intervention for HI because of its central role in systemic glucose homeostasis through regulation of glycogen storage, gluconeogenesis, and suppression of insulin secretion through production of the hepatokine, kisspeptin1 (KISS1). Protein Kinase A (PKA) is a serine/threonine kinase that is inactive while bound to a dimer composed of regulatory subunits (e.g. Prkar1a) [6]. When plasma glucose concentration falls below 65–70 mg/dL, glucagon is secreted from pancreatic alpha cells and binds to its receptor on hepatocytes, which leads to binding of cAMP to the regulatory subunits, conformational changes, and the release of active PKA [6, 7]. Active PKA promotes glycogenolysis through glycogen phosphorylase kinase (PhK) and gluconeogenesis through the increased expression of key genes [phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a, PGC1α)] [7–13]. Stimulation of cAMP-PKA-CREB signaling by glucagon upregulates KISS1. Kiss1 is a secreted hepatokine that signals via the kisspeptin receptor on pancreatic β-cells to decrease insulin secretion [14]. Thus, due to the central role played by PKA in glucagon signaling, its disinhibition by specific depletion of Prkar1a in the liver leads to a significant abrogation of glucose stimulated insulin secretion and increased plasma glucose [14].

Given the ability of disinhibited PKA to increase plasma glucose levels, we wanted to evaluate the effect of Prkar1a reduction in the liver on hypoglycemia caused by hyperinsulinemic conditions. We found that siRNA mediated loss of Prkar1a increased ketone levels and induced KISS1 expression in the context of exogenous insulin treatment. Strikingly, in the Sur1^-/- mouse model (lacking the Sulfonylurea receptor1 subunit of the K_ATP channels and thus a model of K_ATP hyperinsulinism), we found that reduction of Prkar1a resulted in a significant decrease in plasma insulin and an attenuation of fasting hypoglycemia. These findings identify a new critical nexus for development of therapies for treatment of hypoglycemia in children of HI.

Materials and methods

Animal studies

Wildtype rodent studies were conducted at Alnylam Pharmaceuticals and Sur1^-/- mice studies were conducted at the Children’s Hospital of Philadelphia and approved by the Institutional Animal Care and Use Committee (IACUC) of the respective institutions. Method of euthanasia: Inhalation of carbon dioxide (CO2) followed by cervical dislocation.

The generation and genotyping of Sur1^-/- mice were previously described [15]. Sur1^-/- mice are bred and maintained in our mouse colony on a C57Bl/6 genetic background for experiments. 7 male mice, eight to ten weeks old, Sur1^-/- mice were used in each group in all experiments. Mice were maintained on a 12:12-h light-dark cycle and were fed a standard rodent chow diet with free access to food and water. Animal welfare checks were completed once per day. No adverse effects to these experiments were observed or required analgesia.

Wildtype (WT) C57BL/6J female mice were acquired from Jackson laboratories, (Bar Harbor, ME) at 8–10 weeks of age. 6–8 WT mice were used per group in experiments. The each dot in the scatter plots represent one animal each. Mice were allowed to acclimate to a 12:12-h light-dark cycle, housing humidity and temperature for at least 72 hours prior to initiation of the study. Mice were group-housed and maintained on a standard rodent diet (LabDiet, Picolab rodent diet 5053). All animals were provided free access to drinking water. Animal welfare checks at Alnylam are conducted every 24hrs. We did not observe any adverse events at the dose of siRNA used in these studies.

Glycogen staining

For glycogen staining the mice were subcutaneously injected with vehicle control (1X PBS) or indicated doses of Prkar1a siRNA. Glycogen was detected in liver sections following a standardized periodic acid Schiff (PAS) staining technique. Briefly, livers fixed in 10% neutral buffered formalin were embedded in paraffin blocks. 4-micron sections were collected on glass slides, de-paraffinized, incubated with 0.5% periodic acid for 7 min, rinsed in water, and placed in Schiff’s reagent for 15 min. Finally, slides were washed with water and nuclei were stained with Modified Mayer’s Hematoxylin. 1% Diastase was used to verify that staining was specific for glycogen. All reagents were obtained from Rowley Biochemical.

Evaluation of glucose homeostasis

For Sur1^-/- mice random fed plasma glucose and morning fasting (16 hrs) plasma glucose, plasma betahydroxybutyrate and plasma insulin concentrations were measured at baseline and at days 4 and weekly for 3 weeks after treatment. Glucose tolerance testing was carried after a 16 hour fast by administering 2g/kg of dextrose intraperitoneally. Plasma glucose and betahydroxybutyrate concentrations were measured using a hand-held glucose meter (NOVA, Nova Biomedical). Plasma insulin was measured by ELISA (ALPCO; catalogue #80-INSMS-E01).

For wildtype mice, fasting plasma glucose and betahydroxybutyrate were assessed after a 5hr morning fast using blood from a tail nick using handheld glucose meter (ACCU-CHEK Aviva, Roche) or ketone meter (Precision Xtra, Abbott). Fed glucose was measured at the end of the dark cycle. Pyruvate tolerance test was carried out after a 14hr overnight fast by administering 1.5 g/kg sodium pyruvate intraperitoneally.

Osmotic pump implantation

Alzet Micro-osmotic pumps, model 1002 with pumping rate 0.25μl/hr (DURECT Corporation) were filled with Humulin (Eli Lilly) diluted in 1X sterile PBS to allow Insulin release of 0.2 or 0.3U/day. Pumps were implanted subcutaneously under isoflurane anesthesia. The mice were allowed to recover and their plasma glucose was monitored using glucose meter (ACCU-CHEK Aviva, Roche). Plasma insulin levels was measured by ELISA (Crystal Chem, Ultrasensitive mouse insulin ELISA kit, Catalogue #90080, Lot # 16SEUMI411 that detects both human and mouse insulin)

siRNA injection studies

For Prkar1a knockdown studies the mice were subcutaneously injected with vehicle control (1X PBS) or 1 mg/kg Prkar1a siRNA every 2 weeks unless otherwise indicated. The endpoints were assessed from serum or liver tissue 28 days post dosing unless otherwise indicated.

The siRNA targeting Prkar1a was designed, synthesized, and liver targeted by Alnylam Pharmaceuticals, as previously described [16, 17]. The siRNA was designed to target mouse Prkar1a mRNA, NM_021880.

AD-76409 targets position 865–885, 5’-GAUGUAUGAAGAAUUCCUUAGUA-3’

AD-76410 targets position 873–893, 5’-AAGAAUUCCUUAGUAAAGUGUCU-3’

AD-76411 targets position 1394–1414, 5’-AAAAGUUGCUUUAUUGCACCAUU-3’

RNA isolation and qRT-PCR

Total RNA was isolated from liver tissue using the miRNeasy kit (Qiagen) following manufacturer’s protocols. 1ug of RNA from each sample was reverse transcribed using the High capacity Reverse transcription kit (Invitrogen). Quantitative real time PCR was performed on the cDNA using Roche light cycler and the Lightcycler 480 master mix (Roche). All experimental samples were analyzed and normalized with the expression level of a reference gene [calculated by second-derivative maximum by applying the 2−(ΔΔCt) method]. The following Taqman assays (Invitrogen) were used: Prkar1a (Mm00660315_m1), G6PC (Mm00839363_m1), PEPCK (Mm01247058_m1), Ppargc1a (Mm01208835_m1), Kiss1 (Mm03058560_m1), GCK (Mm00439129_m1) and Gapdh (4352339E)

Western blot analysis

Livers were homogenized in RIPA buffer along with protease inhibitors. Total cell lysates denatured by boiling in 2x Laemmli buffer were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The blots were hybridized to specific antibodies overnight at 4°C and the bands were detected using fluorescence imaging using the Licor system. The following antibodies were used: Prkar1a (BD biosciences, Catalog #610609, 1:500), PKAC (BD Biosciences, Catalog #610980, 1:2000), β-actin (Abcam, Catalog #ab8227, 1:2000), PKA substrate (Cell Signaling, Catalog #9624, 1:1000), Fluorescence conjugated secondary antibodies (Licor, Goat anti-rabbit, Catalog #926–32211 and Donkey anti-mouse, Catalog #926–680721:5000).

Statistics

Statistical analyses were performed on GraphPad Prism 6 software. All results are presented as mean ± standard error of the mean (SEM). The level of significance was set at P < 0.05. For multiple measurements data were analyzed using 2-way ANOVA Repeated Measures, Tukey’s multiple comparison test. Single time end points data were analyzed using one-way ANOVA or Student’s t-test.

Results

Loss of Prkar1a activates PKA and downstream liver gluconeogenesis

In order to achieve reduction of Pkar1a in the liver, mice were subcutaneously injected with a liver-targeted siRNA directed against Prkar1a or PBS control. Liver extracts harvested at 10 or 28 days post injection with either 0, 0.5, 1, 3, or 5 mg/kg doses of siRNA revealed a dose dependent suppression of Prkar1a mRNA. The lowest dose of siRNA (0.5 mg/kg) demonstrated a ~60% or ~75% reduction of Prkar1a mRNA expression at 10 and 28 days post initial injection, respectively (Fig 1A). A ~90% reduction of Prkar1a mRNA is achieved by 3 or 5 mg/kg doses at both time points (Fig 1A). Subcutaneous injection of siRNA (1 mg/Kg, bi weekly) directed against Prkar1a also effectively reduced PRKAR1A protein while not having a significant effect on catalytic PKA (PKAc) protein levels (Fig 1B).

Fig 1 — WT mice were injected subcutaneously with siPrkar1a (AD-76410) at the denoted mg/kg dose. Liver extracts were collected from siRNA injected mice or PBS controls at either 10 or 28 days post-injection for (A) qRT-PCR analysis of Prkar1a mRNA expression, or (B) protein for western blot analysis of PRKAR1A and catalytic PKA expression with calculated relative densities normalized to β-actin from liver extracts from WT mice 28 days post-injection. (C) qPCR analysis of mRNA expression of gluconeogenesis targets G6Pase, PEPCK, and Ppargc1a of RNA extracted from liver extracts of bi-weekly siPrkar1a (1 mg/kg) injected WT mice compared to vehicle controls. (D) Glycogen staining of liver tissue in WT mice injected with the denoted dose of siPrkar1a. (E) Pyruvate tolerance test in WT mice administered 21 days after injection with siPrkar1a or PBS control after a 14 hour overnight fast, with calculated area under the curve (AUC). (n = 6 mice/group) Data represent mean +/- SEM. *, p ≤ 0.05; **, p ≤ 0.01 compared to PBS control.

A previous study found increased glycogenolysis and gluconeogenesis in hepatic cells from mice expressing constitutively active PKA [18]. Here we demonstrate that direct loss of the PKA regulatory subunit, Prkar1a, increased PKA activity as evidenced by an increase in phosphorylation of PKA substrates (S1 Fig) and upregulation of expression of downstream targets important for gluconeogenesis: glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and PPARγ coactivator-1α (PGC-1α) (Fig 1C). Functionally, the loss of Prkar1a resulted in an increase in glycogenolysis and gluconeogenesis as observed by a significant reduction in liver glycogen (Fig 1D) and though not statistically significant, a trend of increased conversion of the gluconeogenic precursor pyruvate to glucose in a pyruvate tolerance test (Fig 1E) was observed in mice injected with siPrkar1a. These results demonstrate the essential role of Prkar1a regulation of PKA in glycogenolysis and gluconeogenesis, as well as show that reduction of Prkar1a can mimic the effects of glucagon signaling through the increase of liver glucose output.

Reduction of Prkar1a leads to hyperglycemia in mice

As siRNA mediated loss of Prkar1a caused increased glycogenolysis, we assessed the effect on plasma glucose concentration in mice injected with siPrkar1a. Three different siRNAs directed at Prkar1a resulted in a decrease of Prkar1a transcript with different potencies. AD-76409 and AD-76410 achieved ~90% reduction of Prkar1a mRNA, while AD-76411 only resulted in ~30% loss (Fig 2A). Significant hyperglycemia was observed in both fasting and fed states compared to control starting at 10 days and enduring until 28 days post-injection in wildtype mice treated with siPrkar1a sequences in a manner correlating with the potency of Prkar1a reduction (Fig 2B and 2C). Injection of AD-76410 at different doses (0.5, 1, 3, or 5 mg/Kg) over 28 days at 2 week intervals demonstrated a dose dependent increase in plasma glucose concentration compared to controls. All siPrkar1a doses increased plasma glucose concentration in a dose dependent manner, with the highest doses (3 and 5 mg/Kg) resulting in plasma glucose concentrations of ~400 mg/dL and the lowest dose (0.5 mg/Kg) with plasma glucose concentrations of ~300 mg/dL by day 21 post-injection (Fig 2D) compared to PBS injected control mice in which plasma glucose concentration never exceeded 200 mg/dL. In mice receiving a single dose of siRNA, only the highest doses (5, 3, and 1 mg/Kg) resulted in a significant increase in plasma glucose for the entire 28 days of assessment (S2A Fig). The most potent siRNAs against Prkar1a (AD-76409 and AD-76410) led to a significant upregulation of gluconeogenesis genes PEPCK and PGC-1α (Fig 2E). AD-76409 was also able to significantly increase G6Pase expression. Additionally, ketone levels (β-hydroxybutyrate) trended higher, although not statistically significant in mice treated with the highest siRNA doses (S2B Fig). There was no significant change in weight in these mice compared to controls (S2C Fig). The application of three independent siRNAs directed against Prkar1a and the correlating effects on plasma glucose based dose dependence and potency of reduction indicate the siRNA mediated knockdown of Prkar1a requires reduction of greater than 30% to increase plasma glucose levels.

Fig 2 — WT mice were injected subcutaneously with siRNA (1 mg/kg) directed against Prkar1a (AD-76409, AD-76411, AD-76410) with varying potencies and compared to PBS control. (A) Liver extracts were collected from siRNA injected mice or PBS controls for qRT-PCR analysis of Prkar1a mRNA expression. (B) Fasting plasma glucose levels were assessed after a 16 hour overnight fast at 0, 3, 10, 14, and 28 days post dosing with denoted siPrkar1a siRNAs. (C) Fed plasma glucose levels were assessed with ad libitum feeding at 0, 14, and 28 days post dosing with denoted siPrkar1a siRNAs. (D) Mice were injected with siRNA (AD-76410, 1 mg/kg) directed against Prkar1a every 2 weeks (Q2W) at the denoted mg/kg dose and plasma glucose levels were assessed at 0, 3, 10, 14, 21, and 28 days post dosing. (E) Liver extracts were collected from siRNA (AD-76410, 1 mg/kg) injected mice or PBS controls for RNA and qPCR analysis of mRNA expression of gluconeogenesis targets G6Pase, PEPCK, and Ppargc1a. (n = 6 mice/group) Data represent mean +/- SEM. *, p ≤ 0.05; **, p ≤ 0.01 compared to PBS control.

Prkar1a loss increases plasma ketones during hyperinsulinemic conditions and induces Kiss1 expression

The ability of Prkar1a reduction to significantly increase plasma glucose made it an interesting target for treatment of hypoglycemic conditions. To determine the effectiveness of loss of Prkar1a on hyperinsulinemic hypoglycemia, mice were implanted with an osmotic pump delivering either vehicle, 0.2U or 0.3U/day of insulin 16 days after they were injected with siPrkar1a or control. As expected, insulin administration lowered blood glucose levels (solid lines). In mice pre-treated with Prkar1a siRNA, the baseline plasma glucose was higher (similar to Fig 2B) and we observed a dose dependent attenuation but not a reversal of insulin induced hypoglycemic effects (dotted lines, (Fig 3A). Loss of Prkar1a resulted in suppression of endogenous insulin levels, but as expected, there was no effect on exogenously administered insulin levels (Fig 3B). Interestingly, treatment with siPrkar1a did result in a significant increase in ketones only during insulin-induced hypoglycemic conditions, indicating reversal of the insulin-suppressive effect on ketogenesis (Fig 3C).

Fig 3 — WT mice were injected with siPrkar1a (AD-76410, 1 mg/kg) and a subcutaneous osmotic pump delivering either vehicle (1X PBS), 0.2U, or 0.3 U insulin/day was implanted 16 days (dotted line) post siRNA injection. (A) Plasma glucose levels were assessed 0, 7, 14, 19, 22, 26, and 29 days post dosing with siPrkar1a (AD-76410, 1 mg/kg) after a 5-hour morning fast. (n = 6 mice/group). (B) Plasma insulin levels were assessed at 13 days post osmotic pump implantation after a 5-hour morning fast. (C) Plasma β-hydroxybutyrate levels were assessed at 13 days post pump implantation after a 5-hour morning fast. (D) Liver extracts were collected from siRNA injected mice (AD-76410, 1 mg/kg) or PBS controls with osmotic pump implantation for RNA and qPCR analysis of mRNA expression of Prkar1a and gluconeogenesis targets G6Pase, PEPCK, and Ppargc1a. (E) Liver extracts were collected from siRNA injected (AD-76410, 1 mg/kg) mice or PBS controls with osmotic pump implantation for RNA and assessed by qPCR analysis for Kiss1 mRNA expression level. (n = 6 mice/group) Data represent mean +/- SEM. Statistics was calculated using Tukey’s multiple comparisons test comparing PBS controls to siPrkar1a of same insulin dose. *, p ≤ 0.05; **, p ≤ 0.01.

As seen in our earlier experiments, Prkar1a reduction led to a significant increase in gluconeogenic gene expression including G6pase, PEPCK, and Ppargc1a (Fig 3D). Hypoglycemia will stimulate the release of glucagon to activate hepatic pathways to restore normoglycemia, but most likely no additive effect is seen since maximal activation of these pathways is achieved by either. A previous study identified the hepatic production of the neuro-peptide kisspeptin1 (KISS1) by glucagon stimulation that resulted in the suppression of glucose stimulated insulin secretion in β-cells [14]. Intriguingly, we found a strong induction by more than 40 fold of Kiss1 transcript with loss of Prkar1a (Fig 3E). The ineffectiveness of siPrkar1a to increase plasma glucose in this hypoglycemic model may be due to the use of an exogenous insulin source which is by definition not subject to regulation. We therefore proceeded to evaluate Prkar1a knockdown in the Sur1^-/- mouse, an endogenous hyperinsulinemic model.

Hypoglycemia is attenuated by loss of Prkar1a expression in the Sur1^-/- mouse model of hyperinsulinism

Sur1^-/- mice were injected with PBS or siPrkar1a and glucose homeostasis was evaluated in the course of 3 weeks. Fasting plasma glucose was significantly higher in siPrkar1a-treated compared to PBS-treated Sur1^-/- mice 21 days after injection (Fig 4A). Fed plasma glucose was not significantly different between the two groups (Fig 4B). Absolute insulin concentration was not significantly different in the siPrkar1a treated Sur1^-/- mice (S3A Fig), however, the insulin/ glucose ratio was significantly decreased in the siPrka1a-treated compared to PBS-treated Sur1^-/- mice (Fig 4C). Fasting ketones were not significantly different in siPrkar1a-treated compared to compared to PBS-treated Sur1^-/- mice (S3B Fig). In response to a glucose tolerance test (GTT) siPrkar1a-treated Sur1^-/- mice exhibited a significant increase of plasma glucose compared to controls as assessed by area under the curve (AUC) (Fig 4D). Insulin levels were significantly lower during the GTT in siPrkar1a-treated Sur1^-/- mice, as determined by calculation of area under the curve (AUC) (Fig 4E). Importantly, reduction of Prkar1a led to a significant increase in the transcript levels of PKA targets (PEPCK and Ppargc1a) and pancreatic β-cell signaling hepatokine kisspeptin by more than 10 fold in the liver of Sur1^-/- mice (Fig 4F and 4G). siPrkar1a was not able to overcome the suppression of G6pase in the setting of endogenous hyperinsulinemic condition. However, the ability of Prkar1a reduction to resolve fasting hypoglycemia in the Sur1^-/- hyperinsulinism model exposes new possible therapeutic targets in the liver, PKA and glucagon signaling pathways in the treatment of hyperinsulinemic hypoglycemia.

Fig 4 — *Sur1*^-/- mice were injected subcutaneously with siPrkar1a (AD-76410, 1 mg/kg). (A) Fasting plasma glucose levels were assessed after a 16 hour overnight fast at 8, 15, and 22 days post dosing. (B) Fed plasma glucose levels were measured with ad libitum feeding at 0, 3, 7, 14, and 21 days post dosing. (C) Insulin/ glucose ratios were calculated at day 8, 15, and 21 days post siRNA injection. (D) Glucose tolerance test (GTT) was administered to siPrkar1a (AD-76410, 1 mg/kg) injected mice and controls 21 days post dosing after a 16 hour overnight fast. Area under the curve (AUC) was calculated. (E) Blood samples were collected during GTT time points to assess plasma insulin levels after a 16 hour overnight fast. Liver extracts were collected from *Sur1*^-/- mice injected with siPrkar1a (AD-76410, 1 mg/kg) (green bars) or vehicle control (black bars) and assessed by qPCR for expression of (F) Prkar1a, G6pase, PEPCK (PCK), Ppargc1a or (G) Kiss1. (n = 7) Data represent mean +/- SEM. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 compared to PBS control.

Discussion

Glucagon is used acutely to treat insulin-induced hypoglycemia, but its chemical and physical instability in solution has been a serious limitation for its use for extended periods of time in hormone pumps [19, 20]. Glucagon signaling increases plasma glucose concentration by activating protein kinase A (PKA) signaling that initiates the breakdown of liver glycogen stores and also through hepatic production of Kiss1, which directly suppresses insulin secretion in the β-cell. The present findings using a combination of mouse model of HI (Sur1^-/-) and exogenous insulin administration demonstrate the importance of hepatic signaling in glucose homeostasis and the real potential of exploiting glucagon signaling as an untapped resource for treatment of HI associated hypoglycemia. Evaluation of mice in the context of hyperinsulinemic conditions by use of a mini-osmotic pump to administer exogenous insulin, demonstrated an almost doubling of circulating ketones (Fig 3). In normal fasting individuals, falling glucose concentrations stimulates ketogenesis as an alternative fuel source and this response is particularly important to prevent brain damage during hypoglycemia [21]. However, high levels of circulating insulin, such as that found in HI patients, inhibits ketogenesis and thereby removes this neuro-protective response to hypoglycemia [21–23]. Our results demonstrate that the ketogenic response can in fact be restored in hyperinsulinemic conditions by amplifying the glucagon signaling pathway sufficiently to surpass the inhibitory effects of insulin on ketogenesis.

Examination of the Sur1^-/- hyperinsulinism mouse model in the context of Prkar1a reduction, identified a distinct increase in not only fasting plasma glucose levels, but also a significant decrease in fasting insulin/glucose levels as well (Fig 4). Interestingly, the decrease in insulin concentration in relationship to glucose concentration indicates that the increase in plasma glucose is not solely the result of enhancement of glycogenolysis and gluconeogenesis but may also involve β-cell effects. The latter observation can be explained by the marked upregulation of Kiss1 expression in the liver in both the Sur1^-/- mouse model and the insulin administration model. The liver has previously been shown to have important roles in glucose homeostasis through regulation of glycogen storage, gluconeogenesis, and most interestingly by suppression of insulin secretion in the pancreatic β-cell through glucagon stimulated production of the hepatokine KISS1 [14]. Of note, there have been contradictory reports demonstrating stimulatory effects of kisspeptin on insulin secretion. Assessment of the published work reveals that studies using a physiologic nanomolar concentration of kisspeptin demonstrated an inhibitory effect on insulin secretion [14, 24, 25]. The studies that showed a stimulatory effect on insulin secretion used a supraphysiological dose in the micromolar range (generally 1uM) [26–29]. Further, studies using KISS1R^-/- mice have demonstrated that stimulation with kisspeptin beyond physiological nanomolar concentrations can occur in a KISS1R-independent mechanism [14, 30]. These results indicate potential off-target effects of kisspeptin with use of supraphysiological concentrations.

Measurement of circulating kisspeptin levels in mice and humans has been notably unreliable [31]. Unfortunately, we were unable to find reliable commercial sources for circulating kisspeptin measurements and did not investigate this mechanism further in the current study. However, we did observe decreased circulating endogenous insulin in mice dosed with Prkar1a siRNA (Figs 3B and 4E) supporting the involvement of liver-β-cell crosstalk via Kisspeptin. Glucagon deficiency and blunting of the glucagon counterregulatory response has been observed in both patients with HI and in mouse models of HI [32, 33]. The inhibitory effect of hyperinsulinism on glucagon is two-fold: mutation of the K_ATP channel affects alpha cell membrane potential/ glucagon secretion and high circulating insulin levels directly inhibiting secretion by the alpha-cell [33, 34]. Our results indicate that it is possible to overcome these glucagon inhibitory signals and to alleviate hyperinsulinemic hypoglycemia by amplifying glucagon signaling downstream of the initiating glucagon receptor binding. Direct modification of Prkar1a expression is likely not the best therapeutic option for development in patients as it can result in the formation of myxomas in internal organs due to Carney complex complications [35]. Importantly however, our results indicate that harnessing the mechanisms of amplifying glucagon signaling through the hepatic cAMP-PKA-CREB signaling nexus is a viable option with a plethora of potential for developing therapies for HI associated hypoglycemia.

Supporting information

S1 Fig. Western blot analysis of phospho-PKA substrates in liver extracts from siPrka1a injected WT mice as compared to vehicle injected controls.

WT mice were injected with siRNA (AD-76410, 1mg/kg) directed against Prkar1a or PBS control every 2 weeks until liver tissue was collected 28 days post-injection of initial dose. Western blot analysis of phospho-PKA substrates was completed on the liver extracts (n = 6).

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S2 Fig. Reduction of Prkar1a results in hyperglycemia.

(A) WT mice were injected with siRNAs directed against Prkar1a once at day 0 at the denoted mg/kg dose and plasma glucose levels were assessed at 0, 3, 7, 10, 14, 21, and 28 days post dosing. (B) Mice were injected with siRNAs directed against Prkar1a every 2 weeks at the denoted mg/kg dose and plasma β hydroxyl butyrate levels were assessed at 0, 10, and 28 days post dosing. (C) Mice injected with siPrkar1a every 2 weeks (Q2W) were weighed at 0, 10, 14, 21, and 28 days post dosing. (n = 6 mice/group) Data represent mean +/- SEM.

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S3 Fig. siPrkar1a injection in Sur1^-/- mice.

(A) In Sur1^-/- mice, fasting plasma insulin levels were measured after a 16 hour overnight fast at 8, 15, and 22 days post dosing. (B) Fasting plasma β hydroxyl butyrate levels were measured after a 16 hour overnight fast at 8 and 22 days post dosing. (n = 7) Data represent mean +/- SEM.

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S1 Raw images

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Acknowledgments

We would like to thank Tuyen Nguyen for siRNA screening, Alnylam siRNA synthesis cores for providing siRNAs used in this study, Wendell Davis and Alnylam histology group for glycogen staining and Alnylam vivarium personnel for animal husbandry.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by Alnylam Pharmaceuticals. https://www.alnylam.com MMS, PAH and KF work for Alnylam Pharamaceuticals; MMS, PAH and KF hold shares in Alnylam Pharmaceuticals stock; DDDL received funding from Alnylam Pharmaceuticals to conduct this study. MMS, PAH, KF and DDDL collaborated on the design, data collection, analysis, decision to publish and preparation of the manuscript. JC performed experiments and CAJ analyzed data and wrote the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0236892.r001

Decision Letter 0

Michael Bader

13 May 2020

PONE-D-20-09701

Amplified glucagon signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1-/- mouse model

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors describe the effects of or prkar1a knockdown in the liver in WT C57Bl/6 mice and in SUR-/- mice - a model for neonatal hyperinsulinemic hypoglycemia.

Overall the manuscript has a logical structure and flow, the methods are sound and the data and adequately performed statistical analysis support in large parts the interpretation and conclusions by the authors.

Caveats to the manuscript:

The title indicates that glucagon signaling is amplified in the liver. This is inaccurate because glucagon activates multiple signaling pathways in the liver. The studies have up-regulated the PKA arm of glucagon signaling and not glucagon recptro signaling in the entirety of all possible signaling pathways. This should be corrected in the title.

The method of generating liver targeted siRNA directed to the liver is not provided. It would be difficult to assess whether these methods are adequate and rigorous.

The connection between kisspeptin and insulin secretion is mentioned but the authors do not provide sufficient data to support any connection in their experimental system. This should be clarified in the discussion section.

A time course in the development of increased in kisspeptin production and reduced insulin secretion in SUR -/- would be helpful. Such data was not generated and it may be useful for the authors to discuss this relationship

Figure legends should specify whether the data relates to studies in WT mice or to studies in SUR -/- mice.

Figure S1 legend is insufficient in describing the presented data.

Reviewer #2: Soundarapandian et al have addressed the clinical problem of hyperinsulinism-induced hypoglycemia (HI). This disorder is poorly managed and is currently treated with therapies that include replacement of glucagon and drugs which suppress insulin release. In this study, the authors propose a new approach which involves enhancing hepatic endogenous glucose production (EGP) by targeting the suppression of the regulatory subunit of PKA, Prkar1a. This molecule is a negative regulator of PKA and by disinhibition through siRNA therapeutics they propose to enhance EGP independently of glucagon administration. This is novel and intriguing.

The paper appears to have been put together in haste; some panels are jumbled in Figs 3 and 4, several supplementary figs have been added which could be consolidated, aspects of the supporting literature have been omitted and the methods are incomplete. More worrying is that many of the datasets have potentially valuable trends but fail to reach statistical validity. Thus, despite some promising actions of siPrkar1a on control rodents, the effects in HI mice are unconvincing under most conditions and fail to reach statistical significance despite adequate replicates.

1. Methods/ siPrkar1Controls. (a) There appear to be no publications relating to the (proprietary?) siRNA probesets and the authors have included no information on the sequences of their probes or cited supporting data. This needs to be included. Three probe sets are included AD-76409, AD-76410 and AD-76411 but only AD-76410 (L130) is described as liver-targeted. Please clarify. In some experiments you state which probe was used in the legend, but in others you do not – please clarify throughout. Also, the concentration of probe(s) has not been reported for a number of studies. (b) As Prkar1a is not solely associated with PKA, include additional controls to show that PKA is the only modified protein and not for example AKAPs, ARDGEFs, etc. (c) There is no controls data illustrating that scrambled probesets are ineffective. In the absence of any publications on AD-76409, AD-76410 and AD-76411, please include. (d) Are the actions of the siPrkar1a manipulations reversible. Please include. (e) I am concerned that some of the most dramatic actions – and the statistically significant effects, of siPrkar1a are only seen at the end of the study periods. How confident can we be that hepatic function has not been compromised at this point in time? (f) L135-L136. I disagree, siPrkar1treatment has a positive impact on PKA protein expression – this is not significant, but it cannot be described as ‘no discernible effect’. (g) L156-157 The actions of siPrkar1treatment on glucose levels in the PPT (Fig 1E) are not significant, this needs to me made clear in this sentence. (h) L176-L177 the statement that “AD76409 and AD-76410 led to a significant upregulation of G6Pase, PEPCK, and PGC-1α” is simply not true; AD-76410 had no action on G6Pase.

2. Figures 1D, 2E and 3D shown inconsistent actions of the siRNAs on the expression of the gluconeogenesis targets. This is best exemplified by the data involving AD-76410 and G6Pase expression. This is worrying, please clarify. The profile of targets studied is also different when the investigators used Sur1-/- mice (Fig 4F) for which there is no explanation. Please clarify and make the profiles consistent.

3. siPrkar1treatment appears to induce a consistent increase in Kiss1 gene expression and this is used by the authors to support their hypothesis that siPrkar1has the dual action of enhancing EGP and inhibiting insulin release. However, the is no discussion or citation to the fact that there is a considerable body of literature indicating that kisspeptin has a stimulatory and not inhibitory action on insulin secretion. Can the authors please clarify why this literature is missing from their paper?

4. To support your hypothesis and the data presented in the paper, please assay for kisspeptin in the pre-clinical models.

5. The data modelling HI by exogenous hyperinsulinism has weaknesses and does not fully support the authors. Figure 3A clearly illustrates that whilst siPrkar1treatment enhances plasma glucose, it cannot reverse insulin-induced hypoglycaemia. I agree that there is a trend to abrogate the actions of exogenous insulin, but this is only a trend and not significant. Why does exogenous insulin fail to elevate �-hydroxy butyrate in the control animals (Fig 3B not 3C)? It seems to work OK in the siPrkar1-treated group, but not the controls. Insulin measurements (Fig 3C not 3B) reveal a considerable range of basal (fed?) insulin levels in the mice for which there is no explanation and it is not clear whether the glucose dataset was obtained from fed or fasting mice (Fig 3A)? I also found the insulin measurement dataset confusing; the authors used different ELISA kits to detect human and mouse insulin, but it is not clear which kit has been used for the date in Fig 3C. It appears to me the assay is not able to distinguish the insulins. This needs to be made clear as both the controls and the insulin-pump animals have the same plasma insulin levels and this negates the model which should after all be hyperinsulinemic.

6. The HI Sur1-/- mice datasets detract from the findings. First, the plasma insulin levels are generally lower and not higher than the controls, which is surprising considering these mice are a model of hyperinsulinism. Second, insulin secretion was not suppressed by siPrkar1treatment which goes against their own work (Fig 3) and their explanation for how kisspeptin is relevant to the study. I agree that there is an increase in glucose levels in the mice and that this would be of benefit in a translational capacity. However, this is underpowered as the variance is high and it cannot to linked to an action on the beta-cells in this model of HI. Third, on L244-246 the authors make no comment upon the fact that siPrkar1treatment has either has no action or decreases in the expression of G6Pase which is markedly different to the what happens in non HI mice. Fourth, despite sufficient replications of data, hardly any of the in vivo profiling studies reach statistical significance or validity and this weakens rather than strengthens their arguments. Finally, without including the WT controls, it is hard to conclude the siRNA treatment reversed the hyperinsulinism in these animals. Sur1KO are not hyperinsulinemic but have lost first phase glucose-stimulated insulin secretion. Unlike humans in the fasting state, they are not hyperinsulinemic. Thus, the question whether a Prkar1a knockdown could reverse some effects of hyperinsulinism cannot be addressed in this mouse model. Thus, the authors show impact on glucose production, but not that it has an impact in face of high insulin.

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Reviewer #2: No

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PLoS One. 2020 Jul 31;15(7):e0236892. doi: 10.1371/journal.pone.0236892.r002

Author response to Decision Letter 0

30 Jun 2020

Response to Reviewers:

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We have addressed the PLOS ONE style requirements and edited the manuscript (Title page, titles in sentence format, etc.) and files (dpi, names) to comply.

We have added the requested information to the “Materials and methods” section.

The generation and genotyping of Sur1-/- mice were previously described [1]. Sur1-/- mice are bred and maintained in our mouse colony on a C57Bl/6 genetic background for experiments. 7 male mice, eight to ten weeks old, Sur1-/- mice were used in each group in all experiments. Mice were maintained on a 12:12-h light-dark cycle and were fed a standard rodent chow diet with free access to food and water. Animal welfare checks were completed once per day. No adverse effects to these experiments were observed or required analgesia.

3. In your methods section, please provide the catalog numbers of all antibodies used in your study. In addition, please provide a full description and sequence of the Prkar1a siRNA."

The requested antibody catalog numbers and full description/ sequence of the Prkar1a siRNA have been added to the “Materials and methods” section.

The following antibodies were used: Prkar1a (BD biosciences, Catalog #610609, 1:500), PKAC (BD Biosciences, Catalog #610980, 1:2000), ß-actin (Abcam, Catalog #ab8227, 1:2000), PKA substrate (Cell Signaling, Catalog #9624, 1:1000), Fluorescence conjugated secondary antibodies (Licor, Goat anti-rabbit, Catalog #926-32211 and Donkey anti-mouse, Catalog #926-680721:5000).

The siRNA targeting Prkar1a was designed, synthesized, and liver targeted by Alnylam Pharmaceuticals, as previously described [2, 3]. The siRNA was designed to target mouse Prkar1a mRNA, NM_021880.

AD-76409 targets position 865-885, 5’-GAUGUAUGAAGAAUUCCUUAGUA-3’

AD-76410 targets position 873-893, 5’-AAGAAUUCCUUAGUAAAGUGUCU-3’

AD-76411 targets position 1394-1414, 5’-AAAAGUUGCUUUAUUGCACCAUU-3’

4. Our journal requires that methods are described in enough detail to allow suitably skilled investigators to fully replicate your study. Please provide a more detailed description of your glycogen staining and siRNA injection methods. If materials, methods, and protocols are well established, authors may cite articles where those protocols are described in detail, but the submission should include sufficient information to be understood independent of these references. Please revise your manuscript so that protocols are sufficiently described. For more information please see https://journals.plos.org/plosone/s/submission-guidelines#loc-materials-and-methods.

We have provided more detail for the glycogen staining and siRNA injection methods to the “Materials and methods” section.

For Prkar1a knockdown studies the mice were subcutaneously injected with vehicle control (1X PBS) or 1mg/kg Prkar1a siRNA every 2 weeks unless otherwise indicated. The endpoints were assessed from serum or liver tissue 28 days post dosing unless otherwise indicated.

The siRNA targeting Prkar1a was designed, synthesized, and liver targeted by Alnylam Pharmaceuticals, as previously described [2, 3].

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission.

We have provided the original uncropped and unadjusted blot images in the Supplemental figures.

6. Thank you for stating the following in the Competing Interests section:

"MMS, PAH and KF work for Alnylam Pharmaceuticals; MMS, PAH and KF hold

shares in Alnylam Pharmaceuticals stock; DDDL received funding from Alnylam

Pharmaceuticals to conduct this study."

We confirm that this does not alter our adherence to the PLOS ONE policies on sharing data and materials and have updated our Competing Interests statement.

Reviewer Comments:

Reviewer #1:

1. The title indicates that glucagon signaling is amplified in the liver. This is inaccurate because glucagon activates multiple signaling pathways in the liver. The studies have up-regulated the PKA arm of glucagon signaling and not glucagon receptor signaling in the entirety of all possible signaling pathways. This should be corrected in the title.

We have altered the title to more specifically denote the PKA arm as the active pathway:

Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1-/- mouse model

2. The method of generating liver targeted siRNA directed to the liver is not provided. It would be difficult to assess whether these methods are adequate and rigorous.

We have added references to the methods we used to target the liver in the “Materials and methods” section.

To target the siRNAs to the liver, we used siRNAs conjugated to triantennary N ‐acetylgalactosamine (GalNAc) to induce robust RNAi‐mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). These methods were published previously [2, 3].

3. The connection between kisspeptin and insulin secretion is mentioned but the authors do not provide sufficient data to support any connection in their experimental system. This should be clarified in the discussion section.

We have added further text to the “Discussion” section.

4. A time course in the development of increased in kisspeptin production and reduced insulin secretion in SUR -/- would be helpful. Such data was not generated and it may be useful for the authors to discuss this relationship.

While we agree, we have unfortunately been unable to identify an ELISA kit that would allow for accurate assessment of the plasma KISSPEPTIN levels. The kit used in Song, et al [5] is no longer available for purchase and several other tested kits have not demonstrated good accuracy.

5. Figure legends should specify whether the data relates to studies in WT mice or to studies in SUR -/- mice.

We have edited the figure legends to specify the mice used for the studies.

Figure 1, 2, 3 and Supplemental Figures 1 and 2 are studies completed in WT mice.

Figure 4 and Supplemental Figure 3 are studies completed in Sur1-/- mice.

6. Figure S1 legend is insufficient in describing the presented data.

We have added more details to describe the western blot presented in the Figure S1 figure legend.

Reviewer #2:

(a) We have added the siRNA sequences and liver targeting references to the “Materials and methods” section.

We have edited the figure legends to more clearly identify the siRNA and concentration used.

The siRNAs targeting Prkar1a (AD-76409, AD-76410, and AD-76411) were designed, synthesized, and liver targeted by Alnylam Pharmaceuticals, via conjugation to triantennary N ‐acetylgalactosamine (GalNAc) to induce robust RNAi‐mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR)as previously described [2, 3]. AD-76410 (1mg/kg) was used for experiments unless otherwise noted.

The siRNA was designed to target mouse Prkar1a mRNA, NM_021880.

AD-76409 targets position 865-885, 5’-GAUGUAUGAAGAAUUCCUUAGUA-3’

AD-76410 targets position 873-893, 5’-AAGAAUUCCUUAGUAAAGUGUCU-3’

AD-76411 targets position 1394-1414, 5’-AAAAGUUGCUUUAUUGCACCAUU-3’

(b) In this manuscript, we sought to address whether amplifying signaling downstream of the glucagon receptor in the liver could counteract the effects of hyperinsulinism. Our goal was to activate PKA which is a well-established and central player in the glucagon pathway and activates several key downstream mediators of glucagon, including kisspeptin expression. Guanine nucleotide exchange factors (GEFs) would be associated with cAMP upstream of PRKAR1a inhibition of PKA. The role of A-kinase anchoring proteins (AKAPs) are associated with anchoring PKA to confer compartmentalization of PKA activation. While these proteins have interesting roles in the PKA pathway, investigation of the multitude of proteins identified in the PKA pathway is beyond the scope of our manuscript and is not directly relevant to the hypothesis.

(c) While we did not include a scrambled control probe set, probe AD-76411 serves as a functional negative control due to its inability to decrease the Prkar1a mRNA or plasma glucose levels significantly.

(d) Unfortunately, we do not know if the actions of the siPrkar1a are reversible. Treated mice were euthanized for tissue harvest at the end of the experiment.

(e) The length of time needed to see effects of the siPrkar1a effects are due to the time necessary for significant downstream effects to occur (i.e. changes in transcriptional and protein expression). We are highly confident that hepatic function is not compromised in these experiments. Conditions that cause insulin related hepatic dysfunction such as metabolic syndrome and insulin resistance require years to compromise liver function. The short duration of this experiment (several weeks) is not enough time to develop hepatic dysfunction.

(f) We agree that “no discernible effect” was not the right way to describe the data. We have edited the text to instead say “while not having a significant effect on catalytic (PKAc) protein levels.

(g) We have added the following underlined words to clarify:

Functionally, the loss of Prkar1a resulted in an increase in glycogenolysis and gluconeogenesis as observed by a significant reduction in liver glycogen (Fig 1D) and though not statistically significant, a trend of increased conversion of the gluconeogenic precursor pyruvate to glucose in a pyruvate tolerance test (Fig 1E) was observed in mice injected with siPrkar1a.

(h) We have edited the text to remedy this mistake.

The most potent siRNAs against Prkar1a (AD-76409 and AD-76410) led to a significant upregulation of gluconeogenesis genes G6Pase, PEPCK, and PGC-1α (Fig 2E). AD-76409 also significantly increased G6Pase expression.

In both figures (Figure 1D and 2E) G6Pase shows upregulation, though admittedly, the average is less and not significant in Figure 2E (AD-76410). Variability in transcript levels and a smaller sample size is the likely explanation for the lack of significance with AD-76410. However, it is important to take into consideration that the upward direction of regulation, even if not significant, and the consistency of the upregulation of the other two markers (Ppargc1a and PEPCK) consistently support our hypothesis.

From our data, it appears that siPrkar1a is not able to overcome the suppression of G6pase in the setting of increased insulin (endogenous or exogenous). Importantly though, the upregulation of the other two gluconeogenesis markers remain consistent in the Sur1-/- mouse model.

The profile of targets studied is the same for Figure 1C, 2E, and 4F (G6pase, PEPCK, and Ppargc1a). Inadvertently during the figure creation process, some of the alternate names were used for the figure labels (i.e. Ppargc1a (PGC1α); PEPCK (PCK)). We have edited the figure labels for clarity and consistency.

3. siPrkar1 treatment appears to induce a consistent increase in Kiss1 gene expression and this is used by the authors to support their hypothesis that siPrkar1has the dual action of enhancing EGP and inhibiting insulin release. However, the is no discussion or citation to the fact that there is a considerable body of literature indicating that kisspeptin has a stimulatory and not inhibitory action on insulin secretion. Can the authors please clarify why this literature is missing from their paper?

We are aware of the contradictory studies published that demonstrate opposing effects of treatment with kisspeptin with either an inhibitory or stimulatory action on insulin secretion. One potential proposed explanation was that different isoforms of kisspeptin (KP10 vs KP13) may have different effects on insulin secretion. The 154 amino acid pre-propeptide encoded by Kiss1 is proteolytically processed and results in the 54-amino acid product kisspeptin-54 (KP54, metastin), but also three C-terminal fragments, kisspeptin-14 (KP14), kisspeptin-13 (KP13), and kisspeptin-10 (KP10), which all share the same 10 amino acid amidated sequence. The smallest fragment, KP10, is sufficient to bind and activate the KISS1 receptor (KISS1R) [6]. However, a study comparing KP10 versus KP13 did not demonstrate any functional changes between the two isoforms [7].

The more likely explanation of the contradictory stimulatory/ inhibitory actions of kisspeptin on insulin secretion is the amount of kisspeptin used in the study. Plasma kisspeptin concentrations in HFD and Leprdb/db mice, which was significantly higher than controls, was 0.5 – 1 nM and 7 – 10 nM respectively [5]. Thus, the physiological circulating levels of kisspeptin in mice is measured to be in the very low nanomolar range. Assessment of the published work reveals that studies using a nanomolar concentration of kisspeptin demonstrated an inhibitory effect on insulin secretion [5, 8, 9]. The studies that showed a stimulatory effect on insulin secretion used a supraphysiological dose in the micromolar range (generally 1uM) [7, 10-12]. Further, studies using KISS1R-/- mice have demonstrated that stimulation can occur in a KISS1R-independent mechanism [5, 13]. These results indicate potential off-target effects of kisspeptin with use of supraphysiological concentrations.

We originally did not include discussion of the studies indicating stimulatory effects of kisspeptin, since they appear to be a result of off-target effects by supraphysiological concentrations and our studies involve endogenously expressed kisspeptin. However, we understand the necessity for clarification of the contradictory studies and have included it in our “Discussion” section.

4. To support your hypothesis and the data presented in the paper, please assay for kisspeptin in the pre-clinical models.

Measurement of kisspeptin circulating levels in mice through commercially available assays has been notably unreliable due to large variations in the assay methods, ranges of detection, and lack of clarity about which isoforms of kisspeptin (i.e., KP10, KP13, KP15, KP54) are detected [4]. Unfortunately, we have been unable to identify an ELISA kit that would allow for accurate assessment of the plasma KISSPEPTIN levels in mice. We are limited by the commercially available ELISA kits. The kit used in Song, et al [2] is no longer available for purchase and several other tested kits have not demonstrated good accuracy.

5. The data modelling HI by exogenous hyperinsulinism has weaknesses and does not fully support the authors. Figure 3A clearly illustrates that whilst siPrkar1treatment enhances plasma glucose, it cannot reverse insulin-induced hypoglycaemia. I agree that there is a trend to abrogate the actions of exogenous insulin, but this is only a trend and not significant. Why does exogenous insulin fail to elevate ß-hydroxy butyrate in the control animals (Fig 3B not 3C)? It seems to work OK in the siPrkar1-treated group, but not the controls. Insulin measurements (Fig 3C not 3B) reveal a considerable range of basal (fed?) insulin levels in the mice for which there is no explanation and it is not clear whether the glucose dataset was obtained from fed or fasting mice (Fig 3A)? I also found the insulin measurement dataset confusing; the authors used different ELISA kits to detect human and mouse insulin, but it is not clear which kit has been used for the date in Fig 3C. It appears to me the assay is not able to distinguish the insulins. This needs to be made clear as both the controls and the insulin-pump animals have the same plasma insulin levels and this negates the model which should after all be hyperinsulinemic.

Insulin suppresses lipolysis and ketogenesis, thus, in insulin-mediated hypoglycemia, ß-hydroxybutyrate is suppressed. Our data indicates that during insulin-induced hypoglycemia, downregulation of Prkar1a allows ketogenesis to be activated. Controls (non-insulin treated) are not hypoglycemic, so β-hydroxy butyrate is not expected to be increased.

Plasma insulin concentrations in Figure 3C was assessed in mice fasted for 5 hours as stated in the figure legend. While there are a few outliers, the insulin levels fall within the expected range of plasma insulin. We have corrected the figure placement (Figure 3B is Insulin levels; Figure 3C is β- hydroxybutyrate levels).

Plasma glucose levels in Figure 3A were obtained after a 5 hour fast. This information has been added to the figure legend.

The Insulin ELISA kit used for Figure 3B detects both mouse and human. The insulin can be cleared rapidly, and like other hormones, plasma insulin concentration must be interpreted in the context of the metabolic state, particularly of the plasma glucose concentration. Insulin levels in the Sur1-/- (and also in humans with congenital hyperinsulinism) are not overtly elevated, but fail to be suppressed in the presence of hypoglycemia. Although the plasma insulin concentration is not markedly different between mice treated with insulin or not, there is a clear hypoglycemic effect of the treatment as shown in Figure 3A. We gave the mice the minimum dose of insulin necessary to achieve hypoglycemia, which may not be easy to detect by ELISA. When we optimized this model, we did try higher exogenous insulin levels but the mice became so hypoglycemic that we couldn’t complete experiments. Insulin doses lower than 0.2 U do not induce hypoglycemia.

1) Insulin levels, like other hormones (such as parathyroid hormone in response to calcium) have to be interpreted in the context of the metabolic state. Insulin levels in Sur1-/- mice (and humans with congenital hyperinsulinism) are not overtly elevated, yet they are inappropriate in the setting of hypoglycemia and reflect a marked disturbance in regulated insulin secretion with serious clinical consequences [14, 15]. While the Sur1-/- mouse model’s phenotype is milder compared to the human phenotype, all cardinal features of KATP-hyperinsulinism are reproduced by this model, including fasting hypoglycemia and impaired glucose stimulated insulin secretion. Similarly, isolated islets from the Sur1-/- mouse and human KATP-hyperinsulinism islets exhibit all abnormalities expected from the lack of functional KATP channels: elevated cytosolic calcium, high baseline insulin secretion and impaired glucose stimulated insulin secretion. Thus, we believe that this model is appropriate for these proof-of-concept studies. Furthermore, we have previously used this model for proof-of-concept studies of other potential therapies [16] that were then successfully translated to clinical studies in affected individuals with KATP-hyperinsulinism [17].

2) Insulin secretion was suppressed by siPrkar1a treatment. We have added an area under the curve (AUC) for the plasma insulin levels taken during the GTT (Figure 4E) to make this clearer.

3) While siPrkar1a increases G6Pase in the absence of increased insulin (Figure 1C), in the presence of increased insulin (endogenous or exogenous) siPrkar1a is not able to overcome the suppression of G6pase in the setting of increased insulin (hyperinsulinism or exogenous).

We have added this to the manuscript to describe the effect of insulin and siPrkar1a on G6Pase expression.

4) As we have previously shown, in Sur1-/- mice the hypoglycemic phenotype is triggered by fasting. Although we did not include wild type controls on this experiment, from previous studies, fasting plasma glucose in Sur1-/- is significantly lower than wild type littermates (59.4 ± 1.5 vs 75 ± 1.8 mg/dL, p<0.0001) [16]. In this study, fasting glucose concentrations of control treated mice are similarly low and siPrkar1a significantly increased the fasting glucose 21 days injection (Figure 4A, D). We were remiss to not include the AUC for the plasma insulin levels after fasting in the GTT (Figure 4E), which shows a significant decrease in plasma insulin the Sur1-/- siPrkar1a injected mice.

Overall, we have demonstrated a reversal of the hyperinsulinemic state of the Sur1-/- with siPrkar1a through the significant increase in plasma glucose (Figure 4A and D) as well as a significant decrease in the level of secreted insulin (Figure 4E AUC).

References

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2. Rajeev KG, Nair JK, Jayaraman M, Charisse K, Taneja N, O'Shea J, et al. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo. Chembiochem. 2015;16(6):903-8. doi: 10.1002/cbic.201500023. PubMed PMID: 25786782.

3. Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc. 2014;136(49):16958-61. doi: 10.1021/ja505986a. PubMed PMID: 25434769.

4. Hussain MA, Song WJ, Wolfe A. There is Kisspeptin - And Then There is Kisspeptin. Trends Endocrinol Metab. 2015;26(10):564-72. doi: 10.1016/j.tem.2015.07.008. PubMed PMID: 26412157; PubMed Central PMCID: PMCPMC4587393.

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PLoS One. doi: 10.1371/journal.pone.0236892.r003

Decision Letter 1

Michael Bader

16 Jul 2020

Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1-/- mouse model

PONE-D-20-09701R1

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The short title is misleading. It would be more appropriate to indicate that Prkar1a KD mitigates HI

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PLoS One. doi: 10.1371/journal.pone.0236892.r004

Acceptance letter

Michael Bader

20 Jul 2020

PONE-D-20-09701R1

Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1^-/- mouse model

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Supplementary Materials

S1 Fig. Western blot analysis of phospho-PKA substrates in liver extracts from siPrka1a injected WT mice as compared to vehicle injected controls.

(TIF)

Click here for additional data file.^{(640.7KB, tif)}

S2 Fig. Reduction of Prkar1a results in hyperglycemia.

(TIF)

Click here for additional data file.^{(806.3KB, tif)}

S3 Fig. siPrkar1a injection in Sur1^-/- mice.

(TIF)

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S1 Raw images

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1-/- mouse model

Mangala M Soundarapandian

Christine A Juliana

Jinghua Chai

Patrick A Haslett

Kevin Fitzgerald

Diva D De León

Roles

Abstract

Introduction

Materials and methods

Animal studies

Glycogen staining

Evaluation of glucose homeostasis

Osmotic pump implantation

siRNA injection studies

RNA isolation and qRT-PCR

Western blot analysis

Statistics

Results

Loss of Prkar1a activates PKA and downstream liver gluconeogenesis

Fig 1. siRNA mediated reduction of Prkar1a activates PKA and liver gluconeogenesis.

Reduction of Prkar1a leads to hyperglycemia in mice

Fig 2. Loss of Prkar1a results in hyperglycemia.

Prkar1a loss increases plasma ketones during hyperinsulinemic conditions and induces Kiss1 expression

Fig 3. Loss of Prkar1a increases blood ketones during hyperinsulinemic conditions.

Hypoglycemia is attenuated by loss of Prkar1a expression in the Sur1-/- mouse model of hyperinsulinism

Fig 4. Loss of Prkar1a expression attenuates fasting induced hypoglycemia in Sur1-/- mice.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Michael Bader

Roles

Author response to Decision Letter 0

Decision Letter 1

Michael Bader

Roles

Acceptance letter

Michael Bader

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1^-/- mouse model

Hypoglycemia is attenuated by loss of Prkar1a expression in the Sur1^-/- mouse model of hyperinsulinism

Fig 4. Loss of Prkar1a expression attenuates fasting induced hypoglycemia in Sur1^-/- mice.