Recent antecedent hypoglycemia is a known source of defective glucose counterregulation in diabetes; yet, the mechanisms perpetuating the cycle of progressive α-cell failure and recurrent hypoglycemia remain unknown. Somatostatin has been shown to supress the glucagon response to acute hypoglycemia in rodent models of type 1 diabetes. We hypothesized that somatostatin receptor 2 antagonism (SSTR2a) would restore glucagon counterregulation and delay the onset of insulin-induced hypoglycemia in recurrently hypoglycemic, non-diabetic male rats. Healthy, male, Sprague-Dawley rats (n=39) received bolus injections of insulin (10 U/kg, 8 U/kg, 5 U/kg) on 3 consecutive days to induce hypoglycemia. On day 4, animals were then treated with SSTR2a (10 mg/kg; n=17) or vehicle (n=12) one-hour prior to the induction of hypoglycemia using insulin (5 U/kg). Plasma glucagon level during hypoglycemia was ~30% lower on day 3 (150±75 pg/ml; P<0.01), and 68% lower on day 4 in the vehicle group (70±52 pg/ml; P<0.001) compared to day 1 (219±99 pg/ml). On day 4, SSTR2a prolonged euglycemia by 25±5 min (P<0.05) and restored the plasma glucagon response to hypoglycemia. Hepatic glycogen content of SSTR2a-treated rats was 35% lower than vehicle controls after hypoglycemia induction on day 4 (vehicle: 20±7.0 vs SSTR2a: 13±4.4 µmol/g; P<0.01). SSTR2a treatment reverses the cumulative glucagon deficit resulting from three days of antecedent hypoglycemia in healthy rats. This reversal is associated with decreased hepatic glycogen content and delayed time to hypoglycemic onset. We conclude that recurrent hypoglycemia produces glucagon counterregulatory deficiency in healthy male rats, which can be improved by SSTR2a.

Glucagon (GCN) helps prevent hypoglycemia when blood glucose levels drop; however, recurrent hypoglycemia attenuates GCN counterregulation to subsequent bouts of hypoglycemia. As somatostatin normally inhibits GCN secretion, we tested the hypothesis that a somatostatin receptor type 2 antagonist (SSTR2a), PRL-2903, improves GCN responses attenuated by recurrent hypoglycemia in healthy rats. Healthy male Sprague-Dawley rats (n=22) were made hypoglycemic on three consecutive days (days 1-3, blood glucose 1.7-2.2 mmol/L for ∼2 h) via exogenous insulin administration (10-, 8- and 5- U/kg of Humulin-R on days 1-3, respectively). GCN levels during hypoglycemia on day 3 were significantly lower than on day 1 (117±47 pg/mL vs. 184±77 [mean±SD] pg/mL; P=0.001), highlighting the role of recurrent hypoglycemia in counterregulatory failure. On day 4, rats were treated with either PRL-2903 (10 mg/kg IP; n=13) or vehicle (n=9) 1 h prior to the induction of hypoglycemia with 5 U/kg of R-insulin. GCN levels during hypoglycemia (i.e., glucose ≤3.5 mmol/L) were 2.5-fold higher (109±55 vs. 44±26 pg/mL; P=0.004) compared to vehicle, and time to reach hypoglycemia was 3.2-fold longer (64±45 vs. 20±10 min; P =0.001), with PRL-2903 pre-treatment. Interestingly, C-peptide levels were also lower (P=0.001) with PRL-2903 (0.35±0.22 ng/mL), compared to vehicle (0.63±0.21 ng/mL), inferring a lower insulin secretion during hypoglycemia with PRL-2903 treatment. In conclusion, our data suggests that SSTR2a improves GCN responses following recurrent hypoglycemia, and that this improvement may be associated with a reduction in insulin secretion in healthy rats. Therefore, SSTR2a treatment may be a useful therapeutic approach to improve GCN counterregulatory responses to hypoglycemia. Disclosure M. Riddell: Speaker's Bureau; Self; Medtronic. Consultant; Self; Eli Lilly and Company, JAEB Center For Health Research, Xeris Pharmaceuticals, Inc.. Research Support; Self; Insulet Corporation. Speaker's Bureau; Self; Insulet Corporation. Advisory Panel; Self; Sanofi. Speaker's Bureau; Self; Ascensia Diabetes Care. Stock/Shareholder; Self; Zucara Theraputics. Other Relationship; Self; JDRF. M. Jahangiriesmaili: None. E.R. Mandel: None. C.A. Greenberg: None. A.M. Pasieka: None. T. Teich: None. O. Chan: None. R.T. Liggins: Employee; Self; Zucara Therapeuytics, Inc..

Sodium glucose co-transporter 2 inhibitors (SGLT2i) show promise as an add-on to insulin therapy in patients with type 1 diabetes (T1D), however their effect on glucagon secretion in T1D is not yet known. Increases in glucagon have the potential to protect against hypoglycemia that may develop from insulin overtreatment or exercise. We investigated whether 8 days of SGLT2i treatment (10mg/kg empagliflozin, 2x/day) altered plasma glucagon concentrations in response to voluntary running wheel activity and insulin-induced hypoglycemia in male and female Wistar rats with streptozotocin-induced T1D. SGLT2 inhibition did not alter basal or post-exercise glucagon concentrations and did not affect exercise-associated changes in blood glucose (BG) levels. Additionally, SGLT2 inhibition did not alter voluntary activity levels. However, in males, SGLT2 inhibition lowered plasma glucagon response during an insulin tolerance test (+22.7±30.8 pg/mL vs. +40.4±48.6 pg/mL, p=0.02) and decreased the time taken to reach hypoglycemia (30±13.4 min vs. 63.8±17.2 min, p<0.0001), as compared to placebo. We also observed novel sex differences in the response of BG and glucagon concentrations to voluntary exercise. In female rats alone, voluntary exercise decreased BG concentrations (-2.9±2.6 mM vs. +1.7±1.8 mM, p=0.0003) and led to lower post-exercise glucagon concentrations (37.3±47.9 pg/mL vs. 117.9±86.0 pg/mL, p=0.01), as compared to inactive animals. On average, voluntary activity levels were higher in females than in males (539.2±199.4 m vs. 308.4±215.6 m, p=0.02), however running distances were not correlated with changes in BG levels during exercise (r2=0.02). Our finding of increased hypoglycemia risk with SGLT2i has concerning implications for its use as an adjunct to insulin therapy in T1D. The novel sex differences that we observed should be investigated in individuals with T1D to determine their human applicability and potential mechanisms. Disclosure C.A. Greenberg: None. E.R. Mandel: None. A.M. Pasieka: None. M. Jahangiriesmaili: None. J. Tremblay: None. M. Riddell: Advisory Panel; Self; Xeris Pharmaceuticals, Inc. Research Support; Self; Dexcom, Inc. Speaker's Bureau; Self; Insulet Corporation, Medtronic MiniMed, Inc. Stock/Shareholder; Self; Zucara Therapeutics Inc.