Insulin analogs are key to blood glucose management for millions of people with diabetes. Nonetheless, the risk of hypoglycemia still exists because this insulin remains bioactive at normal or low blood glucose conditions. Here, the aim is to incorporate phenylboronic acids on insulin glargine to create a glucose-responsive designer insulin termed "PBA-F-glargine." It is hypothesized that by inserting a glucose responsive moiety, this designer insulin increases the therapeutic window and reduces the risk of insulin-induced hypoglycemia. Chemical methods are used to incorporate phenylboronic acids into insulin glargine. Biochemical and cell-based assays are used to confirm that the designer insulin PBA-F-glargine preserves insulin bioactivity. In comparison to commercial glargine, in vitro experiments demonstrate that PBA-F-glargine has similar bioactivity and increased solubility that is glucose-dependent. In vivo experiments demonstrate that PBA-F-glargine has 88% bioactivity as compared to glargine at hyperglycemic levels, yet has only 30% bioactivity at euglycemic levels. This threefold difference in bioactivity demonstrates that PBA-F-glargine is responsive to glucose concentrations. In comparison to commercial glargine, PBA-F-glargine reduces iatrogenic hypoglycemia by 15-fold. In conclusion, PBA-F-glargine has a glucose-dependent in vivo bioactivity that markedly reduces the risk of hypoglycemia.

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It is proposed that the impaired counterregulatory response (CRR) to hypoglycemia in insulin deficient diabetes may be due to chronic brain insulin deficiency. To test this hypothesis, streptozotocin-diabetic Sprague-Dawley rats were infused with either insulin (3mU/day) or artificial cerebrospinal fluid (aCSF) bilaterally into the ventromedial hypothalamus (VMH) for 2 weeks and compared to nondiabetic rats. Rats underwent hyperinsulinemic (50 mU.kg.min) hypoglycemic (~45 mg/dl) clamps. Diabetic rats demonstrated an impaired CRR to hypoglycemia noted by an high glucose infusion rate (GIR) and blunted epinephrine and glucagon responses. The defective sympathoadrenal response was restored with chronic infusion of insulin into the VMH. Diabetic rats had decreased VMH Akt phosphorylation and decreased VMH glucose transporter 4 (GLUT4) content, which was also restored with chronic infusion of insulin into the VMH. Separate experiments in non-diabetic rats in which VMH GLUT4 translocation was inhibited with an infusion of indinavir was notable for an impaired CRR to hypoglycemia indicated by increased GIR and diminished epinephrine and glucagon responses. Results suggest that in this model of diabetes, VMH insulin deficiency impairs the sympathoadrenal response to hypoglycemia and chronic VMH insulin infusion is sufficient to normalize the sympathoadrenal response to hypoglycemia, via restoration of VMH GLUT4 expression.

We tested the hypothesis that the correction of ventromedial hypothalamus (VMH) insulin deficiency was sufficient to restore the impaired sympathoadrenal response to hypoglycemia in diabetic rats. Sprague-Dawley rats were injected with either vehicle (nondiabetic controls; CON) or streptozotocin (STZ; 65 mg/kg IP). STZ diabetic rats received osmotic mini-pumps to infuse either artificial cerebrospinal fluid (DIAB) or insulin (3mU/day; DIAB+InsVMH) into the VMH bilaterally. Two weeks later, all three groups underwent hyperinsulinemic (50 mU.kg-1.min-1) hypoglycemic (∼50 mg/dl) clamps. As expected, STZ-diabetic rats showed blunted epinephrine response to hypoglycemia as compared to nondiabetic controls. Chronic infusion of insulin into VMH of diabetic rats normalized the epinephrine response to hypoglycemia and lowered the glucose infusion rate required to maintain hypoglycemia. Examining brain insulin action, it was noted that STZ diabetic rats had decreased Akt phosphorylation and decreased expression of insulin dependent glucose transporter 4 (GLUT4) in VMH by 44 and 40% respectively as compared to the nondiabetic controls. Notably, chronic infusion of insulin to the VMH normalized Akt phosphorylation and GLUT4 expression. In summary, insulin acts chronically in the VMH to preserve the sympathoadrenal response to hypoglycemia, possibly by regulating GLUT4 expression. Disclosure R. Agrawal: None. A. Vieira de Abreu: None. G.T. Durupt: None. S.J. Fisher: None.