Reduction in SGLT1 mRNA Expression in the Ventromedial Hypothalamus Improves the Counterregulatory Responses to Hypoglycemia in Recurrently Hypoglycemic and Diabetic Rats

Abstract

The objective of this study was to determine whether the sodium-glucose transporter SGLT1 in the ventromedial hypothalamus (VMH) plays a role in glucose sensing and in regulating the counterregulatory response to hypoglycemia, and if so, whether knockdown of in the VMH can improve counterregulatory responses to hypoglycemia in diabetic rats or rats exposed to recurrent bouts of hypoglycemia (RH). Normal Sprague-Dawley rats as well as RH or streptozotocin (STZ)-diabetic rats received bilateral VMH microinjections of an adenoassociated viral vector containing either the SGLT1 short hairpin RNA (shRNA) or a scrambled RNA sequence. Subsequently, these rats underwent a hypoglycemic clamp to assess hormone responses. In a subgroup of rats, glucose kinetics was determined using tritiated glucose. The shRNA reduced VMH SGLT1 expression by 53% in nondiabetic rats, and this augmented glucagon and epinephrine responses and hepatic glucose production during hypoglycemia. Similarly, SGLT1 knockdown improved the glucagon and epinephrine responses in RH rats and restored the impaired epinephrine response to hypoglycemia in STZ-diabetic animals. These findings suggest that SGLT1 in the VMH plays a significant role in the detection and activation of counterregulatory responses to hypoglycemia. Inhibition of SGLT1 may offer a potential therapeutic target to diminish the risk of hypoglycemia in diabetes.

Figure 1

Relative change in SGLT1 mRNA expression in the VMH of RH and STZ-diabetic rats compared with nondiabetic, hypoglycemia-naive control rats. Data are presented as mean ± SEM.

Figure 2

Acute hypoglycemia study. A: VMH administration of SGLT1 shRNA reduced SGLT1 mRNA by ∼53%. B: Plasma glucose concentrations during the clamp in control and SGLT1 knockdown (KD) groups. C: Diminished GIRs in SGLT1 KD compared with control animals. Increased plasma glucagon (D) and epinephrine (E) responses to hypoglycemia in SGLT1 KD compared with control rats. F: Hepatic glucose production (HGP) during hypoglycemia was 88% greater in the SGLT1 KD group than in controls. Data are mean ± SEM. *P < 0.05 vs. SGLT1 KD; ***P < 0.001 vs. SGLT1 KD.

Figure 3

RH study. A: SGLT1 shRNA reduced VMH SGLT1 mRNA levels by ∼51% of rats exposed to RH. B: Blood glucose during the 3 days of insulin injection before clamp showed no significant difference between the control and SGLT1 knockdown (KD) groups. C: Plasma glucose concentrations during the clamp in the control and SGLT1 KD groups. D: GIRs during the clamp were diminished in RH + SGLT1 KD rats compared with RH + scrambled AAV. Plasma glucagon (E) and epinephrine (F) levels were increased in SGLT1 KD rats. Data are mean ± SEM. *P < 0.05 vs. RH + SGLT1 KD; **P < 0.01 vs. RH + SGLT1 KD.

Figure 4

Diabetes study. A: SGLT1 shRNA AAV microinjection reduced VMH SGLT1 mRNA levels ∼57%. B: Plasma glucose concentrations during the clamp in control and SGLT1 knockdown (KD) groups. C: GIRs during the clamp were matched in STZ + SGLT1 KD rats compared with STZ + scrambled AAV rats. D: A significant increase in the epinephrine response to hypoglycemia in STZ-diabetic rats was found. *P < 0.05 vs. STZ + SGLT1 KD; **P < 0.01 vs. STZ + SGLT1 KD.

Figure 5

Top panels: Immunohistochemical staining for SGLT1 (red), the astrocytic marker GFAP (green), and the merged image showing colocalization of SGLT1 with GFAP (arrows). Middle and bottom panels: Immunohistochemical staining for SGLT1 (red), the neuronal markers GAD67 and VGLUT2 (green), and the merged image showing colocalization of SGLT1 with GAD67 and VGLUT2 (arrows).

Figure 6

Immunohistochemical images showing that the addition of an SGLT1-specific blocking peptide can reduce the immunoreactive-SGLT1 signal in brain sections (A) compared with brain sections without the blocking peptide (B).