AgRP neuron hyperactivity drives hyperglycemia in a mouse model of type 2 diabetes

Abstract

Growing evidence suggests that the pathogenesis of type 2 diabetes (T2D) involves dysfunctional central mechanisms, and, hence, the brain can be targeted to treat this disease. As an example, a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) can normalize hyperglycemia for weeks or months in rodent models of T2D. Convergent evidence implicates inhibition of a particular subset of neurons as a mediator of this FGF1 effect. Specifically, AgRP neurons, which are located in the hypothalamic arcuate nucleus (ARC) and are hyperactive in Lepob/ob mice and other rodent models of T2D. To investigate whether chronic AgRP neuron inactivation mimics the antidiabetic action of FGF1, we directed an adeno-associated virus (AAV) containing a cre-inducible tetanus toxin-GFP (TeTx-GFP) cassette (or cre-inducible AAV GFP control) to the ARC of obese, diabetic male Lepob/ob mice in which cre recombinase is expressed solely by AgRP neurons (Lepob/ob AgRP-Cre mice). We report that over a 10-wk period of observation, hyperglycemia was fully normalized by AgRP neuron inactivation. In contrast, changes in energy homeostasis parameters (food intake, energy expenditure, body weight, and fat mass) were not observed. We conclude that in diabetic male Lepob/ob mice, AgRP neuron hyperactivity is required for hyperglycemia but is dispensable for obesity.

Keywords: Diabetes; Endocrinology; Metabolism.

Figure 1. Validation of AgRP neuron inactivation.

(A). Schematic depiction for chronic inactivation of AgRP neurons by microinjection of an AAV containing Cre-dependent GFP-fused TeTx delivered bilaterally to the arcuate nucleus (ARC) of Lep^ob/ob AgRP-Cre mice relative to a fluorescent reporter control. Stereological fluorescent images from representative animals showing (B) GFP:TeTx and (C) GFP expression in Lep^ob/ob AgRP-Cre mice. Scale bars: 100 μm. (D) GFP:TeTx Lep^ob/ob AgRP-Cre mice (n = 9) exhibited a blunted refeeding response following an overnight fast when compared with those receiving the cre-inducible GFP controls (n = 9). 3V = third ventricle. Data are expressed as mean ± SEM, P versus GFP control as determined by 2-tailed t-test. *P < 0.05.

Figure 2. Permanent inactivation of AgRP neurons in Lep^ob/ob AgRP-Cre mice induces diabetes remission independent of changes in body weight and food intake.

(A) Nonfasted blood glucose, (B) body weight, and (C) food intake over 10 weeks in Lep^ob/ob AgRP-Cre mice that received a bilateral injection to the arcuate nucleus (ARC) of a Cre-dependent GFP:TeTx (TeTx; n = 9) relative to a GFP control (Control; n = 9). Data are expressed as mean ± SEM, and P versus GFP control was determined by mixed model with the Geisser-Greenhouse correction. *P < 0.05.

Figure 3. Effect of AgRP neuron inactivation on energy homeostasis in Lep^ob/ob AgRP-Cre mice.

(A) Body weight, body composition, including (B) lean mass, (C) fat mass, and (D) percentage of fat, and photoperiod-averaged 24-hour profiles and mean dark and light cycle measures of (E and F) heat production, (G and H) respiratory quotient (RQ), and (I and J) ambulatory activity as determined using indirect calorimetry in Lep^ob/ob AgRP-Cre mice that received a bilateral microinjection of GFP:TeTx (TeTx; n = 9) relative to a GFP control (Control; n = 5–9) to the arcuate nucleus (ARC). Data are expressed as mean ± SEM, versus GFP control as determined by 2-tailed t test.

Figure 4. Effect of AgRP neuron inactivation on glucose tolerance in Lep^ob/ob AgRP-Cre mice.

(A) Blood glucose and (B) plasma insulin levels, (C) changes in blood glucose levels and (D) area under the glucose curve (AUC) during an intraperitoneal glucose tolerance test (ipgtt; 0.5g/kg BW) in Lep^ob/ob AgRP-Cre mice following microinjection of GFP:TeTx (n = 9) or GFP control (n = 8–9) to the arcuate nucleus (ARC). Data are expressed as mean ± SEM, P versus GFP control as determined by 2-tailed t test. *P < 0.05.

Figure 5. Effect of AgRP neuron inactivation on liver metabolism in Lep^ob/ob AgRP-Cre mice.

(A) Liver fat content, (B) liver glycogen content, and (C) hepatic mRNA levels of liver glucoregulatory genes glucokinase (Gck), phosphoenolpyruvate carboxykinase (Pck1), and glucose-6-phosphatase (G6Pc) using real-time PCR in Lep^ob/ob AgRP-Cre mice that received microinjection of GFP:TeTx (n = 7–9) or GFP control (n = 7–9) to the arcuate nucleus (ARC). Data are expressed as mean ± SEM, P versus GFP control as determined by 2-tailed t test. *P < 0.05.

Figure 6. Inactivation of AgRP neurons lowers corticosterone levels in Lep^ob/ob AgRP-Cre mice.

Plasma levels of (A) corticosterone and (B) glucagon during the mid-light cycle in the fed state. Plasma (C) lactate, (D) triglyceride (TG), and (E) free fatty acid levels in either the fed or 6-hr fasted state in Lep^ob/ob AgRP-Cre mice that received microinjection of GFP:TeTx (n = 9) or GFP control (n = 7–9) to the arcuate nucleus (ARC). Data are expressed as mean ± SEM, P versus GFP control as determined by 2-tailed t test. *P < 0.05.