CB1Rs in VMH neurons regulate glucose homeostasis but not body weight

Abstract

Cannabinoid 1 receptor (CB1R) inverse agonists reduce body weight and improve several parameters of glucose homeostasis. However, these drugs have also been associated with deleterious side effects. CB1R expression is widespread in the brain and in peripheral tissues, but whether specific sites of expression can mediate the beneficial metabolic effects of CB1R drugs, while avoiding the untoward side effects, remains unclear. Evidence suggests inverse agonists may act on key sites within the central nervous system to improve metabolism. The ventromedial hypothalamus (VMH) is a critical node regulating energy balance and glucose homeostasis. To determine the contributions of CB1Rs expressed in VMH neurons in regulating metabolic homeostasis, we generated mice lacking CB1Rs in the VMH. We found that the deletion of CB1Rs in the VMH did not affect body weight in chow- and high-fat diet-fed male and female mice. We also found that deletion of CB1Rs in the VMH did not alter weight loss responses induced by the CB1R inverse agonist SR141716. However, we did find that CB1Rs of the VMH regulate parameters of glucose homeostasis independent of body weight in diet-induced obese male mice.NEW & NOTEWORTHY Cannabinoid 1 receptors (CB1Rs) regulate metabolic homeostasis, and CB1R inverse agonists reduce body weight and improve parameters of glucose metabolism. However, the cell populations expressing CB1Rs that regulate metabolic homeostasis remain unclear. CB1Rs are highly expressed in the ventromedial hypothalamic nucleus (VMH), which is a crucial node that regulates metabolism. With CRISPR/Cas9, we generated mice lacking CB1Rs specifically in VMH neurons and found that CB1Rs in VMH neurons are essential for the regulation of glucose metabolism independent of body weight regulation.

Keywords: CB1R; SR141716; VMH; glucose metabolism; metabolic homeostasis.

Graphical abstract

Figure 1.

Generation of conditional cannabinoid 1 receptor (CB1R) knockout mice. A: validation of CB1R^flox/flox × ZP3-Cre (germline Cre) offspring resulting in global CB1R deletion (CB1R^Δ/Δ) as assessed by Cnr1 expression in the brain, epididymal white adipose tissue (WATe), inguinal white adipose tissue (WATi), and gastrocnemius (GAS). CB1R-specific deletion of CB1R from SF1-neurons shown by in situ hybridization (ISH) of Cnr1 mRNA expression. ISH staining indicated by silver grains (black areas/dots) in coronal brain section from (B) CB1R^flox and (C) CB1R^SF1-KO mice. The ventromedial hypothalamus is highlighted by the red dashed box. KO, knockout; WT, wild type. Data are expressed as the mean ± SE. **P < 0.01 between genotypes, assessed by unpaired Student’s t test.

Figure 2.

Deletion of cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus does not alter body weight in chow-fed male mice. Body weight (A), fasting plasma insulin (B), fasting plasma glucagon values (C), glucose tolerance test (GTT; D), and GTT area under the curve (AUC; E) from 11- to 14-wk-old males. Results shown as mean ± SE. *P < 0.05, between genotypes, assessed by unpaired Student’s t test.

Figure 3.

Cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus regulate basal glucose levels independent of the body weight of male mice. A: body weight curves for CB1R^flox versus CB1R^SF1-KO, start of high-fat diet (HFD) feeding indicated by arrow. Following 8–9 wks of HFD feeding, glucose tolerance tests (GTT; B), area under the curve (AUC) for the GTT (C), fasting plasma insulin (D), and fasting plasma glucagon values (E). Results shown as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 between genotypes, assessed by unpaired Student’s t test.

Figure 4.

Deletion of cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus does not alter the efficacy of CB1R inverse agonist SR141716. After 11 wks of HFD, daily injections of Rimonabant (SR141716) were administered for 5 days (IP injections, 10 mg/kg). Weight loss (A), fasting plasma insulin (B), fasting plasma glucagon (C), glucose tolerance test (GTT; D), and area under the curve (AUC; E) for the GTT were determined. Results shown as mean ± SE. *P < 0.05, **P < 0.01 between genotypes, assessed by unpaired Student’s t test. HFD, high-fat diet.

Figure 5.

Deletion of cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus lowers the hepatic glucose production in high-fat diet (HFD)-fed male mice. A: body weight after 8 wks of HFD feeding. B: basal hepatic glucose production (HPG). Insulin sensitivity was determined by hyperinsulinemic-euglycemic clamps to assess the glucose infusion rate (GIR; C) and the rate of glucose disposal (Rd; D). Hepatic gene expression of glycogen synthase (Gys2; E), glucose 6 phosphatase (G6Pase; G6pc; F), phosphoenolpyruvate carboxykinase (PEPCK; Pck1; G), and fibroblast growth factor 21 (FGF21; Fgf21; H). Mice were 16–18 wks old at the time of experiments. Results shown as mean ± SE. *P < 0.05, ***P < 0.001, between genotypes, assessed by unpaired Student’s t test.

Figure 6.

Deletion of cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus does not alter energy expenditure in chow- or high-fat diet (HFD)-fed male mice. Indirect calorimetry measurement of chow-fed mice: oxygen consumption (Vo₂; A) and respiratory exchange ratio (RER; B). C: the mean 24 h food intake for the 72 h of indirect calorimetry measurements. Body composition for chow-fed mice: lean mass (D) and fat mass (E). The mean indirect calorimetry measurement of mice fed HFD for 8–9 wks: oxygen consumption (Vo₂; F) and respiratory exchange ratio (RER; G) for mice fed HFD. H: the mean 24-h food intake for the 72 h of indirect calorimetry measurements. Body composition for HFD-fed mice: lean mass (I) and fat mass (J). Chow- and HFD-fed mice were 16–18 wks old at the time of experiments. For A, B, F, and G, the 12-h lights on (open bar) and 12-h lights off (closed bar) cycles are represented on the x-axis. Data are expressed as the mean ± SE.

Figure 7.

Deletion of cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus does not alter body weight in chow-fed female mice. Body weight (A), lean mass (B), fat mass (C), fasting plasma insulin (D), glucose tolerance test (GTT; E), and insulin tolerance test (ITT; F) from 24- to 27-wk-old female mice. Data are expressed as the mean ± SE.

Figure 8.

Deletion of cannabinoid 1 receptors (CB1Rs) in the ventromedial hypothalamus does not alter body weight in high-fat diet (HFD)-fed female mice. A: body weight curve; HFD is given starting at wk 0. Lean mass (B), fat mass (C), fasting plasma insulin (D), glucose tolerance test (GTT; E), and insulin tolerance test (ITT; F) from 20- to 24-wk-old female mice. Results shown as mean ± SE.