Liver adrenoceptor alpha-1b plays a key role in energy and glucose homeostasis in female mice

Abstract

The liver plays a major role in glucose and lipid homeostasis and acts as a key organ in the pathophysiology of metabolic diseases. Intriguingly, increased sympathetic nervous system (SNS) activity to the liver has been associated with the development and progression of type 2 diabetes and obesity. However, the precise mechanisms by which the SNS regulates hepatic metabolism remain to be defined. Although liver α1-adrenoceptors were suggested to play a role in glucose homeostasis, the specific subtypes involved are unknown mainly because of the limitations of pharmacological tools. Here, we generated and validated a novel mouse model allowing tissue-specific deletion of α-1b adrenoceptor (Adra1b) in hepatocytes to investigate the role of liver ADRA1B in energy and glucose metabolism. We found that selective deletion of Adra1b in mouse liver has limited metabolic impact in lean mice. However, loss of Adra1b in hepatocytes exacerbated diet-induced obesity, insulin resistance, and glucose intolerance in female, but not in male mice. In obese females, this was accompanied by reduced hepatic gluconeogenic capacity and reprogramming of gonadal adipose tissue with hyperleptinemia. Our data highlight sex-dependent mechanisms by which the SNS regulates energy and glucose homeostasis through liver ADRA1B.NEW & NOTEWORTHY The sympathetic nervous system plays an important role in regulating hepatic physiology and metabolism. However, the identity of the adrenoceptors involved in these effects is still elusive. Using CRISPR-Cas9, we developed a novel transgenic tool to study the role of liver α-1b adrenoceptor (ADRA1B). We show that ADRA1B plays a key role in mediating the effects of the sympathetic nervous system on hepatic metabolism, particularly in female mice.

Keywords: adrenergic receptor; glucose metabolism; liver; sex difference; sympathetic nervous system.

Graphical abstract

Figure 1.

Generation and validation of liver-specific Adra1b knock-out mice. A: gene expression analysis of the subtypes of adrenoceptors detected in the liver of C57BL/6J mice (n = 8). B: using CRISPR-Cas9 technology, exon 3 and exon 4 (which contains the start codon) of the Adra1b gene were targeted and flanked by two loxP sites. C: sequences of the two synthetic sgRNA synthetized to insert the LoxP sites. D: sequences of the donor templates consisting of 200 bp ultramer DNA oligonucleotides. E: genotyping of Adra1b^fl/fl, Adra1b^fl/+, and Adra1b^+/+ mice. F: schematic representation of the breeding strategy used to generate Adra1b^LKO mice. G: chromogenic in situ hybridization showing the presence in Adra1b^WT and the absence in Adra1b^LKO, of Adra1b (blue dots). Representative image from n = 2 mice/group. Expression of Adra1b in heart (H) and hypothalamus (I) of Adra1b^WT and Adra1b^LKO mice (n = 8/group). Expression of Adra1b (J), Adra1a (K), Adra2b (L), Adrb1 (M), and Adrb2 (N) in the liver of male and female Adra1b^WT and Adra1b^LKO mice (n = 6–8/group). Significant differences (P < 0.05) between groups (assessed by two-way ANOVA) are presented in each graph (**P < 0.01, ***P < 0.0001). KO, knockout; WT, wild type.

Figure 2.

Loss of liver Adra1b has limited impact in male and female mice fed laboratory chow. A: body weight of 24-wk-old male (n = 8/group) and female (n = 6/group) Adra1b^WT and Adra1b^LKO mice fed laboratory chow. Weight of gonadal white adipose tissue (gWAT, B), inguinal (iWAT, C), brown adipose tissue (BAT, D), heart (E), and liver (F) of mice presented in A. Liver triglyceride (G) and glycogen (H) content (normalized on liver weight) of mice presented in A. I: fasting (4 h) glycemia of mice presented in A. J: glucose tolerance test (GTT) was performed 2 wk before euthanizing the mice. K: area under the curve for the GTT shown in J. L: insulin tolerance test (ITT) was performed 1 wk before euthanizing the mice. M: area under the curve for the ITT shown in L. Significant differences (P < 0.05) between sex and genotype (assessed by two-way ANOVA) are presented in each graph.

Figure 3.

Loss of liver Adra1b exacerbates diet-induced obesity, insulin resistance, and glucose intolerance in female, but not in male mice, fed an obesogenic diet. Body weight curve (A) and final body weight (B) of male Adra1b^WT and Adra1b^LKO mice fed a high-fat diet from 10 to 11 wk of age for 8 wk (n = 5–8/group). C: liver weight of mice presented in A and B. D: glucose tolerance test (GTT) was performed 2 wk before euthanizing the mice. E: area under the curve for the GTT shown in D. F: insulin tolerance test (ITT) was performed 1 wk before euthanizing the mice. G: area under the curve for the ITT shown in L. Body weight curve (H) and final body weight (I) of female Adra1b^WT and Adra1b^LKO mice fed a high-fat diet from 10 to 11 wk of age for 8 wk (n = 9–15/group). J: liver weight of mice presented in H and I. K: glucose tolerance test (GTT) was performed 2 wk before euthanizing the mice. L: area under the curve for the GTT shown in K. M: insulin tolerance test (ITT) was performed 1 wk before euthanizing the mice. N: area under the curve for the ITT shown in M. Significant differences (P < 0.05) between groups (assessed by Student’s t test for I, J, L, and N; by one-way ANOVA for H, K, and N) are presented in each graph. (*P < 0.05, **P < 0.01).

Figure 4.

Loss of liver Adra1b in obese female mice does not impact energy expenditure. Oxygen consumption (V̇o₂, A) and averageV̇o₂ (B) over 3 days. C: average energy expenditure (EE) over 3 days. Respiratory exchange ratio (RER, D) and average RER (E) over 3 days. Food intake over 3 days (F) and average food intake per day (G). Water intake over 3 days (H) and average water intake per day (I) (n = 8/group).

Figure 5.

Pair feeding female Adra1b^LKO mice prevents the exacerbation of obesity and glucose impairments. Daily food intake (A) and cumulative food intake (B) of female Adra1b^WT and Adra1b^LKO mice. The amount of food given to Adra1b^LKO was based on the average eaten by Adra1b^WT the day before. Body weight curve (C) and body weight (D) of Adra1b^WT and pair-fed Adra1b^LKO mice. E: glucose tolerance test (GTT). F: area under the curve for the GTT shown in E. G: insulin tolerance test (ITT). H: area under the curve for the ITT shown in G (n = 7 or 8/group).

Figure 6.

Loss of liver Adra1b affects hepatic gluconeogenic capacity and impairs gonadal white adipose tissue in female mice fed an obesogenic diet. Glycogen (A), triglyceride (B), and cholesterol (C) content in the liver of mice presented in Fig. 3. Data are normalized on liver weight. D: representative hematoxylin and eosin (H&E) staining of the liver of mice presented in Fig. 3. E: analysis of genes involved in gluconeogenesis, de novo lipogeneses, fatty acid transport, and inflammation in the liver. F: weight of gonadal white adipose tissue (gWAT). G: analysis of the expression of genes coding for adrenergic receptors and adipokines, and of genes involved in lipogenesis and lipolysis in gonadal white adipose tissue of mice presented in Fig. 3. H: circulating leptin levels in female Adra1b^WT and Adra1b^LKO mice. Significant differences (P < 0.05) between groups (assessed by Student’s t test) are presented in each graph (n = 9–15/group). (*P < 0.05, **P < 0.01).