Sex-Specific Control of Fat Mass and Counterregulation by Hypothalamic Glucokinase
Article Figures & Tables
Figures
- Figure 1
Inactivation of the Gck gene in Sf1 neurons. A: Structure of the Gck gene, the targeting vector, the floxed Gck allele after flp-mediated removal of the puromycin (Puro) gene, and the recombined Gck allele following Cre-dependent recombination (KO allele). Location of the primers for genotyping are indicated by arrows. B: Brightfield and fluorescence microscopy images of a brain section of an Sf1-Cre;Rosa26tdtomato;Gck+/+ mouse. The VMN (dashed line) and ARC (dotted line) are indicated. C: Following laser-capture microdissection of the VMN or ARC from brains of Sf1-Cre;ROSA26tdtomato;Gck+/+ (Ctrl) and Sf1-Cre;ROSA26tdtomato;Gcklox/lox (KO) mice, RNA was extracted and Gck mRNA was quantified by quantitative RT-PCR analysis. Data are normalized to β-actin expression and set at 1 for VMN Ctrl. nd, not detectable. D: In situ hybridization detection of Gck on coronal hypothalamic sections (Bregma −1.7). Gck was not detected in the VMN (dashed line) of Sf1Gck−/− (KO) mice but was still present in the ARC (dotted line).
- Figure 2
Increased adiposity in female but not male Sf1Gck−/− mice fed a normal chow. A and B: Female and male Ctrl and Sf1Gck−/− (KO) mice were fed normal chow, and their body weight was monitored over 20 weeks (n = 12–20 mice per group). C–F: Fat (C and E) and lean mass (D and F) were measured by EchoMRI in female and male Ctrl and KO mice at 12 and 24 weeks of age (n = 12–20 mice per group). G–I: Gonadal fat (G), inguinal fat (H), and brown adipose tissue (BAT) (I) weights in 26-week-old mice (n = 12–20 mice). J and K: Indirect calorimetry measurement of heat production in female (J) and male (K) Ctrl and KO mice (n = 5–6 mice). hr, hour; ON, overnight. Evaluations used 2-way ANOVA (A and B) and the Student t test (C–K). *P < 0.05; ** P < 0.01; *** P < 0.001.
- Figure 3
Increased adipocyte size in 26-week-old Sf1Gck−/− female mice fed normal chow. Histological sections of gonadal (A) and inguinal (B) fat of Ctrl and Sf1Gck−/− (KO) mice. C and D: Adipocyte size distribution in gonadal (C) and inguinal (D) fat depots of female mice (n = 4, 7–16 images per mouse). E and F: Adipocyte size distribution in gonadal (E) and inguinal (F) fat depots of male mice. Lipolytic and lipogenic gene expression in gonadal (G) and inguinal WAT (H) of female Ctrl and KO mice (n = 7–8 mice). Evaluations used 2-way ANOVA (C–F) and the Student t test (G and H). *P < 0.05; **P < 0.01; ***P < 0.001.
- Figure 4
Normal glucose homeostasis in Sf1Gck−/− mice. Blood glucose in random-fed (A) and overnight (ON)-fasted (B) Ctrl and Sf1Gck−/− (KO) mice at 24 weeks of age. Plasma insulin concentrations in random-fed (C) and ON-fasted (D) Ctrl and KO mice at 27 weeks of age. The same mice displayed a normal intraperitoneal glucose tolerance test (2g/kg) (E and F). n = 8–12 mice. Evaluations used the Student t test (A–D) or 2-way ANOVA (E and F). *P < 0.05.
- Figure 5
Lower glucagon secretion in response to hypoglycemia in female Sf1Gck−/− mice. Blood glucose (A, females; C, males) and plasma glucagon concentrations (B, females; D, males) 1 h after an intraperitoneal injection of saline or insulin (0.3–0.7 U/kg) in Ctrl and Sf1Gck−/− (KO) mice (18–31 weeks old; n = 12–32). E and F: Plasma epinephrine and norepinephrine 1 h after insulin injection to induce hypoglycemia (0.6 U/kg) in 18-week-old female Ctrl and KO mice. G: Blood glucose concentrations during a hypoglycemic-hyperinsulinemic clamp in female Ctrl and KO mice. H: Plasma glucagon concentrations at the end of the clamp (n = 7–8 mice, 18 weeks of age). I: Pancreatic glucagon content (n = 5–6 mice). J: Overnight-fasted plasma glucagon concentrations (n = 9–14 mice). Two-way ANOVA (A–F) and the Student t test (H–J) were used; the effect of insulin is significant in all experiments. *P < 0.05; **P < 0.01.
- Figure 6
No induction of parasympathetic and sympathetic nerve activity by neuroglucopenia in female Sf1Gck−/− mice. A: Parasympathetic nerve activity recordings in Ctrl and Sf1Gck−/− (KO) mice in the basal state and after 2DG injection (600 mg/kg i.p.). B: Sympathetic nerve activity recordings in Ctrl and KO mice in the basal state and after intraperitoneal 2DG injection. C–F: Quantification of the firing rates of the parasympathetic (C and E) and sympathetic (D and F) nerve activities in female and male mice (n = 5–10 mice, 12 weeks of age). sec, second. Two-way ANOVA was used. *P < 0.05; **P < 0.01; ***P < 0.001.
- Figure 7
Gck is not required for the glucose responsiveness of Sf1 GE and GI neurons. Whole-cell patch clamp recordings on Sf1 neurons from Sf1-Cre;ROSA26tdtomato;Gck+/+ (Ctrl) and Sf1-Cre;ROSA26tdtomato;Gcklox/lox (KO) mice. A and B: GI neuron activation by 0.1 mmol/L glucose is detected in Ctrl (A) and KO (B) Sf1 neurons. Similarly, inhibition of GE neurons by 0.1 mmol/L glucose is observed in both Ctrl (C) and KO (D) Sf1 neurons.
Tables
- Table 1
Whole-cell patch clamp recordings of Sf1 neurons from Sf1-Cre;ROSA26tdtomato;Gck+/+ (Ctrl) and Sf1-Cre;ROSA26tdtomato;Gcklox/lox (KO) mice show a similar distribution of GI, GE, and nonresponder neurons in male and female mice
Ctrl mice KO mice Non-Sf1 neurons in the VMN* (female and male) Female Male Female Male GI neurons 3 5 8 6 3 GE neurons 6 7 8 6 4 Nonresponder neurons 3 5 7 4 14 Total 12 17 23 16 21 Data are n.
*Non-Sf1 neurons in the VMN were mainly nonresponder neurons.
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