Hypothalamic POMC deficiency increases circulating adiponectin despite obesity

Abstract

Objective: The steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc^-/-) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc^-/- mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels.

Methods: Circulating adiponectin was measured in obese ArcPomc^-/- mice at ages 4-52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc^-/- mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc^-/- mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc^-/- mice. Finally, we generated compound Adiopoq^-/-:ArcPomc^-/- mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls.

Results: Obese ArcPomc^-/- female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc^-/- female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc^-/- female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc^-/- mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc^-/- female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc^-/- mice did not exacerbate their metabolic abnormalities.

Conclusion: In summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc^-/- mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin.

Keywords: Adiponectin; Melanocortin system; POMC; Sympathetic nervous system.

Figure 1

Increased plasma adiponectin in obese hypothalamic Pomc-deficient mice. Comparison of body weights and circulating adiponectin levels in Pomc^+/+ and ArcPomc^−/− female (A) and male (B) mice on a congenic C57BL/6 J genetic background at the indicated ages (n = 6–10). (C) Immunoblots of plasma protein from 8 to 9-week-old female Pomc^+/+ and ArcPomc^−/− mice with a congenic C57BL/6J genetic background. Total, high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW) adiponectin band intensities were quantified using ImageJ (right) (n = 4). Two-tailed unpaired Student's t-tests were used for comparisons. Data shown are the mean ± s.e.m. of biologically independent samples. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 2

Increased plasma adiponectin in ArcPomc^−/−mice was independent of body weight and fat mass. Restricted feeding was performed on 15-week-old female ArcPomc^−/− mice for 7 weeks (n = 5–8). Body weights (A), body composition (B), and plasma adiponectin (C) before and after food restriction. Pair-feeding was conducted on 4-week-old female ArcPomc^−/− mice for 5 weeks (n = 7–8). Body weights (D), body composition (E), and plasma adiponectin (F) before and after pair-feeding. Two-way ANOVAs or mixed-effects models (depending on the missing values) were used to analyze the effects of treatment and genotype. Post hoc Bonferroni's and Tukey's multiple comparisons were conducted. Data shown are the mean ± s.e.m of biologically independent samples. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 3

Reactivation of Pomc expression in the arcuate nucleus or MTII treatment reduced plasma adiponectin. (A) Schematic representation of the generation of transgenic mice with Cre recombinase-inducible activation of Pomc gene expression in the arcuate nucleus after tamoxifen injection. The inducible ArcPomc^−/− transgenic mice had a loxP-flanked neomycin (neo) resistance cassette inserted upstream of the deleted neuronal Pomc enhancer 2 (nPE2∗) in the Pomc gene. Purple oval, neuronal Pomc enhancer 1 (nPE1); yellow oval, Pomc promoter; and green boxes, Pomc exons. (B) Serial body weights of 41- to 55-week-old female mice at the indicated time points after tamoxifen injection (n = 5–7). (C) Tissue weight 53 days post-tamoxifen injection (n = 5–7). (D) Plasma adiponectin before and after Pomc gene activation in Arc (n = 5–7). (E) Immunoblots of plasma protein from female inducible ArcPomc^−/− and Pomc^+/+ mice pre- and post-activation of Pomc expression in Arc (n = 4). (F) Relative total and high molecular weight adiponectin band intensities (n = 4). (G) Plasma adiponectin levels after melanotan II administration (n = 7, 13–17 weeks old, female). Values were normalized to saline injection within genotypes at respective time points. Two-way ANOVAs with post hoc Bonferroni's and Tukey's multiple comparisons were conducted to examine the genotype and tamoxifen treatment effects. Two-tailed unpaired nonparametric Mann–Whitney tests were used for comparisons of tissue weights. Data shown are the mean ± s.e.m of biologically independent samples. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 4

Reduced central sympathetic outflow to adipose tissue contributed to the elevated level of plasma adiponectin in the ArcPomc^−/−mice. (A) Changes in rectal temperature during 4 h of cold exposure (4 °C) (n = 5, female mice, 30 weeks old). (B) Measurement of catecholamine concentrations in adipose tissue (n = 6–8, female mice, 24–30 weeks old). (C) Immunoblots of total brown fat and subcutaneous fat lysates from Pomc^+/+ and ArcPomc^−/− mice (n = 4–5). Relative tyrosine hydroxylase (TH) band intensity was quantified using ImageJ (right). (D) Plasma adiponectin levels of ArcPomc^−/− mice in response to norepinephrine treatment. Within-subject post-vehicle treatment values were subtracted from the post-norepinephrine treatment values (n = 6–7, female mice, 15–21 weeks old). (E) Plasma adiponectin levels of the ArcPomc^−/− mice in response to propranolol treatment. Within-subject post-vehicle treatment values were subtracted from the post-propranolol treatment values (n = 6–7, female mice, 20–25 weeks old). Two-tailed unpaired Student's t-tests were used to compare changes in body temperature, catecholamine concentrations, and norepinephrine/propranolol treatment effects. Post hoc Bonferroni's multiple comparisons following two-way ANOVAs were used to examine the genotype and sex effects on TH protein quantification. Data shown are the mean ± s.e.m of biologically independent samples. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 5

Subcutaneous fat was the primary source of elevated plasma adiponectin in the ArcPomc^−/−mice. (A) Immunoblots of total subcutaneous and gonadal fat lysates from 26-week-old female mice (left). Adiponectin band intensity was normalized to vinculin (right) (n = 4). (B) Quantitative PCR analysis of adiponectin mRNA expression in the subcutaneous, gonadal, and intrascapular brown fat (n = 6–8, 8–9 weeks old, female). Quantitative PCR analysis of Rapgef3 (C) and Adrb3 (D) in the subcutaneous and gonadal fat (n = 6–8, 8–9 weeks old, female). Two-tailed unpaired Student's t-tests were used to compare the genotype effects. Data shown are the mean ± s.e.m of biologically independent samples. ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 6

Deletion of adiponectin from the ArcPomc^−/−mice had no effect on their metabolic abnormalities. Body weight comparisons of the male (A) and female (E) mice from 4 weeks to 24 weeks of age. Comparisons of the body composition (B: male and F: female) tissue weights (C: male and G: female) and 24-hour food intake (D: male and H: female) between the ArcPomc^−/−:Adipoq^+/+ and ArcPomc^−/−:Adipoq^−/− mice (male, n = 7–11, female, n = 9). (I, J, N, and O) Oral glucose tolerance tests (I: male and N: female) and plasma insulin levels collected at corresponding time points (J: male and O: female). Insulin tolerance tests (K: male and P: female) and HOMA-IR (L: male and Q: female) from the ArcPomc^−/−:Adipoq^+/+ and ArcPomc^−/−:Adipoq^−/− mice (male: n = 6–11 and female: n = 9). Glucose levels from 16-hour fasted, 2-h refed, and 4-h refed mice (M, male: n = 6–10, R, female: n = 9). Two-tailed unpaired Student's t-tests were used to compare genotype effects in body composition, tissue weights, 24-hour food intake, HOMA-IR, fasting, and refed glucose levels. Males: 17–22 weeks old; females: 8–12 weeks old.