Myonectin deletion promotes adipose fat storage and reduces liver steatosis

Abstract

We recently described myonectin (also known as erythroferrone) as a novel skeletal muscle-derived myokine with metabolic functions. Here, we use a genetic mouse model to determine myonectin's requirement for metabolic homeostasis. Female myonectin-deficient mice had larger gonadal fat pads and developed mild insulin resistance when fed a high-fat diet (HFD) and had reduced food intake during refeeding after an unfed period but were otherwise indistinguishable from wild-type littermates. Male mice lacking myonectin, however, had reduced physical activity when fed ad libitum and in the postprandial state but not during the unfed period. When stressed with an HFD, myonectin-knockout male mice had significantly elevated VLDL-triglyceride (TG) and strikingly impaired lipid clearance from circulation following an oral lipid load. Fat distribution between adipose and liver was also altered in myonectin-deficient male mice fed an HFD. Greater fat storage resulted in significantly enlarged adipocytes and was associated with increased postprandial lipoprotein lipase activity in adipose tissue. Parallel to this was a striking reduction in liver steatosis due to significantly reduced TG accumulation. Liver metabolite profiling revealed additional significant changes in bile acids and 1-carbon metabolism pathways. Combined, our data affirm the physiologic importance of myonectin in regulating local and systemic lipid metabolism.-Little, H. C., Rodriguez, S., Lei, X., Tan, S. Y., Stewart, A. N., Sahagun, A., Sarver, D. C., Wong, G. W. Myonectin deletion promotes adipose fat storage and reduces liver steatosis.

Keywords: carbohydrate metabolism; diabetes; lipid metabolism; myokine; obesity.

Figure 1

Myonectin-deficient mouse model. A) Schematic of the gene targeting strategy used to generate myonectin-KO mice. The majority of the myonectin gene (exons 2–7) was replaced with a neomycin resistance gene and lacZ reporter cassette. SA, splice acceptor; pA, polyadenylation signal. B) PCR genotyping showing successful production of WT (+/+), heterozygous (+/−), and homozygous KO (−/−) mice. C) The absence of myonectin transcript in the mouse gastrocnemius muscle was confirmed by quantitative PCR using 2 independent primer pairs. Primer set 1 targets exons 2 and 3, yielding a PCR product size of 143 bp. Primer set 2 targets exons 4 and 6, yielding a PCR product size of 381 bp. WT, n = 6; KO, n = 6 female mice. lacZ, β-galactosidase; loxP, locus of X-over in P1; Neg. Ctrl., negative control; neo, neomycin resistant.

Figure 2

There are no overt carbohydrate metabolism phenotypes in the myonectin-KO mice fed an HFD. A) Body weights of female (WT, n = 8; KO, n = 8) and male (WT, n = 12–13; KO, n = 16) mice over time. Mice were weaned at 3.5 wk of age onto a standard chow diet. At 5 wk of age, the diet was switched to an HFD. B) Body composition analysis of female (WT, n = 7; KO, n = 8) and male (WT, n = 12; KO, n = 16) mice at 28 wk of age. C) Blood glucose levels during intraperitoneal GTTs in 18-wk-old female mice (WT, n = 8; KO, n = 8). D) Blood glucose levels during oral GTT in 19-wk-old male mice (WT, n = 12; KO, n = 12). E) Serum insulin levels in male mice at 0 min before glucose gavage (WT, n = 11; KO, n = 9) and at 15 min after glucose gavage (WT, n = 12; KO, n = 11); for some samples, insulin levels were below the threshold of detection. F, G) Blood glucose levels during ITTs in female mice (WT, n = 7; KO, n = 8) and male mice (WT, n = 11; KO, n = 15) at 23 wk of age. Insulin was injected at a dose of 1.2 U/kg body weight for female mice and 1.5 U/kg body weight for male mice. *P < 0.05.

Figure 3

Impaired lipid tolerance in myonectin-KO male mice fed an HFD. Serum TG (A) and NEFA (B) during lipid tolerance test in male mice (WT, n = 12; KO, n = 16) at 17 wk of age. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Higher postprandial VLDL-TG levels in myonectin-KO male mice fed an HFD. A–C) Serum TG (A), NEFA (B), and cholesterol (C) levels in male mice after an overnight 14-h unfed period ("fasted") or after an unfed period overnight followed by ad libitum refeeding for 2 h (refed). Mice (36 wk of age) were euthanized after refeeding for tissue collection. A total of 2 WT mice did not eat during the refeed period as assessed by no increase in serum TG levels over fasting levels and an empty stomach. Therefore, refed data from these mice were not included in the analysis. Unfed: WT, n = 12; KO, n = 17. Refed: WT, n = 10; KO, n = 17. D, E) Refed serum samples from 10 mice of each genotype were pooled and subjected to FPLC fractionation to separate lipoprotein species. TG (D) and cholesterol (E) levels were measured in each fraction. IDL, intermediate-density lipoprotein. *P < 0.05.

Figure 5

Myonectin deficiency alters lipid distribution in male mice fed an HFD. A–D) WT and myonectin-KO mice were euthanized at 39 wk of age after a 2–4-h unfed period in the morning. Unless otherwise specified, data in this figure are from the analyses of WT (n = 12) and KO (n = 16) male mice. The wet weight of liver (A), visceral (gonadal) fat pad (B), and subcutaneous (inguinal) fat pad (C) are presented along with representative images of H&E-stained liver sections from WT and KO mice (D). E, F) Quantification of TG (E) and cholesterol (F) levels in livers. G) Western blot analysis of Perilipin 2. A total of 2 separate gels were run, each with WT (n = 6) and KO (n = 8) samples. Perilipin 2 (PLIN2) levels were first normalized to HSC70 levels from the same gel and then normalized to the mean WT value. Data were then combined from both gels for analysis. H, I) Representative images of H&E-stained eWAT (H) and iWAT (I) sections from WT and KO male mice. J) Quantification of adipocyte size in eWAT sections (WT, n = 5; KO, n = 5). K) Quantification of adipocyte size in iWAT sections (WT, n = 4; KO, n = 5). *P < 0.05, **P < 0.01.

Figure 6

Steady-state serum and liver metabolite levels in WT and myonectin-KO male mice fed an HFD. Sera and livers were harvested from mice (39 wk old) 2–4 h after food removal. A–D) Quantification of serum TG (A), NEFA (B), cholesterol (C), and ketones (β-hydroxybutyrate) (D) (WT, n = 12; KO, n = 16). E) All liver metabolites that were significantly different (P < 0.05) between WT (n = 10) and myonectin-KO (n = 10) male mice. *P < 0.05.

Figure 7

Postprandial LPL activity in the s.c. (inguinal) fat depot is increased in myonectin-KO male mice fed an HFD. A) LPL activity in eWAT (WT, n = 12; KO, n = 16) and iWAT (WT, n = 12; KO, n = 15) from mice euthanized at 39 wk of age following a 2–4-h unfed period in the morning. B) LPL activity in eWAT and iWAT from mice (WT, n = 10; KO, n = 17) euthanized at 36 wk of age after 2-h refeeding following food removal overnight. C) LPL mRNA expression in the same eWAT (WT, n = 10; KO, n = 16) and iWAT (WT, n = 10; KO, n = 15) samples as in B. D, E) Western blot analysis of LPL in the same iWAT samples as in B. A total of 2 separate gels were run: 1 with 6 WT and 8 KO samples, and the other with 4 WT and 8 KO samples. LPL levels were first normalized to HSC70 levels from the same gel, then normalized to the mean WT value. Data were then combined from both gels for analysis (WT, n = 10; KO, n = 16) (E). Images from 1 gel are shown in D. ***P < 0.001.