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, 20 (7), 1394-402

Adropin Deficiency Is Associated With Increased Adiposity and Insulin Resistance

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Adropin Deficiency Is Associated With Increased Adiposity and Insulin Resistance

K Ganesh Kumar et al. Obesity (Silver Spring).

Abstract

Adropin is a secreted peptide that improves hepatic steatosis and glucose homeostasis when administered to diet-induced obese mice. It is not clear if adropin is a peptide hormone regulated by signals of metabolic state. Moreover, the significance of a decline in adropin expression with obesity with respect to metabolic disease is also not clear. We investigated the regulation of serum adropin by metabolic status and diet. Serum adropin levels were high in chow-fed conditions and were suppressed by fasting and diet-induced obesity (DIO). High adropin levels were observed in mice fed a high-fat low carbohydrate diet, whereas lower levels were observed in mice fed a low-fat high carbohydrate diet. To investigate the role of adropin deficiency in metabolic homeostasis, we generated adropin knockout mice (AdrKO) on the C57BL/6J background. AdrKO displayed a 50%-increase in increase in adiposity, although food intake and energy expenditure were normal. AdrKO also exhibited dyslipidemia and impaired suppression of endogenous glucose production (EndoR(a)) in hyperinsulinemic-euglycemic clamp conditions, suggesting insulin resistance. While homo- and heterozygous carriers of the null adropin allele exhibited normal DIO relative to controls, impaired glucose tolerance associated with weight gain was more severe in both groups. In summary, adropin is a peptide hormone regulated by fasting and feeding. In fed conditions, adropin levels are regulated dietary macronutrients, and increase with dietary fat content. Adropin is not required for regulating food intake, however, its functions impact on adiposity and are involved in preventing insulin resistance, dyslipidemia, and impaired glucose tolerance.

Conflict of interest statement

DISCLOSURE

The authors declared no conflict of interest.

Figures

Figure 1
Figure 1
Serum adropin levels in wild-type mice subjected to various nutritional challenges and in adropin knockout mice (AdrKO). (a) Adropin levels were measured using ELISA in sera collected from wild-type C57BL/6J (B6) mice that were fasted overnight or allowed ad libitum access to rodent chow. No adropin immunoreactivity was observed in sera collected from AdrKO. There were 5–6 mice per group, *P < 0.05 vs. fasted and AdrKO. (b) Measurement of serum adropin in lean B6 mice fed a purified low-fat diet (10% kJ/fat, 70% kJ/carbohydrate) or high-fat diet (60% kJ/fat, 20 kJ%/carbohydrate) for 48 h. All samples were collected at 0900 h from mice housed in a 12 h light:dark cycle, with lights-on from 0600–1800 h, with six mice per group, *P < 0.05 vs. the low-fat diet-fed mice.
Figure 2
Figure 2
Gene targeting strategy used to generate adropin knockout mice (AdrKO). (a) The Enho gene is comprised of two exons, with the adropin open reading frame (ORF) situated in exon 2 (E2). A disrupted Enho allele was generated by inserting loxP sites into the single intron and 3′ of the adropin ORF in exon 2. Expression of Cre-recombinase removes part of the intron and the part of exon 2 containing the open reading frame. Note that this will also remove the neomycin selection cassette (Neo). (b) The transcript produced by the Enho gene is not detected by reverse transcription (RT)-PCR in total RNA samples from the liver, brain, and skeletal muscle of AdrKO. (c) RT-PCR analysis of Dnaic1 and Cntr expression in the central nervous system of WT (+/+), AdrHET (+/−), and AdrKO (−/−).
Figure 3
Figure 3
Adropin knockout mice (KO) weaned onto standard rodent chow exhibit increased adiposity at 9 weeks, but not 6 weeks of age. (a, b) Body weight and adiposity (fat mass as a percent of total body weight) (c, d) are shown for male and female mice at (a, c) 6 weeks and (b, d) 9 weeks of age. At 9 weeks of age, male and female knockout mice exhibit increased fat mass and adiposity; there was no significant difference in any of the parameter measured in heterozygous carriers of the null allele (HET). *P < 0.05 vs. WT. n = 3–5 per group.
Figure 4
Figure 4
Comparison of energy expenditure and insulin sensitivity in male adropin knockout mice (AdrKO) and controls. (a) Respiratory exchange ratio (RER), (b) energy expenditure, and (c) mean hourly movements in the X-axis in 8-week-old control wild-type (WT, n = 8) and AdrKO mice (n = 7). Data shown are the mean of measurements recorded over 48 h, and are presented as mean for the lights-on and dark periods. There was no significant difference in RER or energy expenditure, the reduction in movement of AdrKO in the dark period was statistically significant (*P < 0.05). (dg) Results from the hyperinsulinemic–euglycemic clamp comparing insulin function in WT (n = 8) and AdrKO mice (n = 9). Insulin was infused to generate a physiological increase in (d) serum insulin, and glucose was infused at a variable rate to maintain (e) euglycemia. The glucose infusion rate (GIR) was significantly lower in (f) AdrKO (*P < 0.05). Glucose uptake was reduced by 30–35% in gastrocnemius and diaphragm muscle, but was normal in other (g) muscle types and organs. (h–j) Insulin stimulation of Akt phosphorylation is normal in the liver of AdrKO. (h) Representative western blots showing phosphorylation of Akt on threonine 308 (pAkt, T308), serine 473 (pAkt, S473), and total Akt protein. β-Actin levels were assessed as a control for loading. Quantification of (i) T308 and (j) S473 phosphorylation, expressed as a ratio of total Akt protein (n = 3–5 per group).
Figure 5
Figure 5
Adropin-deficiency does not affect weight gain associated with high-fat diets, but is associated with an increased severity of impaired glucose homeostasis associated with obesity. (a) Body composition of wild-type control (WT), heterozygous carriers of the null adropin allele (HET) and adropin knockout (KO) mice weaned onto chow, and then after 8 weeks on the high-fat diet (60% kJ/fat, HFD) (n = 6/group). Exposure to the HFD resulted in weight gain predominantly due to increased fat mass. Body weight and composition were not significantly different after 8 weeks on HFD; at the start of the study, there was a tendency (P = 0.07) for increased fat mass in adropin knockout mice. (b) Food intake expressed as kJ/day was not significantly affected by genotype. Food intake was recorded over a 1 month period. (c–e) Insulin and glucose measurements recorded at the end of 8 weeks on HFD. Hyperinsulinemia and hyperglycemia were more severe in adropin knockout mice relative to WT. The differences in insulin, blood glucose and homeostasis model assessment–insulin resistance (HOMAIR) between WT and KO were significant (*P < 0.05, **P < 0.001). The differences in insulin, blood glucose, and HOMAIR between HET and KO were not statistically significant. (e) Adropin knockout mice exhibited fasting hypertriglyceridemia (*P < 0.05 vs. WT). (f, g) Impaired glucose tolerance associated with diet-induced obesity is more severe in heterozygous and homozygous carriers of the null adropin allele. Shown are the raw data (f, P < 0.05 when comparing WT vs. *KO only, **WT vs. KO and HET, #WT vs. HET only) and the area under the curve (AUC) above baseline levels (g, *P < 0.05 vs. WT). (h, i) Insulin tolerance tests suggest reduced insulin action in AdrKO carriers of the null adropin allele fed high-fat diet. *P < 0.05, WT vs. KO.
Figure 6
Figure 6
Increased hepatic lipid content and expression of lipogenic genes in adropin knockout mice (AdrKO)-fed high-fat diet (HFD). (a) Total lipid and triglyceride (TG) content (per g wet weight) of liver samples collected from fasted wild type (WT), heterozygous (HET), and homozygous carriers (KO) of the null adropin allele. *P < 0.05 vs. WT. (b) Histological analysis of the liver phenotye of WT mice with AdrKO fed HFD. (c) Expression of genes involved in lipid and carbohydrate metabolism in livers of WT, HET, and KO mice. The expression of stearoyl-CoA desaturase-1 (SCD1) and sterol-regulatory element-binding protein-1c (SREBP1C) was significantly affected by genotype. ACC1, acetyl-CoA carboxylase; CPT1, carnitine palmitoyl transferase-1; FAS, fatty acid synthase; G6p, glucose-6-phosphatase; LPL, lipoprotein lipase; PEPCK, phosphoenolpyruvate carboxykinase; PPARα, peroxisome proliferator- activated receptor α.

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