Basic ScienceHyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis
Introduction
Insulin clearance occurs mostly in the liver and to a lower extent in kidneys and other peripheral tissues [1]. Upon its pulsatile release from pancreatic β-cells into the portal vein [2,3], insulin crosses the sinusoidal endothelium to reach and activate its receptor on the hepatocytic surface membrane [4]. This phosphorylates other substrates and delivers insulin to early/late endosomes for degradation before the receptor recycles back to the surface membrane [5]. In this manner, excess insulin is rapidly removed and maintained at a physiologically higher concentration in the portal vein than in the systemic circulation [6]. Under hyperinsulinemic conditions, the receptor is diverted to lysosomal degradation [7,8] to cause cellular insulin resistance.
Chronic hyperinsulinemia also induces hepatic de novo lipogenesis by activating SREBP1c, a master transcriptional regulator of lipogenic genes [9]. Thus, impaired hepatic insulin clearance leads to a concomitant increase in hepatic steatosis and hepatic insulin resistance.
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a surface membrane substrate of the insulin receptor, promotes insulin clearance [10,11] by taking part of the insulin-insulin receptor complex and increasing its rate of uptake and endosomal targeting [12,13]. Subsequently, it binds to cytosolic fatty acid synthase (FASN), an event that detaches it from the complex to facilitate the dissociation of insulin from its receptor while mediating an inhibitory effect of insulin on FASN [14]. This restricts hepatic de novo lipogenesis and protects the liver against high portal insulin levels. Consistently, mice with null deletion of Ceacam1 gene (Cc1−/−) manifested impaired insulin clearance and chronic hyperinsulinemia at 2 months, followed by insulin resistance, primarily hepatic, at ~6 months of age when propagated on C57BL/6J background [11]. Deletion of Ceacam1 gene induced triacylglycerol production and redistribution to white adipose tissue (WAT). This resulted in hepatic steatosis and visceral obesity at 2 months, followed by lipolysis and mobilization of non-esterified free fatty acids (NEFA) at ~6 months of age [11].
Because of the regulatory effect of CEACAM1 on lipid production (by preventing hyperinsulinemia or mediating insulin downregulation of FASN activity), it is possible that deleting Ceacam1 primarily increased FASN synthesis and hepatic de novo lipogenesis, independently of hyperinsulinemia, to lead to visceral obesity and NEFA mobilization, which causes hepatic insulin resistance (portal hypothesis) [15] that in turn induces a compensatory increase in insulin secretion and ensuing hyperinsulinemia. This model would be consistent with the commonly accepted paradigm of primary insulin resistance causing chronic hyperinsulinemia mainly by inducing a compensatory increase in insulin secretion. However, blocking lipolysis with nicotinic acid did not restore insulin clearance or sensitivity [16]. Together with intact β-cell mass [11], this indicates that hyperinsulinemia in Cc1−/− mice resulted primarily from impaired insulin clearance rather than increased insulin secretion, and that it caused insulin resistance independently of lipolysis.
Cc1−/− mice also developed leptin resistance concomitantly with insulin resistance, basal hyperphagia and decreased spontaneous locomotor activity [17]. Whereas hyperinsulinemia-driven insulin resistance in the hypothalamus caused energy imbalance, as shown by liver-specific rescuing of Ceacam1 in Cc1−/− mice [16], deleting CEACAM1 from the arcuate nucleus region of the hypothalamus, in particular pro-opiomelanocortin (POMC)-expressing neurons, may contribute to the regulation of energy imbalance in Cc1−/− mutants [17].
Given the multiple factors that could contribute to insulin resistance in Cc1−/− mice, including impaired insulin clearance, lipolysis and leptin resistance, it became imperative to identify its primary underlying mechanism with the overarching goal to determine whether it caused or resulted from hyperinsulinemia. To this end, we generated a liver-specific C57BL/6J.Ceacam1 knockout mouse (AlbCre+Cc1fl/fl) and characterized the hepatic and extra-hepatic mechanisms that could be implicated in its altered metabolic phenotype.
Section snippets
Generation of Liver-specific AlbCre+Cc1fl/fl Mice
As in the conditional T cell-specific null mouse [18], the targeting construct inserted a loxP-neo cassette in intron 6 and a loxP fragment in intron 9, deleting a sequence that encodes the cytoplasmic domain [19]. We crossed Cc1loxp/loxp mice with transgenic mice expressing Cre under the transcriptional control of the albumin gene promoter (AlbCre) on C57BL/6J background (Jackson Laboratories, Bar Harbor, ME) (Fig. S1). Heterozygotes were backcrossed >6× with C57BL/6J mice. Offsprings were
Liver-specific Deletion of CEACAM1
qRT-PCR analysis demonstrated that mouse Ceacam1 mRNA (mCc1) was absent in primary hepatocytes from AlbCre+Cc1fl/fl mice (Alb+Cc1fl/fl for simplicity) (Fig. 1A). Immunoblotting with mouse α-CEACAM1 (Ib:α-mCC1) detected CEACAM1 protein in the livers of controls (Alb–Cc1+/+, Alb+Cc1+/+ and Alb–Cc1fl/fl), but not Alb+Cc1fl/fl mutants (Fig. 1B.i). CEACAM1 protein content was intact in other tissues from mutant mice, including kidney, hypothalamus and heart (Fig. 1B.i-ii).
Early Onset of Impaired Insulin Clearance and Hyperinsulinemia in Alb+Cc1fl/fl Mice
Like Cc1−/− [11], Alb+Cc1
Discussion
The cause-effect relationship between insulin resistance and hyperinsulinemia remains elusive [37]. Whereas primary insulin resistance causes hyperinsulinemia mainly by inducing a compensatory increase in insulin secretion [38], evidence in support of the causative role of chronic hyperinsulinemia in insulin resistance is mounting [39,40], in particular when hyperinsulinemia results from impaired insulin clearance [41]. This is underlined by several mechanisms, including downregulation of the
Acknowledgments
We thank M. Kopfman at the Najjar laboratory for her technical assistance in maintaining the mouse lines and assisting in genotyping and phenotyping. We also thank the Osteopathic Heritage Foundation for its John J. Kopchick, PhD, Eminent Research Chair fund to SMN.
Funding
This work was supported by NIH grants: R01-DK054254, R01-DK083850, and R01-HL112248 to SMN, R01-HD081792 to JWH, and 5U2C-DK093000 to JKK. The work was also supported by the Middle-East Diabetes Research Center to HEG and SSG, and the American Heart Association (14POST20480294) to LR.
Duality of Interest
The authors declare no duality of interest.
Author Contributions
HEG, LR, HTM, SSG, IHM, JEH, HLN, and SS researched data. HEG coordinated the research among co-authors, analyzed data and wrote the first draft of the manuscript. JKK analyzed the hyperinsulinemic-euglycemic clamp data. SMN analyzed data and reviewed and revised the manuscript. SMN had full access to all the data of the study and takes responsibility for the conception of the studies and for the integrity and accuracy of data analysis. Original data are available upon request from the senior
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