. 2015 Jan;35(1):26-40.

doi: 10.1128/MCB.00503-14. Epub 2014 Oct 13.

Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis

Affiliations

¹ Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA.
² Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA Internal Medicine, Section of Endocrinology and Metabolism, Yale University School of Medicine, New Haven, Connecticut, USA.
³ Cell Signaling Technology Incorporated, Danvers, Massachusetts, USA.
⁴ Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA Internal Medicine, Section of Endocrinology and Metabolism, Yale University School of Medicine, New Haven, Connecticut, USA Howard Hughes Medical Institute, Yale School of Medicine, New Haven, Connecticut, USA.
⁵ Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA Department of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, USA Program in Integrative Cell Signaling and Neurobiology of Metabolism, Yale University School of Medicine, New Haven, Connecticut, USA anton.bennett@yale.edu.

PMID: 25312648
PMCID: PMC4295383
DOI: 10.1128/MCB.00503-14

Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis

Ahmed Lawan et al. Mol Cell Biol. 2015 Jan.

. 2015 Jan;35(1):26-40.

doi: 10.1128/MCB.00503-14. Epub 2014 Oct 13.

Affiliations

¹ Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA.
² Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA Internal Medicine, Section of Endocrinology and Metabolism, Yale University School of Medicine, New Haven, Connecticut, USA.
³ Cell Signaling Technology Incorporated, Danvers, Massachusetts, USA.
⁴ Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA Internal Medicine, Section of Endocrinology and Metabolism, Yale University School of Medicine, New Haven, Connecticut, USA Howard Hughes Medical Institute, Yale School of Medicine, New Haven, Connecticut, USA.
⁵ Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA Department of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, USA Program in Integrative Cell Signaling and Neurobiology of Metabolism, Yale University School of Medicine, New Haven, Connecticut, USA anton.bennett@yale.edu.

PMID: 25312648
PMCID: PMC4295383
DOI: 10.1128/MCB.00503-14

Abstract

The liver plays a critical role in glucose metabolism and communicates with peripheral tissues to maintain energy homeostasis. Obesity and insulin resistance are highly associated with nonalcoholic fatty liver disease (NAFLD). However, the precise molecular details of NAFLD remain incomplete. The p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) regulate liver metabolism. However, the physiological contribution of MAPK phosphatase 1 (MKP-1) as a nuclear antagonist of both p38 MAPK and JNK in the liver is unknown. Here we show that hepatic MKP-1 becomes overexpressed following high-fat feeding. Liver-specific deletion of MKP-1 enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice while selectively conferring protection from hepatosteatosis upon high-fat feeding. Further, hepatic MKP-1 regulates both interleukin-6 (IL-6) and fibroblast growth factor 21 (FGF21). Mice lacking hepatic MKP-1 exhibit reduced circulating IL-6 and FGF21 levels that were associated with impaired skeletal muscle mitochondrial oxidation and susceptibility to diet-induced obesity. Hence, hepatic MKP-1 serves as a selective regulator of MAPK-dependent signals that contributes to the maintenance of glucose homeostasis and peripheral tissue energy balance. These results also demonstrate that hepatic MKP-1 overexpression in obesity is causally linked to the promotion of hepatosteatosis.

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Figures

**FIG 1**
Generation and characterization of mice with liver-specific deletion of MKP-1. (A) Schematic of the *Dusp1*/MKP-1 gene locus, the targeted construct, and the resulting targeted allele. Cre-mediated excision results in the deletion of exons 2 and 3 of the Mkp-1 gene. The relevant restriction sites are indicated: B, BamHI; E, EcoRI; H, HindIII; S, ScaI. (B) *Mkp-1*^fl/fl and MKP1-LKO genotyping by PCR using genomic DNA. (C) mRNA expression of MKP-1 from chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 6 to 10). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice. (D) Hepatic MKP-1 and MKP-2 protein expression from chow- and HFD-fed mice (upper panel) and *Mkp-1*^fl/fl and MKP1-LKO mice fed an HFD (lower panel). ERK1/2 antibodies were used to detect the expression of ERK1/2 as a loading control.

**FIG 2**
Enhanced hepatic MAPK phosphorylation in MKP1-LKO mice. (A) Liver lysates from chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice were analyzed by immunoblotting with the indicated antibodies. (B) Immunoblots were quantitated by densitometry for the levels of phospho-p38 MAPK/p38 MAPK, phospho-JNK1/2/JNK1/2, and phospho-ERK1/2/ERK1/2, as indicated. Results represent 10 to 13 mice per genotype, and data shown are the means ± SEM; *, P < 0.05, as determined by Student's t test. Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 3**
Increased adiposity, hyperglycemia, and hyperinsulinemia in chow-fed MKP1-LKO mice. (A) Weight curves of chow-fed male *Mkp-1*^fl/fl and MKP1-LKO mice (n = 10 to 12 per genotype). (B) Liver weights and liver-to-body-weight ratio of chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 10 per genotype). (C and D) Representative hematoxylin-and-eosin staining of liver sections (C) and hepatic triglycerides from chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 5 to 8 for each genotype) (D). (E and F) Spectroscopic analysis of total body lean (E) and fat (F) mass of chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 10 per genotype). (G) Serum leptin in chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (H and I) Plasma glucose (H) and insulin (I) in chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 15 per genotype). Data represent the means ± SEM; *, P < 0.05; ***, P < 0.0001 (as determined by Student's t test). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 4**
MKP1-LKO mice are glucose intolerant and exhibit hepatic insulin resistance. (A) Plasma glucose concentration during glucose tolerance tests in overnight fasted chow-fed (left panel) and HFD-fed (right panel) *Mkp-1*^fl/fl and MKP1-LKO mice (n = 15 and n = 8 per genotype for chow- and HFD-fed mice, respectively). (B to E) Hyperinsulinemic-euglycemic clamps were performed on chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice, and the glucose infusion rate (GIR) (B), whole-body glucose uptake (C), skeletal muscle glucose uptake (D), and hepatic endogenous glucose production (EGP) and hepatic insulin action (E) were analyzed (n = 10 per genotype). Data are represented as means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 (as determined by Student's t test and in panel A by analysis of variance [ANOVA] with Bonferroni's posttest for multiple comparisons). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 5**
Hepatic and serum lipid profiles of chow-fed MKP1-LKO mice. (A to E) Hepatic cytosolic diacylglycerol (A), hepatic membrane diacylglycerol (B), total hepatic ceramide (C), hepatic long-chain acyl-CoA (D), and hepatic ceramide species (E) were measured for chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (F to I) Serum low-density lipoprotein (LDL) (F), serum triglycerides (G), serum nonesterified fatty acids (NEFA) (H), and serum high-density lipoprotein (HDL) (I) were measured for chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (J to N) Liver lysates from chow-fed (J and K) and HFD-fed (L, M, and N) *Mkp-1*^fl/fl and MKP1-LKO mice were analyzed by immunoblotting using anti-GRP78, anti-PDI, phospho-eIF2α, and anti-eIF2α antibodies. Immunoblots were quantified by densitometry (n = 4 or 5 mice per genotype). Data represent means ± SEM; ***, P < 0.0001, as determined by Student's t test. Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 6**
Regulation of gluconeogenic genes by hepatic MKP-1. (A to C) mRNA expression of *G6pc*, *Pck1*, and *Pgc1α* from livers of overnight-fasted chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 10 per genotype). Changes in relative luciferase activity in HepG2 hepatoma cells transfected with either pGL3-G6Pc or pGL-3-PCK1 promoter luciferase in the presence or absence of MKP-1 are shown. Constitutively active mutants of the upstream activators of p38 MAPK [MKK6(EE)] and JNK [MKK7(DD)] were overexpressed to induce p38 MAPK and JNK activity, respectively. (D to G) Glucose 6-phosphatase (G6Pase) (D and F) and phosphoenolpyruvate carboxykinase (PEPCK) (E and G). (H) Liver lysates from chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice were analyzed by immunoblotting using phospho-CREB (S133) and CREB antibodies (n = 8 to 10 per genotype). Data represent the means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 (as determined by Student's t test and in panels D to G by analysis of variance [ANOVA] with Bonferroni's posttest for multiple comparisons). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 7**
Impaired STAT-3 phosphorylation and IL-6 expression in MKP1-LKO mice. (A and B) Liver lysates from chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice were analyzed by immunoblotting with phospho-STAT-3 (Y705) (A) or phospho-STAT-3 (S727) (B); STAT-3 and JAK2 antibodies were used as controls (n = 8 to 10 per genotype). (C) Serum IL-6 in chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice. Data represent the means ± SEM; *, P < 0.05; ***, P < 0.0001 (as determined by Student's t test). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 8**
Susceptibility to diet-induced obesity and protection from hepatosteatosis in MKP1-LKO mice. (A) Weight curves of HFD-fed male *Mkp-1*^fl/fl (n = 9) and MKP1-LKO (n = 10) mice for 20 to 24 weeks. (B and C) Spectroscopic analysis of total body lean mass (B) or total body fat mass (C) from HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (D) Blood glucose values from HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (E) Representative hematoxylin-and-eosin and Oil Red O staining of liver sections from HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice. (F) Liver weights of HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (G) Hepatic triglycerides (TG) from HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). (H) Representative hematoxylin-and-eosin staining of skeletal muscle sections from chow- and HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 5 per genotype). (I) Representative hematoxylin-and-eosin staining of epididymal white adipose tissue (eWAT) sections from chow- and HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 5 per genotype). (J to L) Hepatic mRNA expression of PPARγ (J), Srebp1c (K), or PPARα (L) from HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 8 per genotype). Data represent means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 (as determined by Student's t test or in panel A by analysis of variance [ANOVA] with Bonferroni's posttest for multiple comparisons). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 9**
Reduced energy expenditure in MKP1-LKO mice. Chow-fed *Mkp-1*^fl/fl and MKP1-LKO mice were subjected to open-circuit calorimetry. (A) Energy expenditure; (B) oxygen consumption; (C) carbon dioxide production; (D) respiratory exchange ratio; (E) feeding; (F) locomotor activity (n = 8 per genotype). Data represent means ± SEM; *, P < 0.05; **, P < 0.01 (as determined by Student's t test). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 10**
Effects on hypothalamic neuropeptide expression in MKP1-LKO mice. RNA was isolated from the brain of chow-fed (A to C) and HFD-fed (D to F) *Mkp-1*^fl/fl and MKP1-LKO mice, and the expression of POMC, NPY, and AgRP was analyzed by quantitative PCR (n = 5 per genotype). Data are represented as means ± SEM. Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 11**
Hepatic MKP-1 is required for FGF21 expression and skeletal muscle metabolism. (A) Hepatic FGF21 mRNA expression in chow-fed, fasted, and refed mice (n = 8 per genotype). (B) Serum FGF21 levels (n = 8 per genotype). (C) Hepatic β-hyroxybutyrate expression in chow-fed mice (n = 5 per genotype). (D) HMGCS2 mRNA expression in fed and fasted animals (n = 10 per genotype). (E) Relative luciferase activity in hepatocytes derived from *Mkp-1*^fl/fl and MKP1-LKO mice transfected with the FGF21 luciferase promoter (n = 3 or 4 independent experiments). (F) Relative luciferase activity in hepatocytes derived from *Mkp-1*^fl/fl mice transfected with FGF21 luciferase promoter for 48 h, following incubation with vehicle or MAPK inhibitors for 1 h prior to stimulation with glucagon for 6 h (n = 3 or 4). (G) HepG2 cells were cotransfected with the FGF21 luciferase promoter and either the wild type or constitutively active mutants of the upstream activators of p38 MAPK (MKK6EE) and JNK (MKK4EE) or dominant negative mutants of p38 MAPK [MKK6(AA)] and JNK [MKK4(AA)]. HepG2 cells were lysed and immunoblotted for phospho-p38 MAPK and phospho-JNK (n = 3 or 4). Data are represented as means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 (as determined by Student's t test). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice.

**FIG 12**
Hepatic MKP-1 regulates skeletal muscle mitochondrial respiration. (A) mRNA expression of PGC-1α from skeletal muscle of chow and HFD-fed *Mkp-1*^fl/fl and MKP1-LKO mice (n = 5 per genotype). (B) Mitochondrial respiratory function in skeletal muscle of chow (n = 6 per genotype) and HFD-fed (n = 8 per genotype) *Mkp-1*^fl/fl and MKP1-LKO mice. Data are represented as means ± SEM; *, P < 0.05; ***, P < 0.0001 (as determined by Student's t test). Open bars, *Mkp-1*^fl/fl mice; filled bars, MKP1-LKO mice. (C) Integrative mode of metabolic regulation by hepatic MKP-1. In obesity, overexpressed hepatic MKP-1 selectively drives lipogenesis, thereby contributing to the development of hepatosteatosis.

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Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis

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Hepatic mitogen-activated protein kinase phosphatase 1 selectively regulates glucose metabolism and energy homeostasis

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