Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function§

Authors


  • Potential conflict of interest: Nothing to report.

  • §

    This work was supported by Swiss National Foundation Grant 31003A-140930 (to J.-F. D.). J.-F. D. received funding from the European Community's Seventh Framework Programme under grant agreement HEALTH-F2-2009-241762 for the project FLIP. N. B. received a grant from INSERM-Région Aquitaine. J. M. received a fellowship from the European Association for the Study of the Liver.

Abstract

The histidine triad nucleotide-binding (HINT2) protein is a mitochondrial adenosine phosphoramidase expressed in the liver and pancreas. Its physiological function is unknown. To elucidate the role of HINT2 in liver physiology, the mouse Hint2 gene was deleted. Hint2−/− and Hint2+/+ mice were generated in a mixed C57Bl6/J × 129Sv background. At 20 weeks, the phenotypic changes in Hint2−/− relative to Hint2+/+ mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycemia, and an increase in plasma interprandial insulin but a decrease in glucose-stimulated insulin secretion and defective thermoregulation upon fasting. Leptin messenger RNA (mRNA) in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2−/− hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II-III electron transfer was decreased in Hint2−/− mitochondria, which was accompanied by a lower content of coenzyme Q. Hypoxia-inducible factor-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2−/− mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-coenzyme A dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2−/− versus 83% in Hint2+/+). This suggests a greater extent of acetylation in Hint2−/− than in Hint2+/+. Conclusion: Hint2/HINT2 positively regulates mitochondrial lipid metabolism and respiration and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins. (HEPATOLOGY 2013)

The superfamily of histidine triad (HIT) proteins share a distinguishing motif composed of His-ϕ-His-ϕ-His-ϕ–ϕ, where ϕ is a hydrophobic amino acid. Seven human HIT proteins have been identified: three members of the histidine triad nucleotide-binding subfamily (HINT1, HINT2, and HINT3), fragile histidine triad (FHIT), aprataxin, galactose-1-phosphate uridylyltransferase, and scavenger messenger RNA (mRNA) decapping enzyme. The HINT1 gene encodes a 126–amino acid purine nucleotide-binding protein that hydrolyzes AMP-NH2 and lysyl-adenylate.1, 2 HINT1 is expressed ubiquitously and has tumor suppressor properties in the liver. HINT1 mRNA is down-regulated in hepatocellular carcinoma,3, 4 and Hint1−/− mice develop more carcinogen-induced tumors than their wild-type counterparts.5, 6 HINT1 binds to the scaffold protein, POSH, and the HINT1/POSH interaction impairs the ability of c-Jun N-terminal kinase 2 to phosphorylate the transcription factor activator protein-1.7 Ablation of Hint1 protects against hepatic ischemia reperfusion injury.8 HINT2 is 61% identical to HINT1, is expressed in the liver, pancreas,9 and the adrenal gland,10 and has adenosine phosphoramidase activity.9 Like HINT1, the expression of HINT2 mRNA is decreased in hepatocellular carcinoma.9 Unlike HINT1, HINT2 contains a mitochondrial import signal and has been localized exclusively to the mitochondria9 in the vicinity of the contact sites of the inner mitochondrial membrane.10 The biological function of HINT2 is unknown.

In addition to HINT2, liver mitochondria harbor another HIT protein. FHIT does not contain a mitochondrial import signal but is directed from the cytosol to the mitochondria upon interaction with the chaperones heat shock protein (Hsp) 60 and Hsp10.11 The characterization of a knockout Fhit mouse model confirmed the tumor suppressor properties of Fhit12, 13 and its interaction with the flavoprotein ferredoxin reductase to generate a proapoptotic complex. As with the Fhit model, the characterization of a knockout Hint2 model is needed to elucidate the physiological function of Hint2 in the liver.

We postulated that HINT2 contributes to the normal function of hepatic mitochondria. To test this hypothesis, we deleted the Hint2 gene and generated a Hint2−/− mouse strain. The morphology, bioenergetics, and selected metabolic functions of liver mitochondria were compared in Hint2−/− and Hint2+/+ mice and glucose homeostasis was monitored. The characteristics of a HepG2 cell line over- and underexpressing HINT2 were also examined. The results demonstrate that Hint2/HINT2 positively regulates lipid metabolism, mitochondrial respiration, glucose tolerance and response to fasting. These actions can be partly explained by a modulation of the extent of acetylation of selected proteins.

Abbreviations

ADP, adenosine diphosphate; ANOVA, analysis of variance; BAT, brown adipose tissue; CoA, coenzyme A; CPT, carnitine palmitoyltransferase; Drp1, dynamin-related protein 1; ESC, embryonic stem cell; FHIT, fragile histidine triad; GDH, glutamate dehydrogenase; GTT, glucose tolerance test; Hadhsc, L-3-hydroxyacyl-coenzyme A dehydrogenase short chain; Hint, nonhuman hisitidine nucleotide-binding protein; Hint2, histidine triad nucleotide-binding; HINT, human histidine triad nucleotide-binding protein; HIT, histidine triad; Hsp, heat shock protein; ITT, insulin tolerance test; Mfn, mitofusin; mRNA, messenger RNA; NADH, reduced nicotinamide adenine dinucleotide; Opa1, optic atrophy 1; PCR, polymerase chain reaction; WAT, white adipose tissue.

Materials and Methods

Animals.

Hint2−/− mice were generated by homologous recombination in embryonic stem cells (ESCs). To delete the five exons of Hint2, a distal LoxP site was inserted upstream of Hint2 exon 1 and an FRT-neomycin-FRT-LoxP selection cassette was inserted downstream of Hint2 exon 5. The targeting vector in a 129Sv/Pas genetic background was electroporated into 129Sv/Pas ESCs (GenOway, Lyon, France). G418-resistant ESC clones were screened via polymerase chain reaction (PCR) and Southern blotting. Recombined ESC clones were injected into C57BL/6J-derived blastocysts. Germline transmission and deletion of the floxed region (exons 1-5) were assessed after breeding the chimeras with the Cre-expressing C57BL/6J deleter strain. The resulting Hint2 heterozygotes of mixed 129Sv/C57Bl6J genetic background were intercrossed, and constitutive Hint2−/− and control Hint2+/+ mice were selected by PCR and Southern blotting. Mice were subjected to 12-hour light/dark cycles and were fed ad libitum. The 2018 Teklad Global 18% protein diet (Harlan Laboratories Inc, Madison, WI) contained 17% calories derived from fat. Initially, male mice aged 10, 20, and 30 weeks were assessed for phenotypic changes. Additional experimentation was performed on male mice aged 20-28 weeks, except for electron microscopy studies at 50 weeks. Experiments were approved by the University of Bern Animal Care Committee.

Enzymatic Activity.

Activity of L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase short chain (Hadhsc) was measured in isolated liver mitochondria (250 μg/mL). The reaction mixture (37°C, pH 7.0) contained 0.1 M triethanolamine-HCl, 5 mM ethylene diamine tetraacetic acid, 0.45 mM reduced nicotinamide adenine dinucleotide (NADH), and 0.1 mM acetoacetyl-CoA. Enzyme activity was calculated as [(ΔA340nm/minute test − ΔA340nm/minute blank) · (mL)]/6.22, where 6.22 is the extinction coefficient of β-NADH.14 Glutamate dehydrogenase activity (GDH) was measured in tissue lysate using an assay kit that monitored the generation of NADH at 450 nm (Biovision, Mountain View, CA). Carnitine palmitoyltransferase (CPT) activity was measured in liver mitochondria according to Shimoda et al.15 with palmitoyl-CoA as the substrate. Sirtuin 3 activity was measured in liver mitochondria using the Cyclex SIRT3 Deacetylase Fluorometric kit (MBL International, Woburn, MA).

Glucose and Insulin Tolerance Test.

Mice were fasted for 16 hours. Glucose (2 g/kg body weight) or insulin (1 U/kg body weight) was injected intraperitoneally. Glucose concentrations in blood collected from the tail vein were measured before and 15, 30, 60, 90, and 120 minutes after injection (Ascensia Contour; Bayer HealthCare, Basel, Switzerland).

Immunoprecipitation.

Hadhsc and GDH were immunoprecipitated from isolated mitochondria according to Li et al.16 For GDH, the protein A beads were replaced with magnetic Dynabeads M-280 (Life Technologies) (80 μL). Lysine-acetylated proteins were immunoprecipitated according to Hirschey et al.17 in the presence of 10 mM nicotinamide.

Enrichment of Pancreatic Exocrine and Endocrine Fractions.

Islets and exocrine fractions were isolated from the pancreas according to the method described by Zmuda et al.18

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism software. A nonparametric Mann-Whitney U test was selected for comparing two groups. A nonparametric Kruskal-Wallis test with Dunn's post-comparison was selected for multiple group comparisons when the data did not approximate a normal distribution. Parametric analysis of variance (ANOVA) using a Bonferroni's post-test was selected for multiple group comparisons when the sampled data approximated a normal distribution. Data are expressed as the mean ± SD. P values < 0.05 were considered statistically significant and are indicated in the figures as *comparison of −/− versus +/+ under the same experimental conditions and §comparison of different conditions within −/− or +/+ groups.

Additional methods are provided in the Supporting Information. All antibodies are listed in Supporting Tables 1 and 2.

Results

Hint2−/− Mice.

Hint2f/f mice displaying a lox site on each side of the Hint2 gene were bred with mice expressing the Cre recombinase under a ubiquitous promoter (Fig. 1A). Southern blotting revealed that Cre-mediated recombination had excised the Hint2 gene (Fig. 1B). Western blotting confirmed the absence of Hint2 in Hint2−/− livers and pancreas and its presence in Hint2+/+ livers, liver mitochondria, and pancreas (Fig. 1C,E). Immunostaining confirmed the localization of Hint2 in hepatocytes (Fig. 1D) and its constant expression level throughout the 30-week study (Supporting Fig. 1). Hint2−/− mice appeared healthy at birth and were fertile. Hint2−/− mice weighed 26% more than Hint2+/+ mice at 20 weeks (Table 1) and accumulated a greater abundance of retroperitoneal fat (data not shown). Liver weights were slightly higher in Hint2−/− mice at 10 and 20 weeks (Table 1). Plasma levels of alanine aminotransferase and aspartate aminotransferase were normal. Albumin concentrations were normal but increased slightly at 30 weeks in Hint2−/− mice.

Figure 1.

Generation and characterization of Hint2−/− mice. (A) The targeting strategy generated Hint2−/− mice on a mixed 129Sv × C57Bl6J genetic background. A targeting vector in a 129Sv/Pas genetic background was designed. A distal LoxP site upstream of Hint2 exon 1 (blue arrow) and an FRT-neomycin-FRT-LoxP selection cassette downstream of Hint2 exon 5 (blue arrow) were inserted. Germline transmission and deletion in vivo of the floxed region (exons 1-5) were assessed. Chimeras were bred with a Cre-expressing C57BL/6J deleter strain. Exons 1 to 5 of the murine Hint2 gene are represented by stippled boxes; LoxP sites are represented by blue arrowheads and the 5′ probe used to analyze Southern blots is represented by blue boxes. (B) Genomic Southern blot analysis. DNA (20 μg) of liver extracted from the indicated genotypes was digested with NsiI. The Hint2 gene is absent from Hint2−/− mice. (C) Western blot analysis of Hint2 protein expression in Hint2−/− and Hint2+/+ liver homogenates (top), and in cytosol (Cyt.) and mitochondria (Mito.) isolated from Hint2+/+ livers of 9-week-old mice. Actin and Hsp60 were used as loading controls. (D) Immunohistochemistry of Hint2+/+ and Hint2−/− liver sections. Hint2 is expressed only in Hint2+/+ hepatocytes, more abundantly in the pericentral regions. (E) Western blot analysis of Hint2 protein in the pancreas. Hint2 is expressed in homogenate of Hint2+/+ but not Hint2−/− pancreas.

Table 1. Comparison of Metabolic Characteristics in Hint2+/+ and Hint2−/− Mice
 Age
 10 Weeks (n = 11)20 Weeks (n = 16-18)30 Weeks (n = 9-10)
CharacteristicsHint2+/+Hint2−/−Hint2+/+Hint2−/−Hint2+/+Hint2−/−
  • Abbreviations: ALT, alanine aminotransferase AST, aspartate aminotransferase FFA, free fatty acid.

  • *

    P < 0.05,

  • **

    P < 0.01,

  • ***

    P < 0.001 (Mann-Whitney U test).

Body weight, g29.6 ± 0.632.4 ± 1.129.8 ± 0.737.7 ± 1.3***37.3 ± 1.039.9 ± 2.9
Liver weight, g1.5 ± 0.051.7 ± 0.07*1.4 ± 0.051.6 ± 0.07*1.6 ± 0.11.5 ± 0.1
ALT, U/L4.0 ± 0.67.0 ± 0.7*3.0 ± 0.66.0 ± 0.6**9.0 ± 1.211.0 ± 0.8*
AST, U/L34.0 ± 2.134.0 ± 2.032.0 ± 2.941.0 ± 2.0*47.0 ± 4.248.0 ± 4.6
Albumin plasma, g/dL3.2 ± 0.23.1 ± 0.24.1 ± 0.25.8 ± 2.53.5 ± 0.78.0 ± 1.8*
FFA plasma, nmol/μL1.0 ± 0.20.9 ± 0.10. 6 ± 0.10.7 ± 0.10.9 ± 0.11.0 ± 0.2
Triglycerides plasma, nmol/μL1.5 ± 0.21.9 ± 0.21.2 ± 0.21.2 ± 0.21.7 ± 0.10.7 ± 0.1***
Ketone bodies plasma, mM0.3 ± 0.010.3 ± 0.030.2 ± 0.030.2 ± 0.020.3 ± 0.010.3 ± 0.01
Total cholesterol plasma, mg/dL113.7 ± 4.9145.2 ± 10.5*109.5 ± 9.8140.8 ± 8.5*139.5±7.0160.7 ± 9.0
Glucose, mmol/L12.0 ± 1.112.8 ± 1.48.5 ± 0.78.3 ± 0.49.0 ± 0.48.4 ± 0.6
Insulin, ng/mL3.4 ± 1.25.9 ± 3.75.0 ± 2.29.5 ± 1.7**6.2 ± 2.312.8 ± 5.6*
Leptin, ng/mL3.3 ± 1.26.6 ± 5.23.0 ± 1.325.0 ± 6.9**26.2 ± 7.230.6 ± 8.0
Adiponectin, ng/mL4,636.0 ± 362.05,735.0 ± 862.0**4,795.0 ± 653.06,687.0 ± 858.0**5,630.0 ± 931.08,859.0 ± 1,876.0**

Effect of Hint2 Deletion on Liver Morphology and Mitochondria.

Livers of both groups showed an age-associated accumulation of intracytoplasmic lipid vacuoles (Fig. 2A), but this was more acute in Hint2−/− livers, particularly in the pericentral regions. Liver triglycerides at 20 weeks were higher in Hint2−/− mice than in Hint2+/+ mice (P < 0.05) (Fig. 2C). Cholesteryl esters were not different (Fig. 2D).

Figure 2.

Changes in lipid accumulation and histology of Hint2−/− and Hint2+/+ livers. (A) Comparison of hematoxylin and eosin–stained sections of Hint2−/− and Hint2+/+ livers. The box outlines a pericentral region at 10, 20, and 30 weeks. Lipid microvesicular droplets are present occasionally at 10 weeks only in Hint2−/− livers. Large lipid droplets occur frequently only in Hint2−/− livers at 20 weeks. Lipid accumulates in both Hint2−/− and Hint2+/+ livers at 30 weeks. (B) Oil Red O staining of neutral lipids in Hint2−/− and Hint2+/+ livers. The neutral lipids stained by Oil Red O increase in an age-dependent manner in Hint2−/− livers, whereas neutral lipids are detected in Hint2+/+ livers only at 30 weeks. (C) Total liver triglycerides were measured spectrophotometrically. Hint2−/− livers accumulated more triglycerides than Hint2+/+ livers at 20 weeks. *P < 0.05. (D) The content of cholesteryl esters did not differ between Hint2+/+ and Hint2−/− livers. (E) Representative electron microscopy images of mitochondria from livers of Hint2−/− and Hint2+/+ mice at 12 months of age. Hint2+/+ mitochondria appeared tubular or rounded (left panel). Hint2−/− mitochondria were occasionally clustered in perinuclear areas and showed points of local contact and membrane fusion (middle panel). Several mitochondria were deformed, adopting filamentous or concave shapes, often in association with membrane-bound vacuolar structures (right panel). Scale bars = 1 micron.

To determine whether the absence of Hint2 induced structural changes, hepatocyte mitochondria from mice aged 25 and 50 weeks were examined via electron microscopy. Compared with the tubular or oval shapes of Hint2+/+ mitochondria, the Hint2−/− mitochondria appeared clustered and occasionally fused (Fig. 2E). These changes were frequent at 50 weeks but were rare in younger mice.

To determine whether altered expression of mitochondria-shaping proteins could account for the morphological changes, the expressions of optic atrophy 1 (Opa1), mitofusins (Mfn1 and 2), and the cytosolic dynamin-related protein 1 (Drp1) and its receptor on the outer mitochondrial membrane, Fis1, were compared. The expression of fusion protein Opa1 was 1.5-fold higher in Hint2−/− mice than in Hint2+/+ mice, whereas Mfn1 and Mfn2 were not different. Fis1 and Drp1 were slightly lower in Hint2−/− mice (Supporting Fig. 2A,B).

Effect of Hint2 Deletion on Lipid Metabolism and Glycemic Control.

To determine whether the accumulation of lipids was related to defective mitochondrial β-oxidation of fatty acids, the activities of CPT1 and CPT2 and of medium- or short-chain hydroxyacyl-CoA dehydrogenase (Hadhsc), which catalyzes the NAD+-dependent dehydrogenation of 3-hydroxyacyl-CoA in the mitochondrial matrix, were measured. The activity of Hadhsc was decreased by 68% in Hint2−/− mice compared with Hint2+/+ mice (Fig. 3A) without a change in expression of the enzyme (Fig. 3B). The activity of CPT did not change (Supporting Fig. 7A). In plasma, free fatty acid concentrations were not different, triglyceride concentrations were lower in Hint2−/− mice only at 30 weeks, and total cholesterol was slightly higher in Hint2−/− mice (Table 1). Because the Hadhsc enzyme can bind to glutamate dehydrogenase (GDH) in the mitochondrial matrix, which is a potential point of regulation for both enzymes, the activity of GDH was also measured. GDH activity was decreased by 60% in Hint2−/− livers, with no change in GDH expression (Fig. 3C,D). To determine whether the protein-protein interaction of Hadhsc and GDH was disturbed by the absence of Hint2, the co-immunoprecipitation of GDH and Hadhsc was tested. Co-immunoprecipitation was successful in Hint2+/+ and Hint2−/− mitochondria (Fig. 3E,F).

Figure 3.

Activities of β-hydroxyacyl-CoA dehydrogenase short chain (Hadhsc) and glutamate dehydrogenase (GDH) in Hint2+/+ and Hint2−/− livers. (A) Hadhsc activity was measured in mitochondria isolated from Hint2−/− and Hint2+/+ livers. Acetoacetyl-CoA served as the substrate and the change in absorbance of NADH was monitored spectrophotometrically. The values were normalized for mitochondrial protein. Hadhsc activity was decreased by 68% in Hint2−/− mitochondria. *P < 0.05. (B) The expression level of Hadhsc protein was compared in cytosol (C) and mitochondrial (M) fractions of Hint2−/− and Hint2+/+ livers by immunoblotting. The protein expression, normalized for Hsp60, was not different. (C) GDH activity was compared in homogenates of Hint2+/+ and Hint2−/− livers. Glutamate served as the substrate and the generation of NADH was measured spectrophotometrically. GDH activity was decreased by 60% in Hint2−/− livers. **P < 0.01. (D) The expression level of GDH was measured in liver homogenates, normalized for actin. Hint2+/+ and Hint2−/− livers expressed similar amounts of protein. (E and F) Co-immunoprecipitation of Hadhsc and GDH proteins. Mitochondria pellets (1 mg) were lysed in 1% Triton–phosphate-buffered saline. (E) Anti-Hadhsc antibody immunoprecipitated Hadhsc along with pull-down of GDH in both Hint2+/+ and Hint2−/− mitochondria. (F) Anti-GDH antibody immunoprecipitated GDH along with pull-down of Hadhsc in both Hint2+/+ and Hint2−/− mitochondria.

Because the nonfasting interprandial insulin concentrations were two-fold higher in Hint2−/− than in Hint2+/+ mice (Table 1), a glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed and insulin signaling was examined. The GTT yielded higher glucose values in Hint2−/− than in Hint2+/+ mice (area under the curve, 1,378 ± 312 versus 1,021 ± 281 mmol/L × 120 minutes, respectively; P = 0.09) (Fig. 4A). However, random interprandial blood glucose (Table 1) and fasting blood glucose were not different in Hint2−/− versus Hint2+/+ mice (Fig. 4A,C). The phosphorylation of the threonine-serine kinase, Akt, and the expression of downstream targets were measured in liver homogenates, muscle, and white adipose tissue (WAT) of fasted mice after insulin stimulation (Fig. 4B). Insulin induced phosphorylation of Akt at Ser473 and Thr308 in all tissues (Fig. 4B, Supporting Fig. 3A). The downstream target, FoxO1, was phosphorylated (Ser253) in liver and muscle in response to insulin. The phosphorylation of glycogen synthase kinase 3β was increased in muscle in response to insulin in both groups. The hepatic expression of Pck1 was down-regulated by insulin in both groups, slightly more so in Hint2−/− livers (Fig. 4B, Supporting Fig. 3A). The ITT differed in Hint2+/+ and Hint2−/− mice (Fig. 4C, top panel). After similar initial falls in blood glucose, the Hint2+/+ mice were euglycemic at 90 minutes, whereas the Hint2−/− mice remained hypoglycemic. Blood levels of regulatory hormones that counter hypoglycemia were measured after 2 hours. Glucagon was significantly lower in Hint2−/− mice, but the corticosterone level was higher (Fig. 4D). Noradrenaline was also lower in Hint2−/− 2 hours after ITT (2.4 ± 1.5 Hint2−/− versus 9.5 ± 0.76 Hint2+/+, P < 0.05). As expected, Hint2−/− mice were more vulnerable to repeated challenges of insulin-inducing hypoglycemia (Fig. 4C, bottom panel). To test whether a decrease in acute insulin secretion could account for the increase in area under the curve after GTT in Hint2−/− mice, plasma insulin concentrations were measured after a 16-hour fast followed by a glucose load. Insulin secretion was indeed lower in Hint2−/− mice (Fig. 4E). To test whether the Hint2 protein could directly influence glucose-stimulated insulin secretion by virtue of expression in beta cells, Hint2 was localized in the pancreas. Hint2 was expressed in the exocrine enriched fraction of the pancreas along with the marker enzyme α-amylase (Fig. 4F). No Hint2 was detected in the islet cell fraction where Hadhsc was highly expressed. The presence of Hadhsc in the exocrine fraction suggests contamination of the preparation by islet cells, whereas the absence of amylase in the islet cell fraction indicates lack of contamination with acinar cells.

Figure 4.

Comparison of glucose tolerance and insulin signaling pathways in fasted Hint2−/− and Hint2+/+ mice. (A) Glucose tolerance test. Glucose (2 g/kg) was injected intraperitoneally in fasted (16 hours), 21-week-old Hint2−/− (n = 6) and Hint2+/+ mice (n = 6) and blood glucose was measured. The fasting blood glucose (0 minutes) was not different. The area under the glucose-time curve was slightly higher in Hint2−/− than in Hint2+/+ mice (P = 0.09). (B) Insulin signaling in the liver, skeletal muscle (gastrocnemius), and WAT of fasted Hint2−/− and Hint2+/+ mice. Immunoblots of liver, muscle, and WAT were quantified 5 minutes after injection of insulin. The ratios P-Akt/Akt, P-FoxO1/FoxO1, and P-GSK3β/ GSK3β were calculated (Supporting Fig. 3A). The expression levels of phosphoenolpyruvate carboxykinase (Pck1) were normalized to actin (Supporting Fig. 3A). (C) Insulin tolerance test (ITT). After a 16-hour fast, insulin was injected intraperitoneally and blood was collected from the tail vein of 20-week-old Hint2−/− (n = 8) and Hint2+/+ (n = 5) mice (top panel). A separate group of mice (n = 6) were challenged with the ITT three times at weekly intervals (bottom panel). Hint2−/− mice showed impaired recovery from hypoglycemia. *P < 0.05. **P < 0.01. (D) Secretion of counter-regulatory hormones 2 hours after ITT. Plasma levels of glucagon were significantly lower in Hint2−/− mice, whereas the increase in corticosterone levels was higher in Hint2−/− than in Hint2+/+ mice. *P < 0.05. **P < 0.01. (E) Glucose-stimulated insulin secretion. Twenty-week-old fasted (16 hours) mice (n = 6) were injected intraperitoneally with glucose (2 g/kg). Plasma insulin was measured before and 30 minutes after glucose injection. Only Hint2+/+ mice showed significantly increased insulin levels. §P < 0.05 (ANOVA). (F) Immunoblot of Hint2 in pancreatic tissue separated into exocrine and islet cell–enriched fractions. α-Amylase was used as a marker for exocrine cells (top panel). Hadhsc was used as a marker for the islet cell fraction (middle panel). Islet cells may have contaminated the exocrine fraction. Hint2 was only detected in the enriched exocrine fraction of Hint2+/+ pancreas.

Plasma leptin was higher in Hint2−/− mice at 20 weeks, and plasma adiponectin was slightly higher at all points (Table 1). To determine whether the increased fat depots were solely responsible for higher levels of adipocyte hormones, the mRNA levels of adiponectin and leptin were quantified in WAT collected from retroperitoneal fat. In freely fed mice, leptin mRNA was 2.5-fold higher in Hint2−/− than in Hint2+/+ (Fig. 5A), whereas adiponectin mRNA was at equal levels (data not shown).

Figure 5.

The response of Hint2−/− and Hint2+/+ mice to fasting. (A) mRNA expression of leptin in WAT measured by quantitative PCR in Hint2−/− and Hint2+/+ 20 week old mice. Leptin was upregulated in WAT of fed Hint2−/− mice (*P < 0.05, fed Hint2+/+ versus Hint2−/−). (B) Production of ketone bodies after fasting. β-hydroxybutyrate increased significantly in Hint2+/+ and Hint2−/− mice. §§P < 0.01, §§§P < 0.001 (ANOVA). (C) Plasma corticosterone levels in response to fasting. The corticosterone levels were higher in fed Hint2−/− mice than in fed Hint2+/+ mice. Corticosterone increased after fasting. *,§P < 0.05, ***P < 0.001 (ANOVA). (D) Core body temperature of 28-week-old Hint2−/− and Hint2+/+ mice after a 16-hour fast. Only Hint2−/− mice lowered body temperature in response to food deprivation. **,§§P < 0.01(ANOVA). (E) mRNA expression of uncoupling protein 2 (UCP2) measured by quantitative PCR in fed and fasted Hint2−/− and Hint2+/+ WAT and liver. UCP2 was up-regulated in WAT of fed Hint2−/− mice and increased significantly in Hint2+/+ mice after fasting. UCP2 mRNA in liver decreased after fasting in Hint2+/+ mice. §P < 0.05, **P < 0.01 (ANOVA). (F) Immunoblot of Hint2 in WAT and BAT. Hint2 was expressed only in BAT isolated from 50-week-old Hint2+/+ mice. Hint2 was not detected in WAT.

Effect of Hint2 Deletion on Response to Fasting.

To test whether the decrease in hepatic Hadhsc activity caused an intolerance to fasting, the responses of Hint2−/− and Hint2+/+ mice to 16 hours of food deprivation were compared. The decline in blood glucose followed a similar pattern in both groups (Supporting Fig. 7B). β-Hydroxybutyrate increased 4.1-fold in fasting Hint2+/+ and 4.2-fold in fasting Hint2−/− mice, a difference that was not statistically significant (Fig. 5B). Corticosterone plasma levels were higher in Hint2−/− mice than in Hint2+/+ fed mice and increased after fasting (Fig. 5C). Only Hint2−/− mice lowered their body temperatures significantly when deprived of food, suggesting a reduction in basal metabolic energy (Fig. 5D). Both Hint2−/− and Hint2+/+ livers showed a fasting-induced increase in stores of triglycerides, but the changes were not statistically significant (Supporting Fig. 7C). Because UCP2 is known to be up-regulated in a tissue-dependent manner during fasting and can regulate insulin secretion,19 mRNA levels of UCP2 were measured in adipose and liver tissue. In the fed state, UCP2 mRNA was significantly higher in Hint2−/− than in Hint2+/+ WAT (Fig. 5E). After fasting, UCP2 increased in Hint2+/+ WAT but did not increase further in Hint2−/− WAT. In the liver, UCP2 was similar in both fed groups and decreased after fasting in Hint2+/+ (Fig. 5E).

To test for expression of Hint2 in adipose tissue, immunoblotting was performed in WAT and brown adipose tissue (BAT). Hint2 was only detected in BAT (Fig. 5F).

Effect of Hint2 on Mitochondrial Respiration and Oxidative Phosphorylation.

No differences were detected between mitochondria from Hint2−/− and Hint2+/+ livers in the expression of respiratory complexes (Supporting Fig. 4) and in the individual activities of complexes I, II, and III (Fig. 6A). However, the activity of the linked complex II-III was reduced by 60% in Hint2−/− mitochondria. Accordingly, succinate-linked state 3 respiration was decreased by 44%, and pyruvate-linked respiration was decreased by 35% in Hint2−/− mice (Fig. 6B). The content of total coenzyme Q was lower in Hint2−/− livers than in Hint2+/+ livers (Fig. 4C). To determine whether a change in the expression of genes involved in the biosynthesis of coenzyme Q was responsible, the mRNA expression of polyisoprenyl diphosphate synthases 1 and 2, Coq2, Coq3, Coq4, Coq5, Coq6, Coq7, Coq8, and Coq9 was compared in Hint2−/− and Hint2+/+ livers via real-time PCR. Only Coq8 increased 2.5-fold in Hint2−/− at 20 weeks and Coq9 increased 1.6-fold at 10 weeks (data not shown).

Figure 6.

Comparison of respiratory chain activity in Hint2−/− and Hint2+/+ mitochondria and HepG2 cells. (A) Activities of the respiratory complexes I, II, and III and linked complexes II-III were compared in mitochondria from Hint2−/− and Hint2+/+ livers, aged 12 weeks. The linked complex II-III activity was lower in Hint2−/− livers (n = 3 experiments). *P < 0.05. (B) Oxygen consumption during state 3 respiration with pyruvate or succinate as a substrate, as measured by oxymetry. State 3 respiration was lower in Hint2−/− mitochondria (n = 3 experiments). *P < 0.05. (C) Total coenzyme Q levels of mitochondria isolated from Hint2−/− and Hint2+/+ 10-week-old livers. Coenzyme Q9 was measured by high-performance liquid chromatography/electrochemical detection in the presence of succinate as substrate. The total coenzyme Q levels were lower in Hint2−/− mitochondria (n = 3 experiments) *P < 0.05. (D) Effect of HINT2 expression on oxygen consumption in HepG2 cells. Oxygen consumption during state 3 respiration with pyruvate or succinate as substrate was measured by oxymetry in whole, permeabilized HepG2 cells. A lower expression of HINT2 significantly correlated with a decrease in oxygen consumption (n = 4 experiments). *P < 0.05 (HepG2 siRNA-HINT2 versus HepG2 pSup). (E) Oxygen consumption in isolated mitochondria. Oxygen consumption was measured under conditions of adenosine triphosphate synthesis with succinate serving as substrate. Mitochondria isolated from cells underexpressing HINT2 (HepG2 siRNA-HINT2) consumed less oxygen than the control (HepG2 pSup) cells (n = 3 experiments in duplicate). *P < 0.05 (HepG2 siRNA-HINT2 versus HepG2 pSup). The corresponding expression levels of the HINT2 protein in these cell lines is shown in Supporting Fig. 5.

To confirm the link between HINT2 expression and the altered energy metabolism, we generated HepG2 cell lines that expressed varying levels of HINT2 (Supporting Fig. 5). The activities of the individual respiratory chain complexes I, II, III, and IV were not different among the HepG2 variants (data not shown). The silencing of HINT2 (HepG2-siRNA-HINT2) was associated with a 30% decrease in state 3 respiration in the presence of pyruvate and succinate, whereas an overexpression of HINT2 did not influence the state 3 respiration (Fig. 6D). When expressed relative to citrate synthase, oxygen consumption in HepG2-siRNA-HINT2 cells was reduced (Fig. 6E). No differences in state 4 respiration were observed between the cell lines (data not shown).

Reactive Oxygen Species in Hint2−/− Mice.

Hint2−/− hepatocytes produced a 1.5-fold higher level of reactive oxygen species (Supporting Fig. 6A). Activation of hypoxia-inducible transcription factor (Hif) signaling was examined via immunohistochemistry. Activation of Hif-2α but not Hif-1α was higher in Hint2−/− than in Hint2+/+ livers (Supporting Fig. 6B).

Hint2 and Regulation of GDH.

To confirm the link between GDH activity and the absence of Hint2, enzymatic assays were repeated in lysates of HepG2 over- and under-expressing cells. GDH activity increased by 33% in HepG2-HINT2 cells and decreased by 25% in HepG2-siRNA-HINT2 cells relative to controls (Fig. 7A) without a change in enzyme expression (Fig. 7B). In the liver, GDH activity is down-regulated by sirtuin 4–mediated adenosine diphosphate (ADP) ribosylation20 and up-regulated by sirtuin 3–mediated lysine deacetylation.21 To test whether the extent of GDH ADP ribosylation or deacetylation was different in Hint2+/+ and Hint2−/− livers, GDH activity was measured in the presence and absence of snake venom phosphodiesterase, which cleaves the ADP-ribose moiety, and purified sirtuin 3. Phosphodiesterase unmasked similar latent activity in Hint2−/− and Hint2+/+ livers. Sirtuin 3 unmasked a greater extent of latent GDH activity in Hint2−/− livers than in Hint2+/+ livers (126% versus 83%, respectively; P < 0.05) (Fig. 7C). To determine whether the absence of Hint2 changed the extent of acetylation for other mitochondrial proteins, immunoblotting was performed with an antibody against acetylated lysine residues. Several proteins appeared hyperacetylated in Hint2−/− mitochondria, which could not be due to a decrease in expression of sirtuin 3 (Fig. 7D). To determine whether Hint2 itself was acetylated, immunoprecipitation and immunoblotting of acetylated mitochondrial proteins was performed. Hint2 was detected in the Hint2+/+ immunoprecipitate (Fig. 7E), which indicates that Hint2 is either acetylated or binds to acetylated proteins. To test whether the absence of Hint2 affected the enzymatic activity of sirtuin 3 in vitro, a deacetylase assay was performed. The sirtuin 3 deacetylase activity was comparable in Hint2+/+ and Hint2−/− mitochondria (Fig. 7F).

Figure 7.

Effect of Hint2 on regulation of GDH. (A) GDH activity in lysate of HepG2 cells overexpressing and underexpressing HINT2. The activity in cells transfected with control vector was set to 100%. Overexpressing HINT2 cells showed a 33% increase in activity relative to controls. Underexpressing (siRNA-HINT2) cells showed a 25% decrease in activity relative to controls (n = 3 experiments). *P < 0.05, **P < 0.01. (B) Immunoblot of cell lysate from HepG2 cells overexpressing and underexpressing HINT2. GDH was expressed similarly in all cell lines. (C) Effect of posttranslational modification on GDH activity in liver homogenate of 20-week-old Hint2+/+ (n = 6) and Hint2−/− mice (n = 6). Snake venom phosphodiesterase was added to cleave the ADP-ribose moiety from GDH. Purified sirtuin 3 was added to deacetylate GDH. Sirtuin 3 unmasked latent GDH activity in both Hint2+/+ and Hint2−/− livers. §§§P < 0.001. The difference in GDH activity in presence and absence of sirtuin 3 was significantly greater in Hint2−/− than in Hint2+/+ livers. *P < 0.05. (D) Immunoblot of acetylated proteins in Hint2+/+ and Hint2−/− mitochondria. A monoclonal antibody against lysine acetylated proteins detected a greater extent of acetylation in Hint2−/− mitochondria. No difference was detected in the expression levels of sirtuin 3 or sirtuin 4 proteins when normalized for the mitochondrial marker Hsp60. (E) Acetylated proteins were immunoprecipitated from Hint2+/+ and Hint2−/− mitochondria with an anti-lysine acetylation antibody, then immunoblotted. A greater abundance of acetylated proteins were immunoprecipitated from Hint2−/− mitochondria. The Hint2 protein was detected in the pull-down only in Hint2+/+ mitochondria. (F) Sirtuin 3–mediated deacetylase activity in mitochondria isolated from 20-week-old Hint2+/+ (n = 5) and Hint2−/− (n = 5) livers. Deacetylase activity was measured by means of the Cyclex Sirt3 fluorometric assay. Sirtuin 3 activity was not different between Hint2+/+ and Hint2−/− mitochondria.

Discussion

In our model, the absence of Hint2 disturbed the regulation of lipid metabolism, glucose homeostasis, and mitochondrial respiration. The Hint2−/− mice showed an accelerated pattern of hepatic steatosis, a defect in hepatic Hadhsc and GDH activities, a lower glucose tolerance and counter-regulatory response to insulin-provoked hypoglycemia and impaired thermogenesis. Moreover, the mitochondrial electron transport between complex II and complex III was disturbed.

The mechanism by which Hint2 deletion negatively regulates both hepatic Hadhsc and GDH is related to a modification in their state of lysine acetylation.21, 22 Three findings support this conclusion: the addition of sirtuin 3 unmasked a higher fraction of latent GDH activity in Hint2−/− than in Hint2+/+ mitochondria (Fig. 7C), immunoblotting of Hint2−/− mitochondria showed a pattern of hyperacetylation for several proteins, and a greater abundance of acetylated proteins was immunoprecipitated with antiacetylated antibodies, including the Hint2 protein itself, which either associates with acetylated proteins or is itself acetylated. Although the manner in which Hint2 influences the acetylation status in the mitochondria cannot yet be elucidated, a change in expression of sirtuin 3 can be excluded. No change in the enzymatic activity of sirtuin 3 was detected in the assays in vitro. However, sirtuin 3 is an NAD+-dependent enzyme, and either the abundance or the availability of NAD+ may have been changed by the absence of Hint2 in mitochondria. Alternatively, Hint2 may have influenced the acetyl-transferase processes in mitochondria.

A change in the acetylation status of selected proteins could explain several other Hint2−/− phenotypic changes. Hepatic steatosis may be related to an impaired, hyperacetylated Hadhsc protein, since there is an association between Hadhsc deficiency and liver steatosis.23 Moreover, mitochondrial hyperacetylation of multiple proteins due to sirtuin 3 deficiency accelerates the development of metabolic syndrome.24 The impaired thermoregulation Hint2−/− mice could also be explained by an effect on acetylation, since BAT expresses both sirtuin 325 and Hint2 (Fig. 5) and BAT proteins are regulated by acetylation during fasting.26 The reduced respiration in Hint2−/− and silenced HINT2-HepG2 mitochondria could be a primary defect due to the reduced linked complex II-III electron transport and coenzyme Q levels, which in turn could explain the increased reactive oxygen species production.27 Certain components of the electron transport chain are regulated by acetylation,28 which may have been altered in Hint2−/− mitochondria. The cause of the reduced coenzyme Q was not clarified, but a down-regulation of biosynthetic genes at the transcriptional level could be excluded. The appearance of large deformed Hint2−/− mitochondria was an age-dependent feature and different from the structural alterations with cristolysis described in respiratory chain disorders, where fusion and fission were perturbed.29

Because Hint2 was detected solely in the exocrine pancreatic fraction, the two-fold increase in interprandial insulin levels in Hint2−/− mice remains unexplained but was not indicative of insulin resistance (Supporting Fig. 3B). A steatosis-mediated reduction of hepatic insulin clearance was unlikely because insulin was higher in Hint2−/− even after Hint2+/+ livers showed signs of steatosis. The apparent discrepancy between the increase in interprandial insulin and the decrease in glucose-stimulated insulin secretion, which could account for the lower glucose tolerance in Hint2−/− mice, was also not resolved in our experiments, but it is clear that deletion of Hint2 has affected basal and glucose-stimulated insulin secretion in different ways. The up-regulation of leptin mRNA expression in Hint2−/− WAT was possibly secondary to the higher basal insulin and glucocorticoid concentrations.30, 31 The failure of Hint2−/− mice to mount an appropriate counter-regulatory response to hypoglycemia is also not explained, but an impaired hepatic GDH enzyme combined with a lower expression of Pck1 after insulin (Fig. 4B and Supporting Fig. 3A) may have contributed to the poor ITT recovery phase.

The development of hepatic steatosis in the 30-week-old Hint2+/+ mice was unexpected, although hepatic steatosis in a subset of C57Bl6J mice fed a low-fat diet has been reported.32 The diet in our studies provided 17% calories from fat, and this may have contributed to the eventual hepatic lipid accumulation in the C57Bl6J × 129Sv controls. Conversely, an age-dependent decrease in the abundance of FoxO1 protein was noted (Supporting Fig 3B),33 which could have led to a decreased expression of its target, microsomal triglyceride transfer protein, and a reduced hepatic disposition of lipids.34

The generation of the Hint2−/− model has confirmed that Hint2 in the mitochondria of hepatocytes is required for fully competent Hadhsc and GDH enzyme activities. In the absence of Hint2, the acetylation pattern of multiple mitochondrial proteins is changed, hepatic steatosis is accelerated, and glucose tolerance and mitochondrial respiration are affected.

Acknowledgements

We thank Monika Ledermann and Jürg Müller for expert technical assistance. We thank GenOway for support in generating the Hint2+/+ and Hint2−/− mice.

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