AMP-activated protein kinase α1 protects against diet-induced insulin resistance and obesity.

AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1(-/-)) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1(-/-) mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1(-/-) mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1(-/-) mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1(-/-) mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.

AMP-activated protein kinase (AMPK) is a major cellular energy sensor and plays a major role in regulating metabolic homeostasis (1,2). In mammals, AMPK is a heterotrimeric complex with a catalytic subunit (α1 or α2) and two regulatory subunits (β1 or β1 and γ1, γ2, or γ3) (1,2). AMPKα2 is the predominant catalytic form of AMPK in the liver, muscle, and hypothalamus. There is evidence that AMPKα2 is important for the regulation of systemic insulin sensitivity and metabolic homeostasis. In the hypothalamus, AMPKα2 signals regulate food intake and body weight gain (3). Mice globally deficient in AMPKα2 display different metabolic phenotypes when fed different diets (4,5). A lack of AMPKα2 activity in skeletal muscle exacerbates glucose intolerance and the insulin resistance that is caused by high-fat diets (HFDs) (6). In addition, AMPKα2 is required for the effects of many physiologic regulators or pharmaceutical modalities that maintain insulin sensitivity and metabolic homeostasis (7–10).

Mice deficient in AMPKα1 had an increased inflammatory response in an experimental autoimmune encephalomyelitis model (11). AMPKα1 deficiency elevated the levels of reactive oxygen species and oxidized proteins, thereafter shortening the erythrocyte life span in mice (12). Macrophage AMPKα1 has been characterized as a key regulator of inflammatory function (13,14). Its role in protecting against diet-induced metabolic disorders has been hypothesized but not demonstrated (14). The activation of AMPK in adipocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) suppresses adipocyte differentiation and diet-induced obesity (15). However, the activation of AMPK is able to reduce isoproterenol-induced lipolysis; this result is supported by a decrease in adipocyte size and adipose mass in globally deficient in AMPKα1 (AMPKα1−/−) mice (16). To define the physiologic role of AMPKα1 in energy homeostasis, we administered an HFD to AMPKα1−/− mice and then evaluated diet-induced obesity and insulin resistance. We also used bone marrow (BM) transplantation (BMT) to characterize the specific roles of AMPKα1 in macrophages and adipocytes in the regulation of the diet-induced inflammatory response, adiposity, and systemic insulin resistance.

RESEARCH DESIGN AND METHODS

Mice.

The AMPKα1−/− mice were described previously (9). The AMPKα1−/− mice and wild-type (WT) littermates were generated from AMPKα1−/+ breeders in a mixture of C57BL/6 and 129/Sv strains. A mouse 384 single nucleotide polymorphism panel (markers spread across the genome at approximately 7-Mbp intervals; Charles River Laboratories International, Inc., Wilmington, MA) was used to characterize the genetic background of the breeders. Polymorphic markers showed that the heterozygous breeders were a mix of C57BL/6 (48.5%) and 129 (51.5%). Male mice were used in these experiments. All in vivo studies were initiated in mice at age 10 weeks.

The mice were fed an HFD (20 kcal% protein, 20 kcal% carbohydrate, 60 kcal% fat; D12492, Research Diets, New Brunswick, NJ) ad libitum for 12 weeks. Diabetic and/or obese parameters were measured in mice at the end of the 12-week HFD period. For the BMT studies, the recipient mice were lethally irradiated (850 rad) and then intravenously received 5 × 106 BM cells (BMCs) from donor mice, as described previously (17). After 2–4 weeks of recovery for BM reconstitution, the mice were fed an HFD for 12 weeks as described.

After the feeding regimen, the mice were fasted overnight before being killed for the collection of blood and tissue samples. Some mice were fasted for 4 h and used for insulin and glucose tolerance tests and insulin signaling analyses. For some mice, after being killed, the abdomen was quickly opened, and the epididymal, mesenteric, and perinephric fat depots were dissected and weighed as the visceral fat content. After being weighed, the adipose tissue samples were fixed and embedded for histologic and immunohistochemical analyses or were frozen in liquid nitrogen and stored at −80°C until further analysis. All study protocols were reviewed and approved by the institutional animal care and use committees of the University of Minnesota.

Insulin and glucose tolerance tests.

The mice were fasted for 4 h and received an intraperitoneal injection of insulin (1 units/kg) or d-glucose (2 g/kg) (18). For insulin tolerance or glucose tolerance tests, blood samples (5 μL) were collected from the tail vein before and at 30, 60, 90, and 120 min after the insulin or glucose bolus injection.

Analysis of adipose tissue with histologic/immunohistochemical and flow cytometry approaches.

The paraffin-embedded tissue blocks were cut into 5-μm-thick sections and were stained with hematoxylin and eosin. The sections were stained for the expression of F4/80 with rabbit anti-F4/80 (1:100; AbD Serotec, Raleigh, NC), as previously described (18). The fraction of F4/80-expressing cells for each sample was calculated as the sum of the number of nuclei of the F4/80-expressing cells divided by the total number of nuclei in the examined fields. Adipose macrophages were examined by flow cytometry analysis (19,20). Antibodies against F4/80 and integrin alpha X complement component 3 receptor 4 subunit (CD11C) were used to analyze the number of macrophages among the stromal vascular fraction (SVF), and the number of CD11C-positive macrophages among all of the macrophages were analyzed (details provided in the Supplementary Data).

Western blotting to determine inflammatory, insulin, and AMPK signaling.

Frozen tissue samples and isolated/cultured cells were lysed in modified radioimmunoprecipitation assay lysis buffer, separated by SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 and incubated with antibodies diluted in blocking buffer. The blots were incubated with alkaline phosphatase-conjugated secondary antibodies, developed with a chemifluorescence reagent, and scanned with a Storm 860 system (GE Healthcare). The antibodies against inhibitor of κB, p65, arginase, AMPKα1 and AMPKα2, and acetyl-CoA carboxylase (ACC), as well as their phosphorylated forms, were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The antibodies against the various units of the mitogen-activated protein kinase (MAPKs), Akt, and their phosphorylated forms, were from Cell Signaling Technology (Beverly, MA).

RNA isolation, reverse transcription, and real-time PCR.

Total RNA was isolated from frozen tissue samples or cultured cells. Reverse transcription and quantitative real-time PCR were conducted, as previously described (21). The relative amount of each gene in each sample was estimated by the ΔΔCT method. 18Sr RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes and yielded similar results. The results using GAPDH are presented in this report. The sequences of the primers for the cytokines and metabolic transcription factors are reported in Supplementary Table 1.

Cell isolation, culture, and treatment

BM-derived macrophages.

BMCs were collected from AMPKα1−/− mice and WT controls. BMDMs were cultured as described previously (22). Cultured BMDMs were stimulated with recombinant mouse interleukin (IL)-4 (10 ng/mL) for 48–72 h for alternative macrophage activation. To assess cytokine production at the mRNA and protein levels, the cells were stimulated with lipopolysaccharide (LPS; 100 ng/mL) for 2 or 48 h. Supernatants and cells were collected and assayed with enzyme-linked immunosorbent assay kits and real-time RT-PCR, respectively. To determine the inflammatory signaling pathways, the cells were stimulated with LPS (10 ng/mL) and were collected at different time points. The BM-derived macrophages (BMDMs) were also treated with LPS (10 ng/mL) for 2 h and were collected for microarray analysis.

In vitro adipocytes.

Stable AMPKα1 knockdown (AMPKα1-KD) and control (AMPKα1-Ctrl) 3T3-L1 cells were established after transfection with the plasmid expressing short-hairpin RNA against mouse AMPKα1 or a similar-sized RNA with a scramble sequence. To differentiate the 3T3-L1 cells, the 2-day postconfluent cells were incubated in growth medium supplemented with 10 μg/mL insulin, 1 μmol/L dexamethasone, and 0.5 mmol/L 3-isobutyl-1-methyl-xanthine for 48 h, followed by incubation for an additional 6–8 days in growth medium. Adipocyte lipid accumulation was analyzed by oil red O staining. To determine the rate of 14C-glucose incorporation into lipids, each well (6-well plate) of adipocytes was incubated with DMEM supplemented with 1 μCi [U-14C] glucose for 24 h, as previously described (18). To determine changes in the adipocyte insulin signaling, the cells were treated with or without insulin (100 nmol/L) for 30 min. The amount and phosphorylation of Akt were analyzed using Western blots. To analyze adipocyte inflammatory signaling, the amount and phosphorylation of nuclear factor (NF)-κB p65 were examined in adipocytes treated with or without palmitate for 24 h. The samples from similar treatments were also used for the measurement of cytokines and metabolic transcriptional factors using a real-time PCR assay.

Statistical analysis.

Statistical analysis was performed with Instat (GraphPad Software). Data are presented as the mean ± SEM. Data were analyzed with a Student t test to evaluate two-tailed levels of significance. The null hypothesis was rejected at P < 0.05.

RESULTS

Global deficiency of AMPKα1 exacerbates HFD-induced adiposity.

AMPKα1−/− mice fed a low-fat diet exhibited a slight but insignificant increase in body weight compared with AMPKα1+/+ (WT) mice (Supplementary Fig. 1). However, AMPKα1−/− mice fed an HFD gained more body weight than did AMPKα1+/+ mice (Fig. 1). The amount of food intake (grams or kcal intake/day/mouse) of the AMPKα1−/− mice tended to be high compared with that of the control mice (Supplementary Fig. 2), although this increase did not reach statistical significance. At the end of the 12-week HFD period, group differences were not observed in the size or weight of the lungs, hearts, livers, or kidneys, but the epididymal, mesenteric, and perinephric fat masses in the AMPKα1−/− mice were greater than those in the WT mice (Fig. 1). The total visceral fat accounted for 12 and 9% of the whole body weight of the AMPKα1−/− and the WT mice, respectively (Fig. 1). Congruent with this increase in the fat mass, the adipocytes in the HFD-fed AMPKα1−/− mice were markedly larger (as measured by histology) than those in the HFD-fed WT littermates (Fig. 1).

FIG. 1.

HFD-induced adiposity in AMPKα1−/− mice. At 10 weeks of age, the AMPKα1−/− mice and their WT (AMPKα1+/+) littermates were fed an HFD. A: Changes in body weight over a 12-week HFD period. B–D: Changes in visceral fat content. The sum of the epididymal, mesenteric, and perinephric fat masses was estimated as the visceral fat content (scale bar = 1 cm). E: Adipose tissue histology. Sections of the epididymal fat pad were stained with hematoxylin and eosin (scale bar = 50 μm). A–C: Data are the means ± SE; n = 12 mice per group. *P < 0.05 and **P < 0.01 for AMPKα1−/− vs. AMPKα1+/+ mice. B–E: Experiments were conducted in mice at the end of the 12-week HFD period. (A high-quality color representation of this figure is available in the online issue.)

Global deficiency of AMPKα1 aggravates HFD-induced insulin resistance and inflammation.

The circulating levels of glucose and insulin in the AMPKα1−/− mice were higher than those in the WT mice (increases of 22 and 120%, respectively; Fig. 2). In addition, insulin resistance and glucose intolerance in the AMPKα1−/− mice were more severe than in the WT mice (Fig. 2). In adipose tissue, a lower level of insulin-induced Akt phosphorylation was seen in AMPKα1−/− mice compared with WT mice (Fig. 2), whereas in the liver and skeletal muscle, phosphorylated (p-)Akt levels were not significantly different between the two groups (Supplementary Figs. 3).

FIG. 2.

HFD-induced insulin resistance in AMPKα1−/− mice. At age 10 weeks, the AMPKα1−/− mice and their WT (AMPKα1+/+) littermates were fed an HFD, and in vivo experiments were conducted in the mice at the end of the 12-week HFD period. A and B: Levels of glucose and insulin were measured in plasma samples from mice fasted overnight. Blood glucose was measured in mice that were fasted for 4 h and received an intraperitoneal injection of d-glucose (2 g/kg) for a glucose tolerance test (GTT) (C) or insulin (1 units/kg) for an insulin tolerance test (ITT) (D). E: Adipose tissue insulin signaling. Samples of white adipose tissue (WAT) were collected at 5 min after an intravenous injection of insulin (1 units/kg) or PBS into the inferior vena cava. F: Levels of AMPK, ACC, and their phosphorylated forms in adipose tissue. G: Levels of proinflammatory cytokines and transcriptional factors in adipose tissue. Data are the means ± SE; n = 6 mice per group. *P < 0.05 and **P < 0.01 for AMPKα1−/− vs. AMPKα1+/+ mice.

Although the adipose tissue levels of AMPKα2 were equal in the two groups, significant levels of AMPKα1 were detected in the adipose tissue of the WT mice but not in the AMPKα1−/− mice (Fig. 2). The levels of phosphorylated AMPKα and ACC in adipose tissue in the HFD-fed AMPKα1−/− mice were much lower than those in the HFD-fed WT mice (Fig. 2). The mRNA abundance of the genes involved in adipogenesis (PPARγ, AP2, C/EBPα) and the adipokines (leptin and adiponectin) were not affected by AMPK ablation (Fig. 2). However, compared with the HFD-fed AMPKα1+/+ mice, the HFD-fed AMPKα1−/− mice exhibited significant increases in IL-1β, IL-6, and tumor necrosis factor (TNF)-α mRNA in adipose tissue (Fig. 2). Similar elevations of some cytokines were observed in the liver (Supplementary Fig. 3). The anti-inflammatory cytokine IL-10 was also increased at the mRNA level in the livers and adipose tissues of AMPKα1−/− mice compared with WT mice (Fig. 2 and Supplementary Fig. 3). The levels of circulating cytokines in the AMPKα1−/− mice were consistently higher than those in the WT mice (Table 1).

TABLE 1

Inflammatory parameters of mouse circulating blood

Deficiency of AMPKα1 augments the macrophage proinflammatory function.

Immunostaining with macrophage-specific F4/80 antibody revealed a greater number of macrophages in the adipose tissue of the AMPKα1−/− mice than in the control mice (Fig. 3). This result was confirmed by flow cytometry, which also revealed an elevated percentage of CD11c macrophages in the adipose tissue of the AMPKα1−/− mice compared with the percentage in the WT mice (Fig. 3). These data indicate that AMPKα1 deficiency causes an increase in the accumulation of proinflammatory macrophages in the adipose tissue. BMCs were cultured in the presence of macrophage colony-stimulating factor, stimulated with IL-4, and then examined for the expression of arginase 1. No difference was found in the mRNA and protein levels of arginase 1 in the AMPKα1-deficient and WT macrophages (Fig. 3), suggesting that AMPKα1 deficiency does not affect alternative macrophage polarization. An elevated production of inducible nitric oxide synthase, TNF-α, and IL-1β, but not IL-6, was observed in the LPS-stimulated AMPKα1-deficient macrophages compared with the WT macrophages (Fig. 3). Interestingly, the IL-10 levels in AMPKα1-deficient macrophages were also higher than those in the WT macrophages (Fig. 3). In line with these elevations in the proinflammatory cytokine levels, the signaling molecules of NF-κB and MAPKs, which are two pathways critically involved in LPS-induced activities, were phosphorylated earlier or to a greater extent in the AMPKα1-deficient macrophages than they were in the WT macrophages (Fig. 3).

FIG. 3.

Influence of AMPKα1 deficiency on macrophage function. Adipose macrophages were analyzed in the adipose samples of mice at the end of the 12-week HFD period. A: Macrophage infiltration in adipose tissue (scale bar = 50 μm). Sections of the epididymal fat pad were immunostained for F4/80. B: Percentages of adipose macrophages (F4/80+) among the SVF cells or M1 macrophages (CD11c+/F4/80+) among the macrophages (F4/80+). Green histograms showed APC signal in the isotype tubes. C and D (top two panels): mRNA and protein levels of arginase I in BMDMs with or without IL-4 (10 ng/mL) treatment for 48 h. D (bottom two panels)–F: The protein levels of inducible nitric oxide synthase (iNOS), as well as the protein and mRNA levels of TNF-α, IL-1β, IL-6, and IL-10, in BMDMs with or without LPS for 6 or 48 h. A–F: Numeric data are the means ± SE; n = 6 mice per group. *P < 0.05 and **P < 0.01 for AMPKα1−/− vs. AMPKα1+/+ mice. G: Levels of Jun NH2-terminal kinase (JNK), p38, p65, their phosphorylated forms, and inhibitor of κB (IκBα) in AMPKα1+/+ and AMPKα1-deficient macrophages after LPS treatment. The results shown are from one of four experiments. (A high-quality color representation of this figure is available in the online issue.)

We also ran a microarray to identify gene expression patterns influenced by AMPKα. The mRNA levels of macrophage housekeeping genes, which are associated with the signaling of the Toll-like receptors NF-κB and MAPKs, were identical for macrophages from AMPKα1-deficient and WT mice under resting conditions (data not shown). However, cytokines and chemokines were elevated in LPS-treated AMPKα1-deficient macrophages compared with the levels in the WT macrophages (Supplementary Fig. 5). These increases were observed even without LPS treatment (Supplementary Fig. 5). Most of the microarray data were further confirmed by real-time PCR.

Deficiency of AMPKα1 in BMDCs causes insulin resistance but not obesity.

We used BMT to generate two types of chimeric mice: AMPKα1+/+ mice that had received the BMCs of AMPKα1−/− mice (AMPKα1−/− to WT) or AMPKα1+/+ mice (WT to WT). On a 12-week HFD, the body weight gain in both groups of BMT mice was modest and did not show significant differences (Fig. 4). The weight of the visceral fat was also identical between the two groups (Fig. 4). In contrast, the severity of the insulin resistance and glucose intolerance in the AMPKα1−/−–to–WT BMT mice was greater than in the WT–to–WT BMT mice (Fig. 4). In addition, the insulin-induced Akt phosphorylation in the adipose tissue of AMPKα1−/−–to–WT BMT mice was lower than in the WT–to–WT BMT mice (Fig. 4).

FIG. 4.

Metabolic and inflammatory phenotypes of chimeric WT mice with AMPKα1-deficient BMCs. Irradiated WT mice received the BMCs of WT or AMPKα1−/− mice. After a 4-week recovery period, the mice were fed an HFD, and in vivo experiments were conducted in the mice at the end of the 12-week HFD period. A: Body weight over 12 weeks. B and C: Weight of visceral fat content. Blood glucose was measured in mice that were fasted for 4 h and given an intraperitoneal injection of d-glucose (2 g/kg) for glucose tolerance test (GTT) (D) or insulin (1 units/kg) for insulin tolerance test (ITT) (E). F: Adipose tissue insulin signaling. Samples of white adipose tissue (WAT) were collected at 5 min after an intravenous injection of insulin (1 units/kg) or PBS into the inferior vena cava. G: Levels of AMPK, ACC, and their phosphorylated forms in adipose tissue. H: Levels of proinflammatory cytokines and transcriptional factors in adipose tissue. I and J: Numbers of adipose macrophages and percentages of CD11c+ macrophages examined with immunostaining (scale bar = 50 μm) and flow cytometry. Green histograms showed APC signal in the isotype tubes. Numeric data are the means ± SE; n = 14 mice per group (A–E, H–J) and n = 6 mice per group (F and G). *P < 0.05 for AMPKα1−/−–to–WT mice vs. WT–to–WT mice. (A high-quality color representation of this figure is available in the online issue.)

We then examined AMPK signaling in the adipose tissue of these chimeric mice. Western blot analysis did not show any significant differences in the levels of AMPKα1, AMPKα2, ACC isoform 1, or their phosphorylated forms in the two groups of mice (Fig. 4). However, adipose tissue from AMPKα1−/−–to–WT BMT mice had higher mRNA levels of TNF-α, IL-6, and IL-10 (Fig. 4).

We evaluated adipose macrophages using immunostaining and fluorescence-activated cell sorter assays with a macrophage-specific antibody. A very modest increase in the number of macrophages was seen in the adipose tissue of AMPKα1−/−–to–WT BMT mice compared with the WT–to–WT BMT mice (Fig. 4). However, the percentages of the CD11c-expressing macrophages among the adipose macrophages were equal in both groups (Fig. 4), indicating that an in vivo AMPKα1 deficiency in macrophages does not increase monocyte recruitment to adipose tissue.

AMPKα1 KD alters adipocyte metabolic and inflammatory activities.

Stable AMPKα1-KD and AMPKα1-Ctrl 3T3-L1 cells were established and differentiated into adipocytes using a well-established method (18). Western blot analysis showed that the levels of AMPKα1 and p-AMPKα and ACC in AMPKα1-KD adipocytes were lower than in AMPKα1-Ctrl adipocytes (Fig. 5). Oil red O staining showed many more lipid droplets in AMPKα1-KD adipocytes than in AMPKα1-Ctrl cells (Fig. 5). In addition, the rate of 14C-glucose incorporation into lipids in AMPKα1-KD adipocytes was much higher than in AMPKα1-Ctrl cells (Fig. 5).

FIG. 5.

Influence of AMPKα1 KD the regulation of the metabolic and inflammatory activities of adipocytes. Stable AMPKα1-KD (KD1 and KD2) and AMPKα1-Ctl (Ctrl-1 and Ctrl-2) 3T3-L1 cells were established. These cells and nontransfected cells were then differentiated into adipocytes. A: Levels of total AMPK and ACC and p-AMPK and p-ACC. B: Levels of lipid accumulation in adipocytes of different types examined with oil red O staining (scale bar = 500 μm). C: Rates of 14C-glucose incorporation into lipids in AMPKα1-Ctrl and -KD adipocytes. D: Levels of insulin-induced p-Akt in AMPKα1-Ctrl and -KD adipocytes. E: Levels of p65 and p-p65 in AMPKα1-Ctrl and -KD adipocytes after treatment with palmitate (250 μmol/L) for 24 h. F: mRNA levels of cytokines, adipose transcriptional factors, and adipose markers of AMPKα1-Ctrl and -KD adipocytes after treatment with palmitate (Pal; 250 μmol/L) for 24 h. A, B, D and E: Results are from one of four experiments. C and F: Data are the means ± SE; n = 6 per group. *P < 0.05 and **P < 0.01 for AMPKα1-Ctrl vs. AMPKα1-KD adipocytes. (A high-quality color representation of this figure is available in the online issue.)

For cultured adipocytes, increases in the Akt phosphorylation were observed from 5 to 30 min after insulin exposure, and the levels of p-Akt in AMPKα1-KD adipocytes at 30 min after insulin treatment were examined. The p-Akt levels in AMPKα1-KD adipocytes were much lower than in AMPKα1-Ctrl cells (Fig. 5). Even under basal conditions (without insulin treatment), the p-Akt levels were decreased (Fig. 5). In adipocytes treated with palmitate, the extent of the inflammatory response, which was demonstrated by the p-p65 level, was higher in the AMPKα1-KD cells than in the AMPKα1-Ctrl cells (Fig. 5). A real-time PCR assay showed higher mRNA levels of monocyte chemoattractant protein (MCP)-1, IL-1β, TNF-α, and IL-10 in AMPKα1-KD adipocytes than in AMPKα1-Ctrl cells treated with palmitate (Fig. 5). No differences in the adipocyte markers, transcriptional factors, or adipokines were found between the two cell groups.

Deficiency of AMPKα1 in adipocytes leads to obesity and insulin resistance in mice.

To determine the role of adipocyte AMPKα1 in regulating metabolic phenotypes, AMPKα1−/− mice and AMPKα1+/+ mice were irradiated and received the BM of AMPKα1+/+ mice. After a 4-week recovery period, both groups were fed an HFD for 12 weeks. During the first few weeks of the HFD, neither group gained significant body weight. During the last 6 weeks of HFD feeding, however, WT–to–AMPKα1−/− BMT mice gained more body weight than did WT–to–WT BMT mice (Fig. 6), despite similar food intake for both groups (Supplementary Fig. 2B). The weight of the epididymal, mesenteric, and perinephric fat in the WT–to–AMPKα1−/− BMT mice was greater than in the WT–to–WT BMT mice (Fig. 6). The total visceral fat accounted for 10.5 and 7.5% of the whole body weight for the WT–to–AMPKα1−/− BMT mice and the WT–to–WT BMT mice, respectively (Fig. 6). Histologic analysis of the adipose tissue consistently showed larger adipocytes in the WT–to–AMPKα1−/− BMT mice than in the WT–to–WT BMT mice (Fig. 6).

FIG. 6.

Metabolic and inflammatory phenotypes of chimeric AMPKα1−/− mice with WT BMCs. Irradiated AMPKα1−/− mice or WT mice received the BM of WT mice. After a 4-week recovery period, the mice were fed an HFD, and in vivo experiments were conducted in the mice at the end of the 12-week HFD period. A: Changes in body weight over 12 weeks. B and C: Weight of visceral fat content. Blood glucose levels in mice that were fasted for 4 h and received an intraperitoneal injection of d-glucose (2 g/kg) for a glucose tolerance test (GTT) (D) or insulin (1 units/kg) for an insulin tolerance test (E). F: Adipose tissue insulin signaling. Samples of white adipose tissue (WAT) were collected at 5 min after an intravenous injection of insulin (1 units/kg) or PBS into the inferior vena cava. G: Levels of AMPK, ACC, and their phosphorylated forms in adipose tissue. H: Levels of proinflammatory cytokines and transcriptional factors in adipose tissue. I and J: Numbers of adipose macrophages and percentages of CD11c+ macrophages examined with immunostaining (scale bar = 50 μm) and flow cytometry. Green histograms show the APC signal in the isotype tubes. Numeric data are the means ± SE; n = 12 mice per group (A–E, H–J) and n = 6 mice per group (F and G). *P < 0.05 and **P < 0.01 for WT–to–AMPKα1−/− mice vs. WT–to–WT mice. (A high-quality color representation of this figure is available in the online issue.)

Insulin resistance and glucose intolerance in the WT–to–AMPKα1−/− BMT mice were much more severe than in the WT–to–WT BMT mice (Fig. 6). The levels of p-Akt in the adipose tissue of the WT–to–AMPKα1−/− BMT mice were consistently lower than those of the WT–to–WT BMT mice (Fig. 6).

The AMPK signaling in the adipose tissue was examined. Significant levels of p-AMPKα and ACC were observed in the WT–to–WT BMT mice but not in the WT–to–AMPKα1−/− BMT mice (Fig. 6). A real-time PCR assay demonstrated that, compared with the WT–to–WT BMT mice, the WT–to–AMPKα1−/− BMT mice had a significant increase in the mRNA levels of cytokines such as IL-1β, TNF-α, IL-6, and IL-10 (Fig. 6).

The numbers of macrophages and percentages of CD11-expressing macrophages in adipose tissue in the WT–to–AMPKα1−/− BMT mice were much higher than in the WT–to–WT BMT mice (Fig. 6). The extent of these increases was greater than that seen between AMPKα1−/− mice and their WT control mice, indicating that in vivo, AMPKα1-deficient adipocytes are able to recruit more proinflammatory monocytes than WT adipocytes.

DISCUSSION

Many studies have associated AMPKα1 with inflammation (23,24), and inflammation plays a crucial role in the development of diet-induced adiposity and insulin resistance (25–27). Data from other groups have suggested that AMPKα1 promotes macrophage alternative activation and decreases proinflammatory activation (13,14). We have confirmed that AMPKα1-deficient macrophages are highly proinflammatory. However, we also observed that macrophages isolated from AMPKα1-deficient mice did not exhibit significant defects in alternative activation. In addition, the current study indicates a complex role of AMPKα1 in the regulation of inflammation:

First, while producing higher levels of most proinflammatory cytokines, in vitro cultured AMPKα1-deficient macrophages and adipocytes produced the same amount of IL-6 as WT cells did under resting and stimulated conditions.

Second, the anti-inflammatory cytokine IL-10 was robustly upregulated in AMPKα1-deficient macrophages and adipocytes at the cellular level and in HFD-fed AMPKα1−/− mice compared with that in control cells or mice.

Third, the number of adipose macrophages in the WT–to–AMPKα1−/− BMT mice was much higher than in the AMPKα1−/− mice or in the AMPKα1−/−–to–WT BMT mice, suggesting that macrophage AMPKα1 deficiency compromises macrophage infiltration into the adipose tissue. Nevertheless, transferring AMPKα1-deficient BMCs into WT mice did cause systemic insulin resistance, although acceleration in HFD-induced adiposity was not observed in these mice within the experimental timeframe.

Adipocyte AMPKα1 appears to be crucial for the protection of diet-induced insulin resistance and adiposity. The transplantation of WT BMCs to AMPKα1−/− mice did not rescue diet-induced obesity and insulin resistance in the adipose tissue. These data argue in favor of a critical role for AMPKα1 in cells other than BMCs in protecting against diet-induced obesity and insulin resistance. AMPKα1 is mostly abundant in adipose tissue (28,29). Thus, the phenotypes of obesity and insulin resistance in AMPKα1−/− mice and in chimeric AMPKα1−/− mice with WT BMCs may be largely attributable to AMPKα1 deficiency in adipocytes. The inactivation of the downstream signaling of AMPK may be one of the major mechanisms for the obesity phenotype of AMPKα1−/− mice. In the AMPKα1−/− mice, ACC phosphorylation was almost completely abrogated, which was accompanied by increases in the visceral fat mass and size of the adipocytes, without detectable changes in the major markers or transcriptional factors in adipocyte differentiation. The same phenotypes and mechanisms were recapitulated in AMPKα1-KD 3T3-L1 adipocytes, implying that AMPKα1-KD or deletion increases lipid content mainly through fatty acid synthesis by ACC (30). A recent study showed that AICAR reduced adipocyte size via the downregulation of the expression of adipogenic factors in vitro and inhibited HFD-induced adiposity by activating the expression of peroxisome proliferator-activated receptor (PPAR)-γ coactivator 1-α (15). These studies suggest a potential difference in the physiologic and pharmaceutical activities of AMPKα1 or a possible effect of AICAR that is nonspecific for AMPK.

An early study reported that AMPKα1 had an antilipolytic effect on adipocytes in vitro (16). The antilipolytic effect of AMPKα1 in adipocytes was obvious in the presence of β-adrenergic activators. However, the same study showed that in the absence of β-adrenergic activators, a trend of increased lipolysis was seen in adipocytes in which AMPKα1 was specifically activated, and the reverse effect occurred when AMPKα1 was blocked (16). In a more recent study, there was a greater lipid accumulation in adipocytes that differentiated from AMPKα-deficient mouse embryonic fibroblasts than from control mouse embryonic fibroblasts (see Fig. S6 in Djouder et al. [31]).

In contrast to the previous report showing that AMPKα1−/− mice fed a chow diet displayed a small fat pad compared with control mice (16), the AMPKα1−/− mice in the current study showed accelerated adiposity. The mouse breeders used in our study were from the same founders used in previous studies (5,9,32). We used 10-week-old male mice to ensure that the animals could endure the BMT procedure. Therefore, the difference in the mouse age, genetic background, and diet in the two studies may be responsible for the controversial data regarding the role of AMPKα1 in regulating energy homeostasis. To facilitate the reproducibility of the metabolic phenotypes in the AMPKα1−/− mice, information on the genetic background of the mice and the diet formula used in this study are detailed in the .

AMPKα1 deficiency in tissues and cells other than adipose and macrophages may also contribute to the diabetic and/or obese phenotypes observed in the AMPKα1−/− or chimeric mice. AMPKα1 is expressed in various organs, tissues, and cells, including adipose, liver, muscle, brain, heart, endothelial cells, and leukocytes (33). It is highly likely that some of the effects of AMPKα1 deficiency on the diabetic and/or obese phenotypes may be due to a lack of AMPKα1 in some of the other tissues, particularly in the brain, where energy homeostasis is regulated. Additionally, although the role of AMPK α1 deficiency in the macrophages in regulating the inflammatory response in vitro has been studied, in vivo data from chimeric mice actually include the effect of AMPKα1 deficiency in all BMDCs. A lack of AMPK α1 in T cells, B cells, or other BMDC types may equally have contributed to the metabolic phenotype. Notably, lymphocyte populations, including T cells, B cells, natural killer cells, and natural killer T cells, have all been implicated in adipose tissue inflammation and insulin resistance (34–38). Thus, future studies of the role of AMPKα1 in tissues and cells other than adipose and macrophages in determining the diet-induced metabolic phenotype is warranted.

Numerous studies have already shown that AMPK is associated with inflammation, oxidative stress, and apoptosis (39–42). Decreased AMPK activity in adipose tissue has been observed in a wide variety of rodents with obesity and insulin resistance. In recent clinical studies, Gauthier and coworkers (43,44) have reported a decrease in AMPK activity (T172 phosphorylation) and an increase in inflammatory cells in the adipose tissue of massively obese people who are insulin resistant. Revealing the low activity of AMPK in relevant studies as a critical causal factor of inflammation, diabetes and obesity, the current study demonstrates that AMPKα1 is a viable therapeutic target in the treatment of metabolic syndrome.