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. 2008 Nov 26;150(4):1697–1704. doi: 10.1210/en.2008-1409

Hyperphagia and Obesity in Female Mice Lacking Tissue Inhibitor of Metalloproteinase-1

Isabelle Gerin 1, Gwendolyn W Louis 1, Xuan Zhang 1, Tyler C Prestwich 1, T Rajendra Kumar 1, Martin G Myers Jr 1, Ormond A MacDougald 1, Warren B Nothnick 1
PMCID: PMC2659269  PMID: 19036876

Abstract

Certain matrix metalloproteinases and their regulators, the tissue inhibitors of metalloproteinases (TIMPs), are involved in development and remodeling of adipose tissue. In studying Timp1<tm1Pds> mice, which have a null mutation in Timp1 (Timp1−/−), we observed that females exhibit increased body weight by 3 months of age due to increased total body lipid and adipose tissue. Whereas Timp1−/− mice have increased size and number of adipocytes, they also display increased food intake despite hyperleptinemia, suggesting that alterations in hypothalamic leptin action or responsiveness may underlie their weight gain. Indeed, leptin promotes the expression of Timp1 mRNA in the hypothalamus, and leptin signaling via signal transducer and activator of transcription-3 mediates the expression of hypothalamic Timp1. Furthermore, Timp1−/− mice demonstrate increased food intake and altered expression of certain hypothalamic neuropeptide genes prior to elevated weight gain. Thus, whereas previous data suggested roles for matrix metalloproteinases and TIMPs in the regulation of adipose tissue, these data reveal that Timp1 mRNA is induced by leptin in the hypothalamus and that expression and action of Timp1 contributes to the regulation of feeding and energy balance.

Timp1-dependent regulation of hypothalamic physiology represents an important determinant of energy balance that directly influences adiposity.

Obesity increases risk for diabetes and cardiovascular diseases and is a significant health problem throughout the world. Expansion of adipose tissue results when caloric intake chronically exceeds energy expenditure. The matrix metalloproteinase (MMP) system, which consists of MMPs and the tissue inhibitors of metalloproteinases (TIMPs), is important for differentiation and remodeling of adipose tissue (1,2,3) during development and in the expansion of adipose tissue that occurs with obesity. Altered expression of several MMPs and TIMPs is observed in adipose tissue of obese mice (1) and circulation of obese humans (4,5). Furthermore, mice deficient for Mmp3 (6) and Mmp11 (7) exhibit increased adiposity compared with control animals. In addition, male mice treated with MMP inhibitors display reduced body weight and adiposity when exposed to high-fat diets (8,9). This body of work suggests that MMP activity is important locally within adipose tissue but also contributes to the regulation of whole-body energy homeostasis.

The mechanisms that underlie imbalance between energy intake and expenditure can stem from alterations in central nervous system systems that regulate feeding and energy expenditure or alterations in peripheral metabolism. Although many neural systems contribute to the regulation of energy homeostasis, the role of the arcuate nucleus of the hypothalamus (ARC) is well characterized. Within the ARC, at least two distinct neuronal populations regulate energy balance: 1) neurons that coexpress neuropeptide Y (NPY) and agouti-related peptide (AgRP) and 2) neurons that express proopiomelanocortin (POMC) (10,11,12). The ARC POMC neurons are stimulated by leptin and other signals of energy repletion to mediate anorexia and promote energy expenditure (10,11,12,13,14). Leptin and similar signals deactivate NPY/AgRP neurons and suppress expression of these orexigenic (appetite promoting) neuropeptides (15,16,17,18,19,20,21,22). In addition to ARC neurons, circuits elsewhere in the hypothalamus and central nervous system contribute to the regulation of feeding and energy balance (23,24,25), including neurons in the lateral hypothalamus area (LHA) that express hypocretin (HCRT) and melanin-concentrating hormone (MCH) (26,27,28,29,30). MCH treatment or increased MCH expression promotes feeding and positive energy balance. In contrast, whereas acute HCRT treatment promotes hyperphagia, decreased HCRT expression or action also promotes weight gain, consistent with a long-term catabolic action for HCRT. Whereas matrix remodeling processes have not been studied in this context, based on the dynamic rewiring of circuitry in the hypothalamus by leptin (31), a role for MMPs and TIMPs is plausible.

In previous studies conducted in our laboratory (32,33,34,35), we noticed that at 1 yr of age, Timp1-deficient females weigh more than their wild-type counterparts. This observation coupled with the potential role of the metalloproteinase system in the regulation of adipose tissue expansion and whole-body energy homeostasis led us to examine more closely the mechanisms by which Timp1 contributes to overall energy balance in female mice.

Materials and Methods

Animals and experimental design

Timp1<tm1Pds> mice (formerly known as Timp1<tm1Jae>) and referred to within this paper as Timp1−/− mice are maintained on a 129S4/SvJae (formerly known as 129/SvTer) background at the University of Kansas Medical Center and have been previously described (36). Because the Timp1 locus is on the X chromosome, results reported in Figs. 1–5 were from mice generated from null female X null male crosses and wild-type female X wild-type male crosses. Results in Fig. 6 and supplemental Fig. 2, published as supplemental data on The Endocrine Society's Journals Online web site at http://endo.endojournals.org were from crossing ± females, which were full sisters. These sisters were mated to wild-type or null males to generate wild-type and null female cousins. Housing was either under the care of University of Kansas Medical Center Lab Animal Resources or the University of Michigan Unit for Laboratory Management. Timp1−/− and control mice were housed with a regular 12-h light, 12-h dark cycle and ad libitum access to standard rodent chow diet (Laboratory Rodent Diet 5001; LabDiet, St. Louis, MO). Animals with alterations in leptin receptor signaling were produced and maintained in our colony at the University of Michigan. Heterozygote db/+ (Leprdb), s/+ (LeprS1138 or Lepr<tm1Mgmj>) (18), and l/+ (LeprL985 or Lepr<tm2Mgmj>) (18,37) mice on the C57BL/6 background were self-crossed to generate animals homozygous for each altered lepr allele as well as wild-type controls. Animals were genotyped by Taqman-based SNP allelic discrimination assays [reorder numbers 1633916 (LeprS1138 and LeprL985) and 1750407 (Leprdb); Applied Biosystems Inc., Foster City, CA]. For the comparison of leptin-induced gene expression in 8- to 10-wk-old male wild-type and ob/ob (Lepob/ob) mice, animals were purchased from Jackson Labs (Bar Harbor, ME) and acclimated to our facility for at least 1 wk before treatment with leptin (5 mg/kg, ip) for 4 h before the animals were killed. All experiments were approved by the University Committee on Use and Care of Animals at each institution.

Figure 1.

Figure 1

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Development of obesity in female Timp1−/− mice. A, Body weights of wild-type (WT) and Timp1−/− mice at weaning or 3, 6, 9 and 12 months of age. Data are displayed as means ± sd of eight mice per genotype/age. KO, Knockout. B, Analysis of fat and lean mass in wild-type and Timp1−/− mice by DEXA. C, Weights of adipose depots, liver, pancreas, spleen, heart, and lung. Data are displayed as mean ± sd. O-WAT, Ovarian white adipose tissue; P-WAT, perirenal white adipose tissue; D-WAT, dorsolumbar white adipose tissue; I-WAT, interscapular white adipose tissue; BAT, brown adipose tissue. D, DEXA analysis of bone mineral density (BMD). Body weights and composition of wild-type and Timp1−/− mice at 11–13 months of age (n = 8). Statistical significance for each measurement was evaluated with Student's t test within age or end point between genotypes. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Figure 2

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Increased size and number of adipocytes in Timp1−/− mice. A, Size of adipocytes in ovarian adipose tissue was quantified in at least four different randomly chosen microscopic fields (0.58 mm2/field) for each mouse (n = 8/genotype). Average cross-sectional area of adipocytes is shown. B, Number of adipocytes with sizes between 400 and 4,000 μm2 and between 4,000 and 10,000 μm2. Data are reported as mean ± sd. Statistical significance between genotype was evaluated using Student's t test. *, P < 0.05. KO, Knockout; WT, wild type.

Figure 3.

Figure 3

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Glucose tolerance test and serum leptin levels. A, Blood glucose of female wild-type (WT) and Timp1−/− mice at 1 yr of age (n = 7) was measured at the indicated times after an overnight fast and a 1.5 g/kg ip glucose injection. B, Serum leptin concentration from random-fed female wild-type and Timp1−/− mice at 14 months of age (n = 8). Data are presented as mean ± sd. Statistical significance was evaluated with Student's t test. **, P < 0.01. KO, Knockout.

Figure 4.

Figure 4

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Analysis of energy balance of female wild-type (WT) and Timp1−/− mice at 12 months of age. A, Food consumption of individually caged female mice is reported as mean ± sd (n = 8). Statistical significance was evaluated using the Student's t test. **, P < 0.01. B and C, VO2 and respiratory ratio (VCO2/ VO2) were determined using indirect calorimetry and are presented graphically. Average values were obtained at each time point with n = 8/genotype, with each animal measured for 3–5 d. Data are presented as mean ± sd. KO, Knockout.

Figure 5.

Figure 5

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Induction of hypothalamic Timp1 mRNA expression by leptin signaling. Expression of Timp1 mRNA in microdissected ARC C57BL/6 control (WT) (A) or ob/ob (B) animals treated with PBS or leptin (5 mg/kg, ip) for 4 h (n = 8/treatment). Asterisks indicate differences between leptin treatment and PBS for expression of Socs3 or Timp1 mRNAs. C, Expression of Timp1 mRNA in hypothalami of mice carrying genetic alterations in leptin/LepRb signaling (WT, n = 5; ob/ob, n = 4; l/l, n = 6; db/db, n = 4; s/s, n = 6). Expression levels are normalized to Gapdh mRNA and data are expressed as mean ± se relative to WT. Statistical significance was evaluated with Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Figure 6

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Hyperphagia in Timp1−/− mice precedes increases in body weight and adiposity. Body weights (A), ovarian adipose tissue weight (O-WAT; B), inguinal adipose tissue weight (I-WAT; C), serum leptin concentrations (D), and food intake (E) of wild-type (WT) and Timp1−/− mice at 2 and 9 months of age. Data are presented as mean ± sd of 10 mice per genotype/age. F and G, Expression of mRNAs for Timp1 and the indicated neuropeptides in the hypothalamus of wild-type (n = 10) and Timp1−/− (n = 10) mice were determined by quantitative PCR. Expression levels are normalized to Gapdh mRNA and data are expressed as mean ± se. Statistical significance was evaluated with Student's t test. *, P < 0.05; ***, P < 0.001. Other P values are as indicated. KO, Knockout.

Assessment of energy expenditure and food intake

Measurement of oxygen consumption (VO2) with indirect calorimetry was performed on mice at 1 yr of age (n = 8). Animals were maintained on 12 h light, 12-h dark cycles beginning at 0700 and 1900 h, respectively. Mice were acclimated in measuring chambers at room temperature (22 C) for 2–3 d before recording. Measurements of VO2 were recorded every 24 min over 4 d using the Oxymax System (Columbus Instruments, Columbus, OH). Food intake was assessed in mice that were housed individually in cages with wire mesh flooring after acclimation for at least 3 d. Daily food consumption over 3 (Fig. 4) or 7 (Fig. 6) consecutive days was used to calculate average daily food intake (grams per day).

Dual-energy x-ray absorptiometry (DEXA)

Mice were anesthetized using inhaled isoflurane and scanned using the pDEXA PIXImus2 Mouse Densitometer (GE Medical Systems, Madison, WI). System software was used to estimate fat and lean mass, and bone mineral density for each mouse.

Adipocyte histology

Ovarian white adipose tissue from wild-type and Timp1−/− (n = 8) female mice at 14 months of age was dissected and fixed in 10% neutral buffered formalin for at least 48 h. After embedding in paraffin, adipose tissue sections were stained with hematoxylin and eosin. Images were taken on an BX-51 microscope (Olympus, Tokyo, Japan) with an Olympus DP70 color digital camera. Size of adipocytes was quantified in at least four randomly chosen but different microscopic fields (0.58 mm2/field) for each mouse. Adobe Photoshop (San Jose, CA) was used to enhance the contrast of the images before analysis. MetaMorph software (version 6.1; Molecular Devices, Downingtown, PA) was used to calculate cross-sectional area and the number of adipocytes per field with integrated morphometry analysis.

Glucose tolerance test

For the glucose tolerance test, mice were fasted for 16 h and then ip injected with glucose (1.5 g/kg body weight). Blood glucose was measured from tail blood with the OneTouch Ultra glucometer (Lifescan, Burnaby, British Columbia, Canada).

Assessment of serum leptin levels

Blood samples were obtained at 0900 h via tail bleeding from mice at 14 months of age (Fig. 3) or at 0800 h by cardiac puncture from 2- and 9-month old mice (Fig. 6). All mice had free access to food. Serum leptin content was quantified using a mouse leptin ELISA kit (Crystal Chem, Downers Grove, IL) according to the protocol provided by the manufacturer. All serum samples were assayed in duplicate.

Assessment of serum lipid levels

Blood was collected by cardiac puncture at 0800 h from control (n = 5) and null (n = 5) mice with free access to food at 2 months and 9 months of age in two independent cohorts of mice. Concentrations of nonesterified fatty acids in serum were determined using the HR series NEFA-HR (2) kit according to manufacturer's protocol (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Serum cholesterol was determined with the Cholesterol E Reagent kit according to the manufacturer's instructions (Wako Chemicals USA, Richmond, VA). All serum samples were assayed in duplicate.

Quantitative PCR

Mice were deeply anesthetized and hypothalami were isolated and snap frozen whole or were microdissected into various subregions using a mouse brain matrix before snap freezing. Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) reagent and relative RNA concentration was determined by spectrophotometric analysis. One microgram of total RNA was converted to cDNA using oligo(dT) priming and the Superscript first-strand synthesis kit (Invitrogen). Equivalent amounts of cDNA from each animal were subjected to automated fluorescent RT-PCR on an ABI 7700 in triplicate. Gapdh control, Hcrt, Mch, Socs3, and Timp1 primers and probes were as supplied by ABI (Hcrt Mm01964030-s1, Mch Mm01242886-g1, Socs3 Mm00545913-s1, Timp1 Mm00441818-m1). Pomc, Agrp, and Npy primers and probes were as previously described (18). Relative mRNA expression values are calculated by the 2-ΔΔCt method, with normalization of each sample to the average change in cycle threshold value of the controls.

Statistical analysis

Unless indicated otherwise, data are presented as mean ± sd. Data are considered significant with P < 0.05 by two-tailed Student's t test analysis.

Results

Female Timp1−/− mice show an age-dependent increase in body weight

To determine the effects of Timp1 deficiency on body weight gain, we evaluated wild-type and Timp1−/− mice at weaning and followed them up through 12 months of age followed by killing at 14 months of age. At weaning, female mice of both genotypes exhibited similar body weights (Fig. 1A). By 3 months of age, however, Timp1−/− females exhibit significantly greater body weights than their controls (23.8 vs. 20.8 g). This increased body weight of Timp1−/− females continued and became more pronounced through 12 months of age (32.3 vs. 26.0 g). The body weights of male Timp1 null mice or female Timp1 heterozygous mice were not different from controls through 12 months of age (data not shown).

Timp1−/− mice have increased weight of white and brown adipose tissues and increased size and number of adipocytes

To determine the origin of increased body weight in female Timp1−/− mice, analysis of body composition by DEXA was performed and revealed a 1.54-fold increase in total fat mass in the Timp1−/− mice, with no change in lean mass (Fig. 1B). Quantification of adipose depots revealed that elevated total body lipid in Timp1−/− mice was largely accounted for by increased white and brown adipose tissues (Fig. 1C). Weights of white adipose depots in Timp1−/− mice were increased by 1.5- to 2.7-fold. No change was observed for the other organs (liver, pancreas, spleen, heart, and lung). Paradoxically (38), DEXA analysis also showed that bone mineral density was increased by 11% in the Timp1−/− animals (Fig. 1D).

Because increased adipose tissue mass can be caused by differences in cell number as well as cell size, we analyzed sections of white adipose tissue to determine the area of single adipocytes. Compared with wild-type mice, average adipocyte cross sectional area is increased by 1.4-fold in Timp1−/− animals (Fig. 2A). Figure 2B shows the distribution of adipocytes according to their size and the frequency with which they are present in white adipose tissue. Timp1−/− mice contain 38% less small adipocytes (400–4,000 μm2) and 2.3-fold more large adipocytes (4,000–10,000 μm2). Histomorphometric analysis revealed that the increase in adipose tissue weight is only partially (∼50%) due to increased adipocyte volume, suggesting that the number of adipocytes is also increased in adipose tissues of Timp1−/− mice.

Metabolic regulation in Timp1−/− mice

Given the well-known relationship between adiposity and insulin resistance, we performed experiments to investigate glucose homeostasis in Timp1-deficient mice. To assess glucose tolerance, we subjected 1-yr-old female Timp1−/− and control mice to an ip glucose tolerance test (Fig. 3A). This analysis revealed similar glucose tolerance between genotypes, however. Furthermore, serum insulin levels after a 6-h fast were also not statistically different between genotypes (data not shown), suggesting that insulin sensitivity is not significantly altered in these mice despite their increased adiposity. Circulating leptin levels in Timp1−/− mice were elevated by 3.45-fold, consistent with their increased adiposity and suggesting the possibility of leptin resistance in these animals (Fig. 3B).

Energy balance in Timp1−/− female mice

To investigate the basis for the increased adiposity of Timp1−/− females, we measured their daily food intake and energy expenditure (Fig. 4). This analysis revealed that Timp1−/− females exhibited a 20% increase in daily food intake compared with their wild-type counterparts (Fig. 4A), suggesting that hyperphagia contributes to the obesity of these animals. To determine whether energy consumption is also altered in Timp1−/− animals, we continuously monitored VO2 and carbon dioxide production to determine energy use and the respiratory exchange ratio (RER). We observed no difference in VO2 for the Timp1−/− animals compared with the wild-type mice (Fig. 4B), although a slight increase in the RER for the knockout animals was observed for the first 6 h of light (Fig. 4C; P = 0.057). Consistent with this result, RER data were analyzed according to the percent relative cumulative frequency method of Harper and colleagues (39) and a trend toward increased RER at the 10th percentile (P = 0.054) is observed for Timp1 −/− mice. However, the 50th percentile point is not statistically different for RER (P = 0.16), and differences were not observed at any point for VO2 (P = 0.43; data not shown). Whereas these results are consistent with the idea that oxidative burning of lipids is slightly reduced in Timp1−/− mice, the major reason for obesity in these mice is increased food intake.

Leptin regulation of Timp1 mRNA expression in the ARC

The finding of hyperphagic obesity in the face of elevated circulating leptin levels in Timp1−/− mice suggests that leptin action in these animals is impaired. A relationship between leptin and Timp1 has been observed previously in that leptin stimulates Timp1 mRNA and protein expression and Timp1 promoter activity in hepatic stellate cells (40), and leptin may regulate the Timp1 promoter by a specificity protein-1/signal transducer and activator of transcription (Stat)-3-dependent mechanism (41). To examine the potential regulation of Timp1 gene expression by leptin in hypothalamic centers that regulate energy balance, we used semiquantitative RT-PCR to examine expression of Timp1 and Socs3 mRNA in the ARC of wild-type and ob/ob mice after treatment with leptin (5 mg/kg, ip) or vehicle for 4 h (Fig. 5, A and B). As previously demonstrated, leptin treatment acutely stimulates Socs3 mRNA accumulation in wild-type and exquisitely leptin-sensitive ob/ob animals. Similarly, leptin stimulated Timp1 mRNA expression by 2.4-fold and 14.9-fold in wild-type or ob/ob mice, respectively, establishing Timp1 as a likely transcriptional target of leptin in the ARC. We also found that injection of wild-type mice with leptin increases Timp1 mRNA in the LHA (supplemental Fig. 1), providing further support for leptin regulation of Timp1 mRNA in a variety of tissues and cell types (40).

To investigate whether physiologic leptin levels regulate Timp1 and gain insight into potential mechanisms by which leptin might control Timp1 mRNA expression in the hypothalamus, we prepared RNA from the hypothalami of mouse models of altered leptin action. We examined leptin-deficient ob/ob mice, leptin receptor (LepRb) null (thus leptin insensitive) db/db mice, s/s mice (in which LepRb 224 Stat3 signaling is specifically abrogated (18), and l/l animals [in which inhibitory LepRb signals are abrogated, increasing LepRb signaling (37)]. These analyses revealed a 75–83% decrease in Timp1 mRNA in the hypothalamus of ob/ob, db/db, or s/s mice (Fig. 5C), consistent with a role for leptin and LepRb→Stat3 signaling in the physiologic expression of Timp1 in the hypothalamus. Whereas Timp1 mRNA expression in l/l mice was not significantly different from that of controls (P = 0.07), Timp1 expression tended to be elevated in the l/l animals, consistent with the finding of increased leptin action in these mice (37). Taken together, these data demonstrate that hypothalamic expression of Timp1 mRNA is under dynamic control by leptin and suggests that regulation of Timp1 is similar to Socs3 and involves a Stat3-dependent transcriptional mechanism.

Cause of the obese phenotype

To determine whether hyperphagia causes the increase in body weight observed in Timp1 null mice, we performed an experiment in which we evaluated the phenotype of wild-type and Timp1−/− mice before changes in body weight. We also changed the breeding strategy to generate wild-type and Timp1−/− mice from closely related dams (see Materials and Methods). At 2 months of age, differences were not observed between genotypes for body weights (Fig. 6A), glucose tolerance (supplemental Fig. 2), serum nonesterified fatty acids and cholesterol concentrations (supplemental Fig. 2), weights of ovarian and inguinal adipose tissues (Fig. 6, B and C), or serum leptin concentrations (Fig. 6D). However, food intake was increased in Timp1−/− mice by 16% (Fig. 6E) at 2 months of age, indicating that Timp1−/− mice are functionally leptin resistant and that hyperphagia contributes to their weight gain. Whereas glucose tolerance and serum concentrations of nonesterified fatty acids and cholesterol were not altered by genotype at 9 months of age (supplemental Fig. 2), as expected (Figs. 1 and 4), body weight, adiposity, leptin concentrations, and food intake are all significantly increased at this time (Fig. 6). To understand the potential derangements in hypothalamic physiology that might underlie the increased feeding in Timp1−/− mice, we examined hypothalamic mRNA expression for a variety of hypothalamic neuropeptides and signaling mediators known to be involved in the control of energy balance in control and Timp1−/− mice at 2 and 9 months of age (Fig. 6, F and G). We examined mRNA expression of Timp1, the orexigenic ARC neuropeptides Npy and Agrp, the anorexigenic ARC neuropeptide Pomc, the feedback inhibitor Socs3, and the LHA peptides Mch and Hcrt. As expected, detection of Timp1 mRNA in the Timp1−/− animals at all ages was decreased to background levels. In the young animals, which are not fatter than controls and thus not subject to the compensatory changes that may accompany chronic obesity, the expression of Agrp mRNA tended to be lower in Timp1−/− animals relative to controls, and Hcrt expression was significantly reduced by 44.5% (Fig. 6F). In the 9-month-old animals, the expression of Agrp and Hcrt mRNA in Timp1−/− animals continued to trend below that of wild-type animals, and the expression of Npy mRNA was decreased by 44.3% (Fig. 6G). Whereas the decreased expression of orexigenic Agrp (and Npy in older animals) would not be expected to underlie increased feeding, chronically decreased Hcrt levels promote hyperphagic obesity in genetic models and could account for the inception of obesity in Timp1−/− mice. The finding of dysregulated neuropeptide mRNA expression in young and old Timp1−/− suggests the alteration of hypothalamic physiology is due to the lack of Timp1 expression.

Discussion

With an imbalance between energy uptake and expenditure that leads to body weight gain, considerable remodeling of adipose tissue is required to allow the increase in adipocyte size and number that accompanies expansion of adipose mass. It is therefore not surprising that modulation of the balance of MMPs and their inhibitors are reported to influence differentiation and growth of adipocytes and also development of obesity. Unfortunately, the specific regulators of remodeling processes in lean and obese animals are difficult to assign because of the large number of MMPs and TIMPs expressed and regulated in adipose tissue, and presumed to be involved in the enzymatic processing of intercellular matrix (1,2,3). Whereas Chun et al. (3) clearly demonstrated that membrane-type-1 MMP (also known as Mmp14) is required for the coordinate differentiation and maturation of adipocytes from stromal-vascular precursor cells, the pharmacological and genetic studies that point to roles for MMPs in obesity (6,7,8,9) are difficult to interpret without additional analyses of mechanism, including energy balance. It is also unclear as to whether altered circulating levels of MMPs or TIMPs in obese patients are the cause or an effect of obesity (4,5). Importantly, failure of Mmp14 knockout mice to gain weight is associated with altered subcellular distribution of the hypothalamic neuropeptides NPY and AgRP (42), implicating a critical role for extracellular matrix remodeling in regulation of energy balance.

In our study, we recorded body weights of a cohort of wild-type and Timp1−/− mice maintained on a standard chow diet. Starting at 3 months of age, it became apparent that female Timp1−/− mice develop a significantly higher total body weight when compared with control mice of same age, and increased body weight and adipose tissue mass was maintained for at least 1 yr of age (Fig. 1A). Increased adiposity was not caused by changes in ovarian cycling because concentrations of estrogen are not altered by the absence of Timp1 (9.7 vs. 12.4 pg/ml, data not shown). In our studies, no differences in body weight were observed in male Timp1−/− mice on a standard chow diet (data not shown), and this observation is supported by the work of Lijnen et al. (43). Interestingly, these investigators reported that male outcrossed (C57BL/6 × 129/SvJae) Timp1−/− mice are resistant to obesity caused by a high-fat diet. Whereas we did not test effects of a high-fat diet in our experiments, these data suggest that environmental interactions will be important to consider in future studies.

Our experiments demonstrate that leptin acts via the LepRb→Stat3 pathway to promote hypothalamic Timp1 mRNA expression and that Timp1 is required for correct expression of mRNAs for multiple hypothalamic neuropeptides that are crucial for regulation of feeding and energy homeostasis. These results are consistent with the previously reported direct transcriptional regulation of Timp1 mRNA expression by Stat3 in other tissues and systems (41).

Whereas the decrease in Agrp mRNA expression in the Timp1−/− animals was not significant at the level of P < 0.05 in 2- or 9-month-old animals, decreased expression of Agrp is difficult to detect in freely feeding animals, in which the expression of Agrp is already suppressed by leptin and other signals of nutritional repletion (44). Indeed, pooling data from the 2- and 9-month-old groups reveals a statistical decrease in Agrp (as well as Hcrt) mRNA expression, and Agrp mRNA expression is similarly decreased in 14-month-old Timp1−/− animals (data not shown). Agrp expression in fed mice, as in the animals studied herein, is low, and altered Agrp expression becomes relevant for the regulation of feeding and energy balance only in states in which Agrp expression is induced. Thus, decreased Agrp expression in the Timp1−/− animals is unlikely to alter energy homeostasis, although it bespeaks the modest dysregulation of hypothalamic physiology in the Timp1−/− animals. In contrast, the reduction in Hcrt expression may increase feeding and adiposity, as decreased Hcrt action in Hcrt or Hcrt receptor-null animals promotes obesity (45). The attenuation of dysregulated Hcrt expression in older Timp1−/− animals may reflect a compensatory response to increasing obesity and hyperleptinemia.

The mechanism(s) by which Timp1 contributes to the regulation of hypothalamic feeding circuits also remains unclear, although it is tempting to speculate that alterations in synaptic plasticity may play a role. Leptin and nutritional perturbations alter the numbers of inhibitory and excitatory synaptic inputs onto anorectic ARC neurons (31). This process of synaptic plasticity, which mediates long-term potentiation and long-term repression, requires the remodeling of extracellular matrix components as well as the formation and removal of synaptic contacts and the coordinated actions of extracellular proteases, such as the MMPs. Alternative possibilities are that up-regulation of MMP activity in Timp1−/− mice affects cleavage and activity of specific cell surface receptors or that effects of Timp1 are independent of MMP and involve receptor-mediated effects on signaling (46,47,48).

Overall, our findings demonstrate that leptin regulates Timp1 mRNA expression in the hypothalamus via the LepRb 224 Stat3 pathway and that Timp1 is required for the correct regulation of hypothalamic neuropeptides involved in the control of feeding and energy balance. Because Timp1 is required for the regulation of feeding and body energy homeostasis in female animals, Timp1-dependent regulation of hypothalamic physiology represents an important determinant of energy balance that directly influences adiposity.

Supplementary Material

[Supplemental Data]
en.2008-1409_index.html (794B, html)

Acknowledgments

We thank Dr. Guido Bommer for helpful comments with the manuscript.

Footnotes

This work was supported by National Institutes of Health Grants DK62876 and DK51563 (to O.A.M.), DK56731 and DK57768 (to M.G.M.), and in part HD39765 (to W.B.N.). Fellowship support to I.G. was from the Belgian Fonds National de la Recherche Scientifique. Additional support was provided by the Michigan Metabolomics and Obesity Center and the Center for Integrative Genomics. This work used the Morphology and Image Analysis Core and the Cell and Molecular Biology Core of the Michigan Diabetes Research and Training Center funded by Grant NIH5P60 DK20572 from the National Institute of Diabetes and Digestive and Kidney Diseases.

Disclosure Summary: The authors have nothing to disclose.

First Published Online November 26, 2008

Abbreviations: AgRP, Agouti-related peptide; ARC, arcuate nucleus of the hypothalamus; DEXA, dual-energy x-ray absorptiometry; HCRT, hypocretin; LepRb, leptin receptor; LHA, lateral hypothalamus area; MCH, melanin-concentrating hormone; MMP, matrix metalloproteinase; NPY, neuropeptide Y; POMC, proopiomelanocortin; RER, respiratory exchange ratio; Stat, signal transducer and activator of transcription; TIMP, tissue inhibitors of metalloproteinases; VO2, oxygen consumption.

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