Role of aquaporin-7 in the pathophysiological control of fat accumulation in mice

Aquaporins are channels that allow the movement of water across the cell membrane. Some members of the aquaporin family, the aquaglyceroporins, also allow the transport of glycerol, which is involved in the biosynthesis of triglycerides and the maintenance of fasting glucose levels. Aquaporin-7 (AQP7) is a glycerol channel mainly expressed in adipocytes. The deletion of AQP7 gene in mice leads to obesity and type 2 diabetes. AQP7 modulates adipocyte glycerol permeability thereby controlling triglyceride accumulation and fat cell size. Furthermore, the coordinated regulation of fat-specific AQP7 and liver-specific AQP9 may be key to determine glucose metabolism in insulin resistance.     

Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities.

Caveolae organelles and caveolin-1 protein expression are most abundant in adipocytes and endothelial cells. Our initial report on mice lacking caveolin-1 (Cav-1) demonstrated a loss of caveolae and perturbations in endothelial cell function. More recently, however, observation of the Cav-1-deficient cohorts into old age revealed significantly lower body weights, as compared with wild-type controls. These results suggest that Cav-1 null mice may have problems with lipid metabolism and/or adipocyte functioning. To test this hypothesis directly, we placed a cohort of wild-type and Cav-1 null mice on a high fat diet. Interestingly, despite being hyperphagic, Cav-1 null mice show overt resistance to diet-induced obesity. As predicted, adipocytes from Cav-1 null null mice lack caveolae membranes. Early on, a lack of caveolin-1 selectively affects only the female mammary gland fat pad and results in a near complete ablation of the hypo-dermal fat layer. There are also indications of generalized adipose tissue pathology. With increasing age, a systemic decompensation in lipid accumulation occurs resulting in dramatically smaller fat pads, histologically reduced adipocyte cell diameter, and a poorly differentiated/hypercellular white adipose parenchyma. To gain mechanistic insights into this phenotype, we show that, although serum insulin, glucose, and cholesterol levels are entirely normal, Cav-1 null mice have severely elevated triglyceride and free fatty acid levels, especially in the post-prandial state. However, this build-up of triglyceride-rich chylomicrons/very low density lipoproteins is not due to perturbed lipoprotein lipase activity, a major culprit of isolated hypertriglyceridemia. The lean body phenotype and metabolic defects observed in Cav-1 null mice are consistent with the previously proposed functions of caveolin-1 and caveolae in adipocytes. Our results show for the first time a clear role for caveolins in systemic lipid homeostasis in vivo and place caveolin-1/caveolae as major factors in hyperlipidemias and obesity.     

Cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is involved in adipocyte differentiation and hypertrophy, linking to obesity, and insulin resistance

Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor, is induced during adipocyte differentiation; however, its precise contribution to this process is unknown. Using both in vitro and in vivo systems, we show that p21 is crucial for maintaining adipocyte hypertrophy and obesity-induced insulin resistance. The absence of p21 in 3T3-L1 fibroblasts by RNA-mediated interference knockdown or in embryonic fibroblasts from p21(-/-) mice impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet, p21(-/-) mice fed high energy diets had reduced adipose tissue mass and adipocyte size accompanied by a marked improvement in insulin sensitivity. Knockdown of p21 in enlarged epididymal fat of diet-induced obese mice and also in fully differentiated 3T3-L1 adipocytes caused vigorous apoptosis by activating p53. Thus, p21 is involved in both adipocyte differentiation and in protecting hypertrophied adipocytes against apoptosis. Via both of these mechanisms, p21 promotes adipose tissue expansion during high fat diet feeding, leading to increased downstream pathophysiological consequences such as insulin resistance.     

Direct and indirect effects of leptin on adipocyte metabolism

Leptin is hypothesized to function as a negative feedback signal in the regulation of energy balance. It is produced primarily by adipose tissue and circulating concentrations correlate with the size of body fat stores. Administration of exogenous leptin to normal weight, leptin responsive animals inhibits food intake and reduces the size of body fat stores whereas mice that are deficient in either leptin or functional leptin receptors are hyperphagic and obese, consistent with a role for leptin in the control of body weight. This review discusses the effect of leptin on adipocyte metabolism. Because adipocytes express leptin receptors there is the potential for leptin to influence adipocyte metabolism directly. Adipocytes also are insulin responsive and receive sympathetic innervation, therefore leptin can also modify adipocyte metabolism indirectly. Studies published to date suggest that direct activation of adipocyte leptin receptors has little effect on cell metabolism in vivo, but that leptin modifies adipocyte sensitivity to insulin to inhibit lipid accumulation. In vivo administration of leptin leads to a suppression of lipogenesis, an increase in triglyceride hydrolysis and an increase in fatty acid and glucose oxidation. Activation of central leptin receptors also contributes to the development of a catabolic state in adipocytes, but this may vary between different fat depots. Leptin reduces the size of white fat depots by inhibiting cell proliferation both through induction of inhibitory circulating factors and by contributing to sympathetic tone which suppresses adipocyte proliferation. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic (Lep(ob)/Lep(ob) and A(y)/+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.     

Skp2 controls adipocyte proliferation during the development of obesity

The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for approximately 25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (A(y)) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the A(y) mutant mice, although the reduced number of beta cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity.     

Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels

The availability of mice containing an adipocyte lipid-binding protein (ALBP/aP2) gene disruption allowed for a direct examination of the presumed role of lipid-binding proteins in the mobilization and trafficking of intracellular fatty acids. Total body and epididymal fat pad weights, as well as adipose cell morphology, were unaltered in male ALBP/aP2 disrupted mice when compared to their wild-type littermates. Analysis of adipocytes isolated from wild-type and ALBP/aP2 null mice revealed that a selective 40- and 13-fold increase in the level of the keratinocyte lipid-binding protein (KLBP) mRNA and protein, respectively, accompanied the ALBP/aP2 gene disruption. Although KLBP protein was significantly up-regulated, the total lipid-binding protein level decreased 8 -fold as a consequence of the disruption. There was no appreciable difference in the rate of fatty acid influx or esterification in adipocytes of wild-type and ALBP/aP2 null animals. To the contrary, basal lipolysis decreased approximately 40% in ALBP/aP2 nulls as compared to wild-type littermates. The glycerol release from isproterenol-stimulated ALBP/aP2 null fat cells was similarly reduced by approximately 35%. Consistent with a decrease in basal efflux, the non-esterified fatty acid (NEFA) level was nearly 3-fold greater in adipocytes from ALBP/aP2 nulls as compared to wild-type animals. The significant decrease in both basal and isoproterenol-stimulated lipolysis in adipose tissue of ALBP/aP2 null mice supports the model whereby intracellular lipid-binding proteins function as lipid chaperones, facilitating the movement of fatty acids out of the fat cell.     

PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance.

Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.     

Identification of white adipocyte progenitor cells in vivo

The increased white adipose tissue (WAT) mass associated with obesity is the result of both hyperplasia and hypertrophy of adipocytes. However, the mechanisms controlling adipocyte number are unknown in part because the identity of the physiological adipocyte progenitor cells has not been defined in vivo. In this report, we employ a variety of approaches, including a noninvasive assay for following fat mass reconstitution in vivo, to identify a subpopulation of early adipocyte progenitor cells (Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+)) resident in adult WAT. When injected into the residual fat pads of A-Zip lipodystrophic mice, these cells reconstitute a normal WAT depot and rescue the diabetic phenotype that develops in these animals. This report provides the identification of an undifferentiated adipocyte precursor subpopulation resident within the adipose tissue stroma that is capable of proliferating and differentiating into an adipose depot in vivo.     

Activin receptor ALK4 promotes adipose tissue hyperplasia by suppressing differentiation of adipocyte precursors

Adipocyte hyperplasia and hypertrophy are the two main processes contributing to adipose tissue expansion, yet the mechanisms that regulate and balance their involvement in obesity are incompletely understood. Activin B/GDF-3 receptor ALK7 is expressed in mature adipocytes and promotes adipocyte hypertrophy upon nutrient overload by suppressing adrenergic signaling and lipolysis. In contrast, the role of ALK4, the canonical pan-activin receptor, in adipose tissue is unknown. Here, we report that, unlike ALK7, ALK4 is preferentially expressed in adipocyte precursors, where it suppresses differentiation, allowing proliferation and adipose tissue expansion. ALK4 expression in adipose tissue increases upon nutrient overload and positively correlates with fat depot mass and body weight, suggesting a role in adipose tissue hyperplasia during obesity. Mechanistically, ALK4 signaling suppresses expression of CEBPα and PPARγ, two master regulators of adipocyte differentiation. Conversely, ALK4 deletion enhances CEBPα/PPARγ expression and induces premature adipocyte differentiation, which can be rescued by CEBPα knockdown. These results clarify the function of ALK4 in adipose tissue and highlight the contrasting roles of the two activin receptors in the regulation of adipocyte hyperplasia and hypertrophy during obesity.     

Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy

Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy.     

A pericellular collagenase directs the 3-dimensional development of white adipose tissue

White adipose tissue (WAT) serves as the primary energy depot in the body by storing fat. During development, fat cell precursors (i.e., preadipocytes) undergo a hypertrophic response as they mature into lipid-laden adipocytes. However, the mechanisms that regulate adipocyte size and mass remain undefined. Herein, we demonstrate that the membrane-anchored metalloproteinase, MT1-MMP, coordinates adipocyte differentiation in vivo. In the absence of the protease, WAT development is aborted, leaving tissues populated by mini-adipocytes which render null mice lipodystrophic. While MT1-MMP preadipocytes display a cell autonomous defect in vivo, null progenitors retain the ability to differentiate into functional adipocytes during 2-dimensional (2-D) culture. By contrast, within the context of the 3-dimensional (3-D) ECM, normal adipocyte maturation requires a burst in MT1-MMP-mediated proteolysis that modulates pericellular collagen rigidity in a fashion that controls adipogenesis. Hence, MT1-MMP acts as a 3-D-specific adipogenic factor that directs the dynamic adipocyte-ECM interactions critical to WAT development.     

“Spexin improves adipose tissue inflammation and macrophage recruitment in obese mice”

Spexin (SPX) is a novel adipokine related to many metabolic effects, such as gastrointestinal movements, insulin and glucose homeostasis, lipid metabolism and energy balance. This study evaluates the role of SPX in the improvement of the metabolic and inflammatory profile in fructose-rich-diet obese mice. Adult Swiss mice were supplemented or not with fructose (20% in tap water, FRD and CTR, respectively) for 10 weeks. The last ten days, mice were treated or not with SPX (ip. 29 μg/Kg/day, FRD-SPX and CTR-SPX, respectively). A positive correlation was observed between body weight prior to treatment and weight loss after SPX challenge. Moreover, plasma and liver triglycerides and adipose tissue (AT) features (mass, adipocyte hypertrophy, mRNA of leptin) were improved. SPX also induced a reduction in epididymal AT (EAT) expression of TNFα, IL1β and IL6 and an improvement in IL10 and CD206. M1 macrophages in EAT, principally the Ly6C⁻ populations (M1a and M1b), were decreased. Adipocytes from FRD-SPX mice induced less macrophage activation (IL6, mRNA and secretion) than FRD after overnight co-culture with the monocyte cell line (RAW264.7) in stimulated conditions (M1 activation, LPS 100 ng/mL). Finally, in vitro, monocytes pre-incubated with SPX and stimulated with LPS showed decreased inflammatory mRNA markers compared to monocytes with LPS alone. In conclusion, SPX decreased body weight and improved the metabolic profile and adipocyte hypertrophy. Inflammatory Ly6C⁻ macrophages decreased, together with inflammatory marker expression. In vitro studies demonstrate that SPX induced a decrease in M1 macrophage polarization directly or through mature adipocytes.     

Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels

Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3-3.5 wk of age, 10-12 g) gained 49 +/- 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 +/- 4%, AQP1 null mice gained only 4 +/- 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceridemia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [(14)C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radioactivity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice.     

Compensation for an increase in body fat caused by donor transplants into mice

Rodents tend to compensate for experimental obesity in which both adipocyte size and number are increased. In contrast, it was recently reported that Siberian hamsters do not compensate for dorsal subcutaneous transplants of fat, which increase body fat without changing the size of adipocytes. In the first experiment described here we tested whether mice changed the size of their endogenous fat stores 2 or 5 wk after donor fat was added as subcutaneous transplants. Each epididymal fat pad from donor mice was cut in half and placed ventrally in recipient mice, increasing body fat by approximately 10%. After 2 wk, there was no effect of the transplants on the size of endogenous fat depots or the size of adipocytes in epididymal fat depots. There was a substantial decrease in mass and adipocyte size in transplanted fat. Five weeks after surgery the endogenous epididymal and retroperitoneal fat depots of recipient mice were significantly decreased, serum leptin was reduced, and the size of adipocytes in endogenous epididymal fat was significantly reduced, although cell number had not changed. The size of transplanted cells was the same as at 2 wk. In a second experiment, epididymal fat was placed as either dorsal or ventral subcutaneous fat transplants. Five weeks after surgery the endogenous fat depots were decreased in all recipient mice but none of the differences reached statistical significance. These results suggest that mice have mechanisms to maintain total body fat mass that respond to an increase in the number of fat cells present.