Induced adiposity and adipocyte hypertrophy in mice lacking the AMP-activated protein kinase-alpha2 subunit

AMP-activated protein kinase (AMPK) is considered as a cellular energy sensor that regulates glucose and lipid metabolism by phosphorylating key regulatory enzymes. Despite the major role of adipose tissue in regulating energy partitioning in the organism, the role of AMPK in this tissue has not been addressed. In the present study, we subjected AMPKalpha2 knockout (KO) mice to a high-fat diet to examine the effect of AMPK on adipose tissue formation. Compared with the wild type, AMPKalpha2 KO mice exhibited increased body weight and fat mass. The increase in adipose tissue mass was due to the enlargement of the preexisting adipocytes with increased lipid accumulation. However, we did not observe any changes in adipocyte marker expression, such as peroxisome proliferator-activated receptor-gamma, CCAAT/enhancer-binding protein alpha (C/EBPalpha) and adipocyte fatty acid-binding protein (aFABP/aP2), or total cell number. Unlike impaired glucose homeostasis observed on normal diet feeding, when fed a high-fat diet AMPKalpha2 KO mice did not show differences in glucose tolerance and insulin sensitivity compared with wild-type mice. Our results suggest that the increase in lipid storage in adipose tissue in AMPKalpha2 KO mice may have protected these mice from further impairment of glucose homeostasis that normally accompanies high-fat feeding. Our study also demonstrates that lack of AMPKalpha2 subunit may be a factor contributing to the development of obesity.     

Adipocyte metabolism in adipocyte fatty acid binding protein knockout mice (aP2-/-) after short-term high-fat feeding: functional compensation by the keratinocyte [correction of keritinocyte] fatty acid binding protein

Mice null for adipocyte fatty acid binding protein (AFABP) compensate by increasing expression of keratinocyte fatty acid binding protein (KFABP) (Hotamisligil et al. Science 274:1377-1379, 1996). In the present study, AFABP knockout (KO) and wild-type (WT) mice became equally obese on a high-fat diet, as judged by fat pad weights, adipocyte size, and body composition analysis. High-fat feeding led to moderate insulin resistance in both WT and AFABP knockout mice, as indicated by an approximately 2-fold increase in plasma insulin. However, in the high fat-fed mice, plasma glucose levels were approximately 15% lower in the AFABP-KO mice. Adipocytes isolated from AFABP-KO and WT mice fed high- or low-fat diets exhibited similar rates of basal and norepinephrine-stimulated lipolysis and insulin-stimulated rates of glucose conversion to fatty acids and glyceride-glycerol. However, basal glucose conversion to fatty acids was higher in adipocytes of AFABP-KO mice. Adipocyte tumor necrosis factor-alpha release was similarly increased by high-fat diet-induced obesity in both WT and AFABP-KO mice. As assessed by Western blot analysis, the level of KFABP protein in AFABP-KOs was approximately 40% of the level of AFABP in WT controls. The binding affinities of KFABP for long-chain fatty acids were 2- to 4-fold higher than those of AFABP, but the relative affinities for different fatty acids were similar. As for AFABP, the rate of fatty acid transfer from KFABP to model phospholipid vesicles was increased with acceptor membrane concentration and by inclusion of acidic phospholipids, indicating a similar mechanism of transfer. We conclude KFABP can functionally compensate for the absence of AFABP, resulting in no major alterations in adipocyte metabolism or fat accumulation in response to short-term feeding of high-fat diets that result in moderate hyperinsulinemia.     

Sugar,Uric Acid,and the Etiology of Diabetes and Obesity

The intake of added sugars, such as from table sugar (sucrose) and high-fructose corn syrup has increased dramatically in the last hundred years and correlates closely with the rise in obesity, metabolic syndrome, and diabetes. Fructose is a major component of added sugars and is distinct from other sugars in its ability to cause intracellular ATP depletion, nucleotide turnover, and the generation of uric acid. In this article, we revisit the hypothesis that it is this unique aspect of fructose metabolism that accounts for why fructose intake increases the risk for metabolic syndrome. Recent studies show that fructose-induced uric acid generation causes mitochondrial oxidative stress that stimulates fat accumulation independent of excessive caloric intake. These studies challenge the long-standing dogma that “a calorie is just a calorie” and suggest that the metabolic effects of food may matter as much as its energy content. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provides new insights into pathogenesis and therapies for this important disease.     

Involvement of apolipoprotein E in excess fat accumulation and insulin resistance

Although apolipoprotein E (apoE) is well known to play a major role in lipid metabolism, its role in glucose and energy homeostasis remains unclear. Herein, we established apoE-deficient genetically obese Ay (apoE(-/-);Ay/+) mice. ApoE deficiency in Ay mice prevented the development of obesity, with decreased fat accumulation in the liver and adipose tissues. ApoE(-/-);Ay/+ mice exhibited better glucose tolerance than apoE(+/+);Ay/+ mice. Insulin tolerance testing and hyperinsulinemic-euglycemic clamp study revealed marked improvement of insulin sensitivity, despite increased plasma free fatty acid levels. These metabolic phenotypes were reversed by adenoviral replenishment of apoE protein, indicating circulating apoE to be involved in increased adiposity and obesity-related metabolic disorders. Uptake of apoE-lacking VLDL into the liver and adipocytes was markedly inhibited, but adipocytes in apoE(-/-);Ay/+ mice exhibited normal differentiation, suggesting that apoE-dependent VLDL transport is involved in the development of obesity, i.e., surplus fat accumulation. Interestingly, apoE(-/-);Ay/+ mice exhibited decreased food intake and increased energy expenditure. Pair-feeding experiments indicate these phenomena to both contribute to the obesity-resistant phenotypes associated with apoE deficiency. Thus, apoE is involved in maintaining energy homeostasis. ApoE-dependent excess fat accumulation is a promising therapeutic target for the metabolic syndrome.     

Effects of pioglitazone on adipose tissue remodeling within the setting of obesity and insulin resistance

Obesity and dysfunctional energy partitioning can lead to the development of insulin resistance and type 2 diabetes. The antidiabetic thiazolidinediones shift the energy balance toward storage, leading to an increase in whole-body adiposity. These studies examine the effects of pioglitazone (Pio) on adipose tissue physiology, accumulation, and distribution in female Zucker (fa/fa) rats. Pio treatment (up to 28 days) decreased the insulin-resistant and hyperlipidemic states and increased food consumption and whole-body adiposity. Magnetic resonance imaging (MRI) analysis and weights of fat pads demonstrated that the increase in adiposity was not only limited to the major fat depots but also to fat deposition throughout the body. Adipocyte sizing profiles, fat pad histology, and DNA content show that Pio treatment increased the number of small adipocytes because of both the appearance of new adipocytes and the shrinkage and/or disappearance of existing mature adipocytes. The remodeling was time dependent, with new small adipocytes appearing in clusters throughout the fat pad, and accompanied by a three- to fourfold increase in citrate synthase and fatty acid synthase activity. The appearance of new fat cells and the increase in fat mass were depot specific, with a rank order of responsiveness of ovarian > retroperitoneal > subcutaneous. This differential depot effect resulted in a redistribution of the fat mass in the abdominal region such that there was an increase in the visceral:subcutaneous ratio, as confirmed by MRI analysis. Although the increased adiposity is paradoxical to an improvement in insulin sensitivity, the quantitative increase of adipose mass should be viewed in context of the qualitative changes in adipose tissue, including the remodeling of adipocytes to a smaller size with higher lipid storage potential. This shift in energy balance is likely to result in lower circulating free fatty acid levels, ultimately improving insulin sensitivity and the metabolic state.     

HDAC9 Knockout Mice Are Protected From Adipose Tissue Dysfunction and Systemic Metabolic Disease During High-Fat Feeding

During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related disease. Although adipose tissue contains numerous preadipocytes, differentiation into functionally competent adipocytes is insufficient to accommodate the chronic caloric excess and prevent adipocyte overloading. We report for the first time that a chronic high-fat diet (HFD) impairs adipogenic differentiation, leading to accumulation of inefficiently differentiated adipocytes with blunted expression of adipogenic differentiation-specific genes. Preadipocytes from these mice likewise exhibit impaired adipogenic differentiation, and this phenotype persists during in vitro cell culture. HFD-induced impaired adipogenic differentiation is associated with elevated expression of histone deacetylase 9 (HDAC9), an endogenous negative regulator of adipogenic differentiation. Genetic ablation of HDAC9 improves adipogenic differentiation and systemic metabolic state during an HFD, resulting in diminished weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatosteatosis. Moreover, compared with wild-type mice, HDAC9 knockout mice exhibit upregulated expression of beige adipocyte marker genes, particularly during an HFD, in association with increased energy expenditure and adaptive thermogenesis. These results suggest that targeting HDAC9 may be an effective strategy for combating obesity-related metabolic disease.     

Spillover of Fatty Acids During Dietary Fat Storage in Type 2 Diabetes: Relationship to Body Fat Depots and Effects of Weight Loss

Spillover of lipoprotein lipase-generated fatty acids from chylomicrons into the plasma free fatty acid (FFA) pool is an important source of FFA and reflects inefficiency in dietary fat storage. We measured spillover in 13 people with type 2 diabetes using infusions of a [³H]triolein-labeled lipid emulsion and [U-¹³C]oleate during continuous feeding, before and after weight loss. Body fat was measured with dual energy X-ray absorptiometry and computed tomography. Participants lost ∼14% of body weight. There was an ∼38% decrease in meal-suppressed FFA concentration (P < 0.0001) and an ∼23% decrease in oleate flux (P = 0.007). Fractional spillover did not change (P = NS). At baseline, there was a strong negative correlation between spillover and leg fat (r = −0.79, P = 0.001) and a positive correlation with the trunk-to-leg fat ratio (R = 0.56, P = 0.047). These correlations disappeared after weight loss. Baseline leg fat (R = −0.61, P = 0.027) but not trunk fat (R = −0.27, P = 0.38) negatively predicted decreases in spillover with weight loss. These results indicate that spillover, a measure of inefficiency in dietary fat storage, is inversely associated with lower body fat in type 2 diabetes.Free fatty acids (FFAs) mediate insulin resistance (1,2), drive VLDL triglyceride synthesis in the liver (3), and play an important role in the pathogenesis of hypertension (4,5) and diabetes (6). Spillover of lipoprotein lipase (LPL)-generated fatty acids from chylomicrons into the plasma FFA pool is an important source of FFA (7–10) and reflects inefficiency in dietary fat storage. Previous work has shown that the amount of fat taken up in leg fat per gram of tissue increases as a function of leg fat mass, whereas it actually decreases as a function of visceral fat mass and does not change in upper body subcutaneous fat (11). However, it is not clear whether these findings reflect changes in rates of LPL-mediated meal fat hydrolysis, changes in fractional spillover, or both. We therefore undertook a study in people with type 2 diabetes to determine the effects of weight loss on spillover and to investigate potential associations between spillover and body fat depots.     

Endogenous ApoE expression modulates adipocyte triglyceride content and turnover

Apolipoprotein E (apoE) is highly expressed in adipose tissue and adipocytes in which its expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma agonists and tumor necrosis factor-alpha. There is, however, no information regarding a role for endogenous apoE in differentiated adipocyte function. In this report, we define a novel role for apoE in modulating adipocyte lipid metabolism. ApoE(-/-) mice have less body fat and smaller adipocytes compared with wild-type controls. Freshly isolated adipose tissue from apoE(-/-) mice contains lower levels of triglyceride and free fatty acid, and these differences are maintained in cultured adipocytes derived from preadipocytes. Adenoviral expression of apoE in apoE(-/-)-cultured adipocytes increases triglyceride and fatty acid content. During incubation with apoE-containing triglyceride-rich lipoproteins, apoE(-/-) adipose tissue accumulates less triglyceride than wild type. The absence of apoE expression in primary cultured adipocytes also leads to changes in the expression of genes involved in the metabolism/turnover of fatty acids and the triglyceride droplet. Markers of adipocyte differentiation were lower in freshly isolated and cultured apoE(-/-) adipocytes. Importantly, PPAR-gamma-mediated changes in lipid content and gene expression are markedly altered in cultured apoE(-/-) adipocytes. These results establish a novel role for endogenous apoE in adipocyte lipid metabolism and have implications for constructing an integrated model of adipocyte physiology in health and disease.     

Tissue-Specific Inactivation of Type 2 Deiodinase Reveals Multilevel Control of Fatty Acid Oxidation by Thyroid Hormone in the Mouse

Type 2 deiodinase (D2) converts the prohormone thyroxine (T4) to the metabolically active molecule 3,5,3′-triiodothyronine (T3), but its global inactivation unexpectedly lowers the respiratory exchange rate (respiratory quotient [RQ]) and decreases food intake. Here we used FloxD2 mice to generate systemically euthyroid fat-specific (FAT), astrocyte-specific (ASTRO), or skeletal-muscle-specific (SKM) D2 knockout (D2KO) mice that were monitored continuously. The ASTRO-D2KO mice also exhibited lower diurnal RQ and greater contribution of fatty acid oxidation to energy expenditure, but no differences in food intake were observed. In contrast, the FAT-D2KO mouse exhibited sustained (24 h) increase in RQ values, increased food intake, tolerance to glucose, and sensitivity to insulin, all supporting greater contribution of carbohydrate oxidation to energy expenditure. Furthermore, FAT-D2KO animals that were kept on a high-fat diet for 8 weeks gained more body weight and fat, indicating impaired brown adipose tissue (BAT) thermogenesis and/or inability to oxidize the fat excess. Acclimatization of FAT-D2KO mice at thermoneutrality dissipated both features of this phenotype. Muscle D2 does not seem to play a significant metabolic role given that SKM-D2KO animals exhibited no phenotype. The present findings are unique in that they were obtained in systemically euthyroid animals, revealing that brain D2 plays a dominant albeit indirect role in fatty acid oxidation via its sympathetic control of BAT activity. D2-generated T3 in BAT accelerates fatty acid oxidation and protects against diet-induced obesity.     

Free fatty acid storage in human visceral and subcutaneous adipose tissue: role of adipocyte proteins

OBJECTIVE

Because direct adipose tissue free fatty acid (FFA) storage may contribute to body fat distribution, we measured FFA (palmitate) storage rates and fatty acid (FA) storage enzymes/proteins in omental and abdominal subcutaneous fat.

RESEARCH DESIGN AND METHODS

Elective surgery patients received a bolus of [1-¹⁴C]palmitate followed by omental and abdominal subcutaneous fat biopsies to measure direct FFA storage. Long chain acyl-CoA synthetase (ACS) and diacylglycerol acyltransferase activities, CD36, fatty acid-binding protein, and fatty acid transport protein 1 were measured.

RESULTS

Palmitate tracer storage (dpm/g adipose lipid) and calculated palmitate storage rates were greater in omental than abdominal subcutaneous fat in women (1.2 ± 0.8 vs. 0.7 ± 0.4 μmol ⋅ kg adipose lipid⁻¹ ⋅ min⁻¹, P = 0.005) and men (0.7 ± 0.2 vs. 0.2 ± 0.1, P < 0.001), and both were greater in women than men (P < 0.0001). Abdominal subcutaneous adipose tissue palmitate storage rates correlated with ACS activity (women: r = 0.66, P = 0.001; men: r = 0.70, P = 0.007); in men, CD36 was also independently related to palmitate storage rates. The content/activity of FA storage enzymes/proteins in omental fat was dramatically lower in those with more visceral fat. In women, only omental palmitate storage rates were correlated (r = 0.54, P = 0.03) with ACS activity.

CONCLUSIONS

Some adipocyte FA storage factors correlate with direct FFA storage, but sex differences in this process in visceral fat do not account for sex differences in visceral fatness. The reduced storage proteins in those with greater visceral fat suggest that the storage factors we measured are not a predominant cause of visceral adipose tissue accumulation.Excess visceral fat is associated with greater metabolic risk (1,2), whereas preferential lower body fat accumulation is not (3). The mechanisms by which some individuals gain fat in one depot at the expense of another are unknown, but surely relate to an imbalance between storage and release of fatty acids (FAs). The patterns of regional free FA (FFA) release suggest that lipolysis defects cannot explain adipose tissue distribution patterns (4–6). Likewise, meal fat storage patterns do not completely explain regional variations in adipose tissue accumulation (7–11).The direct FFA storage pathway, which is lipoprotein lipase (LPL) independent, exists in both animal (12) and human (13–15) adipose tissue in vivo. Furthermore, qualitative patterns of postabsorptive, direct adipose tissue FFA storage mirror that of body fat distribution (14). Direct FFA storage in men, but not women, is greater in the upper body than lower body subcutaneous fat, and women store FFA more efficiently in subcutaneous fat than men. Hannukainen et al. (16), using positron emission tomography scan technology, found that FFA storage rates are greater in visceral than subcutaneous fat in nonobese men, similar to what we observed in nonobese women (15).Because the direct FFA storage pathway seems to help determine body fat distribution, gaining insight into the adipocyte factors regulating FA incorporation into triacylglycerols may help explain depot differences in fat storage. Potential adipocyte specific rate-limiting steps include 1) facilitated FA transport across the plasma membrane, mediated by proteins such as CD36, plasma membrane-associated fatty acid-binding protein (FABP[pm]), and fatty acid transport protein 1 (FATP1); 2) acylation, which leads to activation/trapping of intracellular FAs, mediated by the activity of a number of fatty acyl-CoA synthetases (ACSs); and 3) the final step of triacylglycerol formation, mediated by diacylglycerol acyltransferase (DGAT).In this study, we measured direct FFA storage into visceral and abdominal subcutaneous adipose tissue using isotope tracer/adipose biopsy techniques. Our hypothesis was that men, typically having more visceral fat than women, would have greater direct FFA storage in omental than abdominal subcutaneous adipose tissue and greater direct omental FFA storage than women. We also measured factors related to the FA storage steps described above and studied their relation to direct FFA storage into adipocyte triglyceride. We hypothesized that depot differences in these factors would correlate with regional, sex-specific FFA storage differences. The results provide evidence for major between-depot and between-individual differences in the FA storage factors that relate to direct FFA storage.     

Cellularity and Adipogenic Profile of the Abdominal Subcutaneous Adipose Tissue From Obese Adolescents: Association With Insulin Resistance and Hepatic Steatosis

OBJECTIVE

We explored whether the distribution of adipose cell size, the estimated total number of adipose cells, and the expression of adipogenic genes in subcutaneous adipose tissue are linked to the phenotype of high visceral and low subcutaneous fat depots in obese adolescents.

RESEARCH DESIGN AND METHODS

A total of 38 adolescents with similar degrees of obesity agreed to have a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test and hyperinsulinemic euglycemic clamp) and imaging studies (MRI, DEXA, ¹H-NMR). Subcutaneous periumbilical adipose cell-size distribution and the estimated total number of subcutaneous adipose cells were obtained from tissue biopsy samples fixed in osmium tetroxide and analyzed by Beckman Coulter Multisizer. The adipogenic capacity was measured by Affymetrix GeneChip and quantitative RT-PCR.

RESULTS

Subjects were divided into two groups: high versus low ratio of visceral to visceral + subcutaneous fat (VAT/[VAT+SAT]). The cell-size distribution curves were significantly different between the high and low VAT/(VAT+SAT) groups, even after adjusting for age, sex, and ethnicity (MANOVA P = 0.035). Surprisingly, the fraction of large adipocytes was significantly lower (P < 0.01) in the group with high VAT/(VAT+SAT), along with the estimated total number of large adipose cells (P < 0.05), while the mean diameter was increased (P < 0.01). From the microarray analyses emerged a lower expression of lipogenesis/adipogenesis markers (sterol regulatory element binding protein-1, acetyl-CoA carboxylase, fatty acid synthase) in the group with high VAT/(VAT+SAT), which was confirmed by RT-PCR.

CONCLUSIONS

A reduced lipo-/adipogenic capacity, fraction, and estimated number of large subcutaneous adipocytes may contribute to the abnormal distribution of abdominal fat and hepatic steatosis, as well as to insulin resistance in obese adolescents.White adipose tissue (WAT) plays a critical role in obesity-related metabolic dysfunctions. Danforth (1) and Shulman (2) raised the hypothesis that inadequate subcutaneous fat stores result in lipid overflow into visceral fat and other nonadipose tissues, which was elegantly explored by Ravussin and Smith (3). Sethi and Vidal-Puig proposed that impaired subcutaneous WAT expandability might cause obesity-associated insulin resistance (4). In adults, increased fat cell size, a marker of impaired adipogenesis, was reported to be related to insulin resistance and predicts the development of type 2 diabetes (5). Recent studies by McLaughlin et al. (6) reported in adults that an increase in the proportion of small adipocytes, but not increased fat cell size, and an impaired expression of markers for adipogenesis are related to insulin resistance. Little is known about adipocyte size and adipogenic capacity during adolescence, a time when the expansion of WAT results from combined adipocyte hypertrophy and hyperplasia. In contrast, adult adipocytes exhibit a remarkably constant turnover (7). Recently, we described a group of obese adolescents presenting with a reduced subcutaneous abdominal fat depot, increased visceral fat, hepatic steatosis, and marked insulin resistance (8). Building on these findings, we asked the following question: is the adipogenic capacity of the abdominal subcutaneous fat depot in obese adolescents associated with a decreased proportion of large adipose cells and reduced expression of genes regulating adipocyte differentiation? We hypothesized that, in some obese adolescents, the lack of expandability of the subcutaneous abdominal fat might be linked to adipocyte size, its adipogenic expression, and the fat accumulation in liver and muscle. To test this hypothesis, we used metabolic and imaging techniques, together with direct measurements of adipocyte size and gene expression, in two groups of obese adolescents with marked differences in the proportion of visceral to subcutaneous abdominal fat.     

Caveolin-1 Expression and Cavin Stability Regulate Caveolae Dynamics in Adipocyte Lipid Store Fluctuation

Adipocytes specialized in the storage of energy as fat are among the most caveolae-enriched cell types. Loss of caveolae produces lipodystrophic diabetes in humans, which cannot be reversed by endothelial rescue of caveolin expression in mice, indicating major importance of adipocyte caveolae. However, how caveolae participate in fat cell functions is poorly understood. We investigated dynamic conditions of lipid store fluctuations and demonstrate reciprocal regulation of caveolae density and fat cell lipid droplet storage. We identified caveolin-1 expression as a crucial step in adipose cell lines and in mice to raise the density of caveolae, to increase adipocyte ability to accommodate larger lipid droplets, and to promote cell expansion by increased glucose utilization. In human subjects enrolled in a trial of 8 weeks of overfeeding to promote fattening, adipocyte expansion response correlated with initial caveolin-1 expression. Conversely, lipid mobilization in cultured adipocytes to induce lipid droplet shrinkage led to biphasic response of cavin-1 with ultimate loss of expression of cavin-1 and -3 and EHD2 by protein degradation, coincident with caveolae disassembly. We have identified the key steps in cavin/caveolin interplay regulating adipocyte caveolae dynamics. Our data establish that caveolae participate in a unique cell response connected to lipid store fluctuation, suggesting lipid-induced mechanotension in adipocytes.     

Role of the fatty acid binding protein mal1 in obesity and insulin resistance

The metabolic syndrome is a cluster of metabolic and inflammatory abnormalities including obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis. The fatty acid binding proteins aP2 (fatty acid binding protein [FABP]-4) and mal1 (FABP5) are closely related and both are expressed in adipocytes. Previous studies in aP2-deficient mice have indicated a significant role for aP2 in obesity-related insulin resistance, type 2 diabetes, and atherosclerosis. However, the biological functions of mal1 are not known. Here, we report the generation of mice with targeted null mutations in the mal1 gene as well as transgenic mice overexpressing mal1 from the aP2 promoter/enhancer to address the role of this FABP in metabolic regulation in the presence or absence of obesity. To address the role of the second adipocyte FABP in metabolic regulation in the presence and deficiency of obesity, absence of mal1 resulted in increased systemic insulin sensitivity in two models of obesity and insulin resistance. Adipocytes isolated from mal1-deficient mice also exhibited enhanced insulin-stimulated glucose transport capacity. In contrast, mice expressing high levels of mal1 in adipose tissue display reduced systemic insulin sensitivity. Hence, our results demonstrate that mal1 modulates adipose tissue function and contributes to systemic glucose metabolism and constitutes a potential therapeutic target in insulin resistance.     

Adipocyte Pseudohypoxia Suppresses Lipolysis and Facilitates Benign Adipose Tissue Expansion

Prolyl hydroxylase enzymes (PHDs) sense cellular oxygen upstream of hypoxia-inducible factor (HIF) signaling, leading to HIF degradation in normoxic conditions. In this study, we demonstrate that adipose PHD2 inhibition plays a key role in the suppression of adipocyte lipolysis. Adipose Phd2 gene ablation in mice enhanced adiposity, with a parallel increase in adipose vascularization associated with reduced circulating nonesterified fatty acid levels and normal glucose homeostasis. Phd2 gene–depleted adipocytes exhibited lower basal lipolysis in normoxia and reduced β-adrenergic–stimulated lipolysis in both normoxia and hypoxia. A selective PHD inhibitor suppressed lipolysis in murine and human adipocytes in vitro and in vivo in mice. PHD2 genetic ablation and pharmacological inhibition attenuated protein levels of the key lipolytic effectors hormone-sensitive lipase and adipose triglyceride lipase (ATGL), suggesting a link between adipocyte oxygen sensing and fatty acid release. PHD2 mRNA levels correlated positively with mRNA levels of AB-hydrolase domain containing-5, an activator of ATGL, and negatively with mRNA levels of lipid droplet proteins, perilipin, and TIP47 in human subcutaneous adipose tissue. Therapeutic pseudohypoxia caused by PHD2 inhibition in adipocytes blunts lipolysis and promotes benign adipose tissue expansion and may have therapeutic applications in obesity or lipodystrophy.     

Visceral adipocyte hypertrophy is associated with dyslipidemia independent of body composition and fat distribution in women

OBJECTIVE

We assessed whether subcutaneous and omental adipocyte hypertrophy are related to metabolic alterations independent of body composition and fat distribution in women.

RESEARCH DESIGN AND METHODS

Mean adipocyte diameter of paired subcutaneous and omental adipose tissue samples was obtained in lean to obese women. Linear regression models predicting adipocyte size in both adipose tissue depots were computed using body composition and fat distribution measures (n = 150). In a given depot, women with larger adipocytes than predicted by the regression were considered as having adipocyte hypertrophy, whereas women with smaller adipocytes than predicted were considered as having adipocyte hyperplasia.

RESULTS

Women characterized by omental adipocyte hypertrophy had higher plasma and VLDL triglyceride levels as well as a higher total-to-HDL cholesterol ratio compared with women characterized by omental adipocyte hyperplasia (P < 0.05). Conversely, women characterized by subcutaneous adipocyte hypertrophy or hyperplasia showed a similar lipid profile. In logistic regression analyses, a 10% enlargement of omental adipocytes increased the risk of hypertriglyceridemia (adjusted odds ratio [OR] 4.06, P < 0.001) independent of body composition and fat distribution measures. A 10% increase in visceral adipocyte number also raised the risk of hypertriglyceridemia (adjusted OR 1.55, P < 0.02). Associations between adipocyte size and homeostasis model assessment of insulin resistance were not significant once adjusted for adiposity and body fat distribution.

CONCLUSIONS

These results suggest that omental, but not subcutaneous, adipocyte hypertrophy is associated with an altered lipid profile independent of body composition and fat distribution in women.Fat tissue expansion under a positive energy imbalance relies on adipocyte hypertrophy (enlargement of existing adipocytes) and adipose tissue hyperplasia (proliferation and differentiation of preadipocytes) (1,2). Several observational studies have demonstrated that mean adipocyte sizes in abdominal subcutaneous and visceral adipose tissue are strongly associated with body composition and fat distribution measures (1,3–5). Adiposity and fat cell size are also intimately related to adipocyte function and to the metabolic alterations associated with obesity. However, factors other than adiposity and fat distribution seem to influence adipocyte size in the subcutaneous and omental adipose tissue depot (4). For example, Weyer et al. (6) have demonstrated that a significant portion of subcutaneous adipocyte size variability is explained by sequence variation in the lamin A/C gene, even after adjustment for body composition. Moreover, changes in adipocyte turnover rates and extracellular matrix composition may also modulate the association between adipocyte size and adiposity (6,7).The large interindividual variability observed in adipocyte size at a given adiposity level suggests that the proneness to fat cell hypertrophy in each fat compartment may differ among individuals. Previous studies have shown that although adiposity and fat distribution are associated with several metabolic alterations, subcutaneous adipocyte size remains an independent predictor of these alterations (1,8). Specifically, enlarged subcutaneous adipocytes were associated with hyperinsulinemia and peripheral insulin resistance independent of adiposity levels (1,5,9–11). More recently, Arner et al. (1) demonstrated that subcutaneous adipocyte hypertrophy was linked to low insulin sensitivity and high insulin levels independent of body composition. This association may arise from the fact that hypertrophic adipocytes are more lipolytic, are more resistant to insulin action than small adipocytes, and have an altered adipokine secretion pattern (12–16).Although visceral adipose tissue accumulation is known as an important predictor of metabolic alterations (17,18), previous studies did not take into account fat distribution in the association between adipocyte size and measures of glucose homeostasis or blood lipids (1,5,9–11,19). Moreover, most of these studies could not consider visceral adipose tissue cellularity (1,5,9,11). The aim of the current study was, therefore, to assess the impact of interindividual variation in abdominal subcutaneous and omental adipocyte size on measures of glucose homeostasis and blood lipid-lipoprotein levels independent of body composition and fat distribution in women. We tested the hypothesis that women characterized by adipocyte hypertrophy in either omental or subcutaneous fat, but with similar values of body composition and fat distribution, would be more likely to present metabolic alterations.     

TM4SF5 Knockout Protects Mice From Diet-Induced Obesity Partly by Regulating Autophagy in Adipose Tissue

Transmembrane 4 L six family member 5 (TM4SF5) functions as a sensor for lysosomal arginine levels and activates the mammalian target of rapamycin complex 1 (mTORC1). While the mTORC1 signaling pathway plays a key role in adipose tissue metabolism, the regulatory function of TM4SF5 in adipocytes remains unclear. In this study we aimed to establish a TM4SF5 knockout (KO) mouse model and investigated the effects of TM4SF5 KO on mTORC1 signaling–mediated autophagy and mitochondrial metabolism in adipose tissue. TM4SF5 expression was higher in inguinal white adipose tissue (iWAT) than in brown adipose tissue and significantly upregulated by a high-fat diet (HFD). TM4SF5 KO reduced mTORC1 activation and enhanced autophagy and lipolysis in adipocytes. RNA sequencing analysis of TM4SF5 KO mouse iWAT showed that the expression of genes involved in peroxisome proliferator–activated receptor α signaling pathways and mitochondrial oxidative metabolism was upregulated. Consequently, TM4SF5 KO reduced adiposity and increased energy expenditure and mitochondrial oxidative metabolism. TM4SF5 KO prevented HFD-induced glucose intolerance and inflammation in adipose tissue. Collectively, the results of our study demonstrate that TM4SF5 regulates autophagy and lipid catabolism in adipose tissue and suggest that TM4SF5 could be therapeutically targeted for the treatment of obesity-related metabolic diseases.     

Storage of circulating free fatty acid in adipose tissue of postabsorptive humans: quantitative measures and implications for body fat distribution

OBJECTIVE

Preferential upper-body fat gain, a typical male pattern, is associated with a greater cardiometabolic risk. Regional differences in lipolysis and meal fat storage cannot explain sex differences in body fat distribution. We examined the potential role of the novel free fatty acid (FFA) storage pathway in determining body fat distribution in postabsorptive humans and whether adipocyte lipogenic proteins (CD36, acyl-CoA synthetases, and diacylglycerol acyltransferase) predict differences in FFA storage.

RESEARCH DESIGN AND METHODS

Rates of postabsorptive FFA (palmitate) storage into upper-body subcutaneous (UBSQ) and lower-body subcutaneous (LBSQ) fat were measured in 28 men and 53 premenopausal women. Stable and radiolabeled palmitate tracers were intravenously infused followed by subcutaneous fat biopsies. Body composition was assessed with a combination of dual-energy X-ray absorptiometry and computed tomography.

RESULTS

Women had greater FFA (palmitate) storage than men in both UBSQ (0.37 ± 0.15 vs. 0.27 ± 0.18 μmol · kg⁻¹ · min⁻¹, P = 0.0001) and LBSQ (0.42 ± 0.19 vs. 0.22 ± 0.11 μmol · kg⁻¹ · min⁻¹, P < 0.0001) fat. Palmitate storage rates were significantly greater in LBSQ than UBSQ fat in women, whereas the opposite was true in men. Plasma palmitate concentration positively predicted palmitate storage in both depots and sexes. Adipocyte CD36 content predicted UBSQ palmitate storage and sex-predicted storage in LBSQ fat. Palmitate storage rates per kilogram fat did not decrease as a function of fat mass, whereas lipolysis did.

CONCLUSIONS

The FFA storage pathway, which had remained undetected in postabsorptive humans until recently, can have considerable, long-term, and sex-specific effects on body fat distribution. It can also offer a way of protecting the body from excessive circulating FFA in obesity.Obesity is a major contributor to chronic disease (1). Furthermore, a recent large cohort study demonstrated a strong positive association between abdominal fat distribution and all-cause mortality independently of general adiposity (2). Participants in the lowest third of BMI (women <23 kg/m²; men <24.9 kg/m²) and the highest quintile of waist or waist-to-hip ratio had the highest relative risk of death. These findings emphasize the importance of body fat distribution not only in overweight/obese individuals but also in normal-weight individuals.Men tend to preferentially store fat in their upper-body region and women in their lower-body region (3). Regional differences in fat accumulation must develop from imbalances in fatty acid storage and/or free fatty acid (FFA) release between fat depots. In both sexes, the upper-body subcutaneous (UBSQ) fat depot is lipolytically more active than lower-body subcutaneous (LBSQ) fat (4,5), suggesting that differences in regional lipolysis cannot explain the sex differences in body fat distribution. In addition to the well-known dietary fat storage pathway, an underappreciated storage pathway in adipose tissue also exists (6–8). It involves the uptake and storage of circulating FFAs in the postabsorptive state. The existence of this process under postabsorptive conditions is counterintuitive considering the rapid efflux of fatty acids due to active lipolysis. Interestingly, the pattern of FFA storage between fat depots corresponds to sex differences in body fat distribution (6). However, FFA storage rates in subcutaneous fat regions have not been quantitatively measured, which would allow direct assessment of its quantitative and physiological importance.Adipose tissue buffers the daily flux of fatty acids in the circulation (9). A remarkable adaptation in obesity is downregulation of lipolysis per unit of fat mass (10–13). This is advantageous because it prevents excessive increases in plasma FFA concentrations. Unfortunately, the enlarged adipose tissue also downregulates dietary fat storage (13). Because adipocytes resist further fat storage, dietary fat may be diverted to nonadipose organs leading to ectopic fat accumulation. It is currently unknown whether postabsorptive FFA storage is also downregulated in obesity or whether the enlarged adipose tissue maintains its ability to store FFA from plasma at rates similar to those in normal-weight individuals.Fatty acid uptake and storage in adipocytes has been well studied in vitro, but limited information is available in vivo. The initial step in fatty acid uptake involves the transmembrane transport of fatty acids. Both passive diffusion and protein-facilitated transport (e.g., fatty acid translocase/CD36, fatty acid-binding proteins, fatty acid transport proteins) are thought to contribute to the transmembrane movement of fatty acids (14–18). The transmembrane transport is intimately coupled to esterification with CoA via the action of acyl-CoA synthetases (ACSs) (19,20). This reaction decreases the intracellular concentration of fatty acids to favor import and is an essential initial step for triglyceride synthesis. Diacylglycerol acyltransferase (DGAT) catalyses the final and only committed step in triglyceride synthesis, the conversion of diacylglycerol to triglyceride, making this a critical step in vivo (21).Our goal was to assess the effect of sex and obesity on FFA storage in UBSQ and LBSQ depots under postabsorptive conditions. We examined the potential importance of the direct FFA storage pathway in redistributing fatty acids between fat depots. We also investigated whether obesity downregulates this pathway, as it does for lipolysis and meal fat storage. Lastly, we studied whether CD36, ACS, and DGAT relate to postabsorptive FFA storage and may constitute limiting steps in this process.