Sarah M. Nicoloro

Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone

Adipose tissue plays a central role in the control of energy homeostasis through the storage and turnover of triglycerides and through the secretion of factors that affect satiety and fuel utilization. Agents that enhance insulin sensitivity, such as rosiglitazone, appear to exert their therapeutic effect through adipose tissue, but the precise mechanisms of their actions are unclear. Rosiglitazone changes the morphological features and protein profiles of mitochondria in 3T3-L1 adipocytes. To examine the relevance of these effects in vivo, we studied white adipocytes from ob/ob mice during the development of obesity and after treatment with rosiglitazone. The levels of approximately 50% of gene transcripts encoding mitochondrial proteins were decreased with the onset of obesity. About half of those genes were upregulated after treatment with rosiglitazone, and this was accompanied by an increase in mitochondrial mass and changes in mitochondrial structure. Functionally, adipocytes from rosiglitazone-treated mice displayed markedly enhanced oxygen consumption and significantly increased palmitate oxidation. These data reveal mitochondrial remodeling and increased energy expenditure in white fat in response to rosiglitazone treatment in vivo and suggest that enhanced lipid utilization in this tissue may affect whole-body energy homeostasis and insulin sensitivity.

Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle—safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of β1,3-d-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 μg kg-1 siRNA directed against tumour necrosis factor α (Tnf-α) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-α levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-α and interleukin-1β production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.

Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone

ABSTRACT White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20- to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.

Cidea is associated with lipid droplets and insulin sensitivity in humans

Storage of energy as triglyceride in large adipose-specific lipid droplets is a fundamental need in all mammals. Efficient sequestration of fat in adipocytes also prevents fatty acid overload in skeletal muscle and liver, which can impair insulin signaling. Here we report that the Cide domain-containing protein Cidea, previously thought to be a mitochondrial protein, colocalizes around lipid droplets with perilipin, a regulator of lipolysis. Cidea-GFP greatly enhances lipid droplet size when ectopically expressed in preadipocytes or COS cells. These results explain previous findings showing that depletion of Cidea with RNAi markedly elevates lipolysis in human adipocytes. Like perilipin, Cidea and the related lipid droplet protein Cidec/FSP27 are controlled by peroxisome proliferator-activated receptor γ (PPARγ). Treatment of lean or obese mice with the PPARγ agonist rosiglitazone markedly up-regulates Cidea expression in white adipose tissue (WAT), increasing lipid deposition. Strikingly, in both omental and s.c. WAT from BMI-matched obese humans, expression of Cidea, Cidec/FSP27, and perilipin correlates positively with insulin sensitivity (HOMA-IR index). Thus, Cidea and other lipid droplet proteins define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance, and its associated comorbidities in humans.

Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

Suppression of oxidative metabolism and mitochondrial biogenesis by the transcriptional corepressor RIP140 in mouse adipocytes

Using an siRNA-based screen, we identified the transcriptional corepressor RIP140 as a negative regulator of insulin-responsive hexose uptake and oxidative metabolism in 3T3-L1 adipocytes. Affymetrix GeneChip profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, TCA cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidative phosphorylation in these cells. Conversely, we show that reexpression of RIP140 in mouse embryonic fibroblasts derived from RIP140-null mice downregulates expression of many of these same genes. Consistent with these microarray data, RIP140 gene silencing in cultured adipocytes increased both conversion of [14C]glucose to CO2 and mitochondrial oxygen consumption. RIP140-null mice, previously reported to resist weight gain on a high-fat diet, are shown here to display enhanced glucose tolerance and enhanced responsiveness to insulin compared with matched wild-type mice upon high-fat feeding. Mechanistically, RIP140 was found to require the nuclear receptor ERRalpha to regulate hexose uptake and mitochondrial proteins SDHB and CoxVb, although it likely acts through other nuclear receptors as well. We conclude that RIP140 is a major suppressor of adipocyte oxidative metabolism and mitochondrial biogenesis, as well as a negative regulator of whole-body glucose tolerance and energy expenditure in mice.

Depot-Specific Differences and Insufficient Subcutaneous Adipose Tissue Angiogenesis in Human Obesity

Background— Adipose tissue expands in response to excess caloric intake, but individuals prone to deposit visceral instead of subcutaneous adipose tissue have higher risk of metabolic disease. The role of angiogenesis in the expandability of human adipose tissue depots is unknown. The objective of this study was to measure angiogenesis in visceral and subcutaneous adipose tissue and to establish whether there is a relationship between obesity, metabolic status, and the angiogenic properties of these depots. Methods and Results— Angiogenic capacity was determined by quantifying capillary branch formation from human adipose tissue explants embedded in Matrigel, and capillary density was assessed by immunohistochemistry. Subcutaneous adipose tissue had a greater angiogenic capacity than visceral tissue, even after normalization to its higher initial capillary density. Gene array analyses revealed significant differences in expression of angiogenic genes between depots, including an increased subcutaneous expression of angiopoietin-like protein 4, which is proangiogenic in an adipose tissue context. Subcutaneous capillary density and angiogenic capacity decreased with morbid obesity, and subcutaneous, but not visceral, adipose tissue angiogenic capacity correlated negatively with insulin sensitivity. Conclusions— These data imply that subcutaneous adipose tissue has a higher capacity to expand its capillary network than visceral tissue, but this capacity decreases with morbid obesity. The decrease correlates with insulin resistance, suggesting that impairment of subcutaneous adipose tissue angiogenesis may contribute to metabolic disease pathogenesis.

Conventional kinesin KIF5B mediates insulin‐stimulated GLUT4 movements on microtubules

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4–yellow fluorescent protein (YFP) move along tubulin–cyan fluorescent protein‐labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)‐kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long‐range movements of GLUT4–YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co‐localized with perinuclear GLUT4. Dominant‐negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc‐tagged GLUT4–green fluorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4‐containing membranes on microtubules via conventional kinesin through a PI3‐kinase‐independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.

Enhanced angiogenesis in obesity and in response to PPARγ activators through adipocyte VEGF and ANGPTL4 production

PPARgamma activators such as rosiglitazone (RSG) stimulate adipocyte differentiation and increase subcutaneous adipose tissue mass. However, in addition to preadipocyte differentiation, adipose tissue expansion requires neovascularization to support increased adipocyte numbers. Paradoxically, endothelial cell growth and differentiation is potently inhibited by RSG in vitro, raising the question of how this drug can induce an increase in adipose tissue mass while inhibiting angiogenesis. We find that adipose tissue from mice treated with RSG have increased capillary density. To determine whether adipose tissue angiogenesis was stimulated by RSG, we developed a novel assay to study angiogenic sprout formation ex vivo. Angiogenic sprout formation from equally sized adipose tissue fragments, but not from aorta rings, was greatly increased by obesity and by TZD treatment in vivo. To define the mechanism involved in RSG-stimulated angiogenesis in adipose tissue, the expression of proangiogenic factors by adipocytes was examined. Expression of VEGFA and VEGFB, as well as of the angiopoietin-like factor-4 (ANGPTL4), was stimulated by in vivo treatment with RSG. To define the potential role of these factors, we analyzed their effects on endothelial cell growth and differentiation in vitro. We found that ANGPTL4 stimulates endothelial cell growth and tubule formation, albeit more weakly than VEGF. However, ANGPTL4 mitigates the growth inhibitory actions of RSG on endothelial cells in the presence or absence of VEGF. Thus, the interplay between VEGF and ANGPTL4 could lead to a net expansion of the adipose tissue capillary network, required for adipose tissue growth, in response to PPARgamma activators.

Body mass index-independent inflammation in omental adipose tissue associated with insulin resistance in morbid obesity.

BACKGROUND Obesity is a strong risk factor for resistance to insulin-mediated glucose disposal, a precursor of type 2 diabetes and other disorders. However, not all obese individuals are insulin resistant. We sought to identify the molecular pathways that might cause obesity-associated insulin resistance in humans by studying the morbidly obese who were insulin sensitive versus insulin resistant, thereby eliminating obesity as a variable. METHODS Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from similarly obese patients undergoing gastric bypass surgery. RESULTS Gene sets related to chemokine activity and chemokine receptor binding were identified as most highly expressed in the omental tissue from insulin-resistant compared with insulin-sensitive subjects, independent of the body mass index. These upregulated genes included chemokines (C-C motif) ligand 2, 3, 4, and 18 and interleukin-8/(CC-X motif) ligand 8 and were not differentially expressed in the subcutaneous adipose tissues between the 2 groups of subjects. Insulin resistance, but not the body mass index, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size, in these morbidly obese subjects. CONCLUSION Our findings have demonstrated that inflammation of the omental adipose tissue is strongly associated with insulin resistance in human obesity even in subjects with similar body mass index values.