Adilson L. Guilherme

Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes

Acquired resistance to the action of insulin to stimulate glucose transport in skeletal muscle is associated with obesity and promotes the development of type 2 diabetes. In skeletal muscle, insulin resistance can result from high levels of circulating fatty acids that disrupt insulin signalling pathways. However, the severity of insulin resistance varies greatly among obese people. Here we postulate that this variability might reflect differences in levels of lipid-droplet proteins that promote the sequestration of fatty acids within adipocytes in the form of triglycerides, thereby lowering exposure of skeletal muscle to the inhibitory effects of fatty acids.

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.

Tumor Necrosis Factor α (TNFα) Stimulates Map4k4 Expression through TNFα Receptor 1 Signaling to c-Jun and Activating Transcription Factor 2

Tumor necrosis factor α (TNFα) is a cytokine secreted by macrophages and adipocytes that contributes to the low grade inflammation and insulin resistance observed in obesity. TNFα signaling decreases peroxisome proliferator-activated receptor γ and glucose transporter isoform 4 (GLUT4) expression in adipocytes, impairing insulin action, and this is mediated in part by the yeast Ste20 protein kinase ortholog Map4k4. Here we show that Map4k4 expression is selectively up-regulated by TNFα, whereas the expression of the protein kinases JNK1/2, ERK1/2, p38 stress-activated protein kinase, and mitogen-activated protein kinase kinases 4/7 shows little or no response. Furthermore, the cytokines interleukin 1β (IL-1β) and IL-6 as well as lipopolysaccharide fail to increase Map4k4 mRNA levels in cultured adipocytes under conditions where TNFα elicits a 3-fold effect. Using agonistic and antagonistic antibodies and small interfering RNA (siRNA) against TNFα receptor 1 (TNFR1) and TNFα receptor 2 (TNFR2), we show that TNFR1, but not TNFR2, mediates the increase in Map4k4 expression. TNFR1, but not TNFR2, also mediates a potent effect of TNFα on the phosphorylation of JNK1/2 and p38 stress-activated protein kinase and their downstream transcription factor substrates c-Jun and activating transcription factor 2 (ATF2). siRNA-based depletion of c-Jun and ATF2 attenuated TNFα action on Map4k4 mRNA expression. Consistent with this concept, the phosphorylation of ATF2 along with the expression and phosphorylation of c-Jun by TNFα signaling was more robust and prolonged compared with that of IL-1β, which failed to modulate Map4k4. These data reveal that TNFα selectively stimulates the expression of a key component of its own signaling pathway, Map4k4, through a TNFR1-dependent mechanism that targets the transcription factors c-Jun and ATF2.

RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27.

Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)‐based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest116: 125, 2006) and the mitogen‐activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA103: 2087, 2006) as negative regulators of insulin‐responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140‐null mice resist weight gain on a high‐fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140‐sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor‐α (TNF‐α). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity.

The v-SNARE Vti1a regulates insulin-stimulated glucose transport and Acrp30 secretion in 3T3-L1 adipocytes.

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

A major role of insulin in promoting obesity- associated adipose tissue inflammation

Objective Adipose tissue (AT) inflammation is associated with systemic insulin resistance and hyperinsulinemia in obese rodents and humans. A longstanding concept is that hyperinsulinemia may promote systemic insulin resistance through downregulation of its receptor on target tissues. Here we tested the novel hypothesis that insulin also impairs systemic insulin sensitivity by specifically enhancing adipose inflammation. Methods Circulating insulin levels were reduced by about 50% in diet-induced and genetically obese mice by treatments with diazoxide or streptozotocin, respectively. We then examined AT crown-like structures, macrophage markers and pro-inflammatory cytokine expression in AT. AT lipogenesis and systemic insulin sensitivity was also monitored. Conversely, insulin was infused into lean mice to determine its affects on the above parameters. Results Lowering circulating insulin levels in obese mice by streptozotocin treatment decreased macrophage content in AT, enhancing insulin stimulated Akt phosphorylation and de novo lipogenesis (DNL). Moreover, responsiveness of blood glucose levels to injected insulin was improved by streptozotocin and diazoxide treatments of obese mice without changes in body weight. Remarkably, even in lean mice, infusion of insulin under constant euglycemic conditions stimulated expression of cytokines in AT. Consistent with these findings, insulin treatment of 3T3-L1 adipocytes caused a 10-fold increase in CCL2 mRNA levels within 6 h, which was blocked by the ERK inhibitor PD98059. Conclusion Taken together, these results indicate that obesity-associated hyperinsulinemia unexpectedly drives AT inflammation in obese mice, which in turn contributes to factors that suppress insulin-stimulated adipocyte DNL and systemic insulin sensitivity.

Tumor Necrosis Factor-α Induces Caspase-mediated Cleavage of Peroxisome Proliferator-activated Receptor γ in Adipocytes

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFα) in adipose tissue of obese subjects potently suppresses the expression of PPARγ and attenuates adipocyte functions. Here we show that PPARγ is a substrate of caspase-3 and caspase-6 during TNFα receptor signaling in adipocytes, and the consequent PPARγ cleavage disrupts its nuclear localization. TNFα treatment of 3T3-L1 adipocytes decreases full-length PPARγ while increasing the level of a 45-kDa immunoreactive PPARγ fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARγ protein in response to TNFα in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARγ cleavage product. Dispersion of nuclear PPARγ to the cytoplasm in response to TNFα treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFα down-regulates PPARγ protein and PPARγ-mediated metabolic processes in adipose cells.

Map4k4 Negatively Regulates Peroxisome Proliferator-activated Receptor (PPAR) γ Protein Translation by Suppressing the Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Cultured Adipocytes

The receptor peroxisome proliferator-activated receptor γ (PPARγ) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARγ expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARγ1 and PPARγ2 proteins in 3T3-L1 adipocytes without affecting PPARγ mRNA levels, suggesting that Map4k4 regulates PPARγ at a post-transcriptional step. PPARγ degradation rates are remarkably rapid as measured in the presence of cycloheximide (t½ = 2 h), but silencing Map4k4 had no effect on PPARγ degradation. However, depletion of Map4k4 significantly enhances [35S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARγ protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARγ protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARγ post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.