Nitrite augments glucose uptake in adipocytes through the protein kinase A-dependent stimulation of mitochondrial fusion

Though it is well accepted that adipose tissue is central in the regulation of glycemic homeostasis, the molecular mechanisms governing adipocyte glucose uptake remain unclear. Recent studies demonstrate that mitochondrial dynamics (fission and fusion) regulate lipid accumulation and differentiation in adipocytes. However, the role of mitochondrial dynamics in glucose homeostasis has not been explored. The nitric oxide oxidation products nitrite and nitrate are endogenous signaling molecules and dietary constituents that have recently been shown to modulate glucose metabolism, prevent weight gain, and reverse the development of metabolic syndrome in mice. Although the mechanism of this protection is unclear, the mitochondrion is a known subcellular target for nitrite signaling. Thus, we hypothesize that nitrite modulates mitochondrial dynamics and function to regulate glucose uptake in adipocytes. Herein, we demonstrate that nitrite significantly increases glucose uptake in differentiated murine adipocytes through a mechanism dependent on mitochondrial fusion. Specifically, nitrite promotes mitochondrial fusion by increasing the profusion protein mitofusin 1 while concomitantly activating protein kinase A (PKA), which phosphorylates and inhibits the profission protein dynamin-related protein 1 (Drp1). Functionally, this signaling augments cellular respiration, fatty acid oxidation, mitochondrial oxidant production, and glucose uptake. Importantly, inhibition of PKA or Drp1 significantly attenuates nitrite-induced mitochondrial respiration and glucose uptake. These findings demonstrate that mitochondria play an essential metabolic role in adipocytes, show a novel role for both nitrite and mitochondrial fusion in regulating adipocyte glucose homeostasis, and have implications for the potential therapeutic use of nitrite and mitochondrial modulators in glycemic regulation.     

Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.     

Inhibition of uncoupling protein 2 by genipin reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Uncoupling protein 2 (UCP2) was reported to be involved in insulin-glucose homeostasis, based on well established event that inhibition of UCP2 stimulates insulin secretion in pancreatic β-cells. However, the role of UCP2 on insulin-stimulated glucose uptake in adipose tissue, which is an indispensable process in insulin-glucose homeostasis, remains unknown. In this study, UCP2 was inhibited by genipin in 3T3-L1 adipocytes, which increased mitochondrial membrane potential, intracellular ATP level and production of reactive oxygen species (ROS). Importantly, insulin-stimulated glucose uptake in 3T3-L1 adipocytes was largely impaired in the presence of genipin, and recovered by CCCP, a mitochondrial uncoupler. Furthermore, genipin leaded to suppression of insulin signal transduction through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). These results suggest that mitochondrial uncoupling in adipocytes positively regulates insulin-stimulated glucose uptake in adipocytes, and UCP2 may play an important role in insulin resistance.     

Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.     

Regulation of leptin secretion from white adipocytes by free fatty acids

Norepinephrine stimulates lipolysis and concurrently inhibits insulin-stimulated leptin secretion from white adipocytes. To assess whether there is a cause-effect relationship between these two metabolic events, the effects of fatty acids were investigated in isolated rat adipocytes incubated in buffer containing low (0.1%) and high (4%) albumin concentrations. Palmitic acid (1 mM) mimicked the inhibitory effects of norepinephrine (1 microM) on insulin (10 nM)-stimulated leptin secretion, but only at low albumin concentrations. Studies investigating the effects of the chain length of saturated fatty acids [from butyric (C4) to stearic (C18) acids] revealed that only fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion. Long-chain mono- and polyunsaturated fatty acids constitutively present in adipocyte triglyceride stores (oleic, linoleic, gamma-linolenic, palmitoleic, eicosapentanoic, and docosahexanoic acids) also completely suppressed leptin secretion. Saturated and unsaturated fatty acids inhibited insulin-stimulated leptin secretion with the same potency and without any significant effect on basal secretion. On the other hand, inhibitors of mitochondrial fatty acid oxidation (palmoxirate, 2-bromopalmitate, 2-bromocaproate) attenuated the stimulatory effects of insulin on leptin release without reversing the effects of fatty acids or norepinephrine, suggesting that fatty acids do not need to be oxidized by the mitochondria to inhibit leptin release. These results demonstrate that long-chain fatty acids mimic the effects of norepinephrine on leptin secretion and suggest that they may play a regulatory role as messengers between stimulation of lipolysis by norepinephrine and inhibition of leptin secretion.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O₂ consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O(2) consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.     

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.     

Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes

Heavy alcohol consumption is an independent risk factor for type 2 diabetes. Although the exact mechanism by which alcohol contributes to the increased risk is unknown, impaired glucose disposal is a likely target. Insulin-stimulated glucose disposal in adipocytes is regulated by two separate and independent pathways, the PI3K pathway and the Cbl/TC10 pathway. Previous studies suggest that chronic ethanol feeding impairs insulin-stimulated glucose transport in adipocytes in a PI3K-independent manner. In search of potential targets of ethanol that would affect insulin-stimulated glucose transport, we investigated the effects of 4-wk ethanol feeding to male Wistar rats on the Cbl/TC10 pathway in isolated adipocytes. Chronic ethanol feeding inhibited insulin-stimulated cCbl phosphorylation compared with pair feeding. Insulin receptor and Akt/PKB phosphorylation were not affected by ethanol feeding. Chronic ethanol exposure also impaired cCbl and TC10 recruitment to a lipid raft fraction isolated from adipocytes by detergent extraction. Furthermore, chronic ethanol feeding increased the amount of activated TC10 and filamentous actin in adipocytes at baseline and abrogated the ability of insulin to further activate TC10 or polymerize actin. These results demonstrate that the impairment in insulin-stimulated glucose transport observed in adipocytes after chronic ethanol feeding to rats is associated with a disruption of insulin-mediated Cbl/TC10 signaling and actin polymerization.     

Differential effect of catecholamines and MPP(+) on membrane permeability in brain mitochondria and cell viability in PC12 cells.

The present study examined the effect of dopamine, 6-hydroxydopamine (6-OHDA), and MPP(+) on the membrane permeability transition in brain mitochondria and on viability in PC12 cells. Dopamine and 6-hydroxydopamine induced the swelling and membrane potential change in mitochondria, which was inhibited by addition of antioxidant enzymes, SOD and catalase. In contrast, antioxidant enzymes did not reduce the effect of MPP(+) on mitochondrial swelling and membrane potential. Catecholamines enhanced the Ca(2+) uptake and release by mitochondria, and the addition of MPP(+) induced Ca(2+) release. Catecholamines induced a thiol oxidation in mitochondria that was decreased by antioxidant enzymes. MPP(+) showed a little effect on the cytochrome c release from mitochondria and did not induce thiol oxidation. Catecholamines and MPP(+) induced a cell death, including apoptosis, in PC12 cells that was inhibited by addition of antioxidant enzymes. The result suggests that the oxidation of dopamine and 6-hydroxydopamine could modulate the membrane permeability in brain mitochondria and induce PC12 cell death, which may be ascribed to oxidative stress. MPP(+) appears to exert a toxic effect on neuronal cells by the action, which is different from catecholamines.     

Glut-4 is translocated to both caveolae and non-caveolar lipid rafts, but is partially internalized through caveolae in insulin-stimulated adipocytes

Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.     

Repletion of atypical protein kinase C following RNA interference-mediated depletion restores insulin-stimulated glucose transport

The role of atypical protein kinase C (aPKC) in insulin-stimulated glucose transport in myocytes and adipocytes is controversial. Whereas studies involving the use of adenovirally mediated expression of kinase-inactive aPKC in L6 myocytes and 3T3/L1 and human adipocytes, and data from knock-out of aPKC in adipocytes derived from mouse embryonic stem cells and subsequently derived adipocytes, suggest that aPKCs are required for insulin-stimulated glucose transport, recent findings in studies of aPKC knockdown by small interfering RNA (RNAi) in 3T3/L1 adipocytes are conflicting. Moreover, there are no reports of aPKC knockdown in myocytes, wherein insulin effects on glucose transport are particularly relevant for understanding whole body glucose disposal. Presently, we exploited the fact that L6 myotubes and 3T3/L1 adipocytes have substantially different (30% nonhomology) major aPKCs, viz. PKC-zeta in L6 myotubes and PKC-lambda in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-zeta, but not PKC-lambda, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-lambda. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-lambda, but not PKC-zeta, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-zeta. These findings in knockdown and, more convincingly, rescue studies, strongly support the hypothesis that aPKCs are required for insulin-stimulated glucose transport in myocytes and adipocytes.     

Palmitic acid stimulates glucose incorporation in the adipocyte by a mechanism likely involving intracellular calcium

The effect of palmitic acid on basal and insulin-stimulated incorporation of glucose into rat adipocytes was studied. Palmitic acid (2.40 mM) stimulated basal as well as insulin-stimulated glucose incorporation in rat adipocytes three and twofold, respectively. Similar degrees of stimulation of basal glucose oxidation by palmitate were also observed. The ability of palmitic acid to stimulate glucose uptake was additive with respect to the stimulation induced by insulin and was proportional to the palmitic acid concentration between 0.15 mM and 2.40 mM. Stimulation of glucose incorporation by palmitic acid was inhibited by preincubating the cells with quin2-AM, which accumulates intracellularly yielding the trapped chelator form. quin2, which binds intracellular Ca2+.The concentration of quin2-AM required for half-maximal inhibition of palmitic acid stimulated glucose incorporation was 3.8 +/- 1.2 microM (mean +/- SEM). The inhibition of palmitic acid-stimulated glucose incorporation by quin2-AM (10 microM) was overcome by incubating cells with the Ca2+ ionophore, A23187, in the presence of extracellular Ca2+ (2.6 mM). Chelation of extracellular Ca2+ with EGTA did not significantly affect the magnitude of palmitic acid-stimulated glucose incorporation. Dantrolene (12.5-100 microM) failed to affect basal or palmitic acid-stimulated glucose incorporation. These findings suggest that palmitic acid stimulates incorporation of glucose in the adipocyte by a mechanism dependent upon intracellular but not extracellular Ca2+.     

Single-step bioluminescence analyses of enzymes,using Cibacrone Blue chromatography for removal of interfering dehydrogenases

Summary Both insulin and lithium ion stimulated cytochalasin B-sensitive glucose transport in isolated rat adipocytes. As a result of enhanced glucose transport, the incorporation of [¹⁴C]glucose into CO₂, glycogen and lipid were increased by both agents. However, the action of these two agents was distinguished. Cytochalasin B decreased insulin-stimulated glucose oxidation, glycogenesis and lipogenesis. In contrast, lithium-stimulated glycogenesis was decreased but lithium-stimulated glucose oxidation and lipogenesis were completely blocked. These results indicate that lithium ion in rat adipocytes has a specific insulin-like effect on glycogenesis without affecting glucose oxidation and lipogenesis.Established Investigator of the American Diabetes Association.     

Inhibition of insulin receptor phosphorylation by indomethacin

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the -subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the -subunit of the solubilized insulin receptor by 6007o and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-¹⁴C]-methyl-D glucose transport by 50070 (P < 0.01), D-[6-¹⁴C]-glucose oxidation by 5007o (P < 0.01), D-[6-¹⁴C]-glucose conversion to lipid by 30010 (P < 0.01), and D-[1-¹⁴C]-glucose conversion to lipid by 6007o (P<0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-¹⁴C]-methyl-D-glucose transport by 75070 (P<0.01), D-[6-¹⁴C]-glucose oxidation by 20% (P<0.05), D-[1-¹⁴C]-glucose oxidation by 35070 (P<0.01), D-[6-¹⁴C] glucose conversion to lipid by 25010 (P<0.01), and D-[1-¹⁴C] glucose conversion to lipid by 4501o (P<0.01). In contrast, insulin binding to its receptor, basal D-[1-¹⁴C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions. This supports the hypothesis that insulin's metabolic effects are linked to activation of the insulin receptor protein kinase and indicates that there may be heterogeneity in the mechanisms of intracellular metabolic control by insulin.     

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.     

Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes

Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20 mmol/l glucose for 4 days), and metabolism of [¹⁴C]oleic acid (OA) and [¹⁴C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO₂, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5 mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5 mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction.     

Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation

Mitochondria have been shown to be impaired in insulin resistance-related diseases but have not been extensively studied during the first steps of adipose cell development. This study was designed to determine the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. 3T3-L1 preadipocytes were differentiated into adipocytes without using glitazone compounds. At days?0, 3, 6, 9, and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Compared to preadipocytes (day?0), new adipocytes (days?6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9?days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1α and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, and CPT1) was increased. Reactive oxygen species (ROS) production displayed a nadir at day?6 with a concomitant increase in antioxidant enzyme gene expression. This 3T3-L1-based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.     

Distinct functions of Ulk1 and Ulk2 in the regulation of lipid metabolism in adipocytes

ULK1 (unc-51 like kinase 1) is a serine/threonine protein kinase that plays a key role in regulating the induction of autophagy. Recent studies using autophagy-defective mouse models, such as atg5- or atg7-deficient mice, revealed an important function of autophagy in adipocyte differentiation. Suppression of adipogenesis in autophagy-defective conditions has made it difficult to study the roles of autophagy in metabolism of differentiated adipocytes. In this study, we established autophagy defective-differentiated 3T3-L1 adipocytes, and investigated the roles of Ulk1 and its close homolog Ulk2 in lipid and glucose metabolism using the established adipocytes. Through knockdown approaches, we determined that Ulk1 and Ulk2 are important for basal and MTORC1 inhibition-induced autophagy, basal lipolysis, and mitochondrial respiration. However, unlike other autophagy genes (Atg5, Atg13, Rb1cc1/Fip200, and Becn1) Ulk1 was dispensable for adipogenesis without affecting the expression of CCAAT/enhancer binding protein α (CEBPA) and peroxisome proliferation-activated receptor gamma (PPARG). Ulk1 knockdown reduced fatty acid oxidation and enhanced fatty acid uptake, the metabolic changes that could contribute to adipogenesis, whereas Ulk2 knockdown had opposing effects. We also found that the expression levels of insulin receptor (INSR), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (SLC2A4/GLUT4) were increased in Ulk1-silenced adipocytes, which was accompanied by upregulation of insulin-stimulated glucose uptake. These results suggest that ULK1, albeit its important autophagic role, regulates lipid metabolism and glucose uptake in adipocytes distinctly from other autophagy proteins.