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Very-low-density lipoprotein receptor deficiency prevents obesity-induced cardiac lipotoxicity
Mohamed Gharib1, Huan Tao2 and Thomas Fungwe3, Tahar Hajri1*
1From the Department of Surgery, Hackensack University Medical Center, NJ 07601 2Division of Cardiovascular Medicine, Atherosclerosis Research Unit, Vanderbilt University, Nashville TN 37212 3Nutritional Sciences, College of Nursing and Allied Health Sciences, Howard University, Washington DC 20059
Running Title: Vldlr expression and obesity-induced lipotoxicity
*Address correspondence to: Tahar Hajri, Department of Surgery, Jurist Research building, Room 432, 40 Prospect Avenue, Hackensack, NJ 07601. Email address: [email protected]
Key words: Obesity, VLDLR, Oxidative Stress, Lipotoxicity, Insulin Resistance, Heart __________________________________________________________________________________________ ABSTRACT
Obesity is associated with excess lipid deposition in non-adipose tissues, leading to lipotoxicity characterized by increased oxidative stress, inflammation, and insulin resistance. Very-low-density lipoprotein receptor (VLDLR), a member of the LDL receptor family, binds and increases the catabolism of apolipoprotein E-triglyceride–rich lipoproteins. Although the VLDLR is highly expressed in the heart, its role in obesity-associated lipotoxicity is unclear. Here, we used lean WT, VLDLR-deficient (VLDLR-/-), genetically obese leptin-deficient (Lepob/ob), and leptin–VLDLR double-null (Lepob/ob VLDLR-/-) mice to determine the impact of VLDLR deficiency on obesity-induced cardiac lipotoxicity. While insulin sensitivity and glucose uptake were reduced in the hearts of Lepob/ob mice, VLDLR expression was upregulated and was associated with increased VLDL uptake and excess lipid deposition. These changes were accompanied by upregulation of cardiac NADPH oxidase (Nox)
expression and increased production of Nox-dependent superoxides. Silencing the VLDLR in Lepob/ob mice reduced VLDL uptake and prevented excess lipid deposition. Moreover, VLDLR deficiency reduced superoxide overproduction and normalized glucose uptake. In isolated cardiomyocytes, VLDLR deficiency prevented VLDL-mediated induction of Nox activity and superoxide overproduction while improving insulin sensitivity and glucose uptake. Of note, compared with Lepob/ob mice, the Lepob/ob VLDLR-/- mice had significantly improved heart performance and energetic reserves. Our findings indicate that VLDLR deficiency reduces cardiac lipotoxicity in obese mice. This effect appears to be linked to the role of VLDLR in VLDL uptake, which triggers a cascade of events leading to insulin resistance and superoxide overproduction.
http://www.jbc.org/cgi/doi/10.1074/jbc.M117.813303The latest version is at JBC Papers in Press. Published on April 9, 2018 as Manuscript M117.813303
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2 INTRODUCTION
Obesity is associated with deposition of excess lipids in non-adipose tissues, commonly called ectopic fat, which instigates metabolic disorders and cell dysfunction referred to as lipotoxicity. Obesity is also accompanied with a state of oxidative stress, defined as excess production of reactive oxygen species (ROS) relative to antioxidant defense (1). Increased oxidative stress and insulin resistance are characteristics of lipotoxicity widely reported in obese humans and animal models (2,3).
Triglyceride (TG)-rich lipoproteins, including chylomicrons and very low density lipoproteins (VLDL), are the primary carriers of TGs in the circulation, and as such represent a rich source of fatty acid (FA)-derived energy for the body. The value of this source is mostly important for the heart where energy needs are predominantly derived from FA oxidation (3,4). Cardiac lipid metabolism is greatly affected by obesity and diabetes (3-6), conditions under which blood concentration of TG-rich lipoproteins is elevated leading to increased lipid supply. Because the heart’s ability to store lipids is limited, the increase of lipid influx may lead to cardiac lipotoxicity (7-10).
In addition to substrate availability, the heart is equipped with multiple regulatory mechanisms that contribute to maintain a sustained supply of FAs. These mechanism(s) include a high lipolytic ability and the presence of multiple cell membrane receptors that facilitate lipid uptake (3,4). Among these receptors is the very low density lipoprotein receptor (VLDLR), a member of the low-density receptor family, which regulates the catabolism and uptake of TG-rich lipoprotein (11). Several investigations (12-15) have shown that VLDLR impacts the catabolism of TG-rich lipoprotein at multiple levels. First, VLDLR is required for optimal functioning of lipoprotein lipase (LPL) either by increasing LPL and TG-rich lipoprotein interactions at the capillary surface (16) or by serving as helper for LPL transportation to the luminal surface of vascular endothelial cells (17). Second, VLDLR binds and internalizes TG-rich lipoprotein remnants through specific binding of apolipoprotein E (18). Through this dual role, VLDLR controls the availability of lipids to the cell both as FA liberated from TG-rich lipoproteins and
as remnant particles (13,15,19). Although VLDLR is highly expressed in the heart, it is not known if its expression is altered by obesity and whether it participates in obesity-induced lipotoxicity. In this study, we investigated the impact of VLDLR expression on obesity-induced lipid deposition, insulin resistance and oxidative stress.
RESULTS
Weight and metabolic parameters: As expected, body weight of Lepob/ob mice was significantly higher than lean WT mice, but was reduced in Lepob/obVLDLR-/- mice (Table 1). In addition, weights of liver, fat depots and heart were significantly higher in obese Lepob/ob mice than lean WT mice. All these parameters, except liver weight, were significantly reduced in Lepob/obVLDLR-/-
mice. Because of the difference in body weight, the ratio heart mass-to-body weight was significantly lower in Lepob/ob mice than WT, VLDLR-/- or Lepob/ob VLDLR-/- mice. However, the ratio heart weight-to-tibia length, a more reliable index, was about 21% higher in Lepob/ob mice than WT indicating a mild cardiac hypertrophy. This ratio was relatively reduced in Lepob/obVLDLR-/- mice (Table 1). These differences were detected in both male and female mice, and there was no apparent sex-specific impact of VLDLR deficiency on tested parameters (Table 1). Compared to WT mice, the concentrations of total cholesterol in plasma and lipoprotein classes were higher in Lepob/ob and Lepob/obVLDLR-/- mice. The concentrations of plasma TG were significantly higher in VLDLR-/- and Lepob/obVLDLR-/- mice than their respective controls, but FFA concentrations were comparable in all groups (Table 1). Compared to WT mice, fasting serum insulin level was lower in VLDLR-/- mice and was strongly increased in Lepob/ob mice, but was normalized in Lepob/obVLDLR-/- mice. Moreover, VLDLR deficiency reduced obesity-induced insulin resistance as revealed by the improvement of GTT (Figure 1A) and ITT (Figure 1B) in Lepob/obVLDLR-/-
mice.
VLDLR deficiency increased glucose uptake: In light of the results of GTT and ITT, we examined the impact VLDLR deficiency on glucose uptake using glucose analogue 2-deoxyglucose. While cardiac glucose uptake was higher in VLDLR-/- mice compared to WT mice, it was
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3 significantly reduced in Lepob/ob mice, but was normalized in Lepob/obVLDLR-/- mice (Figure 1C). Significant increase of glucose uptake was also noticed in skeletal muscle and adipose tissue, but not in liver of Lepob/obVLDLR-/- mice (Figure 1C). Improvement of insulin resistance in the heart of Lepob/obVLDLR-/- mice was also demonstrated in isolated cardiomyocytes showing a marked increase of insulin-induced phosphorylation of Akt and IRS1 proteins (Figure 1D) accompanied by a significant enhancement of 2-deoxyglucose uptake (Figure 1E).
VLDLR deficiency prevented obesity-
induced excess lipid deposition in the heart: In a second step, we examined the impact of VLDLR expression on cardiac lipid content (Figure 2A). Phospholipid contents were similar in all groups. Compared to WT mice, FA and TG contents were lower in VLDLR-/- mice, but were considerably increased in Lepob/ob mice. These parameters, however, were normalized in Lepob/obVLDLR-/-mice (Figure 2A). To gain further information about the molecular mechanisms that could explain increased lipid accumulation in Lepob/ob mice, we assessed the expression of key proteins. Interestingly, protein level of VLDLR was markedly higher in the heart of Lepob/ob mice than WT mice (Figure 2B). Similarly, mRNA abundance of vldlr as well as lrp, cd36 and fatp1 was significantly increased in the heart of Lepob/ob mice; however, the expression of the three latter genes was modestly reduced in Lepob/obVLDLR-/- mice (Figure 2C).
VLDLR deficiency reduced VLDL clearance and tissue uptake but did not alter albumin-bound FA uptake: Next, we examined the clearance of plasma VLDL and tissue uptake using [3H-TG, 14C-COE]-double labeled VLDL (Figure 3). While 3H label traced the catabolism of VLDL-TG, 14C-COE provided information on the uptake of VLDL remnants. Plasma clearance of 3H-TG (Figure 3A) and 14C-COE (Figure 3B) was clearly delayed in VLDLR-/- and Lepob/obVLDLR-/- mice compared to WT and Lepob/ob mice, resulting in lower uptake of 3H (Figure 3C) and 14C (Figure 3D) in the heart. In addition, uptake of 3H and 14C were lower in skeletal muscles and adipose tissue but not in liver of VLDLR-/- and Lepob/obVLDLR-/- mice compared to their respective controls (Figure 3C and 3D). The distribution VLDL-derived 3H in myocardial lipids (Figure 4A) indicated that the proportions of 3H
recovered in FA, diglycerides (DG) and TG were higher in Lepob/ob than WT mice, indicating that part of VLDL-derived FA was channeled towards the esterification pathway thus increasing cardiac lipid content. Silencing VLDLR in Lepob/ob mice reduced significantly the proportions of 3H recovered in cardiac FA, DG and TG (Figure 4A). In addition to VLDL-derived lipids, albumin-bound FAs that circulate in the bloodstream represent another source of lipids readily taken by the heart. To assess whether VLDLR deficiency altered albumin-bound FA uptake, we examined the in vivo uptake of non-degradable fatty acid analogue 125I-BMIPP complexed with albumin. Myocardial uptake of 125I-BMIPP was comparable between WT and VLDLR-/- mice, but was significantly higher in Lepob/ob mice (Figure 4B). There was a modest (21%) reduction of myocardial uptake FA in Lepob/obVLDLR mice, but was not significantly different from that of Lepob/ob mice. Uptake 125I-BMIPP in skeletal muscle, adipose tissue and liver follow similar pattern than in the heart but with smaller magnitude.
VLDLR deficiency improved heart performance in Lepob/ob mice: Compared to WT mice, cardiac performance parameters were not significantly changed in VLDLR-/- mice, but were negatively altered in obese Lepob/ob mice as reflected by a significant reduction of fractional shortening (FS) and increased left ventricular end diastolic dimension (LVIDd) (Table 2). These parameters were notably improved in Lepob/obVLDLR-/- mice. In association with this improvement, contents of glycogen and adenosine 5'-triphosphate (ATP) in hearts were significantly higher in Lepob/ob VLDLR-
/- mice compared to Lepob/ob mice, although they remained about 26 to 29% lower than WT mice (Table 3). Microscopic analysis of cardiac sections did not show a significant difference between groups in gross morphology or collagen deposition (results not shown).
VLDLR deficiency reduced obesity-associated oxidative stress: To assess oxidative stress, we examined tissue contents of hydrogen and lipid peroxides, both of which are used as time-integrated markers of oxidative stress. Compared to WT mice, cardiac contents of hydrogen peroxides (Figure 5A) and lipid peroxides (Figure 5B) were modestly reduced in VLDLR-/- mice, but were strongly increased in Lepob/ob mice. All of these
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4 markers were reduced in Lepob/ob VLDLR-/- mice to levels comparable to WT mice.
VLDLR deficiency reduced superoxide production: Several pathways are capable of generating reactive oxygen species (ROS), some of which are associated with mitochondrial activity (20), others are linked to enzyme activity (21,22). To examine the contribution of these pathways, we measured superoxide production in cardiac tissue homogenates using lucigenin chemiluminescent superoxide assay in the presence of specific inhibitors (Figure 5C). Measurements without inhibitors (basal) provided total superoxides and showed that production was markedly higher in hearts of Lepob/ob than WT and VLDLR-/- mice; but was significantly reduced in Lepob/obVLDLR-/- hearts. The addition of SOD strongly inhibited lucigenin signal in all groups, thereby confirming that the signals measured in the assay were in fact superoxide-induced chemiluminescence. Inhibitors of nitric oxide synthase (L-NAME) and xanthine oxidase (oxypurinol) did not significantly alter superoxide production, while mitochondrial electron transport inhibitor rotenone reduced superoxides in WT (-28% from basal), VLDLR-/- (-12%), Lepob/ob (-39%) and Lepob/obVLDLR-/- (-19%) hearts. Interestingly, Nox inhibitor (VAS2870) strongly reduced superoxide production in all groups, but the inhibitory effect was more pronounced in WT (-73 % from basal) and Lepob/ob (-79%) mice than VLDLR-/- (-48%) and Lepob/obVLDLR-/- mice (-47%). These experiments also show that the effects of Nox inhibitor VAS2870 was more pronounced than that of mitochondrial inhibitor rotenone.
NADPH oxidase expression was upregulated in obese mice and was reduced by VLDLR deficiency: To further examine the role of VLDLR in obesity-induced oxidative stress, we tested the expression of Nox. Interestingly, protein levels of Nox2, Nox4, and p22phox were noticeably higher in the heart of Lepob/ob mice, but was reduced in Lepob/obVLDLR-/- mice (Figure 6A and 6B). These changes were also reflected at the level of mRNA abundance showing higher expression in Lepob/ob mice and a significant reduction in Lepob/obVLDLR-/- mice (Figure 6C).
VLDLR mediated VLDL-induced ROS production in cardiomyocytes: Because VLDL are
the natural ligand of VLDLR, we questioned the involvement of VLDL in ROS production. To answer this question, cardiomyocytes were cultured with VLDL, and then total intracellular ROS (tROS) production was examined with CMH2DCFDA probe (Figure 7A). Compared to untreated cells, chronic exposure to VLDL induced a marked increase of ROS production in cardiomyocytes of Lepob/ob and WT mice (3.2 to 3.6 fold increase), but significantly less in VLDLR-/- and Lepob/obVLDLR-/- (about 1.6 fold increase) mice.
VLDLR deficiency reduced the production of Nox-derived superoxides: Having shown that VLDLR mediates VLDL-induced ROS production and knowing that VLDLR deficiency reduced Nox expression, we question the role of Nox in VLDL-induced superoxide production. According, VLDL-induced superoxide production was tested in cardiomyocytes cultured with or without membrane permeable Nox inhibitor VA285 using lucigenin chemiluminescence procedure and NADPH as a stimulator of superoxide production and indicator of Nox activity (Figure 7B and 7C). While NADPH induced superoxide production, the addition of SOD strongly inhibited lucigenin signal in all groups confirming that the signal measured was superoxide-induced chemiluminescence. Chronic treatment with VLDL induced a strong increase of superoxide production in Lepob/ob cardiomyocytes but relatively less in Lepob/obVLDLR-/- cardiomyocytes. Similarly, superoxide production was lower in VLDLR-/- cardiomyocytes compared to WT cardiomyocytes (Figure 7B). Treatment with VAS2870 reduced VLDL-induced superoxide production in all groups but the strongest inhibitory effect was in Lepob/ob cardiomyocytes (about 3 fold reduction compared to VLDL alone) followed by WT and Lepob/obVLDLR-/- cardiomyocytes, while the lowest effect was in VLDLR-/- cardiomyocytes (Figure 7C). These results indicate that higher production of superoxides in Lepob/ob cardiomyocytes is mostly related to Nox activity in agreement with protein upregulation shown in Figure 6.
VLDLR mediated VLDL-induced translocation of p47Phox and p67Phox to the cell membrane: The translocation of p47Phox and p67Phox subunits from the cytosol to cell membrane is required to form active Nox2 enzymatic complex (23). Therefore, the ratio of cell membrane-to-
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5 cytosol is indicative of protein complex activation. In the absence of VLDL, the amounts of p47Phox (Figure 8A) and p67Phox (Figure 8C) in the cell membrane fraction were low in both Lepob/ob and Lepob/obVLDLR-/- cardiomyocytes. VLDL overload, however, noticeably increased the amounts p47Phox
and p67Phox in plasma membranes of Lepob/ob
cardiomyocytes but was clearly less effective in Lepob/obVLDLR-/- cardiomyocytes (Figures 8A and 8C). Consequently, the ratio membrane-to-cytosol of these proteins was markedly lower in Lepob/obVLDLR-/- cardiomyocytes (Figures 8B and 8D).
VLDLR deficiency prevented VLDL-induced
insulin resistance in cardiomyocytes: To assess the effects of VLDL and VLDLR expression on insulin sensitivity, we examined insulin-induced glucose uptake in cardiomyocytes of Lepob/ob and Lepob/obVLDLR-/- exposed to VLDL (Figure 9). Basal glucose uptake (without insulin) was low in both lepob/ob and lepob/ob VLDLR-/- cardiomyocytes, and was not significantly altered by VLDL exposure (Figure 9A). However, insulin-stimulated glucose uptake was markedly lower (-110%) in Lepob/ob cardiomyocytes than lepob/obVLDLR-/- cardiomyocytes, and was further reduced (-63%) by VLDL exposure compared to a modest reduction (-21%) in lepob/obVLDLR-/- cardiomyocytes. In agreement with this, while pAkt was low in both lepob/ob and lepob/obVLDLR-/- untreated cells (Figure 9B), insulin-induced Akt phosphorylation was markedly higher in lepob/obVLDLR-/- cardiomyocytes than lepob/ob cardiomyocytes and was not significantly altered by VLDL exposure (Figure 9C). Hence, by contrast to Lepob/ob cells, the ratio of active-to-total Akt (pAkt-to-tAkt) was higher in Lepob/ob VLDLR-/- cells after insulin stimulation, and was not significantly altered by VLDL load (Figure 9D).
DISCUSSION
In this study, we asked the question whether VLDLR expression impacts obesity-associated lipotoxicity. The results indicate that upregulation of VLDLR expression in the heart of obese mice is associated with excess lipid deposition, insulin resistance and oxidative stress, whereas silencing VLDLR expression markedly reduces cardiac lipid deposition and prevents lipotoxicity including
improvement of insulin sensitivity and reduction of superoxide overproduction. The role of VLDLR was further demonstrated in isolated cardiomyocytes in which VLDLR deficiency reduced VLDL-induced insulin resistance and superoxide overproduction.
The process of ectopic lipid deposition has been linked to increased availability of lipids in blood as well as upregulation of several genes required for cellular lipid uptake and processing (2-4). We now provide new evidence demonstrating that VLDLR is another potential mediator of obesity-induced lipid deposition in the heart. VLDLR regulation of lipid deposition is linked to VLDL lipids and not to albumin-bound FA in that knockdown of VLDLR expression reduced VLDL uptake and prevented lipid deposition but did not significantly alter the uptake of albumin-bound FAs. Although the expression of LRP1, another member of the LDLR family that can mediate TG-rich lipoprotein uptake (24), was not significantly altered by VLDLR deficiency, it did not fully compensate for the loss of VLDLR expression. All together, these results confer to VLDLR a prominent role in mediating- VLDL-induced lipid deposition.
Obesity-associated lipotoxicity induces multiple metabolic dysfunctions among which insulin resistance, a major factor in the pathogenesis of type 2 diabetes. Previous investigations have shown that utilization of TG-rich lipoproteins is increased in diabetes and insulin-resistance state (5,25). Our results are in line with these studies and further highlight the role of VLDLR in obesity-induced insulin resistance. In obese mice, upregulation of VLDLR expression in the heart increased VLDL uptake creating a mismatch between the supply and utilization of FAs leading eventually to excess lipid accumulation and insulin resistance. By contrast, VLDLR deficiency reduced TG-rich lipoprotein uptake and induced a marked improvement of insulin resistance and glucose uptake. In agreement with this, exposure of cardiomyocytes to VLDL induced significant reduction of insulin-stimulated glucose uptake in Lepob/ob cells, but had clearly less effect on Lepob/obVLDLR-/- cells. Currently, there is no evidence to directly implicate VLDLR in insulin signaling pathway, but one possible mechanism of action could be related to the role of VLDLR in controlling the rate of TG-rich lipoprotein uptake and intracellular lipid deposition. The causative
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6 effect of lipids in the development of insulin resistance has been widely reported (26,27) and was linked to increased concentration of intracellular DGs and FAs, among other lipids and lipid-derivatives (28,29). It is thus possible that down regulation or deficiency of VLDLR improves obesity-associated insulin resistance indirectly though the reduction of toxic lipids.
While the function of VLDLR in peripheral tissues - to facilitate TG-rich lipoprotein catabolism and uptake - is well described (15-18), its role in obesity-induced oxidative stress remains poorly understood. The present study provides new findings that establish a link between VLDLR expression and Nox-dependent superoxide production. While upregulation of VLDLR was associated with upregulation of Nox expression and increased oxidative stress in the heart of obese mice, VLDLR deficiency reduced VLDL uptake and alleviated Nox-dependent superoxide production. In addition and in agreement with previous findings in endothelial (30) and beta cells (31), exposure of cardiomyocytes to VLDL induced Nox-dependent superoxide production. Silencing VLDLR, however, reduced VLDL uptake and abrogated VLDL-induced superoxide production. These results demonstrate that VLDLR mediates VLDL-induced superoxide production, and highlight the evidence of a regulatory mechanisms between VLDLR expression and Nox-dependent superoxide production, a mechanism that may contribute to the increase of oxidative stress in obesity.
Increasing evidence indicate that obesity is associated with both excess lipid deposition and cardiac dysfunction (32,33), but whether this association is a direct cause – effect relationship is still debated. In line with previous reports (34-36), cardiac steatosis in Lepob/ob mice is associated with mild cardiac hypertrophy and dysfunction. Although the impact of VLDLR expression on cardiac function is not evident in lean mice, it seems that VLDLR deficiency preserves cardiac function under physiological and metabolic stress associated with obesity, as revealed in this study, or under hypoxic ischemia reported in a previous study (14). The question whether this protective effect is linked to the improvement of cardiac lipotoxicity is not clear and require further investigation. Noteworthy, reduction of cardiac performance in obese mice has been associated with a depletion of energetic
reserves (34,36). This is possibly related to the switch of substrate utilization associated to insulin resistance and (36). It is thus conceivable that improvement of insulin sensitivity in Lepob/obVLDLR-/- mice restores energy reserves and improves cardiac function. The pathogenesis of cardiac hypertrophy and dysfunction is complex, involving multiple factors that may extend far beyond lipid infiltration (8). Interestingly, in this study the reduction of cardiac performance in Lepob/ob mice is associated with the induction of Nox expression. Of note, target overexpression of Nox2 or Nox4 to cardiomyocytes induced cardiac hypertrophy and dysfunction (37-39). All together, these findings suggest the existence of possible regulatory mechanisms between Nox expression and cardiac function that may also be linked to lipotoxicity.
Nox is a family of membrane-bound enzyme complexes that transfer electrons from NADPH to oxygen, generating superoxides (23,40). Multiple isoforms have been identified, among which are Nox2 and Nox4, the two most abundant isoforms in myocytes (40). Activation of Nox2 and Nox4 require the assembly of a multiple-unit complex that includes membrane-bound p22Phox. In addition, and in difference to Nox4, Nox2 activation necessitates the association of cytosolic activators p47phox and p67phox to the complex, a mechanism that requires relocation and phosphorylation of these sub-units to the membrane (23,40). Therefore, Nox activation is under the control of two mechanisms: chronic upregulation of expression and acute increase of enzymatic complex formation secondary to the translocation and assembly of regulatory subunits (23). In the present study, upregulation of Nox2, Nox4 and p22Phox in obese mice was associated with increased activity, a result which is in line with previous studies in mice with genetic or diet-induced obesity (41-43). Moreover, our study provides new findings in that VLDLR deficiency normalized Nox2 and Nox4 expression suggesting that pre-translational regulation is involved. Furthermore, VLDL increased membrane-associated p47phox and p67phox in Lepob/ob cardiomyocytes, but did not significantly alter membrane-cytosol distribution of these proteins in Lepob/obVLDLR-/- cardiomyocytes. These results indicate that VLDL may also enhance Nox2 activity acutely by increasing the translocation and assembly of required subunits, and that VLDLR
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7 is required for this process. Of interest to these results, it has been reported that FAs induce Nox2 activity (44) and increase acutely the translocation of cytosolic subunits p47phox and p67phox to the cell membrane (45,46). Because VLDLR deficiency suppresses VLDL uptake, and hence diminishes intracellular FAs, it may also delay the translocation of cytosolic subunits and thus reduces acutely Nox2 complex formation.
In conclusion, the current study provides evidence that VLDLR expression promotes cardiac lipotoxicity in obese mice. In addition, experiments in cardiomyocytes demonstrate that VLDLR-mediated VLDL uptake triggers a cascade of events leading to insulin resistance and induction of Nox-dependent superoxide production. Such mechanism highlights the role of VLDLR in obesity-induced lipotoxicity.
EXPERIMENTAL PROCEDURES:
Animals: Wild type (WT), VLDLR null (VLDLR-/-) and Lepob/+ (all with C57Bl/6 background) mice were obtained from Jackson laboratory (Bar Harbor, ME). Leptin and VLDLR double null mice were generated by breeding VLDLR-/- with Lepb/+ mice, and double heterozygotes were then mated to generate Lepob/obVLDLR-/- mice. Breeding male and female heterozygous Lepb/+ mice generated homozygotes Lepob/ob mice. All mice were fed chow diet, and experiments were conducted in 5-6 month-old WT, VLDLR-/-, Lepob/ob and Lepob/obVLDLR-/- mice. WT mice were used as reference lean controls and Lepob/ob mice served as control obese for Lepob/obVLDLR-/- mice. Preliminary experiments showed that there was no gender-specific effect of VLDL deficiency. Therefore, experiments were conducted in both males and females in comparable proportions. All procedures were approved by the Institutional Animal Care and Use Committee of Vanderbilt University and Hackensack University Medical Center University.
Glucose and Insulin Tolerance Tests: These tests were performed as previously described (47), starting with glucose tolerance test (GTT), and then 7 days later insulin tolerance test (ITT).
Echocardiography measurement: Cardiac performance was assessed at Vanderbilt Core Center with non-invasive transthoracic echocardiography on un-anesthetized conscious mice sing an echocardiogram with a 35 MHz probe (Acuson Corp., Mountain View, CA) according to the procedure described earlier (48,49). Pulse Doppler images were collected with the apical four-chamber view to record the mitral Doppler flow spectra. All data and images were saved and analyzed by an Advanced Cardiovascular Package Software to determine Intraventricular septum (IVS), left ventricular internal diameter (LVID), left ventricular posterior wall (LVPW), EF% and FS% under Long-axis M-mode. Mitral valve Doppler was used to establish MV E/A ratio MV decel time.
Heart section and staining: Hearts were collected, fixed in 4% paraformalin, embedded with paraffin and then cut transversally into 5 μm-thick cross sections. Then, section were stained with Hematoxylin & Eosin, Masson’s trichrome or Picrosirius Red, and sections were examined with bright-field microscope equipped with Olympus camera.
Tissue collection and lipid extraction: Mice were subjected to an overnight fast and then anesthetized with an intra-peritoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. For removal of any residual blood, hearts were perfused through the aorta with saline, and tissues were excised, immediately frozen in liquid nitrogen and stored at -80°C for later analysis. Lipids were extracted from tissues with chloroform, methanol, and 0.9% NaCl as described earlier (47). Lipid extract was dried under a nitrogen stream, and the residue was dissolved in 2-propanol. Lipids in tissue extracts and plasma were measured enzymatically and plasma insulin was assessed with ELISA procedure (19) (47). Cardiac glycogen and ATP contents were measured in freeze-clamped tissues. Glycogen was assayed enzymatically after hydrolysis into glucose residues as reported earlier (47), and ATP was measured by enzymatic assay using commercial kit (Sigma, St Louis, MO) as described by Irie et al (50).
Plasma lipoprotein isolation: Plasma lipoprotein fractions were isolated from pooled plasma by discontinuous density gradient ultracentrifugation according as reported elsewhere (51). Lipoprotein classes were collected according to
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8 their density: VLDL (density < 1.006 g/L), low density lipoprotein (LDL) and high density lipoprotein 1 (HDL1) (1.006<1.063 g/L) and HDL (1.063<d<1.21 g/L). Total cholesterol was assayed by enzymatic assay (Sigma)
Isolation and treatment of adult cardiomyocytes: Cardiomyocytes were isolated using the Langendorff perfusion system as previously reported (46,52). Then, myocytes were either seeded onto laminin-coated coverslips for culture, or left in suspension and used within 2 hours of isolation to investigate metabolite uptake (46).
Assessment of glucose uptake: Glucose tissue uptake was measured in vivo using 2-deoxy-[3H]-glucose (PerkinElmer Life Sciences) using the procedure previously detailed (46,47,52). Non-insulin-dependent and insulin-dependent glucose uptake was measured with the same tracer (0.5 μCi/ml) in cardiomyocytes suspension pre-incubated in medium with or without insulin (100 nM) for 15 min at 37°C (47,53). Deoxy-glucose uptake was found to be linear with time for at least 40 min. To assess the effects of VLDL and VLDLR on insulin sensitivity, another set of experiments were conducted in cardiomyocytes plated in laminin-coated plates. Cells were pre-incubated for 12h in culture medium with or without VLDL (final concentration: 250 M of TG). Mouse VLDL were separated from pooled plasma of WT mice with ultracentrifugation as described previously (13). After VLDL pre-incubation, cells were washed and incubated in medium with or without insulin (100 nM) for 15min. Then, glucose uptake was assayed as described above.
Assessment of VLDL uptake and incorporation of VLDL-derived fatty acid in cellular lipids: Blood clearance and tissue uptake of VLDL was assessed in vivo using double labeled VLDL as previously described (13). Mouse VLDL were double labeled by a two-step procedure: first labeling with [3H]-TG VLDL in vivo by intravenous injection of [3H]-palmitate, and then in vitro incorporation of [14]-cholesteryl oleoyl ether ([14C]-COE) as previously described (13). Kinetic of double labeled ([3H]-TG, ([14C]-COE) VLDL, blood and tissue collection, and radioactivity measurement was conducted as described previously (13). Radioactivity recovered in plasma was expressed as
percent of initial (30 seconds) counts. Incorporation of VLDL-derived 3H radioactivity in cellular lipids was assessed after extraction and separation of lipid classes with thin-layer chromatography as previously described (46,52). Final results were normalized to total proteins determined by the Bradford method (13).
Assessment of albumin-bound fatty acid uptake: The uptake of FA was examined using FA analogue β-methyl-p-123I- Iodophenyl-Pentadecanoic Acid 125I-BMIPP as previously published (46,52). Briefly, each mice was injected in the lateral tail vein with 200 μl of the radioisotope solution of [125I]-BMIPP (15 μCi). Collection of blood and tissues, measurements of radioactivity and calculation of uptake were performed as previously described (52).
Measurement of oxidative stress markers in the heart: Homogenates were prepared by mixing frozen hearts with ice-cold 0.1 M phosphate buffer (pH 7.5) containing 1 mM Na2EDTA and 500 mM butylated hydroxytoluene in acetonitrile to the prevent formation auto-oxidation. Supernatant, collected from homogenates after centrifugation at 2,000 rpm for 5 min at 4 °C, was used to test lipid peroxidation products (LPO) and hydrogen peroxides with enzymatic tests as previously described (46).
NADPH-dependent superoxide assay: NADPH-dependent superoxide production was measured in tissue homogenates with the lucigenin chemiluminescent assay using electron donor NADPH and lucigenin as previously detailed (46). To assess potential sources of superoxide, homogenates were pre-incubated for 15 min with the following agents prior to addition of NADPH: nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME, 100 μM), xanthine oxidase inhibitor oxypurinol (100 μM), complex I mitochondrial electron chain inhibitor rotenone (20 μM) and NADPH oxidase (Nox) inhibitor VAS2870 (10 nM). In addition, superoxide scavenger superoxide dismutase (SOD, 200 U/mL) was used as a positive control to confirm the specificity of superoxide detection. Superoxide production was expressed as arbitrary light units after subtraction of background reading set as reactions without NADPH.
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Quantification of reactive oxygen species in cardiomyocytes: Estimation of reactive oxygen species (ROS) production was performed using the membrane-permeable fluorescent probes 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) (46). After reaction with ROS, H2DCFDA was converted to a highly fluorescent 2',7'-dichlorofluorescein (DCF) and used to assess total cell ROS (tROS). To examine the effect of VLDL, cells were cultured for 12 h in low glucose medium with or without VLDL (final concentration: 250 M of TG) and then incubated with CM-H2DCFDA (1 μM) for 30 min at 37°C. Positive control preparations consisted of cells cultured in medium plus hydrogen peroxide (H2O2 (40 nmol/ml). After incubation, cells were washed with PBS and fluorescence was quantified using a fluorescence microplate reader with excitation at 485 nm and emission at 530 nm (46). The DCF fluorescent intensity was expressed in arbitrary units (a.u.) after background subtraction.
Cell treatment and fractionation: Cardiomyocytes seeded onto laminin-coated dishes were cultured in medium with or without VLDL for 12h as described above. Cells were then homogenized in ice-cold buffer and supernatant collected after centrifugation at 600 g for 5 min was re-centrifuged at 12,000 g for 15 min to sediment mitochondria. The resulting supernatant was transferred into another tube and centrifuged for 20 min at 30,000 g at 4°C to separate membrane fraction in the pellet and cytosol fraction in the supernatant. Both membrane and cytosol fractions
were used for western blot detection of p47phox and p67phox.
Immunoblotting and protein determination: Tissue proteins were analyzed by Western blotting as previously described (13,19) and the following primary antibodies were applied: VLDLR, phospho(Ser473)-Akt, Akt, IRS1, phosphor(Tyr608)-IRS-1, Nox2, Nox4, p22phox and -Actin. The specificity and reproducibility of these antibodies were validated prior to this study (13,19,46). The list of antibodies is reported in Supplemental data – Table S1). Band intensity for each protein was analyzed by densitometry (ImageJ version 1.37), and corrections were made using β-actin intensity reading.
Gene expression: Quantitative polymerase chain reaction (qPCR) of selected genes was performed using SYBR Green Supermix with iTaqDNA polymerase on the IQ5 thermocycler, and specifically designed and optimized oligonucleotides, which sequences are reported in Supplementary data -Table S2. Data of qPCR were obtained as threshold cycle (CT) values and difference in the CT values (ΔCT) was derived from the specific gene tested and CT of the control gene (β-actin) according to the equation 2[CTactin
− CTtarget gene] as described previously (13).
Statistical analysis: Averaged values are presented as means ± SD. Statistical significance between groups was performed by One-way ANOVA test followed by Tukey’s test using GraphPad Prism 4 software (GraphPad Software).
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ACKNOWLEDGMENT
This work was supported by grants from the American Heart Association (Award AHA0730356N) and
Hackensack University Medical Center (HUMC).
CONFLICT Of INTEREST
The authors declare that they have no conflicts of interest with the contents of this article
AUTHOR CONTRIBUTIONS
M.G, H.T and T.H. designed and performed the experiments, and analyzed and interpreted data. M.G and T.H.
wrote the manuscript. T.F. interpreted the data, and reviewed and edited manuscript. All authors approved the
final version of the manuscript.
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Table 1: Body and organ weights, and plasma parameters for lean (WT), VLDLR null (VLDLR-/-), leptin null (Lepob/ob) and leptin VLDLR double null (Lepob/obVLDLR-/-) mice. Data were generated from n = 18-17 female (f) and male (m) mice as indicated in the table. Plasma lipoprotein classes were separated from pooled plasma by gradient ultracentrifugation as described in the Experimental procedures. Cholesterol lipoprotein analysis was performed in n = 5 samples per group. Results are expressed as mean ± SD. Statistical significance was performed by One-way ANOVA test followed by Tukey’s test. Statistical difference between VLDLR-/- and WT mice is indicated with asterisks, * p < 0.05. Statistical difference between Lepob/ob and Lepob/ob VLDLR-/- mice is indicated with small alphabetic letters with a p < 0.01 and b p < 0.05. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.01 and B p < 0.05
Genotype WT VLDLR-/- Lepob/ob Lepob/obVLDLR-/-
N (f/m) 18 (10/8) 18 (9/9) 17 (11/7) 17 (10/7)
Body weight final (g) 29 + 6 24 + 5* 67 + 10A 38 + 9a
Liver weight (g) 2.4 + 0.8 2.9 + 0.7 3.8 + 1.0B 3.9 + 0.7
Gonadal fat weight (g) 2.05 + 0.7 1.16 + 0.43* 3.54 + 1.2A 2.36 + 0.9a
Peri-renal fat weight (g) 0.92 + 0.30 0.56 + 0.31* 1.74 + 0.52A 1.09 + 0.34a
Heart weight (mg) 124 + 9 125 + 11 147 + 15B 129 + 10b
Heart weight-to-tibia length (mg/mm) 7.9 + 0.8 8.0 + 1.6 9.6 + 1.5B 8.1 + 1.5b
Triglycerides (mg/dl) 92 + 22 169 + 37* 109 + 24 188 + 38b
Total cholesterol (mg/dl 85 + 14 93 + 19 142 + 35A 143 + 18
VLDL cholesterol (mg/dl) 3.8 + 0.3 4.9 + 0.4 7.2 + 0.8B 6.1 + 1.1
LDL-HDL1 cholesterol (mg/dl) 6.2 + 0.4 5.6 + 0.5 38.1 + 2.9A 36.4 + 2.8
HDL cholesterol (mg/dl) 74 + 6 83 + 6 97 + 7 100 + 7
Free fatty acids (mmol/L) 1.04 + 0.16 1.13 + 0.23 0.93 + 0.21 1.08 + 0.23
Glucose (mg/dl) 97 + 21 89 + 17 105 + 23 99 + 16
Insulin (ng/ml) 4.2 + 2.3 2.1 + 0.7 16.9 + 5.7A 6.5 + 3.5a
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TABLE 2: Cardiac function parameters of conscious mice
WT VLDLR-/- Lepob/ob Lepob/obVLDLR-/-
6 6 8 8
Heart rate (bpm) 611 + 26 605 + 12 575 + 23 597 + 21
LV mass (mg) 94 + 10 89 + 5 132 + 15A 104 + 12a
FS (%) 53 + 7 51 + 5 39 + 3A 48 + 5a
IVSd (mm) 0.79 + 0.09 0.88 + 0.10 0.89 + 0.10 0.91 + 0.13
LVIDd (mm) 2.91 + 0.10 3.14 + 0.23 3.83 + 0.11A 3.01 + 0.17a
LVPWd (mm) 0.81 + 0.08 0.80 + 0.07 0.68 + 0.14 0.74 + 0.13
IVSs (mm) 1.60 + 0.11 1.47 + 0.09 1.35 + 0.24 1.55 + 0.21
LVIDs (mm) 1.37 + 0.43 1.41 + 0.30 2.36 + 1.04 1.51 + 0.49
LVPWs (mm) 1.05 + 0.14 1.09 + 0.15 0.96 + 0.13 1.04 + 0.11
FS indicates fractional shortening; LV mass, left ventricular mass; IVSd, interventricular septal thickness in diastole; LVIDd, LV end-diastolic dimension; LVPWd, LV posterior wall thickness in diastole; IVSs, interventricular septal thickness in systole; LVIDs, LV end-systolic dimension; LVPWs, LV posterior wall thickness in systole. Data are Mean ± SD, n = 6-8 per group. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.05. Statistical difference between Lepob/ob and Lepob/ob VLDLR-/- mice is indicated with small alphabetic letters with a p < 0.05.
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17 TABLE 3: Cardiac glycogen and ATP contents of conscious mice. Data are Mean ± SD, n = 6-8 per group. Statistical difference between WT and VLDLR-/- mice are indicated with and asterisk, * p < 0.05. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.01. Statistical difference between Lepob/ob and Lepob/ob VLDLR-/- mice is indicated with small alphabetic letters with a p < 0.05
WT VLDLR-/- Lepob/ob Lepob/obVLDLR-/-
8 8 6 6
Glycogen (g glucose/mg protein) 72 + 11 53 + 9* 27 + 10A 53 + 12 a
ATP (mol/g weight) 6.2 + 1.6 4.3 + 0.6* 2.7 + 0.7A 4.4 + 0.9 a
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18 Figure legends Fig 1. Glucose metabolism parameters. A) Glucose tolerance test (GTT) in overnight fasted control wild type (WT), VLDLR null (VLDLR-/-) leptin null (Lepob/ob) and leptin and VLDLR double null (Lepob/obVLDLR-/-) mice. Inset panel A shows areas under the glucose tolerance curves. B) Insulin tolerance test (ITT) in 4-h fasted mice. Inset panel B shows areas under the insulin tolerance curves. Results were generated from n = 6 per group and presented as mean + SD. Statistical analysis was performed by One-way ANOVA test followed by Tukey’s test. Statistical difference between VLDLR-/- and WT is indicated with an asterisk * p < 0.05. Statistical difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with a small alphabetic letter, a p < 0.05. C) Uptake of 3H-2-deoxyglucose in the heart of mice, n = 5 – 7 per group. Statistical difference between VLDLR-/- and WT is indicated with an asterisk * p < 0.05. Statistical difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with a small alphabetic letter, c p < 0.05. Statistical difference between Lepob/ob and WT mice is indicated with a capital alphabetic letter with A p < 0.01. D) Representative blots of phosphorylated (p) and total (t) Akt and IRS1 and uptake of 3H-2-deoxyglucose in isolated cardiomyocytes cultured with or without insulin as described in Experimental procedures. Statistical difference between insulin-treated and untreated cells of the same genotype (Panel E) is indicated with pound signs, # # p < 0.01 and # p < 0.05. Statistical significance between insulin-treated VLDLR-/- and WT cells is indicated with asterisk,* p < 0.05. Statistical significance between insulin-treated Lepob/ob and lepob/obVLDLR-/- cells is indicated with a small alphabetic letter, a p < 0.05 Fig 2. Lipid contents and protein expression in cardiac muscle. A) Myocardial lipid contents (n = 8 per group), B) representative western blot and C) relative expression of selected genes assayed by qPCR (n = 12). Results are presented as mean ± SD. Statistical difference between VLDLR-/- and WT mice is indicated with asterisks with ** p < 0.01, and * p < 0.05. Statistical difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letters with a p < 0.01 and b p < 0.05. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.01 and B p < 0.01 Figure 3: Plasma clearance and tissue uptake of double labeled VLDL. A) Plasma decay of 3H-TG and B) 14C-COE, and tissue uptake of 3H-TG (C) and 14C-COE (D) in mice that received intravenous injection of double labeled VLDL. Experiments were conducted as described in Experimental procedures and results are means + SD with n = 6 per group. Statistical difference between VLDLR-/- and WT is indicated with asterisks ** p < 0.01, and * p < 0.05. Statistical difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letters with a p < 0.001, b p < 0.01 and c p < 0.05. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.05. Figure 4: Incorporation of VLDL-TG in cardiac lipids and organ uptake of albumin-bound [125I]-BMIPP. A) Distribution of VLDL-derived 3H in cardiac lipids was determined following extraction and separation with thin layer chromatography. Polar lipids included phospholipids and monoglycerides. Results are presented as mean + SD with n = 6 per group. Statistical difference between VLDLR-/- and WT is indicated with an asterisk * p < 0.05. Statistical difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letters with a p < 0.01. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.01 and B p < 0.05. B) [125I]-BMIPP distribution in organs following injected of [125I]-BMIPP complexed with albumin. Tissues were removed 2 h after injection. Uptake is expressed as percent of injected dose per gram tissue. Means + SD with n = 6 per group. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with and B p < 0.05.
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19 Figure 5: Oxidative stress markers and superoxide production: A) Myocardial contents of hydrogen peroxides and B) lipid peroxides (n = 6 per group). C) NADPH-dependent and SOD-inhibitable superoxide was measured in heart homogenates by the lucigenin chemiluminescent method. Measurements were performed in preparations without inhibitors (basal) or in presence of superoxide dismutase (SOD) used to determine the specific of measurements, L-NAME (nitric oxide synthase inhibitor), Oxypurinol (oxidase inhibitor), Rotenone (complex I mitochondrial electron chain inhibitor) and VAS280 inhibitor of NADPH oxidase (Nox). Measurements were conducted in triplicates from an n = 5 per group. Results are presented as mean ± SD. Statistical difference between VLDLR-/- and WT is indicated with asterisks * p < 0.05 and significance difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letters a p < 0.01. Statistical difference between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.01 and B p < 0.05. Figure 6: NADPH oxidase (Nox) expression. A) Representative blots, B) ratio of optic density of proteins (n = 5 per group), and C) relative expression of cardiac Nox 2, Nox 4 and p22Phox genes (n = 10-12 per group). Results are mean + SD. Statistical difference between Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letters with a p < 0.01, b p < 0.05 and statistical differences between Lepob/ob and WT mice is indicated with capital alphabetic letters with A p < 0.01 and B p < 0.05 Figure 7. Superoxide production in cardiomyocyte culture with or without VLDL. A) Production of reactive oxygen species (ROS) measured with CM-H2DCF/DA in cardiomyocytes cultured with or without VLDL as described in Experimental procedures. Superoxide production determined by lucigenin chemiluminescent method in cardiomyocytes cultured with VLDL alone (B) or in presence of Nox inhibitor VAS2870 (C). Data presented in panel A, B and C were mean + SD of triplicates from two experiments. Statistical difference between untreated and VLDL-treated cells of the same genotype are indicated with pound symbol, # # p < 0.01 and # p < 0.05. Statistical difference between VLDL-treated cells of Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letter a p < 0.05. Statistical differences between VLDL-treated cells of WT and Lepob/ob mice are indicated with capital alphabetic letters with A p < 0.05 Figure 8: Effects of VLDL on the distribution of Nox subunits in membrane and cytosol fractions. Representative blots (A and C) and means of membrane-to-cytosol ratios of optic density (B and D) of p47phox (A and B), p67phox (C and D) proteins in cardiomyocyte culture with or without VLDL. Data were generated from two experiments performed in triplicates and results are mean + SD. Statistical difference between untreated and VLDL-treated cells of the same genotype are indicated with pound symbol, # p < 0.05. Statistical difference between VLDL-treated cells of Lepob/ob and Lepob/obVLDLR-/- mice is indicated with small alphabetic letter a p < 0.01 Figure 9: Effects of VLDL on insulin-induced glucose uptake and Akt phosphorylation. 3H-2-deoxyglucose uptake (A), representative blots (B and C) of phosphorylated (p) and total (t) Akt, and ratio of pAkt-to-tAkt optic density (D) in isolated cardiomyocytes cultured with or without insulin and VLDL as described in Experimental procedures. Data were generated from two experiments performed in triplicates and results are mean + SD. Statistical difference between VLDL-treated and untreated cells of the same genotype is indicated with pound signs, # p < 0.01. Statistical significance between insulin-treated Lepob/ob and Lepob/obVLDLR-/- cells is indicated with a small alphabetic letter, a p < 0.01. Statistical significance between VLDL and insulin-treated Lepob/ob and Lepob/obVLDLR-/- cells is indicated with a capital alphabetic letter, A p < 0.01
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Mohamed Gharib, Huan Tao, Thomas Fungwe and Tahar Hajrilipotoxicity
Very low density lipoprotein receptor deficiency prevents obesity-induced cardiac
published online April 9, 2018J. Biol. Chem.
10.1074/jbc.M117.813303Access the most updated version of this article at doi:
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