The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3

THE EFFECT OF NUCLEOSIDE/NUCLEOTIDE REVERSE TRANSCRIPTASE INHIBITORS AND PROTEASE INHIBITORS ON LIPOPROTEIN LIPASE

Khawar Anwar1 , Sultan Zeb2, Abir Hussain 3, Ayub Jan4, Tahir S Pillay5 , Akbar Said Jan6 1Department of Bio-Chemistry, Shahida Islam Medical & Dental College, Lodhran 2Department of Anatomy, Northwest School of Medicine, Peshawar. 3Department of Community Dentistry, Frontier Medical & Dental College, Abbottabad. 4Public Health Student Abdul Wali Khan University, Mardan 5Departments of Chemical Pathology, Faculty of Health Sciences, University of Pretoria, Pretoria, S. Africa. 6Department of Anaesthesiology, Northwest School of Medicine, Peshawar. ABSTRACT In the treatment of HIV/AIDS, protease inhibitors (PIs) and nucleoside/nucleotide analog reverse transcriptase inhibitors (NRTIs) are the major components of highly active antiretroviral therapy (HAART). The side effects of these drugs include various metabolic disorders, including insulin resistance, , and . The precise mechanistic basis of these remains mostly unknown. Objectives :In this study, we aimed to understand the enzymatic activity of lipoprotein lipase (LPL) under the influence of NRTIs and PIs. Material and Methods: Chinese hamster ovary cells transfected with the human insulin receptor (CHO-IR) were used for the first time to study the effects of NRTIs and PIs on the synthesis and release of LPL. The high level of expression of insulin receptor facilitated sensitive detection of any alteration in the phosphorylation of signaling proteins as compared to 3T3-L1 adipocytes. LPL activity in the supernatant and cell lysate was measured using a colorimetric method em- ploying para-nitrophenyl butyrate(pNPB) as a substrate. The most commonly used ARVs were tested. These included four PIs and six NRTIs. Results :The results showed that NRTIs stavudine and emtricitabine significantly inhibited the LPL activity from the CHO-IR cells. PIs indinavir and nelfinavir were also found to decrease LPL activity extracellularly when added to the assay reaction in vitro.Similarly, nelfinavir and atazanavir sulfate inhibited the activity of the LPL from the CHO-IR cells after 16-hour treatment. This suggested that these drugs may interfere with the enzyme activity intracellularly either at the level of its synthesis or its transportation from the cytoplasm to the cell surface. Conclusion :This finding suggests that protease inhibitors may play a role in inhibiting lipopro- tein lipase activity in vivo, and may thereby induce metabolic disorders in HIV-positive patients being treated with protease inhibitors Key Word: Lipoprotein lipase, Nucleoside/nucleotide reverse transcriptase inhibitors,Protease inhibitors Correspondence: Introduction Dr. Khawer Anwar, Assistant Professor, Bio-Chemistry Department, Shahida Lipoprotein lipase (LPL) is a central enzyme Islam Medical and Dental College Lodhran in lipid metabolism and transport. It is produced in Email: [email protected] Contact: +92-334-1140700 numerous cell types, but the skeletal muscle and the

Available Online 30-September 2019 at http://www.jkcd.edu.pk DOI: https://doi.org/10.33279/2307-3934.2019.9126 1 The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3 parenchyma of are the significant sites LPL release from cells is increased by both of synthesis and physiological actions. insulin and heparin in a dose and time-dependent manner. The mechanisms by which insulin and The physiological location of LPL-mediated hy- heparin release LPL from the cells are not precisely drolysis of lipoproteins is at the capillary endothelial known. Some researchers have observed that heparin cell, but functional LPL is first synthesized by the acts as a secretagogue, thereby suggesting that the parenchymal cells and then translocated to its site released LPL is at first present in intracellular secre- of action 1-3. tory vesicles, which fuse with the plasma membrane There are four main levels of regulation, depend- upon stimulation with heparin. Other data indicate ing upon the location of LPL synthesis in different that there is a heparin-binding site on the LPL dimer, tissues. These are at the transcriptional, posttran- which binds to the heparin sulphate-proteoglycan scriptional, translational, and post-translational extracellular matrix. Heparin treatment of cells shifts levels. These four levels are locally controlled by the plasma membrane-bound LPL by competition different factors in different tissues. This local con- with the HSP, resulting in the release of LPL into trol on LPL synthesis enables tissues to maintain a the cell culture medium 14-16. balance in lipid metabolism, and it also modulates Intracellular glucose levels also accelerate LPL other cellular functions and hormonal effects on the synthesis in adipocytes, and its boosting effect on local tissues 4. LPL is mainly associated with glycosylation of LPL plays an important role in lipid metabolism LPL, which is very important for the regular LPL and transport. It catalyzes the hydrolysis of Triacyl- enzymatic activity and its release. Glucose also glycerol (TAG) attached to circulating chylomicrons enhances the effects of insulin to increase LPL syn- and Very Low-Density Lipoprotein (VLDL). The thesis, but there is no evidence that glucose affects resulting reaction produces non-esterified fatty the transcriptional level (mRNA) of LPL synthesis, acids (NEFA) and 2-monoacylglycerol for tissue as observed with insulin 17,18. Different studies asso- utilization 3,5-7. NEFAs in white adipose tissue are ciate insulin resistance with decreased LPL activity, re-esterified for energy storage as TAG 5. Addition- hypertriglyceridaemia, elevated cholesterol and ally, fatty acids are oxidized to provide an energy chylomicronaemia19,20 source in the heart and to regulate thermogenesis in A recent study in insulin-resistant offspring brown adipose tissue 5. of type 2 diabetes parents revealed a link between Role of insulin, heparin, and glucose in lipo- insulin resistance and decreased mitochondrial con- protein lipase regulation tent, as evidenced by decreased mRNA and protein 21 As discussed above, the regulation of LPL syn- expression of LPL in muscle biopsies . Usually, thesis is essential for normal homeostasis of lipid free fatty acid delivery into skeletal muscle results in metabolism, and insulin is the primary regulator of mitochondrial biogenesis through activation of per- LPL synthesis in adipocytes and the translocation oxisome proliferator–activated receptor (PPAR)-δ. from there to the luminal domain of the endothelial The primary role of LPL here is to hydrolyze the cells. Generally insulin increases LPL activity but serum triglycerides and deliver free fatty acids to synthesis of LPL involves many steps including the muscle cells, as described above. LPL gene transcription, mRNA processing, trans- In many disorders such as diabetes, athero- port and translation, posttranslational modification sclerosis, and , hypertriglyceridaemia is a (glycosylation) “activation” or “inactivation”, and characteristic feature. A well-known cause of genetic finally secretion 8-10. Insulin affects different steps hypertriglyceridaemia is the deficiency of lipoprotein of LPL synthesis in adipocytes such as during the lipase. In humans, type I hyperlipoproteinaemia, process of differentiation by enhancing gene tran- resulting from LPL deficiency, is a rare autosomal scription 11 while in fully differentiated adipocytes it recessive disease. It is characterized by low or no regulates LPL synthesis by increasing mRNA levels LPL activity resulting in hypertriglyceridaemia, de- and enzyme activity by both posttranscriptional and creased levels of HDL cholesterol, lipaemiaretinalis, post-translational modifications of LPL 11-13. and pancreatitis. There are three naturally occurring

2 The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3 mutations in LPL that affect lipid transport and activity in adipocytes and serum or plasma. Howev- metabolism and result in hypertriglyceridaemia and er, these methods are either expensive or laborious obesity in mice 22,23. and cumbersome. By contrast, an earlier method for assaying LPL in milk established that LPL-catalysed Disturbances in body fat distribution, dyslipid- hydrolysis of p-nitrophenyl butyrate (pNPB) could emia and insulin resistance, diabetes, and athero- be measured relatively easily and cheaply using the sclerosis in HIV patients are associated with ARV. appearance of p-nitrophenyl phosphate as an end However, the exact mechanisms of these changes product 34. have not been fully elucidated. HIV infection with acute and chronic effects of some antiretroviral drugs Preparation of CHO-IR cells for the assay of on regional fat distribution correlates with these side released lipoprotein lipase effects 24-26. It has already been reported that HIV CHO-IR cells were cultured and serum-starved, infection itself affects triglyceride metabolism and as described in sections 2.2.1 and 2.2.2. The cells lipoprotein lipase activity 27. were washed with PBS (2 ml) twice and left in an Hypertriglyceridaemia and impaired insulin additional fresh PBS (500 μl) containing insulin sensitivity can be observed even in HIV-negative (100ng/ml) (17nM) for one h at 37 °C in an incuba- subjects treated for short periods of time using pro- tor, to stimulate LPL activity. After one h, heparin tease inhibitors but fat redistribution, on the other sodium-Fresenius (final concentration 100 U/ml) was hand, becomes apparent only after several months added for a further two h to release the enzyme from of treatment and this is most often related to dyslip- the cell surface 35-38. The cell culture supernatants idemia and insulin resistance 26,28,29. were then collected in order to assay for LPL activity. The remaining cells in the well were lysed using lysis With the advent of HAART(Highly Activated buffer and measured for total protein concentration, Anti-Retroviral Therapy), it has become well known as described in section 2.2.3. that the two-drug families; the NRTI (Nucleoside Re- verse Transcriptase Inhibitors) and the PIs (Protease The purified LPL standard was derived from Inhibitors) are often associated with lipid abnormal- Pseudomonas sp (Fluka, Buschs, Switzerland) in a ities and body fat distribution, yet the cellular and range of concentrations from 2.13 U/mg to 213 U/ biochemical mechanisms underlying these effects are mg. Enzyme activity was also measured after the not well understood 26,30. The NNRTI component of addition of phenylmethylsulphonyl fluoride (PMSF) HAART also contributes to dyslipidemia, although (1 mM), to confirm that the assessed enzyme activity the relationship to lipodystrophy is unclear 31-33. was attributed to the LPL, as PMSF is known to in- hibit LPL activity 39. PMSF (10 mM) was prepared Materials and Methods in ethanol. The rationale for measuring lipoprotein lipase Assay of lipoprotein lipase activity in CHO cells LPL activity in supernatants from CHO-IR cells In CHO cells, LPL is consistently produced in- was determined as follows 34: dependently of the cell differentiation stage 16. The increased level of expression of insulin receptors Cell culture supernatants (100 μl) were mixed also makes it easy to modulate the insulin response. with 900 μl buffer (0.1 M sodium phosphate, mono- Moreover, since this model system also used for the basic, anhydrous, pH 7.2; 0.9 % sodium chloride; 0.5 study of insulin signaling, it appeared prudent to % (v/v) Triton X-100 (900 μl)) 34,40. pNPB (10 μl of use this cell line to quantify the regulation of LPL 50 mM in acetonitrile at 1 % (v/v) final concentra- activity by insulin and ARVs. We, therefore, ana- tion) was then added. The reactions were incubated lyzed the effect of HPIs and NRTIs on LPL activity in cuvettes at 37 °C in a water bath 34 for specific in CHO cells, transfected with insulin receptors to periods. Absorbance was then measured using a understand whether ARVs could potentially modu- visible light spectrophotometer at 400 nm. late lipid metabolism through LPL and modulate the Enzyme activity was calculated using the fol- LPL response to insulin. lowing formula 39 : Several methods have been used to measure LPL

3 The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3

1.01 = volume (in ml) of incubation mixture df = dilution factor (0.1) 0.0148 = μM extinction coefficient of p-nitro- phenol at 400 nm 0.1 = volume (in ml) of enzyme used Units/mg protein = (units/ml supernatant) ÷ (mg protein/ml supernatant) Unit definition:

One unit will release 1.0 mole of p-nitrophenol Fig 4.2 (a) phosphate per minute at pH 7.2 and 37° C when using p-nitrophenyl butyrate as the substrate. For this experiment, six NRTIs and four PIs that are commonly used in the treatment regimes in South Africa were examined for their effects on lipoprotein lipase secretion. CHO-IR cells were serum-starved and treated with HIV protease inhibitors and NRTIs for 16 h. The drugs were used at concentrations close to their peak serum concentration (Cmax) values: Zidovudine (AZT) 10 µM, efavirenz 20µM, entrici- tubine 20µM, atazanavir sulphate 20µM 41, stavudine 10µM 42, lamivudine 10µM 43, tenofovir 1 µM 44. The cells were then stimulated with insulin to increase LPL activity and treated with heparin to release LPL. The LPL activity in 100µl of the cell culture supernatants was measured after 30 min of Fig 4.2 (b) incubation in a water bath at 37 °C. In addition to measuring whether these drugs influence LPL activity in cells, the same concen- tration of the drugs was added directly to the assay reaction after recovering the untreated (no drug) cell culture supernatants from cells stimulated with both insulin and heparin. This was done to determine if the drugs inhibited LPL activity directly. These were then incubated at 37 °C in a water bath for 30 min.

RESULTS Figure 4.3: Effect of NRTIs on LPL activity after adding directly in supernatants of CHO-IR cells. Changes in LPL activity measured in CHO-IR cells after insulin and heparin. The LPL-catalysed CHO-IR cells were grown in 6-well plates and upon hydrolysis of pNPB was successfully measured in 70-80% confluency the cells were serum starved for 16 h, followed by exposure to both insulin (100)ng/ml) and supernatants of CHO-IR cells stimulated with both heparin. LPL activity was measured in duplicate in two in- insulin and heparin (Figure 4.2.a). Cells which were dependent experiments. Data are shown as the mean and stimulated with both 100 ng/ml insulin and heparin S.E.M. (n=2). C= control, H= Heparin, I = insulin, AZT* = sodium-Fresenius (final concentration 100U/ml) zidovudine, EFV*= efavirenz, FTC* = emtricitabine, d4T* had significantly increased LPL activity almost 4 = stavudine, 3TC*= lamivudine and, TDF*= tenofovir. p*< fold , with detectable enzyme release from the cell 0.05 using unpaired t test (*) indicate surface from 5 min (p = 0.0009) up to 30 min (p =

4 The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3

Figure 4.6: LPL activity in supernatants of CHO-IR cells treated for 16 hours with HPIs. CHO-IR cells were grown in 6-well plates and upon 70-80 Figure 4.4: LPL activity in supernatants of CHO-IR cells % confluency the cells were treated with HPIs indicated in treated for 16 hours with NRTIs. serum starvation medium for 16 h, followed by exposure to both insulin (100ng/ml) and heparin. LPL activity was CHO-IR Cells were grown to 70-80 % confluency, usually measured in duplicate in two independent experiments. taking 24 to 48 hours in 6-well plates .The cells were then Data are shown as the mean and S.E.M. (n=2). C= control, serum starved for 16 h in the absence or presence of the H= Heparin, I = insulin, IND = indinavir, NFV= nelfinavir nucleoside reverse transcriptase inhibitors, followed by ex- ATV = atazanavirsulfate, RTV = ritonavir. p*< 0.05 using posure to both 100ng/ml insulin (17nM) and heparin. LPL unpaired t-test. activity was measured in duplicate as two independent ex- periments. Data are shown as the mean and S.E.M. (n=2). ten ng (1.7nM) was used to stimulate the cells.A C= control, H= Heparin, I = insulin, AZT = zidovudine, statistically insignificant (p=0.0625) low activity of FTC = emtricitabine, EFV= efavirenz, d4T = stavudine, LPL was measured in the supernatants as shown in 3TC= lamivudine and, TDF= tenofovir. p*< 0.05 using Figure 4.2(b). unpaired t-test Effect of nucleoside/nucleotide reverse tran- scriptase inhibitors on lipoprotein lipase activity in supernatant from the CHO-IR cells. LPL activity was measured in CHOIR treated with Zidovudine (AZT) Efavirenz (EFV), Emtricitabine (FTC), Atazanavir- sulfate (ATV), Stavudine(d4T), Lamivudine(3TC) and, Tenofovir (TDF) for 16 h using the optimized assay conditions. LPL activity increased threefold Figure 4.5: Effect of HPIs on LPL activity after adding (p=0.0009) when cells were exposed to both 100 HPIs directly in supernatants of CHO-IR cells.CHO- ng/ml of insulin and heparin, compared to that of IR cells were grown in 6-well plates and upon 70-80% the unstimulated cell culture supernatants with confluency the cells were serum starved for 16 h, followed heparin or insulin. The effect showed by NRTIs on by exposure to both100ng/ml (17nM) insulin and heparin. LPL activity compared to the enzyme activity in the LPL activity was measured in duplicate in two indepen- dent experiments. Data are shown as the mean and S.E.M. untreated cells exposed to both insulin and heparin in (n=2). C= control, H= Heparin, I = insulin, IND* = indina- two different experiments is discussed below. vir, NFV*= nelfinavir ATV* = atazanavirsulfate, RTV* = ritonavir. p*< 0.05 using unpaired t-test .(*) indicate that In the first experiment, the NRTIs were added drugs were added directly in the reaction assay before directly to the supernatant of the untreated cell, those incubation. treated with insulin and heparin to observe the effects of the drugs on the lipoprotein lipase activity direct- 0.0129) incubation and then at 45 min (p=0.0127). ly after secretion. NRTIs did not alter LPL activity The cells which were stimulated with insulin only significantly in the supernatants. A small decrease also showed increased LPL after 30 min (p = 0.0361) (3TC* p=0.130, d4T*p=0.141, AZT* p=0.394, up to 45 min (p = 0.0197) incubation, as shown in FTC* p=0.127, EFV* p=0.378, TDF* p=0.126) in figure 4.2(b). In order to observe the effect of insulin the LPL activity was observed, although these dif- concentration on LPL activity, a low dose of insulin, ferences were not statistically significant as shown

5 The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3 in Figure 4.3. were used to measure LPL activity using a colorimet- ric method employing pNPB as a substrate. LPL has In order to examine the effect of the NRTIs in been shown to catalyze the hydrolysis of short-chain the synthesis and secretion of LPL, CHO-IR cells fatty acyl esters such as tributyrin, p-nitrophenyl were treated for 16 hours with the NRTIs in the acetate, and pNPBin Vivo 34. This substrate is more serum-free medium. After treatment, two drugs convenient to use than lipid-soluble substrates, as stavudine (p=0.0191) and emtricitabine (p=0.0173) the product of LPL-catalysed hydrolysis of pNPB, resulted in significant decreases in the measured p-nitrophenol, absorbs light strongly at 400 nm.40. LPL activity from the cells. The remaining NRTIs, zidovudine (10µM), lamivudine, tenofovir (1µM), CHO cells provide a convenient model to study and efavirenz (20 µM) resulted in decreased LPL intracellular synthesis and translocation of lipopro- activity, but this was not statistically significant as tein lipase. A significant advantage of CHO cells is shown in Figure 4.4. that the synthesis of LPL is constant in these cells and is not associated with the differentiation pro- Effect of protease inhibitors on lipoprotein lipase cesses of the cells. Another advantage compared activity in supernatant from the CHO-IR cells with adipocyte cell lines, which have been used in CHO-IR cells were serum-starved for 16 h previous studies, is that the CHO cells make the (without drugs), and supernatants were collected cell fractionation process easy because they do not after exposure with insulin (100ng/ml) and heparin. contain large fat-droplets. 16. Moreover, since the cell LPL activity in these supernatants was measured expresses large number of receptors, it is very easy after adding the HPIs directly to the assays reaction to modulate the insulin dose-response. before the incubation. Four HPIs indinavir (50µM), CHO-IR cells treated with heparin and insu- nelfinavir (30µM), atazanavir (20µm) and ritonavir lin showed a measurable 3-fold increase in LPL (30µM) were used in this experiment, and the re- activity. Insulin plays a crucial dose-dependent sulting differences in LPL activity were compared role in LPL synthesis and significantly affects the against heparin and insulin-induced cell supernatants posttranscriptional and posttranslational levels with without any addition of drugs, as shown in Figure minimal effects at mRNA level during differentiation 4.5 below. A significant decrease in LPL activity was of adipocytes 11,45,46. These results indicate that the observed in the presence of nelfinavir (p=.0115) and insulin-induced synthesis of LPL is dose-dependent, indinavir (p=.0221), while a small but insignificant and it is significant (p=0.0135) increases in the LPL decreased in LPL activity was observed in the pres- activity in the presence of high concentration (17nM) ence of atazanavir and ritonavir. of insulin while no significant (p=0.0628) increase In order to evaluate the effect of PIs on the intra- is observed with low insulin concentrations 10ng/ml cellular synthesis and secretion of the LPL, CHO-IR (1.7nM). Insulin does not cause the release of LPL cells were treated with the drugs for 16 h; then, the from the cell surface. However, cells stimulated with supernatant was collected, followed by exposure insulin did show significant increases in LPL activity to the insulin and heparin. A high concentration of in CHO-IR cells, even in the absence of heparin. insulin (100ng /ml) was used in this experiment to The results also show that NRTIs did not change exercise the maximum capacity of the cells to se- LPL activity in the supernatant. Stavudine and em- cret enzyme. LPL activity was measured and then tricitabine significantly inhibited the LPL activity compared with the untreated cell supernatant (only from the CHO-IR cells after treatment. Stavudine, heparin and insulin exposed). A significant decrease saquinavir, indinavir, efavirenz, and tenofovir are the in LPL activity of nelfinavir (p=.0193) and ataza- ARVs most likely to cause lipodystrophy (buffalo navir sulfate (p=0069) was observed while smaller hump), and for this reason, stavudine is no longer (statistically insignificant) decreases in LPL were considered an appropriate treatment for most patients observed with indinavir and ritonavir treated cell in developed countries and is no longer recommend- supernatants (Figure4.6). ed by the WHO. However, due to its low price, it is Discussion still widely used in the developing world. Moreover, In this study, for the first time, CHO-IR cells fat loss on long term exposure, one of the major

6 The effect of nucleoside/nucleotide reverse... JKCD September 2019, Vol. 9, No. 3 side-effects of stavudine, is associated with buffalo The hydrolysis and release of lipids for storage from hump 47. triglyceride-rich lipoproteins such as chylomicrons and VLDLs, and their subsequent storage in adipose Major PIs such as indinavir, nelfinavir, atazana- tissue, cannot take place without lipoprotein lipase. vir sulfate, and ritonavir are still being used in South Therefore regulation of LPL expression is critically Africa, and these were used in this study as well 48. important for normal lipid-lipoprotein homeosta- Indinavir and nelfinavir were also found to decrease sis19,20,57. LPL activity when added to the assay reaction in vitro. This indicates that the drugs also inhibit LPL Conclusion activity extracellularly. Similarly nelfinavir and This study indicates that LPL activity can be atazanavir sulfate inhibited the activity of the LPL readily assayed in CHO-IR cells using pNPB as a from the CHO-IR cells after 16 h treatment. This substrate. Furthermore, this assay and CHO-IR cells suggests that these drugs may interfere with the en- can be used to analyze the effects of PIs and NRTIs zyme activity intracellularly either at the level of its on LPL activity. This is the first study to report that synthesis or its transportation from cytoplasm to the stavudine, emtricitabine, indinaviratazanavir sulfate, cell surface. Nelfinavir was found to be the only drug and nelfinavir significantly decrease LPL activity. that inhibited LPL activity intracellularly as well as extracellularly. This finding suggests that protease References inhibitors may play a role in inhibiting LPL activity 1. Camps, L. et al. Lipoprotein lipase in lungs, spleen, and in vivo, and may thereby induce metabolic disorders liver: synthesis and distribution. Journal of lipid research in HIV-positive patients being treated with PIs. 32, 1877-1888 (1991). A previous study elucidating the possible 2. Camps, L., Reina, M., Llobera, M., Vilaro, S. & Ol- mechanism of severe hypertriglyceridaemia caused ivecrona, T. Lipoprotein lipase: cellular origin and func- tional distribution. The American journal of physiology by ritonavir described the decreased LPL activity 258, C673-681 (1990). responsible for the decrease in TG clearance and decreased fatty acid uptake from VLDL and albumin 3. Braun, J. E. & Severson, D. L. Regulation of the synthe- sis, processing and translocation of lipoprotein lipase. in adipocyte tissue. Also, ritonavir decreased the total The Biochemical journal 287 ( Pt 2), 337-347 (1992). LPL activity in the plasma 49. 4. Wang, H. & Eckel, R. H. Lipoprotein lipase: from gene Similarly, few other studies describe a rela- to obesity. American journal of physiology. Endocri- tionship between the intracellular lipid content nology and metabolism 297, E271-288, doi:10.1152/ and insulin resistance in muscle cells 50,51. A recent ajpendo.90920.2008 (2009). study revealed that decreased expression of LPL in 5. Cryer, A. Tissue lipoprotein lipase activity and its action insulin-resistant muscle cells results in decreased in lipoprotein metabolism. The International journal of free fatty acid influx in the cells, which is a potent biochemistry 13, 525-541 (1981). stimulator of mitochondrial biogenesis through 6. Wang, C. S., Hartsuck, J. & McConathy, W. J. Structure activation of PPAR-δ 21. Similarly, Tissue-specific and functional properties of lipoprotein lipase. Biochim- overexpression of LPL plays a role in development ica et biophysica acta 1123, 1-17 (1992). of tissue-specific insulin resistance like in skeletal 7. Enerback, S. & Gimble, J. M. Lipoprotein lipase gene muscle and liver. Increased intracellular lipid (dia- expression: physiological regulators at the transcrip- cylglycerol and ceramides) which activate the novel tional and post-transcriptional level. Biochimica et biophysica acta 1169, 107-125 (1993). and conventional PKC,s (PKCθ, PKCδ, and PKCβ) responsible of insulin resistance by affecting the 8. Vannier, C., Amri, E. Z., Etienne, J., Negrel, R. & Ail- insulin signalling pathway in to the cells 52-56. haud, G. Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation of LPL deficiency or overexpression is critical in the enzyme. The Journal of biological chemistry 260, the regulation of lipid metabolism, and changes may 4424-4431 (1985). induce metabolic disorders such as hypertriglyceri- 9. Olivecrona, T., Chernick, S. S., Bengtsson-Olivecrona, daemia, chylomicronaemia, pancreatitis, atheroscle- G., Garrison, M. & Scow, R. O. Synthesis and secretion rosis, coronary artery disease and lipodystrophy as- of lipoprotein lipase in 3T3-L1 adipocytes. Demonstra- sociated with insulin resistance and diabetes mellitus. tion of inactive forms of lipase in cells. The Journal of

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