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It is challenging to deliver molecules to the brain for diagnosis and treatment of brain diseases. This is primarily because of the presence of the blood-brain barrier ( BBB), which restricts the entry of many molecules into the brain. In this study, cyclic- ADT peptides ( ADTC1, ADTC5, and ADTC6) have been shown to modify the BBB to enhance the delivery of marker molecules [e.g., 14 C-mannitol, gadolinium-diethylenetriaminepentacetate ( Gd- DTPA)] to the brain via the paracellular pathways of the BBB. The hypothesis is that these peptides modulate cadherin interactions in the adherens junctions of the vascular endothelial cells forming the BBB to increase paracellular drug permeation. In vitro studies indicated that ADTC5 had the best profile to inhibit adherens junction resealing in Madin- Darby canine kidney cell monolayers in a concentration-dependent manner ( IC50 = 0.3 mM) with a maximal response at 0.4 mM. Under the current experimental conditions, ADTC5 improved the delivery of 14 C-mannitol to the brain about twofold compared with the negative control in the in situ rat brain perfusion model. Furthermore, ADTC5 peptide increased in vivo delivery of Gd- DTPA to the brain of Balb/c mice when administered intravenously. In conclusion, ADTC5 has the potential to improve delivery of diagnostic and therapeutic agents to the brain. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1065-1075, 2015 [ABSTRACT FROM AUTHOR]

Background: Although the current combination of surgery, radiation, and chemotherapy is used in the breast-cancer setting, the administration of the anticancer drugs doxorubicin and trastuzumab is associated with an increased risk of developing heart failure. The aim of this study is to determine whether dietary flaxseed is comparable and/or synergistic with the angiotensin-converting enzyme inhibitor perindopril in the treatment of doxorubicin- and trastuzumab-mediated cardiotoxicity., Methods: In a chronic in vivo murine model (n = 110), doxorubicin and trastuzumab (8 mg/kg and 3 mg/kg, respectively) were administered weekly for 3 weeks. Following this period, the mice were randomized to daily consumption of a 10% flaxseed supplemented diet, administration of perindopril (3 mg/kg) via oral gavage, or a combination of both flaxseed and perindopril for an additional 3 weeks., Results: In mice treated with doxorubicin and trastuzumab, the left ventricular ejection fraction decreased from 74% ± 4% at baseline to 30% ± 2% at week 6. Treatment with either flaxseed or perindopril, or with flaxseed and perindopril improved left ventricular ejection fraction to 52% ± 4%, 54% ± 4%, and 55% ± 3%, respectively ( P < 0.05). Although histologic analyses confirmed significant loss of sarcomere integrity and vacuolization in the doxorubicin- and trastuzumab-treated mice, treatment with flaxseed or perindopril, or with flaxseed and perindopril improved myocyte integrity. Finally, the level of Bcl-2 interacting protein 3, high-mobility group box 1 protein expression, and the levels of select oxylipins, were significantly elevated in mice receiving doxorubicin and trastuzumab; these markers were attenuated by treatment with either flaxseed or perindopril, or with flaxseed and perindopril., Conclusions: Flaxseed was equivalent to perindopril at improving cardiovascular remodelling by reducing biomarkers of inflammation, mitochondrial damage, and cell death., (© 2024 The Authors.)

Introduction: Hypoxic persistent pulmonary hypertension in the newborn (PPHN) is usually treated with oxygen and inhaled nitric oxide (NO), both pulmonary arterial relaxants. But treatment failure with NO occurs in 25% of cases. We previously demonstrated that 72 h exposure to hypoxia, modeling PPHN, sensitized pulmonary artery smooth muscle cells (PASMC) to the contractile agonist thromboxane and inhibited relaxant adenylyl cyclase (AC) activity. Methods: In this study, we examined the effects of sodium nitroprusside (SNP), as NO donor, on the thromboxane-mediated contraction and NO-independent relaxation pathways and on reactive oxygen species (ROS) accumulation in PASMC. In addition, we examined the effect of the peroxynitrite scavenger 5,10,15,20-Tetrakis (4-sulfonatophenyl)porphyrinato Iron (III) (FeTPPS) on these processes. Results: Exposure of PASMC to 72 h hypoxia increased total intracellular ROS compared to normoxic control cells and this was mitigated by treatment of cells with either SNP or FeTPPS. Total protein nitrosylation was increased in hypoxic PASMC compared to controls. Both normoxic and hypoxic cells treated with SNP exhibited increased total protein nitrosylation and intracellular nitrite; this was reduced by treatment with FeTPPS. While cell viability and mitochondrial number were unchanged by hypoxia, mitochondrial activity was decreased compared to controls; addition of FeTPPS did not alter this. Basal and maximal mitochondrial metabolism and ATP turnover were reduced in hypoxic PASMC compared to controls. Hypoxic PASMC had higher basal Ca2+, and a heightened peak Ca2+ response to thromboxane challenge compared to controls. Addition of SNP further elevated the peak Ca2+ response, while addition of FeTPPS brought peak Ca2+ response down to control levels. AC mediated relaxation was impaired in hypoxic PASMC compared to controls but was normalized following treatment with FeTPPS. Addition of SNP inhibited adenylyl cyclase activity in both normoxic and hypoxic PASMC. Moreover, addition of the Ca2+ chelator BAPTA improved AC activity, but the effect was minimal. Discussion: We conclude that NO independently augments contraction and inhibits relaxation pathways in hypoxic PASMC, in part by a mechanism involving nitrogen radical formation and protein nitrosylation. These observations may partially explain impaired effectiveness of NO when treating hypoxic pulmonary hypertension., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hinton, Thliveris, Hatch and Dakshinamurti.)

Background: Two anti-cancer agents, doxorubicin (DOX) and trastuzumab (TRZ), are commonly used in the management of breast cancer in women. Despite their efficacy in reducing the morbidity and mortality of individuals with breast cancer, the use of these agents is limited by adverse cardiotoxic side effects. Both the nutraceutical agent flaxseed (FLX) and the pharmaceutical drug perindopril (PER) have been studied individually in the prevention of chemotherapy-mediated cardiac dysfunction. The objective of this study was to determine whether the prophylactic administration of FLX is comparable and/or synergistic with PER in preventing DOX + TRZ-induced cardiotoxicity., Methods: Over a six-week period, 81 wild-type C57Bl/6 female mice (8-12 weeks old) were randomized to receive regular chow (RC) or 10% FLX-supplemented diets with or without PER (3 mg/kg/week; oral gavage). Starting at week 4, mice were randomized to receive a weekly injection of saline or DOX (8 mg/kg) + TRZ (3 mg/kg). Serial echocardiography was conducted weekly and histological and biochemical analyses were performed at the end of the study., Results: In mice treated with RC + DOX + TRZ, left ventricular ejection (LVEF) decreased from 75 ± 2% at baseline to 37 ± 3% at week 6. However, prophylactic treatment with either FLX, PER, or FLX + PER partially preserved left ventricular systolic function with LVEF values of 61 ± 2%, 62 ± 2%, and 64 ± 2%, respectively. The administration of FLX, PER, or FLX + PER was also partially cardioprotective in preserving cardiomyocyte integrity and attenuating the expression of the inflammatory biomarker NF-κB due to DOX + TRZ administration., Conclusion: FLX was equivalent to PER at preventing DOX + TRZ-induced cardiotoxicity in a chronic in vivo murine model.

Although doxorubicin (DOX) is an effective anti-cancer drug with cytotoxicity in a variety of different tumors, its effectiveness in treating glioblastoma multiforme (GBM) is constrained by insufficient penetration across the blood-brain barrier (BBB). In this study, biocompatible magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a carrier of DOX for GBM chemotherapy. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) released DOX within 4 days with the capability of an accelerated release in acidic microenvironments. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) demonstrated an efficient uptake in mouse brain-derived microvessel endothelial, bEnd.3, Madin-Darby canine kidney transfected with multi-drug resistant protein 1 (MDCK-MDR1), and human U251 GBM cells. The DOX-EDT-IONPs could augment DOX's uptake in U251 cells by 2.8-fold and significantly inhibited U251 cell proliferation. Moreover, the DOX-EDT-IONPs were found to be effective in apoptotic-induced GBM cell death (over 90%) within 48 h of treatment. Gene expression studies revealed a significant downregulation of TOP II and Ku70, crucial enzymes for DNA repair and replication, as well as MiR-155 oncogene, concomitant with an upregulation of caspase 3 and tumor suppressors i.e., p53, MEG3 and GAS5, in U251 cells upon treatment with DOX-EDT-IONPs. An in vitro MDCK-MDR1-GBM co-culture model was used to assess the BBB permeability and anti-tumor activity of the DOX-EDT-IONPs and DOX treatments. While DOX-EDT-IONP showed improved permeability of DOX across MDCK-MDR1 monolayers compared to DOX alone, cytotoxicity in U251 cells was similar in both treatment groups. Using a cadherin binding peptide (ADTC5) to transiently open tight junctions, in combination with an external magnetic field, significantly enhanced both DOX-EDT-IONP permeability and cytotoxicity in the MDCK-MDR1-GBM co-culture model. Therefore, the combination of magnetic enhanced convective diffusion and the cadherin binding peptide for transiently opening the BBB tight junctions are expected to enhance the efficacy of GBM chemotherapy using the DOX-EDT-IONPs. In general, the developed approach enables the chemotherapeutic to overcome both BBB and multidrug resistance (MDR) glioma cells while providing site-specific magnetic targeting.

Although anticancer systemic therapy agents clearly lead to improved survival in patients with cancer, these can come at the cost of serious complications including cardiotoxicity. Two types of targeted systemic therapies currently in use for colorectal cancer (CRC) and renal cell cancer (RCC), respectively, include the vascular endothelial growth factor inhibitor bevacizumab (BVZ) and the tyrosine kinase inhibitor sunitinib (SNT). Despite the beneficial effects of BVZ and SNT in improving clinical outcomes in the settings of CRC and RCC, there is an increased risk of cardiac dysfunction. The aim of the present study was to determine whether prophylactic administration of renin-angiotensin system (RAS) inhibitors would attenuate the cardiotoxic side effects of BVZ or SNT in a chronic in vivo murine model. A total of 194 wild-type C57Bl/6 male mice received: 1) 0.9% saline, 2) BVZ (10 mg·kg -1 ·wk -1 ), or 3) SNT (40 mg·kg -1 ·day -1 ) for 4 wk. Within each arm, mice received daily prophylactic treatment with hydralazine (0.05 mg/ml), aliskiren (50 mg/kg), perindopril (4 mg/kg), or valsartan (2 mg/kg). Although hydralazine effectively lowered blood pressure in BVZ- or SNT-treated mice, it did not prevent left ventricular systolic dysfunction. Prophylactic administration of aliskiren, perindopril, or valsartan prevented adverse cardiovascular remodeling in mice treated with either BVZ or SNT. The addition of RAS antagonists also downregulated expression of phosphorylated p38 and Bcl-2-like 19-kDa interacting protein 3 in SNT-treated mice. In our chronic in vivo murine model, RAS antagonists partially attenuated the development of BVZ- or SNT-mediated cardiac dysfunction. Future clinical studies are warranted to investigate the cardioprotective effects of prophylactic treatment with RAS inhibitors in the settings of CRC and RCC. NEW & NOTEWORTHY In the evolving field of cardio-oncology, bevacizumab and sunitinib improve clinical outcomes in the settings of metastatic colorectal cancer and renal cell cancer, respectively. These anticancer drugs, however, are associated with an increased risk of cardiotoxicity. The prophylactic administration of renin-angiotensin system antagonists is partially cardioprotective against bevacizumab- and sunitinib-mediated cardiac dysfunction.

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells significantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co-treatment with TMZ significantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not significantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is significantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death., Competing Interests: The authors declare that they have no conflict of interest.

Oligodendroglial cell death and demyelination are hallmarks of neurotrauma and multiple sclerosis that cause axonal damage and functional impairments. Remyelination remains a challenge as the ability of endogenous precursor cells for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating conditions. Here, in a rat model of lysolecithin lysophosphatidyl-choline (LPC)-induced focal demyelination, we report that Neuregulin-1 (Nrg-1), an important factor for oligodendrocytes and myelination, is dysregulated in demyelinating lesions and its bio-availability can promote oligodendrogenesis and remyelination. We delivered recombinant human Nrg-1β1 (rhNrg-1β1) intraspinally in the vicinity of LPC demyelinating lesion in a sustained manner using poly lactic-co-glycolic acid microcarriers. Availability of Nrg-1 promoted generation and maturation of new oligodendrocytes, and accelerated endogenous remyelination by both oligodendrocyte and Schwann cell populations in demyelinating foci. Importantly, Nrg-1 enhanced myelin thickness in newly remyelinated spinal cord axons. Our complementary in vitro studies also provided direct evidence that Nrg-1 significantly promotes maturation of new oligodendrocytes and facilitates their transition to a myelinating phenotype. Nrg-1 therapy remarkably attenuated the upregulated expression chondroitin sulfate proteoglycans (CSPGs) specific glycosaminoglycans in the extracellular matrix of demyelinating foci and promoted interleukin-10 (IL-10) production by immune cells. CSPGs and IL-10 are known to negatively and positively regulate remyelination, respectively. We found that Nrg-1 effects are mediated through ErbB2 and ErbB4 receptor activation. Our work provides novel evidence that dysregulated levels of Nrg-1 in demyelinating lesions of the spinal cord pose a challenge to endogenous remyelination, and appear to be an underlying cause of myelin thinning in newly remyelinated axons., (© 2017 Wiley Periodicals, Inc.)

Background: In the breast cancer setting, anticancer therapies including doxorubicin (DOX) and trastuzumab (TRZ) are associated with a significantly increased risk of cardiotoxicity. Despite the increasing support for the role of oxidative stress (OS) in its pathophysiology, we still do not have an optimal antioxidant for the prevention of DOX + TRZ-mediated cardiac dysfunction. The objective of this study was to investigate whether the novel antioxidant N-acetylcysteine amide (NACA) can attenuate DOX + TRZ-induced heart failure in a murine model., Methods: A total of 100 C57Bl/6 female mice received 1 of the following drug regimens: (1) saline, (2) NACA, (3) DOX, (4) TRZ, (5) DOX + TRZ, (6) NACA + DOX, (7) NACA + TRZ, and (8) NACA + DOX + TRZ. Serial echocardiography was performed over a 10-day study period, after which the mice were killed for histologic and biochemical analyses., Results: In mice receiving DOX, the left ventricular ejection fraction (LVEF) decreased from 73% ± 4% to 43% ± 2% on day 10. In mice receiving DOX + TRZ, the LVEF decreased from 72% ± 3% to 32% ± 2% on day 10. Prophylactic administration of NACA to mice receiving DOX or DOX + TRZ was cardioprotective, with an LVEF of 62% ± 3% and 55% ± 3% on day 10, respectively. Histologic and biochemical analyses demonstrated a loss of cellular integrity, increased OS, and increased cardiac apoptosis in mice treated with DOX + TRZ, which was attenuated by the prophylactic administration of NACA., Conclusions: NACA attenuated the cardiotoxic side effects of DOX + TRZ in a murine model of chemotherapy-induced cardiac dysfunction by decreasing OS and apoptosis., (Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Effective treatment of brain disorders requires a focus on improving drug permeability across the blood-brain barrier (BBB). Herein, we examined the pharmacokinetic properties of negatively charged iron oxide nanoparticles (IONPs) and the capability of using lysophosphatidic acid (LPA) to transiently disrupt the tight junctions and allow IONPs to enter the brain. Under normal conditions, IONPs had a plasma half-life of six minutes, with the liver and spleen being the major organs of deposition. Treatment with LPA enhanced accumulation of IONPs in the brain and spleen (approximately 4-fold vs. control). LPA and IONP treated mice revealed no sign of peripheral immune cell infiltration in the brain and no significant activation of microglia or astrocytes. These studies show improved delivery efficiency of IONPs following LPA administration. Our findings suggest transient disruption of the BBB may be a safe and effective method for increasing IONP delivery to the brain., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19 kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3(-/-) mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy.

Background: Aminosilane-coated iron oxide nanoparticles (AmS-IONPs) have been widely used in constructing complex and multifunctional drug delivery systems. However, the biocompatibility and uptake characteristics of AmS-IONPs in central nervous system (CNS)-relevant cells are unknown. The purpose of this study was to determine the effect of surface charge and magnetic field on toxicity and uptake of AmS-IONPs in CNS-relevant cell types., Methods: The toxicity and uptake profile of positively charged AmS-IONPs and negatively charged COOH-AmS-IONPs of similar size were examined using a mouse brain microvessel endothelial cell line (bEnd.3) and primary cultured mouse astrocytes and neurons. Cell accumulation of IONPs was examined using the ferrozine assay, and cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay., Results: No toxicity was observed in bEnd.3 cells at concentrations up to 200 μg/mL for either AmS-IONPs or COOH-AmS-IONPs. AmS-IONPs at concentrations above 200 μg/mL reduced neuron viability by 50% in the presence or absence of a magnetic field, while only 20% reductions in viability were observed with COOH-AmS-IONPs. Similar concentrations of AmS-IONPs in astrocyte cultures reduced viability to 75% but only in the presence of a magnetic field, while exposure to COOH-AmS-IONPs reduced viability to 65% and 35% in the absence and presence of a magnetic field, respectively. Cellular accumulation of AmS-IONPs was greater in all cell types examined compared to COOH-AmS-IONPs. Rank order of cellular uptake for AmS-IONPs was astrocytes > bEnd.3 > neurons. Accumulation of COOH-AmS-IONPs was minimal and similar in magnitude in different cell types. Magnetic field exposure enhanced cellular accumulation of both AmS- and COOH-AmS-IONPs., Conclusion: Both IONP compositions were nontoxic at concentrations below 100 μg/mL in all cell types examined. At doses above 100 μg/mL, neurons were more sensitive to AmS-IONPs, whereas astrocytes were more vulnerable toward COOH-AmS-IONPs. Toxicity appears to be dependent on the surface coating as opposed to the amount of iron-oxide present in the cell.

Vanadium can induce potent hypoglycemic effects in type 1 and type 2 diabetes mellitus animals, but toxic adverse effects have inhibited the translation of these findings. Administration of vanadate in a black tea decoction has shown impressive hypoglycemic effects without evidence of toxicity in short-term studies. The purpose of this study was to investigate the hypoglycemic action and the toxic adverse effects of a tea/vanadate (T/V) decoction in diabetic rats over a 14-month treatment period. Streptozotocin-induced type 1 diabetes mellitus rats were orally gavaged with 40 mg sodium vanadate in a black tea decoction only when blood glucose levels were greater than 10 mmol/L. Glycemic status and liver and kidney function were monitored over 14 months. All of the diabetic rats in this treatment group (n = 25) required treatment with the T/V decoction at the start of the study to reduce blood glucose levels to less than 10 mmol/L. Diarrhea was uncommon among the T/V-treated animals during the first week of T/V treatment and was absent thereafter. There was no evidence of liver or kidney dysfunction or injury. From 2 to 6 months, fewer animals required the T/V treatment to maintain their blood glucose levels. After 9 months of treatment, none of the diabetic animals required any T/V to maintain their blood glucose levels at less than 10 mmol/L. Oral administration of a T/V decoction provides safe, long-acting hypoglycemic effects in type 1 diabetes mellitus rats. The typical glycemic signs of diabetes were absent for the last 5 months of the study., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although amino acid deficiencies are known to occur in diabetes patients and are considered to contribute to the occurrence of cardiomyopathy, the mechanisms of the impact of the restoration of amino acids on improved cardiac function are not completely understood. Accordingly, the present study was conducted to examine the beneficial effects of dietary supplementation of taurine, arginine and carnitine, individually or in combination, in an experimental model of chronic diabetes. For inducing diabetes, rats received a single injection of streptozotocin (65 mg/kg body weight). Experimental animals were treated (by oral gavage) daily for three weeks with amino acids before the induction of diabetes; this treatment was continued for an additional eight-week period. Diabetes was observed to induce cardiac dysfunction, myocardial cell damage, and changes in plasma glucose and lipid levels. Treatment of diabetic animals with taurine, unlike carnitine or arginine, attenuated alterations in cardiac function, as evidenced by echocardiography and in vivo catheterization techniques. Taurine, carnitine and arginine, individually or in combination, attenuated diabetes-induced cell damage as revealed by electron microscopy. While carnitine alone reduced plasma levels of triglycerides with an increase in high-density lipoprotein cholesterol, none of the amino acids, alone or in combination, had an effect on myocardial glycogen content, lipid accumulation or hyperglycemia. These results suggest that dietary supplementation of taurine attenuates diabetes-induced changes in cardiac contractile function and ultrastructure without any alterations in plasma lipid and glucose levels.