<i>SLCA71</i> promoter activity in response to insulin.

<p>(a) Luciferase (<i>Luc</i>) reporter constructs containing serial truncations of <i>SLC7A1</i> promoter (−1606 bp (pGL3-hCAT-1⁻¹⁶⁰⁶) or −650 bp (pGL3-hCAT-1⁻¹⁶⁰⁶) from the transcriptional start point) were transfected in primary cultures of HUVECs incubated (8 hours) without (plain bars) or with (dashed bars) 10 μmol/L nitrobenzylthioinosine (NBTI). Cell transfection was done in the absence or presence of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L), along with <i>Renilla</i> reporter plasmid, and assayed for <i>Firefly</i> and <i>Renilla</i> luciferase activity, respectively. Results depict ratio of <i>Firefly</i>/<i>Renilla</i> luciferase activity. Cells were also transfected with the empty pGL3-basic vector or pGL3-control vector (SV40 pGL3) as negative or positive controls, respectively. (b) Fraction of <i>SLC7A1</i> reporter constructs activity inhibited (sensitive) by ZM-241385 in absence (Basal) or presence of insulin or NBTI as in (a). (c) <i>SLC7A1</i> reporter constructs fraction activity non-inhibited (insensitive) by ZM-241385 as in (a). In (a), *<i>P</i><0.05 versus Control, †<i>P</i><0.05 versus corresponding values in the presence of ZM-241385. In (b), *<i>P</i><0.05 versus corresponding Basal, †<i>P</i><0.05 versus values in pGL3-hCAT-1⁻¹⁶⁰⁶ in the presence of insulin, ‡<i>P</i><0.05 versus values in pGL3-hCAT-1⁻⁶⁵⁰ in the presence of insulin or insulin + NBTI. Values are mean ± SEM (n = 6).</p

L-Arginine transport and extracellular adenosine concentration.

<p>(a) Overall (300 µmol/L) L-arginine transport in HUVECs cultured (8 hours) in absence (white bars) or presence (black bars) of 1 nmol/L insulin, without (–NBTI) or with (+NBTI) 10 µmol/L nitrobenzylthioinosine (NBTI). Cells were co-incubated in culture medium without (–) or with (+) CGS-21680 (30 nmol/L) or ZM-21385 (10 nmol/L). (b) L-Arginine transport in the absence or presence of NECA (10 nmol/L) or DPCPX (10 nmol/L) in cells as in (a). (c) Adenosine concentration in the culture medium in cells as in (a). *<i>P</i><0.05 versus cells in the absence of NBTI, CGS-21680 and ZM-241385 (control). Values are mean ± SEM (n = 6–18).</p

hCAT-2A/B protein abundance in response to insulin and adenosine receptor agonists and antagonists.

<p>(a) Western blots (representative of other 6 experiments) for hCAT-2A/B in HUVECs incubated (8 hours) without (–NBTI) or with (+NBTI) 10 μmol/L nitrobenzylthioinosine (NBTI), in the absence (–) or presence (+) of 1 nmol/L insulin, CGS-21680 (30 nmol/L) and/or ZM-241385 (10 nmol/L) (ß-actin is internal control). <i>Lower panel</i>: hCAT-2A/B/ß-actin ratio densitometries from data in absence (white bars) or presence (black bars) of NBTI normalized to 1 in cells in the absence of NBTI, insulin, CGS-21680 or ZM-241385 (control). Values are mean ± SEM (n = 6).</p

Proposed model for insulin action requiring adenosine receptors on L-arginine transport in human fetal endothelial cells.

<p>Human umbilical vein endothelial cells respond to insulin via activation of insulin receptors (IR) leading to a reduced (–) adenosine uptake via the human equilibrative nucleoside transporters (hENTs). Insulin reduced adenosine uptake leading to extracellular accumulation of this nucleoside, which turns into activation of A_2A adenosine receptors (A_2A) at the plasma membrane. Activation of these membrane receptors leads to a mechanism mediated by transcription factors acting as activators (+) between −1606 and −650 bp from the transcription start point of <i>SLC7A1</i> (for hCAT-1) gene promoter. Alternatively, insulin activates transcription factors acting as activators (+) between −650 bp and the transcription start point of <i>SLC7A1</i>. This phenomenon increases (+) by adenosine receptor-activated transcription factors increasing hCAT-1 mRNA expression and protein abundance resulting in stimulation of L-arginine transport by HUVECs in response to insulin.</p

Clinical characteristics of patients and newborns.

<p>Data are mean ± SEM (range). OGTT, oral glucose tolerance test; BMI, body mass index.</p

hCAT-1 protein abundance in response to insulin and adenosine receptor agonists and antagonists.

<p>(a) Western blots (representative of other 16 experiments) for hCAT-1 in HUVECs incubated (8 hours) without (–NBTI) or with (+NBTI) 10 μmol/L nitrobenzylthioinosine (NBTI), in the absence (–) or presence (+) of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L) (ß-actin is internal control). <i>Lower panel</i>: hCAT-1/ß-actin ratio densitometries from data in absence (white bars) or presence (black bars) of NBTI, normalized to 1 in cells in the absence of NBTI, insulin and ZM-241385 (control). (b) Western blots for hCAT-1 in the absence or presence of insulin, CGS-21680 and/or ZM-241385 as in (a). <i>Lower panel</i>: hCAT-1/ß-actin ratio densitometries from data in absence (white bars) or presence (black bars) of NBTI normalized to 1 in cells in absence of NBTI, insulin, CGS-21680 and ZM-241385 (control). (c) hCAT-1 mRNA expression relative to 28S rRNA (internal reference) as in (a) and (b). *<i>P</i><0.05 versus values in control. Values are mean ± SEM (n = 16).</p

L-Arginine transport kinetics.

<p>L-Arginine transport kinetics (3 μCi/mL L-[³H] arginine, 1 minute, 37°C) in HUVECs cultured (8 hours) without (–NBTI; a, c) or with (+NBTI; b, d) 10 μmol/L nitrobenzylthioinosine (NBTI). (a, b) Saturable L-arginine transport in absence (control) or presence of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L) or CGS-21680 (30 nmol/L) as indicated. (c, d) Eadie-Hofstee plot of the L-arginine transport data in (a) and (b), respectively. Values are mean ± SEM (n = 6–18).</p

Gugulipid causes hypercholesterolemia leading to endothelial dysfunction, increased atherosclerosis, and premature death by ischemic heart disease in male mice

<div><p>For proper cholesterol metabolism, normal expression and function of scavenger receptor class B type I (SR-BI), a high-density lipoprotein (HDL) receptor, is required. Among the factors that regulate overall cholesterol homeostasis and HDL metabolism, the nuclear farnesoid X receptor plays an important role. Guggulsterone, a bioactive compound present in the natural product gugulipid, is an antagonist of this receptor. This natural product is widely used globally as a natural lipid-lowering agent, although its anti-atherogenic cardiovascular benefit in animal models or humans is unknown. The aim of this study was to determine the effects of gugulipid on cholesterol homeostasis and development of mild and severe atherosclerosis in male mice. For this purpose, we evaluated the impact of gugulipid treatment on liver histology, plasma lipoprotein cholesterol, endothelial function, and development of atherosclerosis and/or ischemic heart disease in wild-type mice; apolipoprotein E knockout mice, a model of atherosclerosis without ischemic complications; and SR-B1 knockout and atherogenic–diet-fed apolipoprotein E hypomorphic (SR-BI KO/ApoER61^h/h) mice, a model of lethal ischemic heart disease due to severe atherosclerosis. Gugulipid administration was associated with histological abnormalities in liver, increased alanine aminotransferase levels, lower hepatic SR-BI content, hypercholesterolemia due to increased HDL cholesterol levels, endothelial dysfunction, enhanced atherosclerosis, and accelerated death in animals with severe ischemic heart disease. In conclusion, our data show important adverse effects of gugulipid intake on HDL metabolism and atherosclerosis in male mice, suggesting potential and unknown deleterious effects on cardiovascular health in humans. In addition, these findings reemphasize the need for rigorous preclinical and clinical studies to provide guidance on the consumption of natural products and regulation of their use in the general population.</p></div

Effect of gugulipid on plasma cholesterol and hepatic SR-BI and LDL receptor (LDL-R) level in male ApoE KO mice.

<p>(A) Plasma total cholesterol and (B) western blot analysis (left) and quantitation (right) of hepatic LDL-R and SR-BI in control atherogenic diet-fed and gugulipid-treated atherogenic diet-fed mice. AD: atherogenic diet; G: gugulipid. Two independent experimental sets were used; n = 6 in each set. Values represent mean ± SD. *<i>P</i><0.05.</p

Effect of gugulipid on plasma cholesterol and hepatic SR-BI and LDL receptor (LDL-R) level in male ApoE KO mice.

<p>(A) Plasma total cholesterol and (B) western blot analysis (left) and quantitation (right) of hepatic LDL-R and SR-BI in control atherogenic diet-fed and gugulipid-treated atherogenic diet-fed mice. AD: atherogenic diet; G: gugulipid. Two independent experimental sets were used; n = 6 in each set. Values represent mean ± SD. *<i>P</i><0.05.</p