Correction: Paszkiewicz et al. A Peripheral CB1R Antagonist Increases Lipolysis, Oxygen Consumption Rate, and Markers of Beiging in 3T3-L1 Adipocytes Similar to RIM, Suggesting That Central Effects Can Be Avoided. Int. J. Mol. Sci. 2020, 21, 6639.

The authors wish to make the following corrections to this paper [...].

A Peripheral CB1R Antagonist Increases Lipolysis, Oxygen Consumption Rate, and Markers of Beiging in 3T3-L1 Adipocytes Similar to RIM, Suggesting that Central Effects Can Be Avoided.

With the increased prevalence of obesity and related co-morbidities, such as type 2 diabetes (T2D), worldwide, improvements in pharmacological treatments are necessary. The brain- and peripheral-cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been shown to induce weight loss and improve glucose homeostasis. We have previously demonstrated that RIM promotes adipose tissue beiging and decreased adipocyte cell size, even during maintenance on a high-fat diet. Given the adverse side-effects of brain-penetrance with RIM, in this study we aimed to determine the site of action for a non-brain-penetrating CB1R antagonist AM6545. By using in vitro assays, we demonstrated the direct effects of this non-brain-penetrating CB1R antagonist on cultured adipocytes. Specifically, we showed, for the first time, that AM6545 significantly increases markers of adipose tissue beiging, mitochondrial biogenesis, and lipolysis in 3T3-L1 adipocytes. In addition, the oxygen consumption rate (OCR), consisting of baseline respiratory rate, proton leak, maximal respiratory capacity, and ATP synthase activity, was greater for cells exposed to AM6545, demonstrating greater mitochondrial uncoupling. Using a lipolysis inhibitor during real-time OCR measurements, we determined that the impact of CB1R antagonism on adipocytes is driven by increased lipolysis. Thus, our data suggest the direct role of CB1R antagonism on adipocytes does not require brain penetrance, supporting the importance of focus on peripheral CB1R antagonism pharmacology for reducing the incidence of obesity and T2D.

Activation of NPRs and UCP1-independent pathway following CB1R antagonist treatment is associated with adipose tissue beiging in fat-fed male dogs.

CB1 receptor (CB1R) antagonism improves the deleterious effects of a high-fat diet (HFD) by reducing body fat mass and adipocyte cell size. Previous studies demonstrated that the beneficial effects of the CB1R antagonist rimonabant (RIM) in white adipose tissue (WAT) are partially due to an increase of mitochondria numbers and upregulation thermogenesis markers, suggesting an induction of WAT beiging. However, the molecular mechanism by which CB1R antagonism induces weight loss and WAT beiging is unclear. In this study, we probed for genes associated with beiging and explored longitudinal molecular mechanisms by which the beiging process occurs. HFD dogs received either RIM (HFD+RIM) or placebo (PL) (HFD+PL) for 16 wk. Several genes involved in beiging were increased in HFD+RIM compared with pre-fat, HFD, and HFD+PL. We evaluated lipolysis and its regulators including natriuretic peptide (NP) and its receptors (NPRs), ß-1 and ß-3 adrenergic receptor (ß1R, ß3R) genes. These genes were increased in WAT depots, accompanied by an increase in lipolysis in HFD+RIM. In addition, RIM decreased markers of inflammation and increased adiponectin receptors in WAT. We observed a small but significant increase in UCP1; therefore, we evaluated the newly discovered UCP1-independent thermogenesis pathway. We confirmed that SERCA2b and RYR2, the two key genes involved in this pathway, were upregulated in the WAT. Our data suggest that the upregulation of NPRs, ß-1R and ß-3R, lipolysis, and SERCA2b and RYR2 may be one of the mechanisms by which RIM promotes beiging and overall the improvement of metabolic homeostasis induced by RIM.

CB1R antagonist increases hepatic insulin clearance in fat-fed dogs likely via upregulation of liver adiponectin receptors.

The improvement of hepatic insulin sensitivity by the cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been recently been reported to be due to upregulation of adiponectin. Several studies demonstrated that improvement in insulin clearance accompanies the enhancement of hepatic insulin sensitivity. However, the effects of RIM on hepatic insulin clearance (HIC) have not been fully explored. The aim of this study was to explore the molecular mechanism(s) by which RIM affects HIC, specifically to determine whether upregulation of liver adiponectin receptors (ADRs) and other key genes regulated by adiponectin mediate the effects. To induce insulin resistance in skeletal muscle and liver, dogs were fed a hypercaloric high-fat diet (HFD) for 6 wk. Thereafter, while still maintained on a HFD, animals received RIM (HFD+RIM; n = 11) or placebo (HFD+PL; n = 9) for an additional 16 wk. HIC, calculated as the metabolic clearance rate (MCR), was estimated from the euglycemic-hyperinsulinemic clamp. The HFD+PL group showed a decrease in MCR; in contrast, the HFD+RIM group increased MCR. Consistently, the expression of genes involved in HIC, CEACAM-1 and IDE, as well as gene expression of liver ADRs, were increased in the HFD+RIM group, but not in the HFD+PL group. We also found a positive correlation between CEACAM-1 and the insulin-degrading enzyme IDE with ADRs. Interestingly, expression of liver genes regulated by adiponectin and involved in lipid oxidation were increased in the HFD+RIM group. We conclude that in fat-fed dogs RIM enhances HIC, which appears to be linked to an upregulation of the adiponectin pathway.

Hepatic portal vein denervation impairs oral glucose tolerance but not exenatide's effect on glycemia.

The hepatoportal area is an important glucohomeostatic metabolic sensor, sensing hypoglycemia, hyperglycemia, and hormones such as glucagon-like peptide-1 (GLP-1). We have reported previously that activation of hepatoportal sensors by intraportal infusion of glucose and GLP-1 or by subcutaneous administration of GLP-1 receptor activator exenatide and of intraportal glucose improved glycemia independent of corresponding changes in pancreatic hormones. It is not clear whether this effect is mediated via the portal vein (PV) or by direct action on the liver itself. To test whether receptors in the PV mediate exenatide's beneficial effect on glucose tolerance, we performed 1) paired oral glucose tolerance tests (OGTT) with and without exenatide and 2) intravenous glucose tolerance tests before and after PV denervation in canines. Denervation of the portal vein affected oral glucose tolerance; post-denervation (POST-DEN) OGTT glucose and insulin AUC were 50% higher than before denervation (P = 0.01). However, portal denervation did not impair exenatide's effect to improve oral glucose tolerance (exenatide effect: 48 ± 12 mmol·l⁻¹·min before vs. 64 ± 26 mmol·l⁻¹·min after, P = 0.67). There were no changes in insulin sensitivity or secretion during IVGTTs. Portal vein sensing might play a role in controlling oral glucose tolerance during physiological conditions but not in pharmacological activation of GLP-1 receptors by exenatide.

Nasal tau immunotherapy clears intracellular tau pathology and improves cognitive functions in aged tauopathy mice.

Pathological tau aggregates cause cognitive decline in neurodegenerative tauopathies, including Alzheimer's disease (AD). These aggregates are prevalent within intracellular compartments. Current tau immunotherapies have shown limited efficacy in clearing intracellular tau aggregates and improving cognition in clinical trials. In this study, we developed toxic tau conformation-specific monoclonal antibody-2 (TTCM2), which selectively recognized pathological tau aggregates in brain tissues from patients with AD, dementia with Lewy bodies (DLB), and progressive supranuclear palsy (PSP). TTCM2 potently inhibited tau-seeding activity, an essential mechanism underlying tauopathy progression. To effectively target intracellular tau aggregates and ensure rapid delivery to the brain, TTCM2 was loaded in micelles (TTCM2-ms) and administered through the intranasal route. We found that intranasally administered TTCM2-ms efficiently entered the brain in hTau-tauopathy mice, targeting pathological tau in intracellular compartments. Moreover, a single intranasal dose of TTCM2-ms effectively cleared pathological tau, elevated synaptic proteins, and improved cognitive functions in aged tauopathy mice. Mechanistic studies revealed that TTCM2-ms cleared intracellular, synaptic, and seed-competent tau aggregates through tripartite motif-containing 21 (TRIM21), an intracellular antibody receptor and E3 ubiquitin ligase known to facilitate proteasomal degradation of cytosolic antibody-bound proteins. TRIM21 was found to be essential for TTCM2-ms-mediated clearance of tau pathology. Our study collectively provides evidence of the effectiveness of nasal tau immunotherapy in targeting and clearing intracellular tau pathology through TRIM21 and enhancing cognition in aged tauopathy mice. This study could be valuable in designing effective tau immunotherapies for AD and other tauopathies.

Bowen's disease of the nipple in a young man with AIDS: a case report.

Bowen's disease, or squamous cell carcinoma in situ (SCCIS) of the skin, is a malignant neoplasm restricted to the epidermis, without evidence of dermal invasion. It usually develops in sun-exposed area of skin, but other sites can also be affected. Bowen's disease of the nipple is extremely rare and has thus far been reported only in women. We present the case of Bowen's disease of the nipple in an HIV-positive male patient who presented with a scaly lesion on nipple for one year. He also had past genital infection with human papillomavirus, but he was found to be negative for high-risk subtypes. Biopsy of the lesion revealed SCCIS of the nipple areola complex, with extension into the underlying lactiferous ducts of the breast. There was no evidence of invasive carcinoma. The patient was treated with a simple mastectomy with sentinel lymph node biopsy. With the advent of highly active antiretroviral treatment, chronic non-HIV related conditions have become more important, male breast cancer being one of them. To the best of our knowledge, this is the first reported case in the worldwide literature of Bowen's disease of the nipple in a young immunocompromised male patient. More aggressive therapy in HIV-positive male patients presenting with precancerous and cancerous breast lesions is recommended.

Insulin Access to Skeletal Muscle is Preserved in Obesity Induced by Polyunsaturated Diet.

OBJECTIVE: Diets high in saturated fat induce obesity and insulin resistance and impair insulin access to skeletal muscle, leading to reduced insulin levels at the muscle cell surface available to bind insulin receptors and induce glucose uptake. In contrast, diets supplemented with polyunsaturated fat improve insulin sensitivity (SI) and reduce the risk for type 2 diabetes. It was hypothesized that a diet high in polyunsaturated fat would preserve SI and insulin access to muscle, as compared with a diet high in saturated fat. METHODS: After 12 weeks of control, saturated (LARD), or polyunsaturated (salmon oil [SO]) high-fat diet feeding, muscle SI and insulin access to skeletal muscle were measured by using lymph, a surrogate of skeletal muscle interstitial fluid. RESULTS: Both high-fat diets induced similar weight gain, yet only LARD impaired SI. Hyperinsulinemia in the LARD group did not induce an increase in basal interstitial insulin, suggesting reduced insulin access to muscle after LARD, but not after SO. CONCLUSIONS: A diet high in polyunsaturated fat does not impair insulin access to muscle interstitium or induce insulin resistance as observed with a saturated fat diet, despite similar weight gain. Future studies should determine whether dietary SO supplementation improves impairments in insulin access to skeletal muscle.

Indirect Regulation of Endogenous Glucose Production by Insulin: The Single Gateway Hypothesis Revisited.

On the basis of studies that investigated the intraportal versus systemic insulin infusion and transendothelial transport of insulin, we proposed the "single gateway hypothesis," which supposes an indirect regulation of hepatic glucose production by insulin; the rate-limiting transport of insulin across the adipose tissue capillaries is responsible for the slow suppression of free fatty acids (FFAs), which in turn is responsible for delayed suppression of hepatic endogenous glucose production (EGP) during insulin infusion. Preventing the fall in plasma FFAs during insulin infusion either by administering intralipids or by inhibiting adipose tissue lipolysis led to failure in EGP suppression, thus supporting our hypothesis. More recently, mice lacking hepatic Foxo1 in addition to Akt1 and Akt2 (L-AktFoxo1TKO), all required for insulin signaling, surprisingly showed normal glycemia. Inhibiting the fall of plasma FFAs in these mice prevented the suppression of EGP during a clamp, reaffirming that the site of insulin action to control EGP is extrahepatic. Measuring whole-body turnover rates of glucose and FFAs in L-AktFoxo1TKO mice also confirmed that hepatic EGP was regulated by insulin-mediated control of FFAs. The knockout mouse model in combination with sophisticated molecular techniques confirmed our physiological findings and the single gateway hypothesis.

Renal Denervation Reverses Hepatic Insulin Resistance Induced by High-Fat Diet.

Activation of the sympathetic nervous system (SNS) constitutes a putative mechanism of obesity-induced insulin resistance. Thus, we hypothesized that inhibiting the SNS by using renal denervation (RDN) will improve insulin sensitivity (SI) in a nonhypertensive obese canine model. SI was measured using euglycemic-hyperinsulinemic clamp (EGC), before (week 0 [w0]) and after 6 weeks of high-fat diet (w6-HFD) feeding and after either RDN (HFD + RDN) or sham surgery (HFD + sham). As expected, HFD induced insulin resistance in the liver (sham 2.5 ± 0.6 vs. 0.7 ± 0.6 × 10-4 dL ⋅ kg-1 ⋅ min-1 ⋅ pmol/L-1 at w0 vs. w6-HFD [P < 0.05], respectively; HFD + RDN 1.6 ± 0.3 vs. 0.5 ± 0.3 × 10-4 dL ⋅ kg-1 ⋅ min-1 ⋅ pmol/L-1 at w0 vs. w6-HFD [P < 0.001], respectively). In sham animals, this insulin resistance persisted, yet RDN completely normalized hepatic SI in HFD-fed animals (1.8 ± 0.3 × 10-4 dL ⋅ kg-1 ⋅ min-1 ⋅ pmol/L-1 at HFD + RDN [P < 0.001] vs. w6-HFD, [P not significant] vs. w0) by reducing hepatic gluconeogenic genes, including G6Pase, PEPCK, and FOXO1. The data suggest that RDN downregulated hepatic gluconeogenesis primarily by upregulating liver X receptor α through the natriuretic peptide pathway. In conclusion, bilateral RDN completely normalizes hepatic SI in obese canines. These preclinical data implicate a novel mechanistic role for the renal nerves in the regulation of insulin action specifically at the level of the liver and show that the renal nerves constitute a new therapeutic target to counteract insulin resistance.

Insulin access to skeletal muscle is impaired during the early stages of diet-induced obesity.

OBJECTIVE: Insulin must move from the blood to the interstitium to initiate signaling, yet access to the interstitium may be impaired in cases of insulin resistance, such as obesity. This study investigated whether consuming a short- and long-term high-fat diet (HFD) impairs insulin access to skeletal muscle, the major site of insulin-mediated glucose uptake. METHODS: Male mongrel dogs were divided into three groups consisting of control diet (n = 16), short-term (n = 8), and long-term HFD (n = 8). Insulin sensitivity was measured with intravenous glucose tolerance tests. A hyperinsulinemic euglycemic clamp was performed in each animal at the conclusion of the study. During the clamp, lymph fluid was measured as a representation of the interstitial space to assess insulin access to muscle. RESULTS: Short- and long-term HFD induced obesity and reduced insulin sensitivity. Lymph insulin concentrations were approximately 50% of plasma insulin concentrations under control conditions. Long-term HFD caused fasting plasma hyperinsulinemia; however, interstitial insulin concentrations were not increased, suggesting impaired insulin access to muscle. CONCLUSIONS: A HFD rapidly induces insulin resistance at the muscle and impairs insulin access under basal insulin concentrations. Hyperinsulinemia induced by a long-term HFD may be a compensatory mechanism necessary to maintain healthy insulin levels in muscle interstitium.

Exenatide Treatment Alone Improves ß-Cell Function in a Canine Model of Pre-Diabetes.

BACKGROUND: Exenatide's effects on glucose metabolism have been studied extensively in diabetes but not in pre-diabetes. OBJECTIVE: We examined the chronic effects of exenatide alone on glucose metabolism in pre-diabetic canines. DESIGN AND METHODS: After 10 weeks of high-fat diet (HFD), adult dogs received one injection of streptozotocin (STZ, 18.5 mg/kg). After induction of pre-diabetes, while maintained on HFD, animals were randomized to receive either exenatide (n = 7) or placebo (n = 7) for 12 weeks. ß-Cell function was calculated from the intravenous glucose tolerance test (IVGTT, expressed as the acute insulin response, AIRG), the oral glucose tolerance test (OGTT, insulinogenic index) and the graded-hyperglycemic clamp (clamp insulinogenic index). Whole-body insulin sensitivity was assessed by the IVGTT. At the end of the study, pancreatic islets were isolated to assess ß-cell function in vitro. RESULTS: OGTT: STZ caused an increase in glycemia at 120 min by 22.0% (interquartile range, IQR, 31.5%) (P = 0.011). IVGTT: This protocol also showed a reduction in glucose tolerance by 48.8% (IQR, 36.9%) (P = 0.002). AIRG decreased by 54.0% (IQR, 40.7%) (P = 0.010), leading to mild fasting hyperglycemia (P = 0.039). Exenatide, compared with placebo, decreased body weight (P<0.001) without altering food intake, fasting glycemia, insulinemia, glycated hemoglobin A1c, or glucose tolerance. Exenatide, compared with placebo, increased both OGTT- (P = 0.040) and clamp-based insulinogenic indexes (P = 0.016), improved insulin secretion in vitro (P = 0.041), but had no noticeable effect on insulin sensitivity (P = 0.405). CONCLUSIONS: In pre-diabetic canines, 12-week exenatide treatment improved ß-cell function but not glucose tolerance or insulin sensitivity. These findings demonstrate partial beneficial metabolic effects of exenatide alone on an animal model of pre-diabetes.

Increase in visceral fat per se does not induce insulin resistance in the canine model.

OBJECTIVES: To determine whether a selective increase of visceral adipose tissue content will result in insulin resistance. METHODS: Sympathetic denervation of the omental fat was performed under general inhalant anesthesia by injecting 6-hydroxydopamine in the omental fat of lean mongrel dogs (n = 11). In the conscious animal, whole-body insulin sensitivity was assessed by the minimal model (SI ) and the euglycemic hyperinsulinemic clamp (SICLAMP ). Changes in abdominal fat were monitored by magnetic resonance. All assessments were determined before (Wk0) and 2 weeks (Wk2) after denervation. Data are medians (upper and lower interquartile). RESULTS: Denervation of omental fat resulted in increased percentage (and content) of visceral fat [Wk0: 10.2% (8.5-11.4); Wk2: 12.4% (10.4-13.6); P < 0.01]. Abdominal subcutaneous fat remained unchanged. However, no changes were found in SI [Wk0: 4.7 (mU/l)(-1) min(-1) (3.1-8.8); Wk2: 5.3 (mU/l)(-1) min(-1) (4.5-7.2); P = 0.59] or SICLAMP [Wk0: 42.0 × 10(-4) dl kg(-1) min(-1) (mU/l)(-1) (41.0-51.0); Wk2: 40.0 × 10(-4) dl kg(-1) min(-1) (mU/l) (-1) (34.0-52.0); P = 0.67]. CONCLUSIONS: Despite a selective increase in visceral adiposity in dogs, insulin sensitivity in vivo did not change, which argues against the concept that accumulation of visceral adipose tissue contributes to insulin resistance.

High-fat diet-induced insulin resistance does not increase plasma anandamide levels or potentiate anandamide insulinotropic effect in isolated canine islets.

BACKGROUND: Obesity has been associated with elevated plasma anandamide levels. In addition, anandamide has been shown to stimulate insulin secretion in vitro, suggesting that anandamide might be linked to hyperinsulinemia. OBJECTIVE: To determine whether high-fat diet-induced insulin resistance increases anandamide levels and potentiates the insulinotropic effect of anandamide in isolated pancreatic islets. DESIGN AND METHODS: Dogs were fed a high-fat diet (n = 9) for 22 weeks. Abdominal fat depot was quantified by MRI. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp. Fasting plasma endocannabinoid levels were analyzed by liquid chromatography-mass spectrometry. All metabolic assessments were performed before and after fat diet regimen. At the end of the study, pancreatic islets were isolated prior to euthanasia to test the in vitro effect of anandamide on islet hormones. mRNA expression of cannabinoid receptors was determined in intact islets. The findings in vitro were compared with those from animals fed a control diet (n = 7). RESULTS: Prolonged fat feeding increased abdominal fat content by 81.3±21.6% (mean±S.E.M, P<0.01). In vivo insulin sensitivity decreased by 31.3±12.1% (P<0.05), concomitant with a decrease in plasma 2-arachidonoyl glycerol (from 39.1±5.2 to 15.7±2.0 nmol/L) but not anandamide, oleoyl ethanolamide, linoleoyl ethanolamide, or palmitoyl ethanolamide. In control-diet animals (body weight: 28.8±1.0 kg), islets incubated with anandamide had a higher basal and glucose-stimulated insulin secretion as compared with no treatment. Islets from fat-fed animals (34.5±1.3 kg; P<0.05 versus control) did not exhibit further potentiation of anandamide-induced insulin secretion as compared with control-diet animals. Glucagon but not somatostatin secretion in vitro was also increased in response to anandamide, but there was no difference between groups (P = 0.705). No differences in gene expression of CB1R or CB2R between groups were found. CONCLUSIONS: In canines, high-fat diet-induced insulin resistance does not alter plasma anandamide levels or further potentiate the insulinotropic effect of anandamide in vitro.

Essentiality of portal vein receptors in hypoglycemic counterregulation: direct proof via denervation in male canines.

A major issue of in the treatment of diabetes is the risk of hypoglycemia. Hypoglycemia is detected both centrally and peripherally in the porto-hepatic area. The portal locus for hypoglycemic detection was originally described using the "local irrigation of the liver" approach in a canine model. Further work using portal vein denervation (DEN) in a rodent model characterized portal hypoglycemic sensing in detail. However, recent controversy about the relevance of rodent findings to large animals and humans prompted us to investigate the effect of portal DEN on the hypoglycemic response in the canine, a species with multiple similarities to human glucose homeostasis. Hypoglycemic hyperinsulinemic clamps were performed in male canines, before (PRE) and after (POST) portal vein DEN or sham surgery (CON, control). Insulin (30 pmol/kg·min) and glucose (variable) were infused to slowly decrease systemic glycemia to 50 mg/dL over 160 minutes. The average plasma glucose during clamp steady state was: 2.9 ± 0.1 mmol DEN-PRE, 2.9 ± 0.2 mmol DEN-POST, 2.9 ± 0.1 mmol CON-PRE, and 2.8 ± 0.0 mmol CON-POST. There were no significant differences in plasma insulin between DEN and CON, PRE and POST experiments. The epinephrine response to hypoglycemia was reduced by 62% in DEN but not in CON. Steady-state cortisol was 46% lower after DEN but not after CON. Our study shows, in a large animal model, that surgical disconnection of the portal vein from the afferent pathway of the hypoglycemic counterregulatory circuitry results in a substantial suppression of the epinephrine response and a significant impact on cortisol response. These findings directly demonstrate an essential role for the portal vein in sensing hypoglycemia and relating glycemic information to the central nervous system.

Large size cells in the visceral adipose depot predict insulin resistance in the canine model.

Adipocyte size plays a key role in the development of insulin resistance. We examined longitudinal changes in adipocyte size and distribution in visceral (VIS) and subcutaneous (SQ) fat during obesity-induced insulin resistance and after treatment with CB-1 receptor antagonist, rimonabant (RIM) in canines. We also examined whether adipocyte size and/or distribution is predictive of insulin resistance. Adipocyte morphology was assessed by direct microscopy and analysis of digital images in previously studied animals 6 weeks after high-fat diet (HFD) and 16 weeks of HFD + placebo (PL; n = 8) or HFD + RIM (1.25 mg/kg/day; n = 11). At 6 weeks, mean adipocyte diameter increased in both depots with a bimodal pattern only in VIS. Sixteen weeks of HFD+PL resulted in four normally distributed cell populations in VIS and a bimodal pattern in SQ. Multilevel mixed-effects linear regression with random-effects model of repeated measures showed that size combined with share of adipocytes >75 µm in VIS only was related to hepatic insulin resistance. VIS adipocytes >75 µm were predictive of whole body and hepatic insulin resistance. In contrast, there was no predictive power of SQ adipocytes >75 µm regarding insulin resistance. RIM prevented the formation of large cells, normalizing to pre-fat status in both depots. The appearance of hypertrophic adipocytes in VIS is a critical predictor of insulin resistance, supporting the deleterious effects of increased VIS adiposity in the pathogenesis of insulin resistance.

TNF-alpha downregulates vascular endothelial Flk-1 expression in human melanoma xenograft model.

High-dose TNF with melphalan has significant antitumor activity in regional perfusion of the limbs and liver in human malignancies. TNF is believed to target tumor vasculature, but the precise molecular mechanism is unknown. The present study demonstrates that TNF downregulates the VEGF receptor, fetal liver kinase-1 (Flk-1), on tumor endothelium in a human melanoma xenograft model. NIH1286 human melanoma cells were transduced with a 720-bp fragment of the human VEGF(121) gene to develop well-vascularized tumors that served as an amplified system for measuring Flk-1 expression changes. We injected 5 x 10(6) cells subcutaneously, each of two distinct single cell clones (NIH1286/3 and NIH1286/15), into athymic nude mice to produce tumors approximately 10 mm in size. Each animal then received either BSA or TNF in BSA by tail vein. Tumors harvested at different time points post-TNF were analyzed for Flk-1 mRNA and protein expression. Data obtained showed that intravascular TNF downregulated Flk-1 expression in tumor endothelial cells. This effect could contribute to the antitumor activity of TNF known to target tumor vasculature.