Citations

Abstract Expression of neuropilin-1 (NRP-1) has been shown in many cancer cells, but its molecular effect on tumorigenesis is largely unknown. In this report, we show that in aggressive types of renal cell carcinoma (RCC), NRP-1 is expressed at a high level. We show that after knockdown of NRP-1 by short hairpin RNA, RCC cells express significantly lower levels of MDM-2 and p63 proteins but higher levels of p53, and exhibit reduced migration and invasion. When implanted in mice, RCC cells with a reduced NRP-1 level have a statistically significant smaller tumor-forming ability than control cells. Also, NRP-1 knockdown RCC cells exhibit a more differentiated phenotype, as evidenced by the expression of epithelial-specific and kidney-specific cadherins, and the inhibition of sonic hedgehog expression participated in this effect. Inhibition of sonic hedgehog expression can be reversed by ΔNp63α overexpression. Our study reveals that NRP-1 helps maintain an undifferentiated phenotype in cancer cells. [Cancer Res 2008;68(21):8667–72]

OBJECTIVE—To determine the contribution of liver and viscera to splanchnic cortisol production in humans. RESEARCH DESIGN AND METHODS—D4 cortisol was infused intravenously; arterial, portal venous, and hepatic venous blood was sampled; and liver and visceral fat were biopsied in subjects undergoing bariatric surgery. RESULTS—Ratios of arterial and portal vein D4 cortisol/cortisoltotal (0.06 ± 0.01 vs. 0.06 ± 0.01) and D4 cortisol/D3 cortisol (1.80 ± 0.14 vs. 1.84 ± 0.14) did not differ, indicating that no visceral cortisol production or conversion of D4 cortisol to D3 cortisol via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) occurred. Conversely, ratios of both D4 cortisol to cortisoltotal (0.05 ± 0.01; P < 0.05) and D4 cortisol to D3 cortisol (1.33 ± 0.11; P < 0.001) were lower in the hepatic vein than in the portal vein, indicating production of both cortisol and D3 cortisol by the liver. The viscera did not produce either cortisol (−8.1 ± 2.6 μg/min) or D3 cortisol (−0.2 ± 0.1 μg/min). In contrast, the liver produced both cortisol (22.7 ± 3.90 μg/min) and D3 cortisol (1.9 ± 0.4 μg/min) and accounted for all splanchnic cortisol and D3 cortisol production. Additionally, 11β-HSD-1 mRNA was approximately ninefold higher (P < 0.01) in liver than in visceral fat. Although 11β-HSD-2 gene expression was very low in visceral fat, the viscera released cortisone (P < 0.001) and D3 cortisone (P < 0.01) into the portal vein. CONCLUSIONS—The liver accounts for all splanchnic cortisol production in obese nondiabetic humans. In contrast, the viscera releases cortisone into the portal vein, thereby providing substrate for intrahepatic cortisol production.

OBJECTIVE—Current criteria for the diagnosis of diabetic ketoacidosis (DKA) are limited by their nonspecificity (serum bicarbonate [HCO3] and pH) and qualitative nature (the presence of ketonemia/ketonuria). The present study was undertaken to determine whether quantitative measurement of a ketone body anion could be used to diagnose DKA. RESEARCH DESIGN AND METHODS—A retrospective review of records from hospitalized diabetic patients was undertaken to determine the concentration of serum β-hydroxybutyrate (βOHB) that corresponds to a HCO3 level of 18 mEq/l, the threshold value for diagnosis in recently published consensus criteria. Simultaneous admission βOHB and HCO3 values were recorded from 466 encounters, 129 in children and 337 in adults. RESULTS—A HCO3 level of 18 mEq/l corresponded with βOHB levels of 3.0 and 3.8 mmol/l in children and adults, respectively. With the use of these threshold βOHB values to define DKA, there was substantial discordance (∼≥20%) between βOHB and conventional diagnostic criteria using HCO3, pH, and glucose. In patients with DKA, there was no correlation between HCO3 and glucose levels on admission and a significant but weak correlation between βOHB and glucose levels (P < 0.001). CONCLUSIONS—Where available, serum βOHB levels ≥3.0 and ≥3.8 mmol/l in children and adults, respectively, in the presence of uncontrolled diabetes can be used to diagnose DKA and may be superior to the serum HCO3 level for that purpose. The marked variability in the relationship between βOHB and HCO3 is probably due to the presence of other acid-base disturbances, especially hyperchloremic, nonanion gap acidosis.

OBJECTIVE—To determine mechanisms by which pioglitazone and metformin effect hepatic and extra-hepatic insulin action. RESEARCH DESIGN AND METHODS—Thirty-one subjects with type 2 diabetes were randomly assigned to pioglitazone (45 mg) or metformin (2,000 mg) for 4 months. RESULTS—Glucose was clamped before and after therapy at ∼5 mmol/l, insulin raised to ∼180 pmol/l, C-peptide suppressed with somatostatin, glucagon replaced at ∼75 pg/ml, and glycerol maintained at ∼200 mmol/l to ensure comparable and equal portal concentrations on all occasions. Insulin-induced stimulation of glucose disappearance did not differ before and after treatment with either pioglitazone (23 ± 3 vs. 24 ± 2 μmol · kg−1 · min−1) or metformin (22 ± 2 vs. 24 ± 3 μmol · kg−1 · min−1). In contrast, pioglitazone enhanced (P < 0.01) insulin-induced suppression of both glucose production (6.0 ± 1.0 vs. 0.2 ± 1.6 μmol · kg−1 · min−1) and gluconeogenesis (n = 11; 4.5 ± 0.9 vs. 0.8 ± 1.2 μmol · kg−1 · min−1). Metformin did not alter either suppression of glucose production (5.8 ± 1.0 vs. 5.0 ± 0.8 μmol · kg−1 · min−1) or gluconeogenesis (n = 9; 3.7 ± 0.8 vs. 2.6 ± 0.7 μmol · kg−1 · min−1). Insulin-induced suppression of free fatty acids was greater (P < 0.05) after treatment with pioglitazone (0.14 ± 0.03 vs. 0.06 ± 0.01 mmol/l) but unchanged with metformin (0.12 ± 0.03 vs. 0.15 ± 0.07 mmol/l). CONCLUSIONS—Thus, relative to metformin, pioglitazone improves hepatic insulin action in people with type 2 diabetes, partly by enhancing insulin-induced suppression of gluconeogenesis. On the other hand, both drugs have comparable effects on insulin-induced stimulation of glucose uptake.

OBJECTIVE—We sought to determine whether, and if so the mechanism by which, testosterone replacement improves carbohydrate tolerance. RESEARCH DESIGN AND METHODS—Fifty-five elderly men with relative testosterone deficiency ingested a labeled mixed meal and underwent a frequently sampled labeled intravenous glucose tolerance test before and after either placebo or treatment with testosterone patch (5 mg/day) for 2 years. RESULTS—Despite restoring bioavailable testosterone to values observed in young men, the change (24 months minus baseline values) in fasting and postprandial glucose, insulin, and C-peptide concentrations and meal appearance, glucose disposal, and endogenous glucose production were virtually identical to those observed after 2 years of placebo. The change over time in insulin and C-peptide concentrations post–intravenous glucose injection also did not differ. Furthermore, the change over time in insulin action and glucose effectiveness (measured with the unlabeled and labeled “oral” and “intravenous” minimal models), as well as insulin secretion and hepatic insulin clearance (measured with the C-peptide model), did not differ in the testosterone and placebo groups. CONCLUSIONS—We conclude that 2 years of treatment with testosterone in elderly men does not improve carbohydrate tolerance or alter insulin secretion, insulin action, glucose effectiveness, hepatic insulin clearance, or the pattern of postprandial glucose metabolism. Thus, testosterone deficiency is unlikely the cause of the age-associated deterioration in glucose tolerance commonly observed in elderly men.

Glucocorticoids are potent regulators of protein, fat, and carbohydrate metabolism. To determine if cortisol production occurs within the splanchnic bed in humans, 11 nondiabetic subjects were studied using the hepatic/leg catheterization method along with an infusion of [9,11,12,12-2H4] cortisol (D4-cortisol) as proposed by Andrews et al. In the fasting state, there was net release (P < 0.05) of cortisol from the splanchnic bed (6.1 ± 2.6 μg/min) and net uptake (P < 0.05) by the leg (1.7 ± 0.7 μg/min). This, along with cortisol production by other tissues (e.g., the adrenals), resulted in a total-body cortisol appearance rate of 18.1 ± 1.9 μg/min. Fractional splanchnic D4-cortisol extraction averaged 12.9 ± 1.3% (P < 0.001), splanchnic cortisol uptake 14.8 ± 2.0 μg/min (P < 0.001), and splanchnic cortisol production 22.2 ± 3.3 μg/min (P < 0.001). On the other hand, fractional leg D4-cortisol extraction averaged 5.6 ± 1.8% (P < 0.02), leg cortisol uptake 2.3 ± 0.7 μg/min (P < 0.01), and leg cortisol production 0.4 ± 0.4 μg/min, which did not differ from zero. Because D4-cortisol loses a deuterium during conversion to [9,12,12-2H3] cortisone (D3-cortisone), which in turn generates [9,12,122H3] cortisol (D3-cortisol) via 11-β hydroxysteroid dehydrogenase (11β-HSD) type 1, D3-cortisol production can be used as an index of 11β-HSD type 1 activity. Net splanchnic D3-cortisol release (3.9 ± 0.4 μg/min) and splanchnic D3-cortisol production (7.1 ± 0.7 μg/min) occurred (P < 0.01) in all subjects. In contrast, there was minimal leg D3-cortisol production (0.04 ± 0.01 μg/min), resulting in a strong correlation between splanchnic D3-cortisol production and total-body 3D-cortisol production in both the fasting state (r = 0.84; P < 0.02) and during an infusion of insulin (r = 0.97; P < 0.01). Thus, splanchnic production of cortisol occurs in nondiabetic humans at rates approximating that which occurs in the remainder of the body. These data support the possibility that alterations in splanchnic cortisol production contribute to visceral fat accumulation and the hepatic insulin resistance of obesity or type 2 diabetes.

To determine whether the insulin dose-response curves for suppression of endogenous glucose production (EGP) and stimulation of splanchnic glucose uptake (SGU) differ in nondiabetic humans and are abnormal in type 2 diabetes, 14 nondiabetic and 12 diabetic subjects were studied. Glucose was clamped at ∼9.5 mmol/l and endogenous hormone secretion inhibited by somatostatin, while glucagon and growth hormone were replaced by an exogenous infusion. Insulin was progressively increased from ∼150 to ∼350 and ∼700 pmol/l by means of an exogenous insulin infusion, while EGP, SGU, and leg glucose uptake (LGU) were measured using the splanchnic and leg catheterization methods, combined with a [3-3H]glucose infusion. In nondiabetic subjects, an increase in insulin from ∼150 to ∼350 pmol/l resulted in maximal suppression of EGP, whereas SGU continued to increase (P < 0.001) when insulin was increased to ∼700 pmol/l. In contrast, EGP progressively decreased (P < 0.001) and SGU progressively increased (P < 0.001) in the diabetic subjects as insulin increased from ∼150 to ∼700 pmol/l. Although EGP was higher (P < 0.01) in the diabetic than nondiabetic subjects only at the lowest insulin concentration, SGU was lower (P < 0.01) in the diabetic subjects at all insulin concentrations tested. On the other hand, in contrast to LGU and overall glucose disposal, the increment in SGU in response to both increments in insulin did not differ in the diabetic and nondiabetic subjects, implying a right shifted but parallel dose-response curve. These data indicate that the dose-response curves for suppression of glucose production and stimulation of glucose uptake differ in nondiabetic subjects and are abnormal in people with type 2 diabetes. Taken together, these data also suggest that agents that enhance SGU in diabetic patients (e.g. glucokinase activators) are likely to improve glucose tolerance.

The present study sought to determine whether elevated plasma free fatty acids (FFAs) alter the splanchnic and muscle glucose metabolism in women. To do so, FFAs were increased in seven women by an 8-h Intralipid/heparin (IL/hep) infusion, and the results were compared with those observed in nine women who were infused with glycerol alone. Glucose was clamped at ∼8.3 mmol/l and insulin was increased to ∼300 pmol/l to stimulate both muscle and hepatic glucose uptake. Insulin secretion was inhibited with somatostatin. Leg and splanchnic glucose metabolism were assessed using a combined catheter and tracer dilution approach. The glucose infusion rates required to maintain target plasma glucose concentrations were lower (P < 0.01) during IL/hep than glycerol infusion (30.8 ± 2.6 vs. 65.0 ± 7.9 μmol · kg−1 · min−1). Whole-body glucose disappearance (37.0 ± 2.2 vs. 70.9 ± 8.7 μmol · kg−1 · min−1; P < 0.001) and leg glucose uptake (24.3 ± 4.2 vs. 59.6 ± 10.0 μmol · kg fat-free mass of the leg−1 · min−1; P < 0.02) were also lower, whereas splanchnic glucose production (8.2 ± 0.8 vs. 4.3 ± 0.7 μmol · kg−1 · min−1; P < 0.01) was higher during IL/hep than glycerol infusion. We conclude that in the presence of combined hyperinsulinemia and hyperglycemia, elevated FFAs impair glucose metabolism in women by inhibiting whole- body glucose disposal, muscle glucose uptake, and suppression of splanchnic glucose production.

The present study sought to determine whether elevated plasma free fatty acids (FFAs) alter the ability of insulin and glucose to regulate splanchnic as well as muscle glucose metabolism. To do so, FFAs were increased in 10 subjects to ∼1 mmol/l by an 8-h Intralipid/heparin (IL/Hep) infusion, whereas they fell to levels near the detection limit of the assay (<0.05 mmol/l) in 13 other subjects who were infused with glycerol alone at rates sufficient to either match (n = 5, low glycerol) or double (n = 8, high glycerol) the plasma glycerol concentrations observed during the IL/Hep infusion. Glucose was clamped at ∼8.3 mmol/l, and insulin was increased to ∼300 pmol/l to stimulate both muscle and hepatic glucose uptake. Insulin secretion was inhibited with somatostatin. Leg and splanchnic glucose metabolism were assessed using a combined catheter and tracer dilution approach. Leg glucose uptake (21.7 ± 3.5 vs. 48.3 ± 9.3 and 57.8 ± 11.7 μmol · kg−1 leg · min−1) was lower (P < 0.001) during IL/Hep than the low- or high-glycerol infusions, confirming that elevated FFAs caused insulin resistance in muscle. IL/Hep did not alter splanchnic glucose uptake or the contribution of the extracellular direct pathway to UDP-glucose flux. On the other hand, total UDP-glucose flux (13.2 ± 1.7 and 12.5 ± 1.0 vs. 8.1 ± 0.5 μmol · kg−1 · min−1) and flux via the indirect intracellular pathway (8.4 ± 1.2 and 8.1 ± 0.6 vs. 4.8 ± 0.05 μmol · kg−1 · min−1) were greater (P < 0.05) during both the IL/Hep and high-glycerol infusions than the low-glycerol infusion. In contrast, only IL/Hep increased (P < 0.05) splanchnic glucose production, indicating that elevated FFAs impaired the ability of the liver to autoregulate. Splanchnic insulin extraction, directly measured using the arterial and hepatic vein catheters, did not differ (67 ± 3 vs. 71 ± 5 vs. 69 ± 1%) during IL/Hep and high- and low-glycerol infusions. We conclude that elevated FFAs exert multiple effects on glucose metabolism. They inhibit insulin- and glucose-induced stimulation of muscle glucose uptake and suppression of splanchnic glucose production. They increase the contribution of the indirect pathway to glycogen synthesis and impair hepatic autoregulation. On the other hand, they do not alter either splanchnic glucose uptake or splanchnic insulin extraction in nondiabetic humans.