Monitoring the effects of bevacizumab beyond progression in a murine colorectal cancer model: a functional imaging approach.

Clinical studies have shown that bevacizumab beyond progression to first line therapy is beneficial for overall survival in advanced stage colorectal cancer. We studied the utility of several functional imaging modalities to assess the efficacy of bevacizumab beyond progression (BBP). All BALB/c mice with s.c. LS174T xenografts were treated with capecitabine, oxaliplatin and bevacizumab combination therapy. Tumor volume was assessed using caliper measurements. Increase of 1.5 times the initial volume on two subsequent measurements, was considered progression. In half of the mice bevacizumab treatment was continued (n = 13) after progressive disease was established, while the others received saline injections (n = 12). Within 3 days after progression, multi-modal imaging was performed using FDG-PET, diffusion weighted imaging, T2* and dynamic contrast enhanced MRI. Measurements were repeated 7 and 10 days after the first measurements. Afterwards, tumors were analyzed for expression of carbonic anhydrase IX, glucose transporter 1, 9 F1 to stain the vasculature and Ki67 to assess proliferation. In the BBP group tumor growth after progression was reduced compared to the control group (p < 0.01). FDG-PET showed a trend towards lower FDG uptake in the BBP group (p = 0.08). DWI, T2* and DCE-MRI parameters were not significantly different between both groups. The immunohistochemical analyses showed higher CAIX-positive fraction (p < 0.01) and lower Ki67 expression (p = 0.06) in the BBP group. The relative vascular area was significantly lower in the BBP group (p = 0.03). GLUT-1 expression and vascular density did not significantly differ between both groups. Bevacizumab after progression resulted in significant changes in the tumor proliferation and microenvironment compared to discontinuation of bevacizumab. FDG-PET may be sensitive to BBP-induced effects.

Bilateral thoracoscopic splanchnicectomy for pain in patients with chronic pancreatitis impairs adrenomedullary but not noradrenergic sympathetic function.

BACKGROUND: Bilateral thoracoscopic splanchnicectomy (BTS) is a well-known technique to alleviate intractable pain in patients with chronic pancreatitis. BTS not only disrupts afferent fibers from the pancreas that mediate pain but also postganglionic sympathetic fibers, which originate in segments T5-T12 and which innervate the vasculature of the liver, pancreas, and the adrenal gland. The purpose of this study was to assess whether and how BTS affects sympathetic noradrenergic and adrenomedullary function in patients with chronic pancreatitis. METHODS: Sixteen patients with chronic pancreatitis for at least 1 year underwent autonomic function testing before and 6 weeks after BTS for intractable pain. Testing was performed during supine rest and during sympathetic stimulation when standing. RESULTS: Supine and standing systolic and diastolic blood pressure were significantly lower post-BTS compared with pre-BTS (P = 0.001). One patient showed orthostatic hypotension after BTS. Baseline plasma norepinephrine levels and plasma norepinephrine responses to sympathetic activation during standing were not reduced by BTS. In contrast, supine plasma epinephrine levels and responses during standing were significantly reduced (P < 0.001). Parasympathetic activity was unaffected by BTS as shown by unaltered Valsalva ratio, I-E difference, and ΔHRmax. CONCLUSIONS: BTS for pain relief in patients with chronic pancreatitis reduced adrenomedullary function, due to disruption of the efferent sympathetic fibers to the adrenal gland. BTS did not affect noradrenergic sympathetic activity, although blood pressure was lower after the sympathectomy.

Validity of free testosterone calculation in pregnant women.

Objective Increased maternal testosterone concentration during pregnancy may affect the fetus. Therefore it is clinically relevant to have a quick and reliable method to determine free testosterone levels. Current calculators for free testosterone are suspected to perform poorly during pregnancy due to suggested competition between high levels of estradiol and free (bio-active) testosterone for sex hormone-binding globulin (SHBG) binding. Therefore, it is claimed that reliable calculation of free testosterone concentration is not possible. However, recent evidence on SHBG-binding sites questions the estradiol effect on the testosterone-SHBG binding during pregnancy. In this study, we investigated whether the free testosterone concentration can be calculated in pregnant women. Design and methods Free testosterone was measured with a specially developed equilibrium dialysis method combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). Free testosterone was also calculated with the formulas of Vermeulen et al. and Ross et al. Results Total and free testosterone measured in healthy men and women were in good agreement with earlier reports. In pregnant women, total testosterone values were higher than in non-pregnant women, whereas free testosterone values were comparable. Calculated free testosterone levels in pregnant women were highly correlated, but marginally higher, compared to measured free testosterone levels. Conclusions We developed an equilibrium dialysis-LC-MS/MS method for the measurement of free testosterone in the low range of pregnant and non-pregnant women. Although during pregnancy total testosterone is increased, this is not the case for free testosterone. The free testosterone formulas perform well in pregnant women.

Early identification of persistent trophoblastic disease with serum hCG concentration ratios.

The objective of the present study was to assess the diagnostic potential of serum human chorionic gonadotropin (hCG) ratios obtained at different intervals after evacuation of hydatidiform mole to diagnose persistent trophoblastic disease (PTD) and to compare its diagnostic accuracy with the current FIGO 2000 criteria as a gold standard. We calculated hCG ratios from serum hCG concentrations of 204 patients (86 with and 118 without PTD) registered with the Dutch Central Registry for Hydatidiform Moles between 1977-2004. The hCG ratios obtained in week 1, 3, and 5 after evacuation identified, respectively, 20%, 52%, and 79% of patients with PTD (median: 3.0 weeks) at the 95% specificity level, while FIGO 2000 criteria identified, respectively, 0%, 16%, and 66% (median: 4.7 weeks). It is concluded that a serum hCG ratio identifies patients with PTD approximately 2 weeks earlier than the internationally accepted FIGO 2000 criteria and identifies more than 75% of patients who develop PTD by the fifth week after evacuation.

Impaired Sertoli cell function in males diagnosed with Noonan syndrome.

In order to study male gonadal function in Noonan syndrome, clinical and laboratory data, including inhibin B, were gathered in nine pubertal males diagnosed with Noonan syndrome. Bilateral testicular maldescent was observed in four, and unilateral cryptorchidism occurred in two. Puberty was delayed in three patients. Luteinising hormone (LH) levels were normal in all patients in our series, while follicle stimulating hormone (FSH) levels were raised in seven. Inhibin B was low in six males and just above the lower limit of normal in two. Importantly, all three men with normal testicular descent displayed signs of Sertoli cell dysfunction, indicating, in contrast to earlier reports, that bilateral cryptorchidism does not seem to be the main contributing factor to impairment of testicular function in Noonan syndrome. These findings suggest different mechanisms of disturbance in male gonadal function, which is frequently associated with Sertoli dysfunction.

Gonadotropins and female sex steroid hormones in cyst fluid and serum from patients with ovarian tumors.

The objective of the present study was to determine the concentrations of LH, FSH, 17beta-estradiol and progesterone in ovarian cyst fluid and serum from patients with benign and malignant ovarian tumors and to assess the correlation of the gonadotropin and female sex steroid hormone concentrations with menopausal and tumor status. Ovarian cyst fluid and blood samples were prospectively collected from 103 patients with ovarian tumors. Seventy-four of the patients had benign ovarian tumors while 29 patients had malignant ovarian tumors. Malignant ovarian tumors showed significantly higher LH and FSH cyst fluid concentrations compared to concentrations from patients with benign tumors. Within the malignant subset, LH and FSH concentrations correlated with increasing FIGO stage and grade. Furthermore, LH and FSH cyst fluid concentrations showed strong correlations (r > 0.62) with serum concentrations in case of malignant tumors, especially in postmenopausal women, but not in case of benign tumors. The highest gonadotropin concentrations were observed in cyst fluid from malignant ovarian tumors. The most probable explanation for this is an increased vascular permeability within the cysts. Supportive evidence for such an increased vascular permeability is our previous finding of significantly higher VEGF concentrations in cyst fluid from malignant ovarian tumors. The possibility of ectopic production of LH and FSH by malignant ovarian tissue cannot completely be ruled out.

[Intraoperative parathyroid hormone measurement in patients with primary hyperparathyroidism; particularly valuable for suspected solitary parathyroid adenoma and re-operation]. / Intraoperatieve bepaling van parathormoon bij primaire hyperparathyreoïdie; vooral waardevol bij vermoeden van solitair bijschildklieradenoom en bij heroperatie.

OBJECTIVE: Analysis of the value of intraoperative parathormone (PTH) measurement in patients with primary hyperparathyroidism. DESIGN: Prospective study. METHOD: Evaluation of the value of intraoperative measurement ofPTH in 75 patients (including 19 patients with multiple endocrine neoplasia(MEN)-1 syndrome) who underwent parathyroidectomy in 2001-2005. RESULTS: The so-called Miami-criterion (PTH concentration 10 min after excision at least 50% below the value measured prior to the first incision) correctly predicted the success of the operation in 91% of the subjects. The success rate was correctly predicted as follows: in subgroups of patients with MEN-1 syndrome, 85%, patients after exclusion of MEN-1, 94%, and patients in whom a solitary adenoma was likely after preoperative localization studies, 97%. In 13% of the total number of operations, PTH-measurements led to further exploration, removal of additional parathyroid tissue and normocalcemia postoperatively. In patients without MEN-1 syndrome, in whom a solitary adenoma was likely on the basis of preoperative investigations, it was possible to limit the operation to a unilateral procedure in 87%. CONCLUSION: In the majority of patients with primary hyperparathyroidism, intraoperative PTH-measurement in combination with preoperative imaging studies leads to patients being cured with a unilateral instead of a bilateral operation.

Identification and characterization of multiple species of tenascin-X in human serum.

We analysed the diversity of tenascin-X (TNX) species in serum and studied parameters that could affect determination of TNX levels in serum. Using western blot analysis we identified at least seven distinct TNX species, ranging from 75 kDa to the presumably full-length 450 kDa form. Purification of serum TNX followed by sequence analysis positively identified two major TNX species of 75 and 140 kDa. We found that serum TNX binds to tropoelastin but not to fibrillar collagens. We conclude that serum TNX is composed of distinct molecular species that retain functional activity.

Transsphenoidal pituitary surgery via the endoscopic technique: results in 35 consecutive patients with Cushing's disease.

OBJECTIVE AND DESIGN: The endoscopic technique has been recently introduced in the field of transsphenoidal pituitary surgery. This technique allows inspection of sellar, supra- and parasellar structures and removal of the tumor under direct visualization, is minimally traumatic and permits easier reoperations. This is the first report on the results of endoscopic surgery for patients with Cushing's disease. Our aim was to retrospectively analyze the results of pituitary surgery in 35 consecutive patients with Cushing's disease operated in our hospital after the introduction of the endoscopic technique (1998-2004). METHODS: Remission was defined as suppression of plasma cortisol (< or =50 nmol/L) after 1 mg dexamethasone overnight determined in the first 3 months after surgery and disappearance of clinical signs and symptoms of hypercortisolism. The patients were followed for an average of 27 months (range 4 to 81 months, median 20 months). RESULTS: Pituitary MRI showed a macroadenoma in 6 patients, a microadenoma in 17 patients and no adenoma in 12 patients. After the initial surgery 27 patients (77%) were in remission. None of the patients had a relapse during follow-up. In the remaining 8 patients hypercortisolemia persisted after surgery. Three of them had a second endoscopic pituitary surgery resulting in remission in two patients. In one patient a second endoscopic pituitary surgery will soon follow. The remaining four patients were treated with radiotherapy postoperatively. Two of them were at the time of data collection in remission. One patient from the remission group had a serious epistaxis and three patients had cerebrospinal fluid leakage, one requiring an external lumbar drain, shortly after surgery. No complications were recorded in the failure group. Postoperatively 34% of all patients required substitution with levothyroxine, 40% required substitution with glucocorticoids, 17% received estrogens or testosterone and 6% still required desmopressin. CONCLUSIONS: Endoscopic transsphenoidal pituitary surgery resulted in our series of patients with Cushings disease in an excellent postoperative remission rate. A randomized clinical trial, comparing endoscopic and conventional pituitary surgery in patients with Cushings disease, is needed to determine the pros and cons of both techniques.

A patient with recurrent hypercortisolism after removal of an ACTH-secreting pituitary adenoma due to an adrenal macronodule.

A 41-yr-old female was referred for signs and symptoms of Cushing's syndrome. Cortisol was not suppressed by 1 mg dexamethasone (0.41 micromol/l). Midnight cortisol and ACTH were 0.44 micromol/l and 18 pmol/l, respectively. Urinary cortisol excretion was 250 nmol/24 h (normal between 30 and 150 nmol/24 h). A magnetic resonance imaging (MRI) revealed a pituitary lesion of 7 mm. ACTH and cortisol levels were unaltered by administration of human CRH and high-dose dexamethasone. Inferior sinus petrosus sampling showed CRH-stimulated ACTH levels of 128.4 (left sinus) vs a peripheral level of 19.2 pmol/l, indicating Cushing's disease. After 4 months of pre-treatment with metyrapone and dexamethasone, endoscopic transsphenoidal resection of an ACTH-positive pituitary adenoma was performed. ACTH levels decreased to 2.6 pmol/l and fasting cortisol was 0.35 micromol/l. Despite clinical regression of Cushing's syndrome and normalization of urinary cortisol, cortisol was not suppressed by 1 mg dexamethasone (0.30 micromol/l). Ten months post-operatively, signs and symptoms of Cushing's syndrome reoccurred. A high dose dexamethasone test according to Liddle resulted in undetectable ACTH, but no suppression of cortisol levels, pointing towards adrenal-dependent Cushing's syndrome. Computed tomography (CT)-scanning showed a left-sided adrenal macronodule. Laparoscopic left adrenalectomy revealed a cortical macronodule (3.5 cm) surrounded by micronodular hyperplasia. Fasting cortisol had decreased to 0.02 micromol/l. Glucocorticoid suppletion was started and tapered over 12 months. Symptoms and signs of hypercortisolism gradually disappeared. This case illustrates, that longstanding ACTH stimulation by a pituitary adenoma can induce unilateral macronodular adrenal hyperplasia with autonomous cortisol production.

Fertility in patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is generally regarded as a paediatric endocrine disease, but nowadays nearly all patients reach adulthood as a result of improved diagnosis and treatment. It is now increasingly recognised that treatment goals shift during life: one of the major treatment goals in childhood and puberty, i.e. normal growth and development, is no longer relevant after childhood, whereas other aspects, such as fertility and side effects of long-term glucocorticoid treatment, become more important in adulthood. This paper focuses on fertility in male and female adult patients with CAH. In males with CAH the fertility rate is reduced compared with the normal population, the most frequent cause being testicular adrenal rest tumours. Development and growth of these tumours is assumed to be ACTH dependent and undertreatment may play an important role. If intensifying glucocorticoid treatment does not lead to tumour decrease, surgical intervention may be considered, but the effect on fertility is not yet known. In females with CAH the degree of fertility depends on the phenotype of the CAH. Most fertility problems are seen in the classic salt-wasting type. Age of menarche and regularity of the menstrual cycle depends on the degree of adrenal suppression. Not only adrenal androgens have to be normalised but also the levels of adrenal progestins (progesterone and 17-OH-progesterone) that interfere with normal ovulatory cycles. The regularity of menstrual cycles can be considered as an important measure of therapeutic control in adolescent females with CAH and therefore as a therapeutic goal from (peri)pubertal years on. Other factors that contribute to impaired fertility in females with CAH are ovarian hyperandrogenism (polycystic ovary syndrome), ovarian adrenal rest tumours, genital surgery and psychological factors. Subfertility in CAH can have its origin already in the peripubertal years and is therefore of interest to the paediatric endocrinologist.

Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). METHODS: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. RESULTS: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). CONCLUSIONS: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategies.

Predictive impact of urokinase-type plasminogen activator: plasminogen activator inhibitor type-1 complex on the efficacy of adjuvant systemic therapy in primary breast cancer.

One of the most thoroughly studied systems in relation to its prognostic relevance in patients with breast cancer, is the plasminogen activation system. This system comprises of, among others, the urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-1). In this study we investigated whether the uPA:PAI-1 complex is associated with the responsiveness of patients with primary breast cancer to adjuvant systemic therapy. Quantitative enzyme-linked immunosorbent assays were used to assess the levels of uPA, PAI-1, and uPA:PAI-1 complex in 1119 tumors of patients with primary invasive breast cancer. These patients were followed for a median follow-up time of 59 months (range, 2-267 months) after the primary diagnosis. Correlations with well-known clinicopathological factors, and univariate and multivariate survival analyses were performed. High uPA:PAI-1 complex levels were correlated with an adverse histological grade, and inversely associated with negative estrogen and progesterone receptor status. High tumor levels of uPA:PAI-1 complex predicted an early relapse in the univariate relapse-free survival analysis (P < 0.001). The multivariate analysis showed that high uPA:PAI-1 complex levels were associated with a decreased relapse-free survival time (P = 0.033), independently of age, tumor size, number of lymph nodes affected, progesterone receptor status, uPA, adjuvant endocrine, and chemotherapy. More important, it was demonstrated that there is a larger benefit from adjuvant chemotherapy for patients with higher versus lower tumor levels of uPA:PAI-1 complex. The results of this study imply that the expression of uPA:PAI-1 complex independently predicts the efficacy of adjuvant chemotherapy in patients with primary breast cancer.

Cyclin-E is a strong predictor of endocrine therapy failure in human breast cancer.

Recently, cyclin-E was reported to be the most prominent prognostic factor for breast cancer outcome described so far, even surpassing axillary nodal involvement. Earlier studies on the prognostic value of cyclin-E in breast cancer, however, yielded heterogeneous results. Therefore, we set out to confirm and extend these results by quantitative Taqman RT-PCR of cyclin-E levels in 277 resectable breast cancers. Cyclin-E levels were not associated with relapse-free survival (RFS) or overall survival (OS) in the total cohort of patients, or in the subset of patients without involved lymph nodes that were not treated with adjuvant systemic therapy. Besides several classical clinicopathological factors, the interaction between cyclin-E and adjuvant endocrine therapy (P=0.01, HR=3.04, 95% CI: 1.30-7.09) was found to contribute significantly in multivariate analyses. Cyclin-E levels were associated with poor RFS specifically in patients treated with adjuvant endocrine therapy (n=108, P=0.001, HR=4.01, 95% CI: 1.76-9.12), independent of estrogen receptor status. In conclusion, cyclin-E is not a pure prognostic factor in breast cancer, but rather a predictor of failure of endocrine therapy. Differences in literature on the presumed prognostic value of cyclin-E may be due to differences in treatment. Assessment of cyclin-E levels can aid in improving adjuvant treatment selection.

Expression of the transcription factor Ets-1 is an independent prognostic marker for relapse-free survival in breast cancer.

The transcription factor Ets-1 regulates the expression of several angiogenic and extracellular matrix remodeling factors, and might be implicated in disease progression of breast cancer. In the present study, the prognostic value of Ets-1 expression was assessed by quantitative real-time fluorescence RT-PCR in 123 sporadic primary breast cancer samples of patients with a median follow-up time of 62 months. Ets-1 expression levels correlated significantly with VEGF and PAI-1 in the same tissue. In univariate (P=0.0011) and multivariate (P=0.005) analyses, Ets-1 expression showed significant prognostic value for relapse-free survival. Ets-1 is a strong, independent predictor of poor prognosis in breast cancer. This seems - at least in part - to be attributable to its role in transcriptional regulation of factors involved in angiogenesis (VEGF), and extracellular matrix remodeling (PAI-1).

Measurement of plasminogen activator system components in plasma and tumor tissue extracts obtained from patients with breast cancer: an EORTC Receptor and Biomarker Group collaboration.

The plasminogen activator (PA) system comprises the 2 serine proteases, urokinase PA (uPA) and tissue PA (tPA), the 2 serpin inhibitors, PAI-1 and PAI-2 and the uPA receptor (uPAR; CD87). High levels of uPA, PAI-1, uPA-PAI-1 complex and uPAR in breast cancer tissue are associated with poor prognosis, while high levels of tPA or PAI-2 correlate with good prognosis. In this study, pre-operative plasma levels of uPA, PAI-1, uPAR, tPA, uPA-PAI-1 complex, and tPA-PAI-1 complex were measured in patients with benign (n=103) and malignant breast disease (n=113) by immunoenzymatic assays (ELISA). While plasma antigen levels of uPA, PAI-1, uPA-PAI-1 complex and uPAR were not significantly different in the 2 groups, antigen levels of tPA and tPA-PAI-1 complex were significantly higher in patients with breast carcinoma compared to the control group. In plasma from the breast cancer patients, uPA levels correlated weakly but significantly with those of tPA (r=0.20, p=0.035) and uPAR (r=0.208, p=0.028). tPA levels correlated strongly with tPA-PAI-1 complex (r=0.972, p=0.0001) while uPA-PAI-1 levels were significantly associated with PAI-1 levels (r=0.534, p<0.0001), tPA levels (r=0.348, p=0.0003) and tPA-PAI-1 levels (r=0.356, p=0.002). However, no significant correlation was found between plasma and tumor tissue levels of uPA, PAI-1, uPA-PAI-1 complex, tPA or tPA-PAI-1. Our findings indicate that determination of these factors in plasma do not reflect their concentration in tumor tissue. Therefore, measurement of PA components in blood cannot be recommended for assessing prognosis in breast cancer.

Hypothalamic-pituitary-adrenal axis activity in patients with rheumatoid arthritis.

OBJECTIVE: To study the hypothalamic-pituitary-adrenal (HPA) axis in patients with rheumatoid arthritis (RA). METHODS: Fifty patients with RA participated in 3 groups: recent onset active RA (n = 20), longstanding active RA (n = 20) and long-standing RA in remission (n = 10), and were compared with 20 healthy controls. The activity of the HPA-axis was assessed under basal conditions and in response to stress (insulin tolerance test, ITT). In addition, patients with recent onset RA underwent a corticotropin releasing hormone (CRH) test and a dexamethasone suppression test. Plasma levels of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IL-6 were also measured. RESULTS: Basal plasma, salivary and urinary cortisol levels and plasma adrenocorticotropic hormone (ACTH) levels were not different between patients with RA and healthy controls. During the ITT, cortisol levels were consistently lower in RA patients than in healthy controls. ACTH levels during the ITT were not different between patients with RA and healthy controls. ACTH and cortisol responses to CRH were assessed only in patients with recent onset RA and were found to be within normal limits. Basal circulating plasma IL-6 levels were significantly higher in patients with active RA than in the other groups. CONCLUSION: Under the standardized conditions of the ITT, patients with RA have decreased plasma cortisol levels compared to healthy controls, despite elevated levels of IL-6. The defect is probably located at the adrenal level and may be of pathogenetic significance for the development of chronic arthritis.

Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane.

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.

Regulation of GLUT1-mediated glucose uptake by PKClambda-PKCbeta(II) interactions in 3T3-L1 adipocytes.

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.

Anti-inflammatory effects of troglitazone in nondiabetic obese subjects independent of changes in insulin sensitivity.

BACKGROUND: Obesity is characterised by insulin resistance and by elevated levels of proinflammatory markers. We investigated whether, in the absence of changes in glucose, thiazolidinediones (TZDs) have anti-inflammatory effects and whether improvement of insulin sensitivity correlates with suppression of inflammatory markers. METHODS: We performed a randomised double-blind placebo-controlled crossover study with troglitazone (400 mg daily for eight weeks) in 15 normoglycaemic obese subjects. We measured plasma high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), leptin, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and tumour necrosis factor-alpha (TNF-alpha) after each of the two treatment periods and in 13 age- and sex-matched lean individuals. RESULTS: Obese subjects were insulin resistant (decreased glucose infusion rate (GIR) during euglycaemic hyperinsulinaemic clamp) and had higher plasma levels of hsCRP, IL-6, leptin, tPA, and PAI-1 compared with lean subjects. TNF-alpha also tended to be higher. Troglitazone improved insulin sensitivity (mean increase in whole body glucose uptake 23.1 +/- 10.5% (p = 0.047)) and normalised plasma concentrations of hsCRP, tPA and TNF-alpha, whereas it did not significantly change IL-6, leptin and PAI-1. Changes in GIR did not correlate with changes in inflammatory markers. CONCLUSION: Troglitazone induces suppression of some of the inflammatory markers that are elevated in normoglycaemic obese subjects. The suppression of inflammatory markers, however, does not correlate with improvement in insulin sensitivity, suggesting involvement of partially differential mechanisms in these effects of TZDs.