Disruption of beta cell acetyl-CoA carboxylase-1 in mice impairs insulin secretion and beta cell mass.

AIMS/HYPOTHESIS: Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. METHODS: Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (ßACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-ßACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. RESULTS: ßACC1KO and tmx-ßACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in ßACC1KO mice but not in tmx-ßACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. CONCLUSIONS/INTERPRETATION: Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood.

High dietary fat and sucrose results in an extensive and time-dependent deterioration in health of multiple physiological systems in mice.

Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD). HFHSD-fed mice rapidly exhibited metabolic alterations, including obesity, hyperleptinemia, physical inactivity, glucose intolerance, peripheral insulin resistance, fasting hyperglycemia, ectopic lipid deposition, and bone deterioration. Prolonged exposure to HFHSD resulted in morbid obesity, ectopic triglyceride deposition in liver and muscle, extensive bone loss, sarcopenia, hyperinsulinemia, and impaired short-term memory. Although many of these defects are typically associated with aging, HFHSD did not alter telomere length in white blood cells, indicating that this diet did not generally promote all aspects of aging. Strikingly, glucose homeostasis was highly dynamic. Glucose intolerance was evident in HFHSD-fed mice after 1 week and was maintained for 24 weeks. Beyond 24 weeks, however, glucose tolerance improved in HFHSD-fed mice, and by 60 weeks, it was indistinguishable from that of chow-fed mice. This improvement coincided with adaptive ß-cell hyperplasia and hyperinsulinemia, without changes in insulin sensitivity in muscle or adipose tissue. Assessment of insulin secretion in isolated islets revealed that leptin, which inhibited insulin secretion in the chow-fed mice, potentiated glucose-stimulated insulin secretion in the HFHSD-fed mice after 60 weeks. Overall, the excessive calorie intake was accompanied by deteriorating function of numerous physiological systems.

Gene expression profiling to predict the risk of locoregional recurrence in breast cancer: a pooled analysis.

The 70-gene signature (MammaPrint) has been developed to predict the risk of distant metastases in breast cancer and select those patients who may benefit from adjuvant treatment. Given the strong association between locoregional and distant recurrence, we hypothesize that the 70-gene signature will also be able to predict the risk of locoregional recurrence (LRR). 1,053 breast cancer patients primarily treated with breast-conserving treatment or mastectomy at the Netherlands Cancer Institute between 1984 and 2006 were included. Adjuvant treatment consisted of radiotherapy, chemotherapy, and/or endocrine therapy as indicated by guidelines used at the time. All patients were included in various 70-gene signature validation studies. After a median follow-up of 8.96 years with 87 LRRs, patients with a high-risk 70-gene signature (n = 492) had an LRR risk of 12.6% (95% CI 9.7-15.8) at 10 years, compared to 6.1% (95% CI 4.1-8.5) for low-risk patients (n = 561; P < 0.001). Adjusting the 70-gene signature in a competing risk model for the clinicopathological factors such as age, tumour size, grade, hormone receptor status, LVI, axillary lymph node involvement, surgical treatment, endocrine treatment, and chemotherapy resulted in a multivariable HR of 1.73 (95% CI 1.02-2.93; P = 0.042). Adding the signature to the model based on clinicopathological factors improved the discrimination, albeit non-significantly [C-index through 10 years changed from 0.731 (95% CI 0.682-0.782) to 0.741 (95% CI 0.693-0.790)]. Calibration of the prognostic models was excellent. The 70-gene signature is an independent prognostic factor for LRR. A significantly lower local recurrence risk was seen in patients with a low-risk 70-gene signature compared to those with high-risk 70-gene signature.

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action. METHODS: DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a (-/-) (Doc2aKO), Doc2b (-/-) (Doc2bKO) and Doc2a (-/-)/Doc2b (-/-) (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy. RESULTS: Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice. CONCLUSIONS/INTERPRETATION: Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.

Identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity.

A critical feature of obesity is enhanced insulin secretion from pancreatic ß-cells, enabling the majority of individuals to maintain glycaemic control despite adiposity and insulin resistance. Surprisingly, the factors coordinating this adaptive ß-cell response with adiposity have not been delineated. Here we show that fatty acid binding protein 4 (FABP4/aP2) is an adipokine released from adipocytes under obesogenic conditions, such as hypoxia, to augment insulin secretion. The insulinotropic action of FABP4 was identified using an in vitro system that recapitulates adipocyte to ß-cell endocrine signalling, with glucose-stimulated insulin secretion (GSIS) as a functional readout, coupled with quantitative proteomics. Exogenous FABP4 potentiated GSIS in vitro and in vivo, and circulating FABP4 levels correlated with GSIS in humans. Insulin inhibited FABP4 release from adipocytes in vitro, in mice and in humans, consistent with feedback regulation. These data suggest that FABP4 and insulin form an endocrine loop coordinating the ß-cell response to obesity.

Genome-wide data reveal novel genes for methotrexate response in a large cohort of juvenile idiopathic arthritis cases.

Clinical response to methotrexate (MTX) treatment for children with juvenile idiopathic arthritis (JIA) displays considerable heterogeneity. Currently, there are no reliable predictors to identify non-responders: earlier identification could lead to a targeted treatment. We genotyped 759 JIA cases from the UK, the Netherlands and Czech Republic. Clinical variables were measured at baseline and 6 months after start of the treatment. In Phase I analysis, samples were analysed for the association with MTX response using ordinal regression of ACR-pedi categories and linear regression of change in clinical variables, and identified 31 genetic regions (P<0.001). Phase II analysis increased SNP density in the most strongly associated regions, identifying 14 regions (P<1 × 10(-5)): three contain genes of particular biological interest (ZMIZ1, TGIF1 and CFTR). These data suggest a role for novel pathways in MTX response and further investigations within associated regions will help to reach our goal of predicting response to MTX in JIA.

Local control, toxicity, and cosmesis in women younger than 50 enrolled onto the American Society of Breast Surgeons MammoSite Radiation Therapy System registry trial.

BACKGROUND: The American Society of Breast Surgeons enrolled women onto a registry trial to prospectively study patients treated with the MammoSite Radiation Therapy System (RTS) breast brachytherapy device. This report examines local recurrence (LR), toxicity, and cosmesis as a function of age in women enrolled onto the trial. METHODS: A total of 1449 primary early-stage breast cancers were treated in 1440 women. Of these, 130 occurred in women younger than 50 years of age. Fisher's exact test was performed to correlate age (<50 vs. > or = 50 years) with toxicity and with cosmesis. The association of age with LR failure times was investigated by fitting a parametric model. RESULTS: Women younger than 50 were more likely to develop fat necrosis: 4.6% (6 of 130) vs. 1.8% (24 of 1319) (P = .0456). Other toxicities were comparable. At 2 years, cosmesis was excellent or good in 87% of assessable women aged <50 years (n = 74) and in 94% of assessable older women (n = 751) (P = .0197). At 3 years, this difference disappeared: excellent or good in 90% (56 of 62) of younger women vs. 93% (573 of 614) of older women (P = .2902). The crude LR rate for the group was 1.7% (25 of 1449). There was no statistically significant difference in LR as a function of age. In women <50, 3.1% (4 of 130) developed a LR; in the older patients, 1.6% (21 of 1319) developed LR (3-year actuarial LR rates, 2.9% vs. 1.7%, respectively; P = .2284). CONCLUSIONS: Accelerated partial breast irradiation with the MammoSite RTS results in low toxicity and produces similar cosmesis and local control at 3 years in women younger than 50 when compared with older women.

Use of methotrexate in juvenile idiopathic arthritis.

Methotrexate (MTX) has transformed the outlook for children with juvenile idiopathic arthritis (JIA). Most of the evidence from uncontrolled clinical trials suggests that MTX is an effective agent for treating active JIA. Data from controlled clinical trials suggests that MTX has statistically significant effects on patient centred disability measures in JIA patients with active arthritis. Although we would like a much larger study directed evidence base for our use of the drug, the studies that have been done are sound and have been followed by a change in clinical expectations and advice that speak of qualitative evidence from clinical practice, confirming the scientifically acquired data. Randomised controlled multicentre trials using sufficient numbers of patients, including functional assessment and quality of life measures, are needed to confirm the long term efficacy and safety of MTX in JIA.

Improved lymphatic mapping technique for breast cancer.

Breast sentinel lymph node biopsy is becoming more common. However, the best injection technique is not well established. Currently the gold standard is peritumoral injection. However, for upper outer quadrant tumors there is considerable axillary "shine through" which makes the identification of the radioactive sentinel lymph node difficult. We undertook a study to compare an injection in Sappey's subareolar plexus to the gold standard of peritumoral injection. Between December 1997 and March 1998, 85 patients with breast cancer were enrolled in the study. All patients were injected with 2 cc of normal saline containing 1.0 mCi of unfiltered technetium sulfur colloid in Sappey's subareolar plexus in the clock position of the breast cancer. In the operating room the patients underwent a peritumoral injection of 5 cc of 1% isosulfan blue. All blue and radioactive lymph nodes were identified and removed. The majority of the tumors were in the upper outer quadrant and were diagnosed by core biopsy. Only half of the patients had palpable tumors and approximately 25% had previous upper outer quadrant biopsy incisions. Peritumoral blue dye injection yielded an identification rate of 94%, with 99% of these being blue and radioactive. Three patients had radioactive lymph nodes with no blue lymph nodes identified. One of these patients had a micrometastasis. Injection in Sappey's subareolar plexus in the clock position of the tumor drained to the same sentinel lymph node as peritumoral injection. This injection technique solved the two major problems confronting the wide adoption of sentinel lymph node biopsy for breast cancer staging. First, it eliminates axillary "shine through" which will allow nonspecialist surgeons to more easily identify the radioactive axillary sentinel lymph node. Second, it allows for easier isotope injection by the technician or nuclear medicine physician, by eliminating the need for three-dimensional localization. This new technique should allow the majority of breast cancer patients who are treated by nonspecialist surgeons to be offered this less morbid, more accurate procedure.

Gamma probe guided biopsy of the sentinel node in malignant melanoma: a multicentre study.

Sentinel lymph node biopsy was attempted in 336 patients with clinically node-negative cutaneous melanoma. All patients were injected with technetium-99m labelled radiocolloid, with 108 patients simultaneously receiving vital blue dye for sentinel node identification. Sentinel lymph nodes were identified in 329 patients, giving a technical success rate of 97.9%. Metastatic disease was identified in 39 (11.9%) of the patients in whom sentinel nodes were found. Patients with negative sentinel nodes were observed and patients with positive sentinel nodes underwent comprehensive lymph node dissection. The presence of metastatic disease in the sentinel nodes and primary tumour depth by Breslow or Clark levels were joint predictors of survival based on Cox proportional hazards modelling. Disease recurrences occurred in 26 (8.8%) patients with negative sentinel lymph nodes, with isolated regional recurrences as the first site in 10 (3.4%). No patients with Clark level II primary tumours were found to have positive sentinel nodes or disease recurrences. One patient with a thin (<0.75 mm) Clark level III primary had metastatic disease in a sentinel node. Patients with metastases confined to the sentinel nodes had similar survival rates regardless of the number of nodes involved.

The details of successful sentinel lymph node staging for breast cancer.

Sentinel lymphadenectomy is an effective and accurate tool for staging breast cancer. In recent years the details of a successful program have become better defined. The authors outline practical considerations for the performance of successful sentinel lymph node staging from a multidisciplinary perspective.

State-of-the-art lymph node staging for breast cancer in the year 2000.

Axillary staging for breast cancer is vitally important for determining appropriate adjuvant hormone and chemotherapy. In the absence of distant metastases, axillary lymph node status remains the most accurate predictor of clinical outcome. Sentinel lymph node biopsy is a minimally invasive approach with enhanced accuracy and less morbidity than conventional axillary dissection. The stage is now set for the sentinel lymphadenectomy staging to move from state-of-the-art care to the standard care in coming years.

Credentialing issues with sentinel lymph node staging for breast cancer.

Sentinel lymphadenectomy (SL) is a minimally invasive approach for staging patients with breast cancer. SL, when performed in lieu of axillary dissection, is associated with less morbidity and is potentially more cost effective and more accurate than the historical axillary dissection in the detection of regional nodal metastases. The credentialing and privileging of SL, as with any surgical procedure, is by the policies of the local hospital or institution. The suggested credentialing criteria for local hospitals has been an area of controversy. Herein the authors outline the credentialing controversy and suggest criteria for the implementation of sentinel lymph node staging for breast cancer.

Preoperative combined modality therapy with paclitaxel, carboplatin, prolonged infusion 5-fluorouracil, and radiation therapy in localized esophageal cancer: preliminary results of a Minnie Pearl Cancer Research Network phase II trial.

PURPOSE: To evaluate the feasibility, toxicity, and therapeutic efficacy of 1-hour paclitaxel, carboplatin, continuous low-dose infusional 5-fluorouracil, and concurrent radiation therapy administered preoperatively in patients with localized esophageal cancer. PATIENT AND METHODS: Forty-nine patients with localized esophageal cancer, of either squamous cell carcinoma or adenocarcinoma histology, were enrolled into this phase II trial. All patients were candidates for surgical resection and received the following neoadjuvant therapy: paclitaxel, 200 mg/m2, 1 hour IV on days 1 and 22; carboplatin, AUC 6.0, IV on days 1 and 22; 5-fluorouracil, 225 mg/m2/day, continuous IV infusion on days 1 to 42; and radiation therapy, 45 Gy, administered by 1.8-Gy daily fractions beginning on day 1 of chemotherapy. Upon completion of this neoadjuvant regimen, patients were reevaluated, and all responding patients were resected within 6 weeks of completing neoadjuvant treatment. RESULTS: Administration of this combined modality regimen was associated with moderate toxicity and was tolerated by most patients. Leukopenia (65%) and esophagitis (31%) were the most common toxicities. Most patients did not require nutritional support. There were no treatment-related deaths during neoadjuvant therapy; however, three patients (9%) experienced postoperative death. Preliminary assessment of treatment efficacy is encouraging, with 17 of 37 evaluable patients (46%) achieving pathologic complete remission and an additional 11 patients (30%) having only microscopic residual disease. CONCLUSIONS: This novel, combined-modality neoadjuvant approach for the treatment of localized esophageal carcinoma is feasible and can be administered with toxicity that compares favorably to previously reported neoadjuvant regimens containing high-dose cisplatin. Preliminary assessment of efficacy is also encouraging, with 46% of patients having pathologic complete response. Further follow-up and larger numbers of patients are required to assess efficacy more definitively.

Functional parathyroid cyst and hypocalciuric hypercalcemia.

Parathyroid cysts are uncommon. They can be divided into functional and nonfunctional cysts depending on whether or not they are associated with hypercalcemia. Functioning cysts are very rare, with fewer than twenty reported cases. We report a case of functioning parathyroid cyst associated with hypocalciuric hypercalcemia. We have been unable to find a similar case previously reported in the literature.

Cognitive behavior therapy, relaxation training, and tricyclic antidepressant medication in the treatment of depression.

Outcomes of seven treatment trials comparing cognitive behavioral therapy to treatment with tricyclic antidepressant medication in major depressive disorder have been quite similar to one another. This led us to question whether treatment outcome in time-limited studies reflected a unique effect of cognitive behavioral therapy. To test the uniqueness hypothesis, relaxation training, a nonpharmacologic, noncognitive treatment, was chosen as a comparison for cognitive behavioral therapy as well as drug therapy. Treatment duration was 16 weeks. The sample of 37 patients treated for major depressive disorder was less depressed than those previously studied. For both cognitive behavioral therapy and relaxation training, outcome of depression was superior to that of tricyclic antidepressant medication by endpoint analysis. The posttreatment scores on the Beck Depression Inventory of 82% of the group receiving cognitive behavioral therapy improved to a Beck Depression Inventory score < or = 9 which was not significantly greater than that for the group receiving relaxation training (73%), so a unique effect was not demonstrated for cognitive behavioral therapy. The outcome for tricyclic antidepressant medication (29% improved to criteria) was significantly worse than that for cognitive behavioral therapy. The patient's pretreatment initial expectancy was not predictive.

Occurrence and prognosis of contralateral carcinoma of the breast.

Of the 4,554 patients who registered at The University of Texas M. D. Anderson Cancer Center, Houston, Texas from 1965 to 1988 with a diagnosis of carcinoma of the breast and who underwent surgical treatment of at least one carcinoma of the breast at this institution, 142 had either a history of a prior carcinoma of the breast (metachronous; n = 55) or a contralateral carcinoma of the breast detected within four months of registration (synchronous; n = 87). We retrospectively studied the records of these 142 patients and found that the occurrence of bilateral carcinoma of the breast was low (3.1 percent), the frequency of metachronous carcinoma of the breast remained relatively constant over time, the nodal status of the second carcinoma of the breast correlated with the method of discovery rather than the stage of the first carcinoma of the breast and survival rates from the second carcinoma of the breast were similar for metachronous and synchronous disease. These data support the role of vigilant surveillance of the contralateral breast with screening at the time of initial diagnosis and during follow-up evaluation. Because the likelihood of detecting a second carcinoma of the breast at an early stage is high, with subsequent good survival rates, the use of prophylactic mastectomy should be very selective and based on the emotional needs of the patient.

The immunogenic properties of drug-resistant murine tumor cells do not correlate with expression of the MDR phenotype.

Alterations in the immunogenic properties of tumor cells frequently accompany selection for multiple-drug-resistant (MDR) variants. Therefore, studies were performed to examine the hypothesis that overexpression of membrane P-glycoprotein, commonly observed in MDR tumor cells, is associated with enhanced immunogenic properties. Immunogenicity was determined by (a) the ability of drug-sensitive parental UV2237M fibrosarcoma cells and drug-resistant UV2237M variant cells to immunize normal mice against rechallenge with parental tumor cells and (b) the ability of normal syngeneic mice to reject cell inocula that caused progressive tumor growth in immunocompromised mice. Variant UV2237M cell lines included subpopulations selected for a six- to ten-fold increase in mRNA for P-glycoprotein and expression of the MDR phenotype (resistance to doxorubicin) and cells sensitive to doxorubicin (and no expression of MDR properties) but resistant to ouabain. All UV2237M drug-resistant cells were highly immunogenic in immunocompetent mice, regardless of their MDR phenotype. Additional studies showed that CT-26 murine adenocarcinoma cells, sensitive or resistant to doxorubicin (expressing high levels of P-glycoprotein), injected into normal syngeneic Balb/c mice produced rapidly growing tumors. The data do not demonstrate a correlation between the immunogenic properties of drug-resistant tumor cells and the expression of P-glycoprotein.