PPARγ gene expression is autoregulated in primary adipocytes: ligand, sumoylation, and isoform specificity.

Being a key-factor in glucose homeostasis, PPARγ transcriptional activity (TA) is of high importance. However, its mediation by ligands and post-translational modifications in insulin target tissues are unclear. We investigated effects of rosiglitazone (Rg) and sumoylation on PPARγ-TA by overexpressing expression vectors and promoter-reporters for PPARγ1 and PPARγ2 in primary rat adipocytes. Wild type (WT) PPARγ1 and PPARγ2 dose-dependently repressed transcription from their promoters to a maximum of 40-50%. PPARγ2 mutants defective in either MAP-kinase phosphorylation (S112A) or the ligand-binding domain (LBD; P495L, L496A/E499A) exhibited decreased repression of PPARγ2 promoter. Rg enhanced repression by S112A, but not by LBD-defective mutants. Sumoylation-defective PPARγ1 mutants K77R and K365R repressed PPARγ2 promoter activity similar to WT, while Rg enhanced repression by K77R but not by K365R. Sumoylation-defective PPARγ2 mutants K107R and K395R exhibited impaired TA and impaired responsiveness to Rg. GLUT4 promoter, previously shown by us to be repressed by WT-PPARγ1 and WT-PPARγ2, was similarly repressed by both sumoylation-defective PPARγ1 mutants, while both sumoylation-defective PPARγ2 mutants exerted reduced repression. Surprisingly, Rg alleviated only WT-PPARγ2-induced repression, while augmenting that induced by WT-PPARγ1 and all sumoylation-defective mutants. Promoter and chromatin immunoprecipitation analyses revealed that PPARγ2 autorepression involves its direct binding to its promoter. In concert with effects at promoter level, Rg decreased endogenous level of PPARγ2 mRNA, while increasing that of GLUT4. We suggest a hypothetical model for PPARγ gene regulation in primary adipocytes that is isoform-specific and Rg/sumoylation-dependent. These findings are important due to the role of PPARγ and Rg in insulin sensitivity.

AHNAK KO mice are protected from diet-induced obesity but are glucose intolerant.

AHNAK is a 700 KD phosphoprotein primarily involved in calcium signaling in various cell types and regulating cytoskeletal organization and cell membrane architecture. AHNAK expression has also been associated with obesity. To investigate the role of AHNAK in regulating metabolic homeostasis, we studied whole body AHNAK knockout mice (KO) on either regular chow or high-fat diet (HFD). KO mice had a leaner phenotype and were resistant to high-fat diet-induced obesity (DIO), as reflected by a reduction in adipose tissue mass in conjunction with higher lean mass compared to wild-type controls (WT). However, KO mice exhibited higher fasting glucose levels, impaired glucose tolerance, and diminished serum insulin levels on either diet. Concomitantly, KO mice on HFD displayed defects in insulin signaling, as evident from reduced Akt phosphorylation and decreased cellular glucose transporter (Glut4) levels. Glucose intolerance and insulin resistance were also associated with changes in expression of genes regulating fat, glucose, and energy metabolism in adipose tissue and liver. Taken together, these data demonstrate that (a) AHNAK is involved in glucose homeostasis and weight balance (b) under normal feeding KO mice are insulin sensitive yet insulin deficient; and (c) AHNAK deletion protects against HFD-induced obesity, but not against HFD-induced insulin resistance and glucose intolerance in vivo.

CYP2E1 impairs GLUT4 gene expression and function: NRF2 as a possible mediator.

Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells.

The safety and efficacy of adding once-daily insulin detemir to oral hypoglycaemic agents in patients with type 2 diabetes in a clinical practice setting in 10 countries.

AIMS: Evaluate the safety and efficacy of once-daily insulin detemir initiated in routine clinical practice in patients with type 2 diabetes mellitus inadequately controlled with oral hypoglycaemic agents (OHAs). METHODS: This large observational study was conducted in 10 countries. Adverse event data (including hypoglycaemia) and glycaemic control were recorded before and 24 weeks following insulin initiation while patients continued routine clinical management. RESULTS: In this study, 17 374 patients (53% male) were included. Mean pre-insulin values (±s.d.) were: age 62 ± 12 years; body mass index (BMI) 29.3 ± 5.4 kg/m(2); diabetes duration 10 ± 7 years; haemoglobin A1c (HbA1c) 8.9 ± 1.6%. During the study, 27 patients experienced serious adverse drug reaction, severe hypoglycaemic events or both; and there were 31 episodes of severe hypoglycaemia in 21 patients. After 24 weeks, HbA1c was 7.5 ± 1.2% (change of -1.3%; p < 0.001) and mean weight change was -0.6 kg (confidence interval -0.7, -0.5 kg, p < 0.001). Daily insulin dose increased from 13 ± 6 U (0.16 ± 0.09 U/kg) to 22 ± 16 U (0.27 ± 0.17U/kg) by 24 weeks. Multivariate regression analysis identified several independent demographic and treatment predictors of end of study HbA1c. CONCLUSIONS: Addition of once-daily insulin detemir to patients with type 2 diabetes mellitus on OHA therapy resulted in few adverse events, significant improvements in glycaemic control, small reductions in weight and low rates of hypoglycaemia. On the basis of this study, concerns about hypoglycaemia or weight gain should not preclude initiation of basal insulin analogues in patients with poor glycaemic control on OHAs.

Improvement in glycemic control by gastric electrical stimulation (TANTALUS) in overweight subjects with type 2 diabetes.

BACKGROUND: The TANTALUS system (MetaCure Ltd.) is a minimally invasive implantable gastric stimulation modality that does not exhibit malabsorptive or restrictive characteristics. The device applies gastric contractility modulation (GCM) signals to the stomach antrum. The signals are delivered in synchronization to the native electrical activity of the stomach during meals. Retrospective analysis of previous studies indicated that type 2 diabetes mellitus (T2DM) subjects on oral medication with hemoglobin A1c (HbA1c) between 7.5% and 9.5% are the population with most potential benefit from the treatment. The current study includes subjects enrolled prospectively within that range of HbA1c. AIM: To prospectively investigate the potential effect of the TANTALUS system on glycemic control and weight in overweight subjects with T2DM. METHODS: In this European multicenter, open-label study, 13 T2DM obese (6 male, 7 female, BMI 37.2 +/- 1.0 kg/m(2), range 30.4-44.0 kg/m(2)) subjects treated with oral antidiabetic medications but with poor glycemic control (HbA1c > or = 7%, range 7.3-9.5%) were implanted laparoscopically with the TANTALUS system. RESULTS: Thirteen subjects that had completed 3 months of treatment showed a significant reduction in HbA1c from 8.0 +/- 0.2% to 6.9 +/- 0.1% (p < 0.05), whereas fasting blood glucose decreased from 175 +/- 6 mg/dL to 127 +/- 8 mg/dL (p < 0.05). The glycemic improvement was accompanied by reduction in weight from 104.4 +/- 4.4 kg to 99.7 +/- 4.8 kg, and in waist circumference from 122.3 +/- 3.2 cm to 117.0 +/- 3.0 cm. CONCLUSIONS: Interim results with the TANTALUS system suggest that this stimulation regime can potentially improve glucose levels and induce moderate weight loss in obese T2DM subjects on oral antidiabetic therapy with poor glycemic control. Further evaluation is required to determine whether this effect is due to induced weight loss and/or due to direct signal-dependent mechanisms.

The proposed terminology 'A(1c)-derived average glucose' is inherently imprecise and should not be adopted.

The proposed use of a more precise standard for glycated (A(1c)) and non-glycated haemoglobin would lead to an A(1c) value, when expressed as a percentage, that is lower than that currently in use. One approach advocated to address the potential confusion that would ensue is to replace 'HbA(1c)' with a new term, 'A(1c)-derived average glucose.' We review evidence from several sources suggesting that A(1c) is, in fact, inherently imprecise as a measure of average glucose, so that the proposed terminology should not be adopted.

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach.

AIMS/HYPOTHESIS: We examined a clinical model of ex vivo transdifferentiation of primary adult hepatocytes to insulin-secreting cells for the treatment of type 1 diabetes. MATERIALS AND METHODS: Isolated rat hepatocytes were transduced in primary culture with a human lentivirus containing pancreatic duodenal homeobox 1 (PDX1, now known as insulin promoter factor 1, homeodomain transcription factor [IPF1]). Insulin expression and secretion of the newly engineered cells were assessed in vitro by RT-PCR, in situ hybridisation, immunostaining and radioimmunoassay. PDX1-transduced hepatocytes were further studied in vivo by injecting them under the renal capsule of diabetic SCID mice. RESULTS: Isolated rat hepatocytes were efficiently transduced with the lentiviral vector, as assessed by green fluorescent reporter gene expression. The transduced cells exhibited insulin at both mRNA (RT-PCR, in situ hybridisation) and protein levels (immunostaining and radioimmunoassay). Moreover, insulin secretion by the engineered cells was dependent on glucose and sulfonylurea. Other beta cell genes, including those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), glucokinase (Gck), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), the potassium inwardly-rectifying channel, subfamily J, member 11 (Kcnj11) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) were also expressed. The PDX1-transduced hepatocytes expressed several pancreatic transcription factors related to early pancreatic endocrine development (endogenous Pdx1, neurogenic differentiation factor 1 [Neurod1], and NK6 transcription factor related, locus 1 [Nkx6-1]) as well as the late-stage pancreatic transcription factors (paired box gene 4 [Pax4], paired box gene 6 [Pax6], and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A [Mafa]). Transplantation of 3 x 10(6) transdifferentiated liver cells under the renal capsule of seven streptozotocin-induced diabetic SCID mice resulted in significant reduction of non-fasting blood glucose levels from 30.7 +/- 1.3 to 8.7 +/- 3.7 mmol/l (mean +/- SEM, p = 0.01), in 6 to 8 weeks. Removal of the graft resulted in severe hyperglycaemia. CONCLUSIONS/INTERPRETATION: Ex vivo lentiviral-mediated PDX1 expression in isolated adult liver cells represents a potential model for type 1 diabetes mellitus therapy.

Analysis of the peroxisome proliferator activated receptor gamma (PPARgamma) gene in HAIRAN syndrome with obesity.

OBJECTIVES: To test the hypothesis that the triad of hyperandrogenism, insulin resistance and acanthosis nigricans (HAIRAN syndrome) in the presence of obesity, also known as type C insulin resistance, is caused by mutations in the gene for peroxisome proliferator activated receptor gamma (PPARgamma), a receptor for the thiazolidinedione drugs that enhance sensitivity to insulin. To investigate possible correlations between mutations in PPARgamma and the degree of insulin resistance. DESIGN: A candidate gene approach to study the molecular basis for a syndrome of obesity; a comparison of genotype with in vivo phenotype. PATIENTS: Fifteen unrelated patients with HAIRAN syndrome and obesity. Controls for the gene analysis: 25 unrelated non-diabetic non-obese individuals. Controls for the metabolic studies: six unrelated patients with type 2 diabetes mellitus and nine unrelated non-diabetic non-obese individuals. MEASUREMENTS: Analysis of polymerase chain reaction (PCR) products of the 7 exons that constitute the entire coding region of both PPARgamma isoforms (PPARgamma1 and PPARgamma2) for single-stranded conformational polymorphisms (SSCP); in exons with variant patterns: restriction fragment length polymorphism (RFLP) analysis; and, where relevant, direct sequencing. Evaluation of insulin resistance using the insulin euglycaemic clamp technique. RESULTS: A synonymous substitution in codon 477 (CACHis --> CATHis) was found in one patient. A missense mutation in codon 12 of PPARgamma2 (CCAPro --> GCAAla) was found in another patient, but not in any of 25 non-diabetic, non-obese control individuals. The patient with the Pro12Ala variant had the highest steady state glucose infusion rate (SSGIR) and most marked suppression of hepatic glucose production rate (HGPR) of all of the patients studied. CONCLUSIONS: Mutations in the PPARgamma gene are unlikely to be major contributors to HAIRAN syndrome with obesity. The Pro12Ala variant may correlate with a lesser degree of insulin resistance in these patients.

L-Glutamic acid gamma-monohydroxamate. A potentiator of vanadium-evoked glucose metabolism in vitro and in vivo.

We report that the vanadium ligand L-Glu(gamma)HXM potentiates the capacity of free vanadium ions to activate glucose uptake and glucose metabolism in rat adipocytes in vitro (by 4-5-fold) and to lower blood glucose levels in hyperglycemic rats in vivo (by 5-7-fold). A molar ratio of two L-Glu(gamma)HXM molecules to one vanadium ion was most effective. Unlike other vanadium ligands that potentiate the insulinomimetic actions of vanadium, L-Glu(gamma)HXM partially activated lipogenesis in rat adipocytes in the absence of exogenous vanadium. This effect was not manifested by D-Glu(gamma)HXM. At 10-20 microM L-Glu(gamma)HXM, lipogenesis was activated 9-21%. This effect was approximately 9-fold higher (140 +/- 15% of maximal insulin response) in adipocytes derived from rats that had been treated with vanadium for several days. Titration of vanadium(IV) with L-Glu(gamma)HXM led to a rapid decrease in the absorbance of vanadium(IV) at 765 nm, and (51)V NMR spectroscopy revealed that the chemical shift of vanadium(IV) at -490 ppm disappeared with the appearance of a signal characteristic to vanadium(V) (-530 ppm) upon adding one equivalent of L-Glu(gamma)HXM. In summary, L-Glu(gamma)HXM is highly active in potentiating vanadium-activated glucose metabolism in vitro and in vivo and facilitating glucose metabolism in rat adipocytes in the absence of exogenous vanadium probably through conversion of trace intracellular vanadium into an active insulinomimetic compound. We propose that the active species is either a 1:1 or 2:1 L-Glu(gamma)HXM vanadium complex in which the endogenous vanadium(IV) has been altered to vanadium(V). Finally we demonstrate that L-Glu(gamma)HXM- and L-Glu(gamma)HXM.vanadium-evoked lipogenesis is arrested by wortmannin and that activation of glucose uptake in rat adipocytes is because of enhanced translocation of GLUT4 from low density microsomes to the plasma membrane.

The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor.

Children with uncontrolled type I (insulin-dependent) diabetes mellitus are characterized by a slow growth rate, which improves upon adequate therapy. While skeletal growth is an energy-consuming process involving high glucose utilization, the role of glucose transporters (GLUT) and their regulation in the bone formation process are not yet fully understood. Thus, we studied both in vivo and in vitro early endochondral bone formation in control and streptozotocin-induced young diabetic mice. Using in situ hybridization and immunohistochemistry techniques, we demonstrated the novel existence of the insulin-sensitive glucose transporter (GLUT4), as well as GLUT1, in juvenile-derived murine mandibular condyles and in the humeral growth plate-two models for endochondral bone formation. Insulin-like growth factor (IGF) I receptors (IGF-I-R), but not insulin receptors (IR), were shown to have cellular distribution similar to GLUT4, being more abundant in mature chondrocytes. Further, in the skeletal growth centers of streptozotocin-induced diabetic mice, GLUT4, IGF-I, and IGF-I and insulin receptor levels, but not GLUT1 were markedly reduced. The decrease in GLUT4 and in IGF-I and insulin receptors was associated with severe histological changes in the mandibular condyles and humeral growth plate. Insulin therapy restored IR levels to normalcy, whereas IGF-I-R and GLUT4 levels were only partially recovered. Thus, GLUT4 and IGF-I-R have a potential role in early bone growth in mice. Further, during early bone growth GLUT4 may be regulated through the IGF-I receptor rather than via the insulin receptor. We propose that skeletal growth retardation in type I diabetes may be associated with reduced expression of the GLUT4 and IGF-I receptor in the bone growth center.

Analysis of the insulin receptor gene tyrosine kinase domain in obese patients with hyperandrogenism, insulin resistance and acanthosis nigricans (type C insulin resistance).

OBJECTIVE: To test the hypothesis that the triad of hyperandrogenism, insulin resistance and acanthosis nigricans (HAIR-AN syndrome) in the presence of obesity, also known as type C insulin resistance (type C), is caused by mutations at the tyrosine kinase domain of the insulin receptor gene. DESIGN: A candidate gene approach to study the molecular basis for a syndrome of obesity. SUBJECTS: 15 patients with type C insulin resistance and 25 control individuals. MEASUREMENTS: Analysis of polymerase chain reaction (PCR) products of exons 17 to 21 of the insulin receptor gene, comprising the tyrosine kinase domain, for single strand conformational polymorphisms (SSCP) and sequence analysis of exons with variant SSCP patterns. RESULTS: A synonymous C to T substitution in position 3 of codon 984, which does not alter the amino acid predicted, was found in one patient and in four of 25 control individuals. CONCLUSION: Type C insulin resistance is not commonly caused by mutations in the tyrosine kinase domain of the insulin receptor gene.

Double-vein jugular/inferior vena cava clamp technique for long-term in vivo studies in rats.

We present a step-by-step manual for chronic cannulation of rats using a simple technique. This concept facilitates repeated clamping of the same rat over a 6-10-week period, providing a completely separate infusion route from blood sampling access which is placed into mixed venous blood in the inferior vena cava. Permanent catheters implanted into the left external jugular vein and the inferior vena cava were used for miniature blood sampling and recycling. The design and running of clamp experiments and other physiological research models are detailed. Long-term reliable venous access, simple installation, and easy after-care of the rats' cannulas are the principal advantages of the procedure described.

The effect of acute insulin-like growth factor-II administration on glucose metabolism in the rat.

The in vivo effects of the insulin-like growth factor-II (IGF-II) on glucose metabolism is not yet well defined. To assess the acute effect of IGF-II administration on whole body glucose utilization and hepatic glucose production, we used the well-established euglycemic clamp technique and compared the effects in awake cannulated rats with those of insulin. Each animal underwent several 90-min euglycemic studies, alternating between IGF-II and insulin. Following IGF-II infusion, tissue glucose uptake was increased to 9.8 +/- 0.6 mg/kg/min (mean +/- SEM), which represented only 14% of the effect of insulin, despite the molar plasma concentration ratio of insulin: IGF-2 being 1:460. IGF-II and insulin infusion reduced hepatic glucose output by 49 and 75%, respectively. Thus, IGF-II, administered acutely, affects glucose homeostasis in a manner very similar to insulin, probably via the insulin receptors, although with significantly lower potency.

Repeated BCG vaccination is more effective than a single dose in preventing diabetes in non-obese diabetic (NOD) mice.

Stimulation of the non-obese diabetic (NOD) mouse immune-system with a single bacillus Calmette-Guerin (BCG) vaccination can inhibit the development of diabetes. The optimal dose, and the time and number of vaccinations is still to be clarified. In this study we evaluated the protective effect of repeated BCG vaccinations on preventing diabetes in NOD mice. 17/32 (53%) of the control group, 8/31 (26%) of the single vaccine-treated (at age 35 days) mice, and 7/23 (30%) of the single vaccine-treated (at age 90 days) mice developed diabetes, and none of the repeated BCG vaccination (at age 35 & 90 days, n = 14) animals developed the disease, up to 250 days of age (p < 0.05, compared with controls and each of the single-vaccination groups). While the severity of insulitis was lower in repeatedly BCG-treated mice at age 120 days as compared with controls and single BCG-vaccination groups, we could not detect significant differences in the Intracellular adhesion molecule-1 (ICAM-1) expression between the various groups. There were no differences in weight gain and blood hematocrit between the different groups. Our report demonstrates that repeated BCG vaccination is safe and more effective than a single dose in preventing type I diabetes in NOD mice. This data should be considered in planning new human trials with BCG.

Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.

The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs.

The IGF-I receptor gene promoter is a molecular target for the Ewing's sarcoma-Wilms' tumor 1 fusion protein.

Desmoplastic small round cell tumor (DSRCT) is an abdominal malignancy in children which is characterized by a recurrent chromosomal translocation, t(11;22)(p13;q12). This rearrangement results in the fusion of the ubiquitously expressed EWS1 gene to the Wilms' tumor suppressor (WT1) gene. The chimeric protein contains the N-terminal domain of EWS1 fused to the DNA-binding domain of WT1, including zinc fingers 2-4. Because WT1 has been shown previously to bind and repress the insulin-like growth factor I (IGF-I-R) promoter, we investigated whether this promoter is, in addition, a target for the aberrant EWS/WT1 transcription factor. EWS/WT1 activated the IGF-I-R promoter approximately 340%, whereas a fusion protein containing a three-amino acid insert (KTS) between zinc fingers 3 and 4 had no effect. On the other hand, expression vectors encoding either WT1 or EWS1 reduced the activity of the promoter to 46 and 58% of control values, respectively. Results of gel shift assays indicate that the binding affinity of EWS/WT1 to a fragment of the 5'-flanking region of the receptor promoter was higher than the affinity of WT1 itself. Consistent with the results of functional assays, the binding of EWS/WT1(+KTS) was significantly reduced. Due to the central role of the IGF-I-R in tumorigenesis, activation of the receptor promoter by EWS/WT1 may constitute a potential mechanism for the etiology and/or progression of DSRCT.

Thyroid function in early normal pregnancy: transient suppression of thyroid-stimulating hormone and stimulation of triiodothyronine.

In order to determine the effect of gestation on thyroid function in healthy subjects, we have prospectively evaluated thyroid function in pregnant individuals undergoing termination of pregnancy, and repeated the tests 2-3 months later. Venous blood was tested for human chorionic gonadotropin (hCG), thyroid-stimulating hormone (TSH), free thyroxine (FT4) and total triiodothyronine (TT3). Early pregnancy thyroid function tests showed a significant decrease (p < 0.001) in TSH and a significant increase (p < 0.001) in TT3 as compared to the nonpregnant state; FT4, however, did not change significantly. In 8 (11.2%) pregnant subjects, TT3 levels were above the normal range for nonpregnant controls. Elevated thyroid function in early pregnancy is transient, and does not usually warrant antithyroid treatment. Thus, any conclusion regarding thyroid function in early pregnancy should be based on pregnant controls rather than general population controls.

The regulation of IGF-I receptor gene expression.

The insulin-like growth factor I (IGF-I) receptor mediates most of the biological effects of IGF-I and -II. Despite its structural similarity to the insulin receptor, the IGF-I receptor is mainly involved in the transduction of growth and differentiation types of signals. The IGF-I receptor gene is constitutively expressed by most cells in the organism as well as in culture, consistent with the role of the IGFs as survival factors. In addition, the expression of the IGF-I receptor gene is modulated by a number of physiological and pathological factors, including developmental stage, nutritional status, hormones, growth disorders and malignancy. The regulatory region of the IGF-I receptor gene has been characterized and shown to display a high level of basal promoter activity. Transcription factor Sp1 is a strong activator of IGF-I receptor gene expression, whereas tumor suppressor WT1 represses its activity. The biological implications of these findings in both normal development and disease are described in this review.

Modulation of the activity of glucose transporters (GLUT) in the aged/obese rat adipocyte: suppressed function, but enhanced intrinsic activity of GLUT.

To study the contribution of glucose transporters (GLUT) to insulin resistance in aging, GLUT intrinsic activity was assessed in a cell-free system. Adipocytes were isolated from 18-month-old rats and young controls and incubated either with or without 7 nM insulin. Plasma membrane (PM) and low density microsomal fractions were prepared from the cells, and GLUT levels were assessed in these fractions before and after reconstitution into liposomes. Glucose transport rates were measured in intact cells and liposomes. Functional and intrinsic activities of GLUT were assessed from the ratio between these transport rates and GLUT levels in the respective fractions. Basal 3-O-methylglucose transport rates were unaffected by aging, which is consistent with unchanged levels of GLUT in PM. Insulin-stimulated glucose transport was 60% lower in aging, as was the extent of GLUT recruitment to PM. The effect of insulin stimulation of GLUT functional activity by 6-fold at PM was attenuated by 40% in aging. Conversely, the basal intrinsic activity of GLUT was significantly enhanced in aging (by 280% and 230% in PM and density microsomal liposomes, respectively) and was further stimulated by insulin by about 160% in PM, compared to only about 117% stimulation in controls. In conclusion, our data show that insulin stimulates the intrinsic activity of GLUT in rat adipocytes, and this activity is further enhanced in aging. Impaired glucose uptake in aging can be attributed to depleted GLUT4 levels and impaired function of GLUT at the cell surface. The discrepancy observed between impaired function and enhanced intrinsic activity of GLUT suggests the presence of additional factors that modulate the full functional expression of GLUT at the cell surface.

Glucose utilization in morbidly obese subjects before and after weight loss by gastric bypass operation.

OBJECTIVE: To investigate the effect of weight reduction induced by gastric bypass operation (GBO), on the peripheral resistance to insulin in extremely obese subjects. DESIGN: A three-stage euglycemic clamp was applied to assess the metabolic clearance rate of glucose (g-MCR) in lean controls and in obese subjects, prior to and 6-12 months post operation. SUBJECTS: Six obese subjects (four obese normoglycemics-ON and two obese non-insulin dependent diabetics-OD) before and after GBO and six healthy, lean controls (LC) were compared. MAIN OUTCOME MEASURES: Body mass index after GBO, metabolic clearance rate of glucose under increasing insulin concentrations. RESULTS: GBO resulted in a significant change in body mass index from a pre-operation value (mean +/- s.d.) of 45.0 +/- 8.5 to 30.4 +/- 5.9 kg m-2 and remained significantly greater than controls (23.3 +/- 2.3 kg m-2). Glucose MCR increased from a mean baseline value of 3.0 +/- 1.6 to 6.7 +/- 3.9 ml kg-1 min-1 at post GBO (P) (P < 0.02). Similar effects were obtained under the two higher insulin concentration. CONCLUSIONS: GBO resulted in a massive reduction in BMI, that still remained above normal controls. This could explain the effect of weight reduction to decrease, but not completely reverse, peripheral resistance to insulin associated with obesity.