Increased expression of ubiquitous enzyme, tissue transglutaminase (TG2) is consistently seen in a variety of models of apoptosis. Here we show that retinoblastoma protein (Rb) is a substrate for the recently identified serine/threonine kinase activity of TG2. Phosphospecific antibodies indicated that TG2 phosphorylated at the critically important Ser780 in Rb. Furthermore, when E2F1 was immunoprecipitated from MCF-7 breast cancer cells and incubated with Rb773–928 fragment, phosphorylation of Rb by TG2 destabilizes the Rb/E2F1 complex. TG2 alone in the absence of ATP had no effect. The TG2 kinase activity was abrogated by high Ca2+ concentrations whereas the cross-linking activity of TG2 was inhibited by ATP. These results suggest that a rise in cytosolic Ca2+ that accompanies many pro-apoptotic stimuli may result in a switch in TG2 functional activity from phosphorylation to cross-linking. This research was supported by funds from the Canadian Institute of Health Research.

IGF binding protein (IGFBP)-3 inhibits cell growth and promotes apoptosis by sequestering free IGFs. In addition IGFBP-3 has IGF-independent, proapoptotic, antiproliferative effects on prostate cancer cells in vitro. Expression of the large T-antigen (Tag) under the long probasin promoter (LPB) in LPB-Tag mice results in prostate tumorigenesis. To investigate the IGF-dependent and IGF-independent effects of IGFBP-3 on prostate tumor growth, we crossed LPB-Tag mice with cytomegalovirus (CMVBP-3) and phosphoglycerate kinase (PGKBP-3) mice that overexpress IGFBP-3 under the cytomegalovirus promoter and the phosphoglycerate kinase promoter, respectively, and also I56G/L80G/L81G-mutant IGFBP-3 (PGKmBP-3) mice that express I56G/L80G/L81G-IGFBP-3, a mutant, that does not bind IGF-I but retains IGF-independent proapoptotic effects in vitro. Prostate tumor size and the steady-state level of p53 were attenuated in LPB-Tag/CMVBP-3 and LPB-Tag/PGKBP-3 mice, compared with LPB-Tag/wild-type (Wt) mice. A more marked effect was observed in LPB-Tag/CMVBP-3, compared with LPB-Tag/ PGKBP-3, reflecting increased levels of transgene expression in CMVBP-3 prostate tissue. No attenuation of tumor growth was observed in LPB-Tag/PGKmBP-3 mice during the early tumor development, indicating that the inhibitory effects of IGFBP-3 were most likely IGF dependent during the initiation of tumorigenesis. At 15 wk of age, epidermal growth factor receptor expression was increased in LPB-Tag/ Wt and LPB-Tag/PGKmBP-3 tissue, compared with LPB-Tag/PGKBP-3. IGF receptor was increased in all transgenic mice, but pAkt expression, a marker of downstream IGF-I action, was increased only in LPB-Tag/Wt and LPB-Tag/PGKmBP-3. After 15 wk of age, a marked reduction in tumor growth was apparent in LPB-Tag/PGKmBP-3 mice, indicating that the IGF-independent effects of IGFBP-3 may be important in inhibiting tumor progression.

Prohibitin (PHB‐1) is a highly conserved protein involved in mitochondrial biogenesis and function. It is secreted in lipid droplets from adipocytes and is present in the circulation. In adipose tissue it functions as a membrane receptor and can target binding partners to the mitochondria. Here we report that PHB‐1 has a hitherto undescribed role as an inhibitor of pyruvate carboxylase (PC). As a consequence, it can modulate insulin‐stimulated glucose and fatty acid oxidation. It had no effect on insulin‐stimulated 2‐deoxglucose uptake by isolated adipocytes but inhibited insulin‐stimulated oxidation of [14C]glucose with a half‐maximal concentration of ≈ 4 nm. It also inhibited oleic acid oxidation in glucose‐depleted adipocytes via depletion of oxaloacetate. In vitro experiments using broken‐cell assays confirmed that PHB‐1 inhibited PC. MALDI‐TOF analysis of proteins identified by cross‐linking of PHB‐1 to adipocyte membranes indicated that PHB‐1 is closely associated with PC and EH domain 2 (EHD2). On the basis of these data, we propose that PHB‐1 is recycled between the extracellular space and the mitochondria by a mechanism involving lipid rafts and EHD2 and can modulate mitochondrial fuel metabolism by inhibition of PC.

BACKGROUND:The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis.OBJECTIVES:We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation.METHODS:(i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively.RESULTS:(i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation.CONCLUSIONS:We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.

Increased expression and activity of the ubiquitous enzyme, tissue transglutaminase (TG2), is consistently seen in a variety of models of apoptosis. The p53 oncoprotein is also involved in apoptosis. Here we investigated the interaction of TG2 with p53 and show that the p53 is a substrate for the recently identified serine/threonine kinase activity of TG2. Phosphospecific antibodies indicated that TG2 phosphorylated p53 at Ser(15) and Ser(20), residues that are critically important in the interaction of p53 with Mdm2. The TG2-induced phosphorylation was abrogated by high Ca(2+) concentrations and inhibited by cystamine, a known inhibitor of TG2 cross-linking activity. Furthermore, we demonstrate that TG2-induced phosphorylation of p53 reduces the ability of p53 to interact with Mdm2. Although TG2 cross-linking activity has been clearly implicated in apoptosis, our observations reported here suggest TG2 modification of p53 could be an additional mechanism whereby TG2 could facilitate apoptosis.

Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function and protection against senescence. There is considerable controversy concerning the function of nuclear-localized PHB. PHB has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. PHB might also function as a cell-surface receptor for an as-yet unidentified ligand. Cell-associated PHB in the gastrointestinal tract has been implicated in protection against infection and inflammation and the induction of apoptosis in other tissues. The diverse array of functions of PHB, together with the emerging evidence that its function can be modulated specifically in certain tissues, suggest that targeting PHB would be a useful therapeutic approach for the treatment of variety of disease states, including inflammation, obesity and cancer.

The majority of insulin-like growth factor-I (IGF-I) in the adult rodent circulation is bound to high affinity IGF binding proteins. We investigated the changes in IGF-I clearance, blood glucose and plasma insulin levels, and tissue 2-deoxyglucose uptake after intravenous administration of the IGF aptamer, NBI-31772, which selectively competes with IGF-I for binding to the IGFBPs, but has no effect at the IGF-I receptor. Clearance of 125I-IGF-I was significantly increased in NBI-31772-treated mice compared with vehicle-treated mice (t1/2 = 45.0 +/- 1.9 vs. 56.3 +/- 3.9 min, respectively; p = 0.021). However, NBI-31772 had no significant effect on glucose levels, and no insulin sparing effect was apparent neither under basal conditions nor during an intravenous glucose challenge. The decline in the specific activity after 3H-2-deoxyglucose administration was significantly less rapid in NBI-31772-treated mice compared with controls, suggesting that the IGF-I aptamer had an inhibitory effect on hepatic gluconeogenesis. In contrast, no insulin-like effect was apparent in other tissues examined. 3H-2-deoxyglucose accumulation was similar in all tissues analyzed, including skeletal muscle, which is thought to be particularly sensitive to IGF-I. These data suggest that the IGF-I aptamer affects clearance of radiolabeled IGF-I from the circulation, but has no marked effects on glucose nor insulin homeostasis. The search for hydrophilic IGF aptamers with longer duration of action that could be used in the treatment of diabetes may be rewarding.

IGFBPs regulate growth and development by regulating IGF transport to tissues and IGF bioavailability to IGF receptors at cell membrane level. IGFBP excess leads predominantly to inhibition of IGF action and growth retardation with impaired organogenesis. Absence of human and also mouse ALS leads to decreased IGF-I levels in circulation and causes mild growth retardation. Although IGFBP KO mice demonstrate relatively minor phenotypes, the possibility of compensatory mechanisms that mask the phenotypic manifestation of lack of individual binding proteins needs to be further investigated. Recent studies of hepatic regeneration in IGFBP-1 KO mice and also with mutant IGFBP-3 Tg mice provide some limited support for the existence of IGF-independent mechanism of action in vivo.

BACKGROUND:Adipose tissue produces both vascular growth factors and inhibitors. Since obesity is associated with expansion of the capillary bed in regional adipose depots the balance between these factors may favor angiogenesis.OBJECTIVE:To investigate the relationship between body mass index and serum concentrations of vascular growth factors in human subjects.METHODS:Vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, soluble VEGF receptor-2 (sVEGFr2), hepatocyte growth factor (HGF), angiopoietin-2, angiogenin and endostatin concentrations were measured in serum collected from 58 lean (24 males, 34 female, mean BMI, 22.2+/-0.3) and 42 overweight and obese (16 males and 26 females, mean BMI, 33.5+/-1.2) subjects after an overnight fast.RESULTS:Sexual dimorphism was apparent in the serum concentrations of VEGF-C, VEFG-D and angiopoietin-2 with significantly higher levels in female compared to male subject. VEGF, VEGF-C, VEGF-D, soluble VEGF receptor-2, angiopoietin-2, angiogenin and endostatin but not HGF were significantly elevated in overweight and obese subjects. Positive correlations between BMI and the serum concentrations of VEGF-C, VEGF-D, sVEGF-R2, angiopoietin-2, angiogenin and endostatin were observed even after adjustment for gender and age.CONCLUSIONS:Increased levels of vascular growth factors as well as the angiogenesis inhibitor endostatin are present in overweight and obese subjects and may contribute to previously documented increased risk of metastatic disease in obese subjects with cancer.

BACKGROUND Growth hormone deficiency (GHD) in adult life has been associated with increased central adiposity, decreased insulin sensitivity, dyslipidaemia and increased risk of cardiovascular disease. The effects of GH replacement on adiponectin and resistin, adipokines which have a role in modulating insulin sensitivity have not been previously reported.AIM To examine the effects of GH replacement on adipokine levels and insulin resistance in GHD patients.DESIGN Seventeen adult GHD patients were examined at baseline and after 1 year of treatment with recombinant human GH (mean dose 0.31 mg/day, range 0.13-0.67 mg/day).RESULTS GH replacement significantly increased IGF-I levels. The mean IGF-I SD score increased from - 1.98 at baseline to 0.76 at study end. GH replacement was associated with a significant reduction in percentage body fat (34.11 +/- 1.33 vs. 30.65 +/- 1.27%, P < 0.0005) and a significant increase in lean body mass (63.57 +/- 1.24 vs. 66.96 +/- 1.18%, P < 0.0004), before and after treatment, respectively. Surprisingly, there was no effect of GH replacement on the plasma levels of leptin, resistin or adiponectin or on plasma lipid profile. Insulin sensitivity did not deteriorate during GH replacement despite the known 'anti-insulin' effect of GH. Fasting glucose, insulin and insulin resistance as calculated using the homeostasis model assessment insulin resistance index (HOMA-R) were unchanged by GH treatment.CONCLUSION These data demonstrate GH replacement in adult subjects with GHD is effective in changing body composition and restoring IGF-I levels over a 12-month period; however, in our study, these changes were not accompanied by changes in adipokine levels or beneficial effects on plasma lipids or insulin resistance.

OBJECTIVE:To examine gender differences and hormonal regulation of resistin, adiponectin, and leptin.RESEARCH METHODS AND PROCEDURES:Plasma levels were measured, and mRNA expression in perigonadal fat was quantified by RNase protection assays.RESULTS:Plasma resistin declined with age despite an increase in adiposity in both genders. In male mice, plasma leptin increased, whereas adiponectin levels were constant. In females, both adiponectin and leptin levels increased with age. Resistin mRNA levels were significantly higher in female than male mice at all ages, whereas leptin and adiponectin mRNA levels were similar in fat from 6-week-old male and female mice, and sexual dimorphism was apparent only in the older mice, with higher levels apparent in females. Castration did not abolish gender differences in plasma levels or resistin, adiponectin, or leptin mRNAs. Castration of male mice did not significantly change adipokine mRNA levels or plasma levels of resistin or leptin; however, adiponectin was significantly increased. Dihydrotestosterone treatment had no effect on adipokine mRNA expression or resistin and adiponectin levels but increased leptin levels. In contrast, ovariectomy significantly increased resistin mRNA abundance and decreased leptin and adiponectin mRNAs. Plasma leptin levels were also increased by ovariectomy, whereas resistin and adiponectin levels were unchanged. Estrogen replacement significantly reduced resistin mRNA and increased leptin and adiponectin mRNA levels but had no effect on plasma adipokine levels.DISCUSSION:The gender differences in adipokine mRNA expression and plasma levels were not ablated by castration and seem to be dependent on other factors in addition to gonadal steroids.

The role of resistin in obesity and insulin resistance in humans is controversial. Therefore, resistin protein was quantitated by ELISA in serum of 27 lean [13 women/14 men, body mass index (BMI) 21.7 +/- 0.4 kg/m(2), age 33 +/- 2 yr] and 50 obese (37 women/13 men, BMI 49.8 +/- 1.5 kg/m(2), age 47 +/- 1 yr) subjects. There was more serum resistin protein in the obese (mean +/- SEM: 5.3 +/- 0.4 ng/ml; range 1.8-17.9) than lean subjects (3.6 +/- 0.4 ng/ml; range 1.5-9.9; P = 0.001). The elevation of serum resistin in obese humans was confirmed by Western blot as was expression of resistin protein in human adipose tissue and isolated adipocytes. There was a significant positive correlation between resistin and BMI (r = 0.37; P = 0.002). Multiple regression analysis with predictors BMI and resistin explained 25% of the variance in homeostasis model assessment of insulin resistance score. BMI was a significant predictor of insulin resistance (P = 0.0002), but resistin adjusted for BMI was not (P = 0.11). The data demonstrate that resistin protein is present in human adipose tissue and blood, and that there is significantly more resistin in the serum of obese subjects. Serum resistin is not a significant predictor of insulin resistance in humans.

AIMS:To investigate whether availability of glucometer reagents increases the frequency of self-blood glucose monitoring (SBGM) and improves glycaemic control in diabetic patients. METHODS:Sixty-two insulin-treated diabetic patients were randomized to two groups, matched for age, gender, education, income, type and duration of diabetes, years of insulin treatment, number of daily insulin injections, and haemoglobin (Hb)A1c. All patients were given a glucometer, but one group (no cost, NC) was provided glucometer test strips free of charge. The other group (control, C) had to purchase strips as they found it necessary. Both groups of patients were followed longitudinally at 2-monthly intervals for 12 months with measurement of blood glucose and HbA1c, and the frequency of SBGM was determined by downloading the glucometer memory. RESULTS:The SBGM frequency was significantly higher in the NC group vs. the C group during the first 4 months (2.0 +/- 0.2 tests/day vs. 1.4 +/- 0.1 tests/day, P<0.025). Mean HbA1c remained stable over the 12 months in the NC group, whereas an increase with time was observed in the C group. The difference in HbA1c between the two groups was significant (P<0.002) after 6 months. Random blood glucose measured at each visit and average glucose recorded by the glucometer were also lower in the NC group vs. the C group (P<0.005). There was a negative correlation between HbA1c and SBGM frequency, and HbA1c in patients testing at least twice a day was lower than in those testing less than twice a day (8.8 +/- 0.2% vs. 9.6 +/- 0.2%, P<0.001). CONCLUSIONS:In this prospective study, having easy access to glucometer strips provided free of charge to patients increased SBGM frequency. The relationship between HbA1c and SBGM frequency supports the view that SBGM is an essential tool in diabetes management.

Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) was identified as a binding partner for insulin-like growth factor binding protein-3 (IGFBP-3) in solubilized T47D and MCF-7 human breast cancer cell membranes. The interaction between AMF/PGI and IGFBP-3 was verified by cross-linking biotinylated IGFBP-3 to intact cells. After solubilization of the membranes, the biotinylated complexes were precipitated with streptavidin-agarose conjugate and analyzed by SDS-PAGE. A Mr ∼80,000 complex was identified when the nitrocellulose membranes were probed either with streptavidin-horseradish peroxidase conjugate or AMF/PGI antiserum confirming the cross-linking of IGFBP-3 to AMF/PGI. The interaction between IGFBP-3 and AMF/PGI was also further confirmed by ligand blotting of purified AMF/PGI using biotinylated IGFBP-3. Both glycosylated and nonglycosylated IGFBP-3 inhibited the catalytic activity of AMF/PGI in a dose-dependent fashion. In addition, IGFBP-3 inhibited the binding of AMF/PGI to breast cancer cells and AMF/PGI-induced migration of both T47D and MCF-7 human breast cancer cells. IGFBP-3 also decreased the phosphorylation of AMF/PGI and reduced the translocation of AMF/PGI to the cell membrane and AMF/PGI. AMF/PGI resulted in a dose-dependent inhibition of IGFBP-3 induced apoptosis in T47D and MCF-7 cells. In summary, we have identified AMF/PGI as a membrane-associated binding partner for IGFBP-3 in breast cancer cells. The ability of IGFBP-3 to bind and inhibit the actions of AMF/PGI may have some role in the antiproliferative proapoptotic effects of IGFBP-3.

BACKGROUND Cushing's syndrome (CS) is associated with central adiposity, insulin resistance and impaired glucose homeostasis. Adipose tissue is thought to regulates glucose homeostasis via circulating adipokines, such as resistin, leptin and adiponectin, although their role in the insulin resistance associated with CS has not been established.DESIGN We examined the relationship between insulin resistance and adipokine levels in CS patients. We compared plasma levels of resistin, leptin and adiponectin in 10 women and four men patients with CS, with 14 health subjects matched for age, gender and body mass index. A subgroup of three women and four men with pituitary-dependent CS were re-examined at least 9 months after curative surgery.RESULTS CS patients had significantly more truncal fat and less lean body mass as assessed by DEXA compared to control subjects. Total cholesterol, triglycerides and insulin resistance, as calculated using the homeostasis model assessment of insulin resistance (HOMA-R), was significantly increased in CS patients. Of the adipokines measured, only resistin was significantly different between female CS patients and female control subjects (5.05 +/- 0.56 vs. 2.91 +/- 0.39 mug/l, P = 0.015). Curative surgery significantly reduced total body fat and truncal fat, leptin, total and low-density lipoprotien (LDL) cholesterol, glucose and HOMA-R. A reduction in both resistin and adiponectin was also observed but the differences between pre- and post-treatment levels did not achieve statistical significance.CONCLUSION Here we report for the first time that resistin levels are significantly elevated in CS patients and may be important in the insulin resistance associated with glucocorticoid excess.

Tissue transglutaminase (TG2) is a ubiquitous enzyme that cross-links glutamine residues with lysine residues, resulting in protein polymerization, cross-linking of dissimilar proteins, and incorporation of diamines and polyamines into proteins. It has not previously been known to have kinase activity. Recently, insulin-like growth factor-binding protein-3 (IGFBP-3) has been reported to be phosphorylated by breast cancer cell membranes. We purified the IGFBP-3 kinase activity from solubilized T47D breast cancer cell membranes using gel filtration, ion-exchange chromatography, and IGFBP-3 affinity chromatography. The fractions containing kinase activity were further purified by high pressure liquid chromatography and analyzed by tandem mass spectroscopy. TG2 was detected in fractions containing kinase activity. Antisera to TG2 and protein A-Sepharose were used to immunoprecipitate TG2 from membrane fractions. The immunoprecipitates retained IGFBP-3 kinase, whereas immunoprecipitation deleted kinase activity in the membrane supernatant. The inhibitors of TG2, cystamine and monodansyl cadaverine, abolished the ability of the T47D cell membrane preparation to phosphorylate IGFBP-3. Both TG2 purified from guinea pig liver and recombinant human TG2 expressed in insect cells were able to phosphorylate IGFBP-3. TG2 kinase activity was inhibited in a concentration-dependent fashion by calcium, which has previously been shown to be important for the cross-linking activity of TG2. These data provide compelling evidence that TG2 has intrinsic kinase activity, a function that has not previously been ascribed to TG2. Furthermore, we provide evidence that TG2 is a major component of the IGFBP-3 kinase activity present on breast cancer cell membranes.