Insulin and cellular stress induced glucose uptake in 3T3-L1 adipocytes

The research on 3T3-L1 adipocytes described in this thesis demonstrates how two different types of cellular stress inducing agents, namely the vicinal thiol binding agent arsenite and the conventional PKC-binding and -activating agent PMA act to increase glucose uptake in these cells. Whereas arsenite uses mainly the insulin-responsive GLUT4 transporter, PMA increases basal glucose transport through the GLUT1 transporter. As described in Chapter 3, arsenite-induced glucose uptake illustrates several requirements needed by any agent acting through GLUT4. These are, a tyrosine kinase activity, p38 MAPK activation and PKC-\lambda activity. Though PI-3' kinase activation is an essential step in insulin-signalling, this step is not required for arsenite-induced glucose uptake. Apparently, the need for tyrosine-kinase activity in arsenite induced glucose uptake resides in the ability to tyrosine-phosphorylate Cbl (see Chapter 3 Fig. 5). A further illustration of the importance of Cbl-tyrosine phosphorylation comes from our studies on rottlerin (Chapter 4). The ATP-depletion mediated by this pharmacological compound does not seem to be responsible for the observed inhibition of GLUT4 translocation (as was postulated by Kayali et al.[1]). Rather, aside from acting as an uncompetitive inhibitor of GLUT4, rottlerin hampers Cbl tyrosine phosphorylation, which leads to a 75% reduction in GLUT4 translocation (see Chapter 4, Fig. 3 and 4). Regrettably, the nature of the arsenite-induced tyrosine-kinase activity remains as of yet unidentified. Though the specific ability of arsenite to induce STAT5a tyrosine-phosphorylation in the mature adipocyte, should provide a straightforward tool to enable its identification (J.L Gonz_lez-Galindo, unpublished observations) Previously it had been demonstrated that insulin-induced p38 MAPK was involved in regulating the amount of glucose taken up by the cell without affecting GLUT4 translocation, suggesting some kind of intrinsic effect on the GLUT4 transporter itself [2]. Our observations on arsenite, a potent activator of p38 MAPK, illustrate a similar phenomenon in GLUT4-mediated stress-induced glucose uptake (see Chapter 3, Fig. 6). Subsequent research, described in a recently submitted manuscript, provides a detailed analysis of the involvement of p38 MAPK. These data demonstrate that p38 MAPK is involved in fine-tuning glucose uptake by regulating the turnover capacity of the GLUT4 transporter. A further note on the fine tuning of GLUT4-mediated glucose uptake comes from the observations on genistein, described in Chapter 5. This research suggests that in GLUT4 the turnover capacity for glucose can also be regulated through an intracellular ATP-binding Walker B motif akin to that described for GLUT1 [3]. Though further research is required to elucidate this mechanism, this theoretical resolution constitutes a significant step forwards towards understanding mechanisms in action after GLUT4 membrane translocation. If these observations are mechanistically linked in the cell remains to be elucidated. Aside from leading to enquiries into the mechanisms of insulin-induced glucose uptake, arsenite also opened up an avenue of more physiological research. We observed that arsenite-induced glucose uptake was sensitive to treatment with the insulin-resistance inducing agent dexamethasone. Subsequent analysis (described in Chapter 7) learned that although PI-3' kinase signalling is affected, in 3T3-L1 adipocytes the signalling pathway downstream is able to absorb this impediment. Rather, MKP-1 and -4 are upregulated in response to dexamethasone. Consequentially p38 MAPK activity is lost, leading to a reduction in glucose uptake. Given that MKP-4 is also upregulated in db/db- and ob/ob-mice [4], and that treatment of db/db mice with a glucocorticoid-receptor antagonist improves blood glucose levels [5;6], attenuation of p38 MAPK-signalling could be an important factor in type II diabetes. To enable the studies described in this chapter, a novel tool had to be developed. 3T3-L1 adipocytes have for long been inaccessible to ectopic expression of DNA. By the application of Lentivirus as described in Chapter 6, a large number of cells can be efficiently and reliably transduced. This novel tool will make the 3T3-L1 adipocyte readily amendable to routine molecular biological techniques, which will be of great benefit to the research field. In contrast to arsenite, PMA does not induce GLUT4 translocation, but acts solely through GLUT1. As illustrated in Chapter 8 of this thesis, in 3T3-L1 adipocytes the earliest and most PMA-sensitive PKC isoform is PKC-\betaII. But rather than activation, it is the concomitant degradation of this isoform which induces GLUT1 translocation. Further research (described in Chapter 9) highlighted the processes involved : First transcription of GLUT1, operating through the classical PKC-ERK-GLUT1 pathway. Second, translocation of GLUT1. This translocation is mediated by PKC-\lambda which is found associated with PKC-\betaII in the basal state. Thus upon degradation of the \betaII-isoform (or disruption of this complex by treatment with myristoylated pseudo-substrate peptides against \betaII) PKC-\lambda is released and free to act as a positional queue inducing translocation of the GLUT1 containing vesicles.UBL - phd migration 201

Lipocalin-2 Deficiency Impairs Thermogenesis and Potentiates Diet-Induced Insulin Resistance in Mice

Lipocalin (LCN) 2 belongs to the lipocalin subfamily of low-molecular mass-secreted proteins that bind small hydrophobic molecules. LCN2 has been recently characterized as an adipose-derived cytokine, and its expression is upregulated in adipose tissue in genetically obese rodents. The objective of this study was to investigate the role of LCN2 in diet-induced insulin resistance and metabolic homeostasis in vivo. Systemic insulin sensitivity, adaptive thermogenesis, and serum metabolic and lipid profile were assessed in LCN2-deficient mice fed a high-fat diet (HFD) or regular chow diet. The molecular disruption of LCN2 in mice resulted in significantly potentiated diet-induced obesity, dyslipidemia, fatty liver disease, and insulin resistance. LCN2(-/-) mice exhibit impaired adaptive thermogenesis and cold intolerance. Gene expression patterns in white and brown adipose tissue, liver, and muscle indicate that LCN2(-/-) mice have increased hepatic gluconeogenesis, decreased mitochondrial oxidative capacity, impaired lipid metabolism, and increased inflammatory state under the HFD condition. LCN2 has a novel role in adaptive thermoregulation and diet-induced insulin resistanc

Cellularity and Adipogenic Profile of the Abdominal Subcutaneous Adipose Tissue From Obese Adolescents: Association With Insulin Resistance and Hepatic Steatosis

We explored whether the distribution of adipose cell size, the estimated total number of adipose cells, and the expression of adipogenic genes in subcutaneous adipose tissue are linked to the phenotype of high visceral and low subcutaneous fat depots in obese adolescents. A total of 38 adolescents with similar degrees of obesity agreed to have a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test and hyperinsulinemic euglycemic clamp) and imaging studies (MRI, DEXA, (1)H-NMR). Subcutaneous periumbilical adipose cell-size distribution and the estimated total number of subcutaneous adipose cells were obtained from tissue biopsy samples fixed in osmium tetroxide and analyzed by Beckman Coulter Multisizer. The adipogenic capacity was measured by Affymetrix GeneChip and quantitative RT-PCR. Subjects were divided into two groups: high versus low ratio of visceral to visceral + subcutaneous fat (VAT/[VAT+SAT]). The cell-size distribution curves were significantly different between the high and low VAT/(VAT+SAT) groups, even after adjusting for age, sex, and ethnicity (MANOVA P = 0.035). Surprisingly, the fraction of large adipocytes was significantly lower (P <0.01) in the group with high VAT/(VAT+SAT), along with the estimated total number of large adipose cells (P <0.05), while the mean diameter was increased (P <0.01). From the microarray analyses emerged a lower expression of lipogenesis/adipogenesis markers (sterol regulatory element binding protein-1, acetyl-CoA carboxylase, fatty acid synthase) in the group with high VAT/(VAT+SAT), which was confirmed by RT-PCR. A reduced lipo-/adipogenic capacity, fraction, and estimated number of large subcutaneous adipocytes may contribute to the abnormal distribution of abdominal fat and hepatic steatosis, as well as to insulin resistance in obese adolescent

Genome-Wide Analysis of Glucocorticoid Receptor Binding Regions in Adipocytes Reveal Gene Network Involved in Triglyceride Homeostasis

Glucocorticoids play important roles in the regulation of distinct aspects of adipocyte biology. Excess glucocorticoids in adipocytes are associated with metabolic disorders, including central obesity, insulin resistance and dyslipidemia. To understand the mechanisms underlying the glucocorticoid action in adipocytes, we used chromatin immunoprecipitation sequencing to isolate genome-wide glucocorticoid receptor (GR) binding regions (GBRs) in 3T3-L1 adipocytes. Furthermore, gene expression analyses were used to identify genes that were regulated by glucocorticoids. Overall, 274 glucocorticoid-regulated genes contain or locate nearby GBR. We found that many GBRs were located in or nearby genes involved in triglyceride (TG) synthesis (Scd-1, 2, 3, GPAT3, GPAT4, Agpat2, Lpin1), lipolysis (Lipe, Mgll), lipid transport (Cd36, Lrp-1, Vldlr, Slc27a2) and storage (S3-12). Gene expression analysis showed that except for Scd-3, the other 13 genes were induced in mouse inguinal fat upon 4-day glucocorticoid treatment. Reporter gene assays showed that except Agpat2, the other 12 glucocorticoid-regulated genes contain at least one GBR that can mediate hormone response. In agreement with the fact that glucocorticoids activated genes in both TG biosynthetic and lipolytic pathways, we confirmed that 4-day glucocorticoid treatment increased TG synthesis and lipolysis concomitantly in inguinal fat. Notably, we found that 9 of these 12 genes were induced in transgenic mice that have constant elevated plasma glucocorticoid levels. These results suggested that a similar mechanism was used to regulate TG homeostasis during chronic glucocorticoid treatment. In summary, our studies have identified molecular components in a glucocorticoid-controlled gene network involved in the regulation of TG homeostasis in adipocytes. Understanding the regulation of this gene network should provide important insight for future therapeutic developments for metabolic diseases

Insulin and cellular stress induced glucose uptake in 3T3-L1 adipocytes

The research on 3T3-L1 adipocytes described in this thesis demonstrates how two different types of cellular stress inducing agents, namely the vicinal thiol binding agent arsenite and the conventional PKC-binding and -activating agent PMA act to increase glucose uptake in these cells. Whereas arsenite uses mainly the insulin-responsive GLUT4 transporter, PMA increases basal glucose transport through the GLUT1 transporter. As described in Chapter 3, arsenite-induced glucose uptake illustrates several requirements needed by any agent acting through GLUT4. These are, a tyrosine kinase activity, p38 MAPK activation and PKC-\lambda activity. Though PI-3' kinase activation is an essential step in insulin-signalling, this step is not required for arsenite-induced glucose uptake. Apparently, the need for tyrosine-kinase activity in arsenite induced glucose uptake resides in the ability to tyrosine-phosphorylate Cbl (see Chapter 3 Fig. 5). A further illustration of the importance of Cbl-tyrosine phosphorylation comes from our studies on rottlerin (Chapter 4). The ATP-depletion mediated by this pharmacological compound does not seem to be responsible for the observed inhibition of GLUT4 translocation (as was postulated by Kayali et al.[1]). Rather, aside from acting as an uncompetitive inhibitor of GLUT4, rottlerin hampers Cbl tyrosine phosphorylation, which leads to a 75% reduction in GLUT4 translocation (see Chapter 4, Fig. 3 and 4). Regrettably, the nature of the arsenite-induced tyrosine-kinase activity remains as of yet unidentified. Though the specific ability of arsenite to induce STAT5a tyrosine-phosphorylation in the mature adipocyte, should provide a straightforward tool to enable its identification (J.L Gonz_lez-Galindo, unpublished observations) Previously it had been demonstrated that insulin-induced p38 MAPK was involved in regulating the amount of glucose taken up by the cell without affecting GLUT4 translocation, suggesting some kind of intrinsic effect on the GLUT4 transporter itself [2]. Our observations on arsenite, a potent activator of p38 MAPK, illustrate a similar phenomenon in GLUT4-mediated stress-induced glucose uptake (see Chapter 3, Fig. 6). Subsequent research, described in a recently submitted manuscript, provides a detailed analysis of the involvement of p38 MAPK. These data demonstrate that p38 MAPK is involved in fine-tuning glucose uptake by regulating the turnover capacity of the GLUT4 transporter. A further note on the fine tuning of GLUT4-mediated glucose uptake comes from the observations on genistein, described in Chapter 5. This research suggests that in GLUT4 the turnover capacity for glucose can also be regulated through an intracellular ATP-binding Walker B motif akin to that described for GLUT1 [3]. Though further research is required to elucidate this mechanism, this theoretical resolution constitutes a significant step forwards towards understanding mechanisms in action after GLUT4 membrane translocation. If these observations are mechanistically linked in the cell remains to be elucidated. Aside from leading to enquiries into the mechanisms of insulin-induced glucose uptake, arsenite also opened up an avenue of more physiological research. We observed that arsenite-induced glucose uptake was sensitive to treatment with the insulin-resistance inducing agent dexamethasone. Subsequent analysis (described in Chapter 7) learned that although PI-3' kinase signalling is affected, in 3T3-L1 adipocytes the signalling pathway downstream is able to absorb this impediment. Rather, MKP-1 and -4 are upregulated in response to dexamethasone. Consequentially p38 MAPK activity is lost, leading to a reduction in glucose uptake. Given that MKP-4 is also upregulated in db/db- and ob/ob-mice [4], and that treatment of db/db mice with a glucocorticoid-receptor antagonist improves blood glucose levels [5;6], attenuation of p38 MAPK-signalling could be an important factor in type II diabetes. To enable the studies described in this chapter, a novel tool had to be developed. 3T3-L1 adipocytes have for long been inaccessible to ectopic expression of DNA. By the application of Lentivirus as described in Chapter 6, a large number of cells can be efficiently and reliably transduced. This novel tool will make the 3T3-L1 adipocyte readily amendable to routine molecular biological techniques, which will be of great benefit to the research field. In contrast to arsenite, PMA does not induce GLUT4 translocation, but acts solely through GLUT1. As illustrated in Chapter 8 of this thesis, in 3T3-L1 adipocytes the earliest and most PMA-sensitive PKC isoform is PKC-\betaII. But rather than activation, it is the concomitant degradation of this isoform which induces GLUT1 translocation. Further research (described in Chapter 9) highlighted the processes involved : First transcription of GLUT1, operating through the classical PKC-ERK-GLUT1 pathway. Second, translocation of GLUT1. This translocation is mediated by PKC-\lambda which is found associated with PKC-\betaII in the basal state. Thus upon degradation of the \betaII-isoform (or disruption of this complex by treatment with myristoylated pseudo-substrate peptides against \betaII) PKC-\lambda is released and free to act as a positional queue inducing translocation of the GLUT1 containing vesicles

Characterization of the Escherichia coli damage-independent UvrBC endonuclease activity

Incision of damaged DNA templates by UvrBC inEscherichia coli depends on UvrA, which loads UvrB on the site of the damage. A 50-base pair 3′ prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5′ to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond. Analysis of a 5′ prenicked cholesterol substrate revealed that the second 5′ incision is efficiently produced by UvrBC independent of UvrA. This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5′ single strand overhang. Incision occurred in the presence of ATP or ADP but not in the absence of cofactor. We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5′ single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3′ incision on damaged substrates. However, the UvrBC incision uses the catalytic site in UvrC which makes the 5′ incision on damaged DNA substrates

Isolation and identification of the human homolog of a new p53-binding protein, Mdmx

Contains fulltext : 26148___.PDF (publisher's version ) (Open Access)Oude waarde dc.rights: (c)RU Radboud Universiteit Nijmegen, 199

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

Contains fulltext : 143690.pdf (Publisher’s version ) (Closed access)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

Rottlerin inhibits multiple steps involved in insulin-induced glucose uptake in 3T3-L1 adipocytes

Recently, it was shown that rottlerin inhibits insulin-stimulated glucose uptake and reduces intracellular adenosine triphosphate (ATP) levels in 3T3-L1 adipocytes, suggesting that these two events are causally linked. However, several other reports show that ATP-depletion induces glucose uptake in both muscle cells and adipocytes. In the present study, the mechanism of inhibition by rottlerin was studied in detail, in order to resolve this apparent discrepancy. It was found that rottlerin strongly reduces insulin-stimulated 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes by a partial inhibition of the translocation of the insulin-responsive GLUT4 glucose transporter towards the plasma membrane (PM). Whereas the insulin-induced phosphatidyl-inositol-3' (PI-3') kinase signaling pathway is unaffected by rottlerin, Cbl tyrosine phosphorylation, which provides an essential, PI-3' kinase-independent signal towards GLUT4 translocation, is markedly attenuated. Furthermore, we also observed a direct inhibitory effect of rottlerin on insulin-induced glucose uptake in 3T3-L1 adipocytes. The direct inhibition of insulin-stimulated 2-DOG uptake by rottlerin displayed characteristics of uncompetitive inhibition: with the K(m(app)) of glucose uptake reduced from 1.6 to 0.9 mM and the V(max(app)) reduced from 5.2 to 1.0 nmol/minmg in the presence of rottlerin. In conclusion, rottlerin inhibits multiple steps involved in insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes. The observed reduction in GLUT4 translocation towards the PM and the uncompetitive inhibition of the glucose transport process provide alternative explanations for the inhibitory effects of rottlerin aside from the effects of rottlerin on intracellular levels of AT