Resistance Invivo Research Articles

Exosomal circ_0032704 confers sorafenib resistance to hepatocellular carcinoma and contributes to cancer malignant progression by modulating the miR-514a-3p/PD-L1 pathway.

This study aims to explore the role of circ_0032704 in sorafenib-resistant hepatocellular carcinoma (HCC). The expression of circ_0032704, miR-514a-3p, and programmed death-ligand 1 (PD-L1) mRNA was detected by quantitative real-time PCR (qPCR). The expression of multidrug resistant-related proteins, migration/invasion-related proteins, exosome-related proteins, and PD-L1 protein was detected by western blot. Cell viability was detected by CCK-8 assay. Cell proliferation, migration, and invasion were assessed by EdU assay, wound healing assay, and transwell assay. The binding between miR-514a-3p and circ_0032704 or PD-L1 was verified by RIP assay, pull-down assay, and dual-luciferase reporter assay. Cell- or serum-derived exosomes were isolated and identified by TEM and NTA. Xenograft models were established to determine the effect of circ_0032704 on drug resistance invivo. Circ_0032704 was overexpressed in sorafenib-resistant HCC tissues and cells. Circ_0032704 knockdown reduced sorafenib resistance in HCC cells and inhibited cell proliferation, migration, and invasion of sorafenib-resistant HCC cells, while these effects were reversed by PD-L1 overexpression. We found that circ_0032704 positively regulated PD-L1 expression via targeting miR-514a-3p. Exosomes with circ_0032704 inhibition reduced sorafenib resistance in HCC cells and inhibited cell proliferation, migration, and invasion of sorafenib-resistant HCC cells. Exosomes with circ_0032704 inhibition also inhibited tumor growth invivo. The expression of circ_0032704 in exosomes was stable and possessed diagnostic value. Circ_0032704 enhanced sorafenib resistance in HCC and promoted the malignant development of sorafenib-resistant HCC. Circ_0032704 could be transported by exosomes, and exosomal circ_0032704 had diagnostic value.

The lipid droplet in cancer: From being a tumor-supporting hallmark to clinical therapy.

Abnormal lipid metabolism, one of the hallmarks in cancer, has gradually emerged as a novel target for cancer treatment. As organelles that store and release excess lipids, lipid droplets (LDs) resemble "gears" and facilitate cancer development in the body. This review discusses the life cycle of LDs, the relationship between abnormal LDs and cancer hallmarks, and the application of LDs in theragnostic and clinical contexts to provide a contemporary understanding of the role of LDs in cancer. A systematic literature search was conducted in PubMed and SPORTDiscus. Retrieve and summarize clinical trials of drugs that target proteins associated with LD formation using the Clinical Trials website. Create a schematic diagram of lipid droplets in the tumor microenvironment using Adobe Illustrator. As one of the top ten hallmarks of cancer, abnormal lipid metabolism caused by excessive generation of LDs interrelates with other hallmarks. The crosstalk between excessive LDs and intracellular free fatty acids (FFAs) promotes an inflammatory environment that supports tumor growth. Moreover, LDs contribute to cancer metastasis and cell death resistance invivo. Statins, as HMGCR inhibitors, are promising to be the pioneering commercially available anti-cancer drugs that target LD formation.

Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System.

Resistin plays an important role in the pathophysiology of obesity-mediated insulin resistance in mice. However, the biology of resistin in humans is quite different from that in rodents. Therefore, the association between resistin and insulin resistance remains unclear in humans. Here, we tested whether and how the endocannabinoid system (ECS) control circulating peripheral blood mononuclear cells (PBMCs) that produce resistin and infiltrate into the adipose tissue, heart, skeletal muscle, and liver, resulting in inflammation and insulin resistance. Using human PBMCs, we investigate whether the ECS is connected to human resistin. To test whether the ECS regulates inflammation and insulin resistance invivo, we used 2 animal models such as "humanized" nonobese diabetic/Shi-severe combined immunodeficient interleukin-2Rγ (null) (NOG) mice and "humanized" resistin mouse models, which mimic human body. In human atheromatous plaques, cannabinoid 1 receptor (CB1R)-positive macrophage was colocalized with the resistin expression. In addition, resistin was exclusively expressed in the sorted CB1R-positive cells from human PBMCs. In CB1R-positive cells, endocannabinoid ligands induced resistin expression via the p38-Sp1 pathway. In both mouse models, a high-fat diet increased the accumulation of endocannabinoid ligands in adipose tissue, which recruited the CB1R-positive cells that secrete resistin, leading to adipose tissue inflammation and insulin resistance. This phenomenon was suppressed by CB1R blockade or in resistin knockout mice. Interestingly, this process was accompanied by mitochondrial change that was induced by resistin treatment. These results provide important insights into the ECS-resistin axis, leading to the development of metabolic diseases. Therefore, the regulation of resistin via the CB1R could be a potential therapeutic strategy for cardiometabolic diseases.

Deep (phospho)proteomics profiling of pre- treatment needle biopsies identifies signatures of treatment resistance in HER2+ breast cancer

Patients with early-stage HER2-overexpressing breast cancer struggle with treatment resistance in 20%-40% of cases. More information is needed to predict HER2 therapy response and resistance invivo. In this study, we perform (phospho)proteomics analysis of pre-treatment HER2+ needle biopsies of early-stage invasive breast cancer to identify molecular signatures predictive of treatment response to trastuzumab, pertuzumab, and chemotherapy. Our data show that accurate quantification of the estrogen receptor (ER) and HER2 biomarkers, combined with the assessment of associated biological features, has the potential to enable better treatment outcome prediction. In addition, we identify cellular mechanisms that potentially precondition tumors to resist therapy. We find proteins with expression changes that correlate with resistance and constitute to a strong predictive signature for treatment success in our patient cohort. Our results highlight the multifactorial nature of drug resistance invivo and demonstrate the necessity of deep tumor profiling.

Multi-omics analysis reveals distinct non-reversion mechanisms of PARPi resistance in BRCA1- versus BRCA2-deficient mammary tumors

BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanismsthat do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance invivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response.

Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity

Insulin resistance is a pathological state often associated with obesity, representing a major risk factor for type 2 diabetes. Limited mechanism-based strategies exist to alleviate insulin resistance. Here, using single-cell transcriptomics, we identify a small, critically important, but previously unexamined cell population, p21Cip1 highly expressing (p21high) cells, which accumulate in adipose tissue with obesity. By leveraging a p21-Cre mouse model, we demonstrate that intermittent clearance of p21high cells can both prevent and alleviate insulin resistance in obese mice. Exclusive inactivation of the NF-κB pathway within p21high cells, without killing them, attenuates insulin resistance. Moreover, fat transplantation experiments establish that p21high cells within fat are sufficient to cause insulin resistance invivo. Importantly, a senolytic cocktail, dasatinib plus quercetin, eliminates p21high cells in human fat exvivo and mitigates insulin resistance following xenotransplantation into immuno-deficient mice. Our findings lay the foundation for pursuing the targeting of p21high cells as a new therapy to alleviate insulin resistance.

The p75NTR and its carboxyl-terminal fragment exert opposing effects on melanoma cell proliferation and apoptosis via modulation of the NF-κB pathway.

The p75 neurotrophin receptor (p75NTR ), a member of the tumor necrosis factor superfamily of receptors, is sensitive to proteolysis and has been observed to be expressed in various cancers. However, the roles of p75NTR and its proteolytic fragments in tumorigenesis remain incompletely understood. Here, we report that the proportion of the p75NTR carboxyl-terminal fragment (p75NTR -CTF) is much higher than that of the full-length p75NTR (p75NTR -FL) in melanoma cells. Whereas p75NTR -FL positively regulates apoptosis, p75NTR -CTF promotes cell proliferation and survival, as well as increasing sorafenib resistance invivo and invitro. Moreover, p75NTR -CTF activates the nuclear factor kappa B pathway and enhances the mRNA and protein levels of its downstream genes c-IAP1/2, FLIP, bFGF, IL8 and VEGF. On the contrary, p75NTR -FL inhibits these processes. Taken together, these findings demonstrate that p75NTR -CTF and p75NTR -FL have opposing functions in melanoma cells, suggesting that the ratio of the two proteins affects the balance between cell death and survival. The presence of distinct p75NTR proteolytic fragments may affect biological outcomes in tumor cells.

Inhibition of MEK1/2 Forestalls the Onset of Acquired Resistance to Entrectinib in Multiple Models of NTRK1-Driven Cancer

NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib invivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance invivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.

1738-P: The Role of Adipocyte YAP in Glucose Homeostasis and Insulin Resistance In Vivo

Adipose tissue is a dynamic metabolic organ that is essential for the regulation of whole-body glucose homeostasis. Adipose tissue hypertrophy with obesity is associated with glucose intolerance and insulin resistance underlying type 2 diabetes. Furthermore, studies suggest fibrosis formation is increased in adipose tissue of mice fed with a high-fat diet (HFD) and results in metabolically dysfunctional adipose tissue. Yes-associated protein 1 (YAP) is a transcription cofactor that promotes fibrosis in the lung, liver, and kidney. However, the role of YAP in adipose tissue and glucose homeostasis is unknown. To understand the role of YAP in vivo under metabolic stress conditions, we assessed how HFD impacts YAP protein levels. We have found that YAP protein levels increase in adipose tissue with weight gain. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. To further investigate the role of YAP in adipose tissue and glucose homeostasis in vivo, we developed adipose tissue-specific YAP knockout (adipose tissue YAP-KO) mice and placed them on both chow and HFD for 12 weeks. On an HFD, adipose tissue YAP-KO mice show improved glucose tolerance compared to controls. While blood glucose levels were lower, adipose tissue YAP-KO mice did not show improvements in insulin sensitivity. Together, these data indicate that YAP increases in adipose tissue with weight gain and disruption of YAP in adipocytes improves glucose tolerance; suggesting that adipocyte YAP plays an important role in modulating glucose homeostasis under metabolic stress conditions. Disclosure D. Han: None. R. Aslam: None. T. Ojha: None. D. Yuen: None. C.T. Luk: None.

1879-P: Targeted Disruption of Muscle-Specific ROCK2 Results in Insulin Resistance In Vivo

Insulin signaling is essential for the maintenance of glucose homeostasis. ROCK2 has been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. However, the in vivo function of ROCK2 in muscle remains to be elucidated. To explore the physiological role of muscle ROCK2 in the regulation of whole-body glucose homeostasis and insulin sensitivity in vivo, mice lacking ROCK2 in skeletal muscle selectively (myogenic-Cre; ROCK2lox/lox) were studied. Here we show that muscle-specific ROCK1-deficient mice displayed insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of muscle-specific ROCK2. These effects were independent of changes in adiposity. To determine the mechanism(s) by which muscle-specific deletion of ROCK2 causes insulin resistance, we measured the ability of insulin to activate downstream distal pathways in skeletal muscle. Insulin-stimulated IRS-1 tyrosine phosphorylation in skeletal muscle was markedly reduced by ∼80% in myogenic-Cre; ROCK2lox/lox mice compared with control (ROCK2lox/lox) mice. Concurrently, impaired insulin-induced phosphorylation of Akt, AS160, GSK, and S6K was found when ROCK2 was specifically deleted in muscle. Of pathological significance, ROCK2 activity in skeletal muscle was markedly decreased in obese type 2 diabetic mice in response to insulin, along with an impairment of IRS-1 phosphorylation. These data suggest that ROCK2 deficiency results in systemic insulin resistance by impairing insulin signaling in skeletal muscle, at the level of IRS-1. Thus, our results identify ROCK2 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes. Disclosure H. Kim: None. R.M. Pereira: None. A.G. Uner: None. H. Lee: None. Y. Kim: None.

8 - The RIPK2 Inhibitor GSK583 Protects Against NOD1-Mediated FFA-Induced Insulin Resistance In Vivo

Obesity-induced insulin resistance (IR) is associated with chronic low-grade inflammation. Elevated free fatty acids (FFA), as seen in obesity, can act as damage associated molecular patterns (DAMPs) to activate certain receptors of the innate immune system. We have previously shown that the nucleotide oligomerization domain (NOD) 1 receptor can be activated by saturated FFA in vitro and in vivo. NOD1 signals through an inflammatory kinase, RIPK2, which can lead to increased cytokine production.

SUN-107 Abdominal Adipose Stem Cell Dysfunction In Vitro May Reflect Adipose Insulin Resistance In Vivo in Normal Weight Polycystic Ovarian Syndrome Women

Subcutaneous (SC) abdominal adipose stem cells (ASCs) of normal-weight polycystic ovary syndrome (PCOS) women show exaggerated development to newly-formed adipocytes in vitro (1). To determine whether this phenomenon in PCOS was related to adipose insulin resistance (adipose-IR) in vivo, 8 normal-weight PCOS women and 8 age- and BMI-balanced controls underwent circulating androgen and fasting insulin/free fatty acid (FFA) measurements. SC abdominal ASCs were isolated by fat biopsy and cultured in adipogenic medium for 12 days. ASC commitment to preadipocytes and newly-formed adipocytes derived from preadipocytes were determined by ZFP423 protein expression on day 0 and cellular lipid content by Oil Red O staining on day 12, respectively (i.e. adipogenesis). Adipose-IR was calculated as the product of fasting circulating FFA and insulin levels (2). Student’s t-test compared serum androgen levels and adipose-IR by female type; partial correlation coefficients examined associations of ZFP423 protein expression and newly-formed adipocyte lipid content with adipose-IR, adjusting for androgen levels. PCOS women had greater serum total testosterone [T] and free [f] T (P<0.005, both androgens) levels and higher adipose-IR (P=0.01) than controls. Newly-formed adipocyte lipid content positively correlated with adipose-IR in all women combined (R=0.57, P=0.02), however, significance was lost when adjusted for serum androgen levels (R=0.44, P=0.10 and R=0.36, P=0.18, for total T and fT, respectively). ZFP423 protein expression was not significantly correlated with adipose-IR in all women combined (R=-0.28, P=0.29), but was negatively correlated with adipose-IR (R=-0.64, P=0.01) after adjusting for total T levels. Using exploratory subgroup analysis, the positive relationship of newly-formed adipocyte lipid content with adipose-IR was significant in PCOS women (R=0.67, P=0.05) but not in controls (R=0.10, P=0.80). Similarly, the negative relationship of ZFP423 protein expression with adipose-IR was characteristic of PCOS women (R=-0.88, P=0.002) but not controls (R=-0.26, P=0.50). Thus, given the limited number of subjects, we hypothesize that altered SC abdominal adipogenesis contributes to increased adipose-IR in normal-weight PCOS women and that this relationship is due in part to hyperandrogenism.

Exosomal transfer of miR-214 mediates gefitinib resistance in non-small cell lung cancer.

Recent studies have demonstrated that exosomal miRNAs mediate as intercellular bio-messengers of drug resistance in lung cancer. Our objective was to investigate whether exosomes derived from gefitinib resistant non-small cell lung cancer cells could confer resistance to its recipient cells. Exosomes were successfully isolated by ultracentrifugation and exosomes morphologies and sizes were determined by transmission electron microscopy and dynamic light scattering analysis. Fluorescent PKH-67 labeled exosomes derived from PC-9GR cells could be taken up and internalized by PC-9 cells. CCK8 measurement showed that PC-9GR-derived exosomes could confer gefitinib resistance in PC-9cells. MiRNA-214 was upregulated in gefitinib resistant PC-9GR cells and its derived exosomes by qPCR analysis. Inhibition of exosomal miR-214 with antagomir reversed gefitinib resistance conferred by PC-9GR-derived exosomes invitro, which was confirmed by flow cytometry analysis and westernblot of apoptotic protein (caspase-3, caspase-3 cleaved, bax) and anti-apoptotic protein (bcl-2). Finally, exosomes enriched with miR-214 antagomir was further confirmed to reverse gefitinib resistance invivo. Our results are the first to show that exosomes derived from gefitinib-resistant PC-9GR cells could transfer resistance to its recipient sensitive PC-9cells, which might be mediated by exosomal transfer of miR-214.

CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo

Transplantation of hematopoietic stem cells (HSCs) with a naturally occurring CCR5 mutation confers a loss of detectable HIV-1 in the patient, making ablation of the CCR5 gene in HSCs an ideal therapy for an HIV-1 cure. Although CCR5 disruption has been attempted in CD4+ Tcells and hematopoietic stem/progenitor cells (HSPCs), efficient gene editing with high specificity and long-term therapeutic potential remains a major challenge for clinical translation. Here, we established a CRISPR/Cas9 gene editing system in human CD34+ HSPCs and achieved efficient CCR5 ablation evaluated in long-term reconstituted NOD/Prkdcscid/IL-2Rγnull mice. The CCR5 disruption efficiency in our system remained robust in secondary transplanted repopulating hematopoietic cells. More importantly, an HIV-1 resistance effect was observed as indicated by significant reduction of virus titration and enrichment of human CD4+ Tcells. Hence, we successfully established a CRISPR/Cas9 mediated CCR5 ablating system in long-term HSCs, which confers HIV-1 resistance invivo. Our study provides evidence for translating CCR5 gene-edited HSC transplantation for an HIV cure to the clinic.

Identification of bona fide alternative renin transcripts expressed along cortical tubules and potential roles in promoting insulin resistance in vivo without significant plasma renin activity elevation.

Renin belongs to a family of aspartyl proteases and is the rate-limiting enzyme in the synthesis of the potent vasoactive peptide angiotensin II. Processing of renal renin has been extensively investigated in juxtaglomerular granular cells, in which prorenin and active renin are present in secretory condensed granules. Previous studies demonstrated alternative renin transcription in rat adrenal glands. Different studies reported novel intracellular forms of renin deduced from novel 5' variants derived from renin mRNA in both mice and humans. Comprehensive detailed studies in genetically engineered mice showed that both a secreted and an intracellular form of renin plays divergent mechanism regulating fluid intake and metabolism by the brain renin-angiotensin system; however, the presence, regulation, and functions of these renin isoforms in kidney and adrenal gland are not fully understood in mice. To investigate the characteristics of renin isoforms in mice, we performed a systematic inventory of renin transcripts of mice with and without a duplication of the renin gene alternatively from previous studies. We discovered a novel isoform of renin of the Ren2 gene, which conserved functionally important residues of the prosegment and incomplete isoforms of the Ren1C/D gene lacking a pre-pro segment. In situ hybridization assays revealed alternative renin isoforms expressed along cortical tubules. Newly generated transgenic mice with systemic overexpression of alternative renin transcript showed enhanced local angiotensin II generation without elevation of plasma renin activity and systemic insulin resistance in vivo, providing new pathophysiological insights into insulin resistance exaggerated by bona fide renin isoform.

Long-term In Vitro Treatment of Human Glioblastoma Cells with Temozolomide Increases Resistance In Vivo through Up-regulation of GLUT Transporter and Aldo-Keto Reductase Enzyme AKR1C Expression

Glioblastoma (GBM) is the most frequent malignant glioma. Treatment of GBM patients is multimodal with maximum surgical resection, followed by concurrent radiation and chemotherapy with the alkylating drug temozolomide (TMZ). The present study aims to identify genes implicated in the acquired resistance of two human GBM cells of astrocytic origin, T98G and U373, to TMZ. Resistance to TMZ was induced by culturing these cells in vitro for months with incremental TMZ concentrations up to 1 mM. Only partial resistance to TMZ has been achieved and was demonstrated in vivo in immunocompromised mice bearing orthotopic U373 and T98G xenografts. Our data show that long-term treatment of human astroglioma cells with TMZ induces increased expression of facilitative glucose transporter/solute carrier GLUT/SLC2A family members, mainly GLUT-3, and of the AKR1C family of proteins. The latter proteins are phase 1 drug-metabolizing enzymes involved in the maintenance of steroid homeostasis, prostaglandin metabolism, and metabolic activation of polycyclic aromatic hydrocarbons. GLUT-3 has been previously suggested to exert roles in GBM neovascularization processes, and TMZ was found to exert antiangiogenic effects in experimental gliomas. AKR1C1 was previously shown to be associated with oncogenic potential, with proproliferative effects similar to AKR1C3 in the latter case. Both AKR1C1 and AKR1C2 proteins are involved in cancer pro-proliferative cell chemoresistance. Selective targeting of GLUT-3 in GBM and/or AKR1C proteins (by means of jasmonates, for example) could thus delay the acquisition of resistance to TMZ of astroglioma cells in the context of prolonged treatment with this drug.

Evaluation of Acute Corneal Barrier Change Induced by Topically Applied Preservatives Using Corneal Transepithelial Electric Resistance In Vivo

We evaluated acute changes in corneal barrier function after instillation with preservatives using corneal transepithelial electric resistance (TER) in vivo and cytotoxicity tests in vitro. The corneal TER of live rabbits was measured using a volt-ohm meter and silver/silver chloride electrodes. The cornea was exposed to the preservatives benzalkonium chloride (BAC; 0.001%, 0.002%, 0.005%, 0.01%, and 0.02%), 0.04% paraben, 0.5% chlorobutanol, 0.005% chlorhexidine digluconate, 2% boric acid, and 0.01% ethylenediaminetetraacetic acid, and then changes in the TER were monitored for 60 seconds. Cultured normal rabbit corneal epithelial cells were exposed to the same preservatives for 60 seconds in vitro, and cell viability was evaluated using the WST-1 assay. The TER instantly decreased and became significantly lower than the control within 10 seconds after instillation with 0.01% and 0.02% BAC (P < 0.01) and within 60 seconds after that with 0.005% BAC (P < 0.01). The TER decreased concomitantly with increasing BAC concentration. Cell viability after instillation with 0.005%, 0.01%, and 0.02% BAC for 60 seconds was significantly lower than that of the control (P < 0.0001). None of the other preservatives significantly altered the TER or cell viability. Decreases in the TER correlated with cell viability (r = 0.94, P < 0.0001). Instillation with BAC immediately disrupted the corneal epithelium. Corneal epithelial cell death is supposed to be associated with a decline in barrier function; thus, corneal TER measurement in vivo can assess the acute toxicity of preservatives added to ophthalmic drugs.

Targeted Disruption of ROCK1 Causes Insulin Resistance in Vivo

Insulin signaling is essential for normal glucose homeostasis. Rho-kinase (ROCK) isoforms have been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. To investigate the physiological role of ROCK1 in the regulation of whole body glucose homeostasis and insulin sensitivity in vivo, we studied mice with global disruption of ROCK1. Here we show that, at 16-18 weeks of age, ROCK1-deficient mice exhibited insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of ROCK1. These effects were independent of changes in adiposity. Interestingly, ROCK1 gene ablation caused a significant increase in glucose-induced insulin secretion, leading to hyperinsulinemia. To determine the mechanism(s) by which deletion of ROCK1 causes insulin resistance, we measured the ability of insulin to activate phosphatidylinositol 3-kinase and multiple distal pathways in skeletal muscle. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 or phospho-tyrosine was also reduced approximately 40% without any alteration in tyrosine phosphorylation of insulin receptor in skeletal muscle. Concurrently, serine phosphorylation of IRS-1 at serine 632/635, which is phosphorylated by ROCK in vitro, was also impaired in these mice. Insulin-induced phosphorylation of Akt, AS160, S6K, and S6 was also decreased in skeletal muscle. These data suggest that ROCK1 deficiency causes systemic insulin resistance by impairing insulin signaling in skeletal muscle. Thus, our results identify ROCK1 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.

Whole-Body Glycolysis Measured by the Deuterated-Glucose Disposal Test Correlates Highly With Insulin Resistance In Vivo

The purpose of this study was to compare an in vivo test of whole-body glycolysis, the deuterated-glucose disposal test (2H-GDT), with insulin sensitivity measured by the euglycemic-hyperinsulinemic glucose clamp and the steady-state plasma glucose (SSPG) test. The 2H-GDT consists of an oral glucose challenge containing deuterated glucose, followed by measurement of heavy water (2H2O) production, which represents whole-body glycolytic disposal of the glucose load. 2H2O production is corrected for ambient insulin concentration as an index of tissue insulin sensitivity. The 2H-GDT was compared with euglycemic-hyperinsulinemic glucose clamps in healthy lean subjects (n = 8) and subjects with the metabolic syndrome (n = 9) and with the SSPG test in overweight (n = 12) and obese (n = 6) subjects. A strong correlation with the clamp was observed for the 75-g and 30-g 2H-GDT (r = 0.95, P < 0.0001 and r = 0.88, P < 0.0001, respectively). The 2H-GDT and clamp studies revealed marked insulin resistance in subjects with metabolic syndrome compared with lean control subjects. The correlation with the clamp was maintained in each group (lean, r = 0.86, P < 0.01; metabolic syndrome, r = 0.81, P < 0.01) for the 75-g test. The 2H-GDT also correlated strongly with the SSPG test (r = -0.87, P < 0.0001) in overweight and obese subjects. The 2H-GDT, which measures whole-body glycolysis in humans in a quantitative manner, correlates highly with the euglycemic-hyperinsulinemic glucose clamp and the SSPG test. Impaired insulin-mediated whole-body glycolysis is a feature of insulin resistance, which provides a means of assessing insulin sensitivity in vivo.

Inactivation of the Myc−Induced DNA Damage Response Promotes Lymphomagenesis and Treatment Resistance In Vivo.

Activation of the DNA damage response (DDR) was recently claimed to serve as a critical anti−cancer barrier in early human tumorigenesis (Bartkova−J et al., and Gorgoulis−VG et al., Nature, 2005). As a consequence, deregulation of the DDR machinery may be required for tumor manifestation.Myc activation is an oncogenic event frequently detected in malignant lymphomas. It is known that constitutive Myc expression provokes apoptosis via the ARF/p53 pathway as a cellular failsafe mechanism to counteract malignant transformation, but the potential role of Myc signaling to the DDR machinery, mediated via the Atm (ataxia telangiectasia mutated) kinase, as another putative tumor suppressive restraint remains still elusive.Utilizing a tamoxifen−regulatable Myc system as well as RNA interference in vitro, we found in cell−based models that acute induction of Myc, associated with an increased production of reactive oxygen species (ROS) and marks of DNA strand breaks, leads to p53 activation which is mainly mediated by Atm and its upstream regulator PP5.To further examine the role of Atm during Myc−driven tumorigenesis in vivo, we intercrossed Atm knockout mice to Eμ−myc transgenic mice that develop B−cell lymphomas. Lymphomas formed significantly faster in the absence of Atm, mostly due to a significant apoptotic defect in Atm−/− lymphomas compared to Atm+/+ derived (referred to as ‘control') lymphomas.In accordance with our in vitro findings, we detected significantly elevated levels of ROS−induced oxidative DNA damage and γ−H2AX foci in Myc−driven lymphomas. Importantly, constitutive expression of Myc selected against components of the Atm−governed DDR in a subset of control lymphomas. Like Atm deficiency, these lesions predetermined a poor response in subsequent cytotoxicity assays in vitro and anticancer therapy in vivo, since DNA damaging treatments also produce apoptosis via PP5/Atm.The role for ROS as inducers of DNA damage during the execution of Myc−induced apoptosis was further substantiated by the finding that Eμ−myc transgenic mice continuously exposed to the ROS scavenger N−acetylcysteine (starting prenatally) developed lymphomas with significantly reduced levels of spontaneous apoptosis and with less marks of DNA damage (i.e. γ−H2AX foci), but a retained ability to respond to anticancer therapy in vitro and in vivo.Hence, Atm eliminates pre−neoplastic lesions by converting oncogenic signaling into apoptosis, while selection for an attenuated Atm response permits lymphoma formation, and, in turn, has profound implications for reduced efficacy of DNA damaging therapies.