Faculty Bibliography

AIMS/HYPOTHESIS/OBJECTIVE:Although it is known that lipid metabolism plays a role in insulin resistance in type 2 diabetes and in obesity, the mechanism is still largely unknown. Apolipoprotein E (ApoE) regulates plasma lipid levels and also plays a role in the uptake of lipids into various tissues. To investigate whether the suppression of whole-particle lipoprotein uptake into tissues affects insulin responsiveness and the diabetic condition, we examined the effect of an ApoE (also known as Apoe) gene deletion in MKR mice, a mouse model of type 2 diabetes. METHODS:ApoE ( -/- ), MKR, ApoE ( -/- )/MKR and control mice were placed on a high-fat, high-cholesterol diet for 16 weeks. Glucose tolerance, serum insulin, blood glucose, insulin tolerance, tissue triacylglycerol content and atherosclerotic lesions were assessed. RESULTS:ApoE ( -/- )/MKR and ApoE ( -/- ) mice showed significantly improved blood glucose, glucose tolerance and insulin sensitivity. Reduced triacylglycerol content in liver and reduced fat accumulation in liver and adipose tissue were found in ApoE ( -/- )/MKR and ApoE ( -/- ) mice compared with control and MKR mice. ApoE ( -/- ) and ApoE ( -/- )/MKR mice demonstrated similarly large atherosclerotic lesions, whereas MKR and control mice had small atherosclerotic lesions. CONCLUSIONS/INTERPRETATION/CONCLUSIONS:We demonstrated that ApoE deficiency abrogates insulin resistance in a mouse model of type 2 diabetes, suggesting that lipid accumulation in tissue is a major cause of insulin resistance in this mouse model.

The insulin-like growth factor I (IGF-I) signaling pathway has been shown to play an important role in several aspects of cancer biology, including metastasis. The aim of this study was to define the contribution of serum (endocrine) and local (tumour microenvironment) IGF-I on osteosarcoma tumour growth and metastasis, a cancer that is known to be dependent on the IGF-I axis. To test this hypothesis, we evaluated the primary tumour growth and metastatic progression of K7M2 murine osteosarcoma cells injected to a genetically engineered mouse [liver-specific IGF-I deficient (LID)] in which serum IGF-I levels are reduced by 75%, while maintaining expression of IGF-I in normal tissues. We first demonstrated that IGF-I in the tumour and the tumour-microenvironment were maintained in the LID mice. Within this designed model, there was no difference in primary tumour growth or in pulmonary metastasis in LID mice compared to control mice. Furthermore, there was no difference in the number or localization of single metastatic cells immediately after their arrival in the lungs of LID mice and control mice, as analysed by single cell video microscopy. Collectively, these data suggest that marked reduction in serum IGF-I is not sufficient to slow the progression of either primary or metastatic models of osteosarcoma.

Type 2 diabetics often demonstrate normal or increased bone mineral density, yet are at increased risk for bone fracture. Furthermore, the anti-diabetic oral thiazolidinediones (PPARgamma agonists) have recently been shown to increase bone fractures. To investigate the etiology of possible structural and/or material quality defects, we have utilized a well-described mouse model of Type 2 diabetes (MKR). MKR mice exhibit muscle hypoplasia from birth with reduced mass by the pre-diabetic age of 3 weeks. A compensatory hyperplasia ensues during early (5 weeks) development; by 6-8 weeks muscle is normal in structure and function. Adult whole-bone mechanical properties were determined by 4-point bending to test susceptibility to fracture. Micro-computed tomography and cortical bone histomorphometry were utilized to assess static and dynamic indices of structure, bone formation and resorption. Osteoclastogenesis assays were performed from bone marrow-derived non-adherent cells. The 8-week and 16-week, but not 3-week, male MKR had slender (i.e., narrow relative to length) femurs that were 20% weaker (p<0.05) relative to WT control femurs. Tissue-level mineral density was not affected. Impaired periosteal expansion during early diabetes resulted from 250% more, and 40% less of the cortical bone surface undergoing resorption and formation, respectively (p<0.05). Greater resorption persisted in adult MKR on both periosteal and endosteal surfaces. Differences were not limited to cortical bone as the distal femur metaphysis of 16 week MKR contained less trabecular bone and trabecular separation was greater than in WT by 60% (p<0.05). At all ages, MKR marrow-derived cultures demonstrated the ability for enhanced osteoclast differentiation in response to M-CSF and RANK-L. Taken together, the MKR mouse model suggests that skeletal fragility in Type 2 diabetes may arise from reduced transverse bone accrual and increased osteoclastogenesis during growth that is accelerated by the diabetic/hyperinsulinemic milieu. Further, these results emphasize the importance of evaluating diabetic bone based on morphology in addition to bone mass.

Morbid obesity is considered a systemic inflammatory state. The objective of this project was to characterize the adipokine, cytokine and chemokine protein profile in serum from control, lean and obese mice. We hypothesized that chemokines and cytokines are altered by caloric restriction and diet-induced obesity as a function of changes in body composition. Six-week-old female C57BL/6N mice (n = 12 per group) were randomized to one of three diets: control (fed ad libitum); lean (30% calorie-restricted regimen relative to control) and diet-induced obese (DIO; high calorie diet, fed ad libitum). Body weight, body composition and food intake were monitored throughout the study. After 10 weeks on the diets, blood samples were collected, and adipokine/cytokine/chemokine serum profiles were measured by antibody array. Lean mice, relative to the control group, displayed increased concentrations of insulin-like growth factor (IGF) binding protein-3, -5 and -6 and adiponectin and decreased IGF-1. These mice also showed increased concentrations of interleukin (IL)-10, IL-12 p40/p70, eotaxin, monocyte chemoattractant protein-5 and SDF-1. In contrast, DIO mice displayed increased leptin, IL-6 and LPS-induced chemokine and decreased concentrations of all chemokines/cytokines measured relative to control mice. As such, these data indicate that DIO may lead to an inflammatory state characterized as a shift towards a T helper lymphocyte type 1-skewed responsiveness. The demonstration of differential adipokine, cytokine and chemokine protein profile in control, lean and DIO mice may have implications for immune responsiveness and risk of disease.

Serum insulin-like growth factor (IGF) -1 is secreted mainly by the liver and circulates bound to IGF-binding proteins (IGFBPs), either as binary complexes or ternary complexes with IGFBP-3 or IGFBP-5 and an acid-labile subunit (ALS). The purpose of this study was to genetically dissect the role of IGF-1 circulatory complexes in somatic growth, skeletal integrity, and metabolism. Phenotypic comparisons of controls and four mouse lines with genetic IGF-1 deficits-liver-specific IGF-1 deficiency (LID), ALS knockout (ALSKO), IGFBP-3 (BP3) knockout, and a triply deficient LID/ALSKO/BP3 line-produced several novel findings. 1) All deficient strains had decreased serum IGF-1 levels, but this neither predicted growth potential or skeletal integrity nor defined growth hormone secretion or metabolic abnormalities. 2) IGF-1 deficiency affected development of both cortical and trabecular bone differently, effects apparently dependent on the presence of different circulating IGF-1 complexes. 3) IGFBP-3 deficiency resulted in increased linear growth. In summary, each IGF-1 complex constituent appears to play a distinct role in determining skeletal phenotype, with different effects on cortical and trabecular bone compartments.

The majority of insulin-like growth factor (IGF)-I and IGF-II circulate in the serum as a complex with the insulin-like growth factor binding protein (IGFBP)-3 or IGFBP-5, and an acid-labile subunit (ALS). The function of ALS is to prolong the half-life of the IGF-I-IGFBP-3/IGFBP-5 binary complexes. Fourteen different mutations of the human IGFALS gene have been identified in 17 patients, suggesting that ALS deficiency may be prevalent in a subset of patients with extraordinarily low serum levels of IGF-I and IGFBP-3 that remain abnormally low upon growth hormone stimulation. Postnatal growth was clearly affected. Commonly, the height standard deviation score before puberty was between -2 and -3, and approximately 1.4 SD shorter than the midparental height SDS. Pubertal delay was found in 50% of the patients. Circulating IGF-II, IGFBP-1, -2 and -3 levels were reduced, with the greatest reduction observed for IGFBP-3. Insulin insensitivity was a common finding, and some patients presented low bone mineral density. Human ALS deficiency represents a unique condition in which the lack of ALS proteins results in the disruption of the entire IGF circulating system. Despite a profound circulating IGF-I deficiency, there is only a mild impact on postnatal growth. The preserved expression of locally produced IGF-I might be responsible for the preservation of linear growth near normal limits.

Current dogma suggests that the positive correlation between obesity and cancer is driven by white adipose tissue that accompanies obesity, possibly through excess secretion of adipokines. However, recent studies in fatless A-Zip/F-1 mice, which have undetectable adipokine levels but display accelerated tumor formation, suggest that adipokines are not required for the enhanced tumor development. The A-Zip/F-1 mice are also diabetic and display elevated circulating levels of other molecules frequently associated with obesity and carcinogenesis: insulin, insulin-like growth factor-1, and inflammatory cytokines. Therefore, we postulate that the pathways associated with insulin resistance and inflammation, rather than adipocyte-derived factors, may represent key prevention or therapeutic targets for disrupting the obesity-cancer link.

Insulin receptor (IR) signaling is considered to be important in growth and development in addition to its major role in metabolic homeostasis. The metabolic role of insulin in carbohydrate and lipid metabolism is extensively studied. In contrast, the role of IR activation during embryogenesis is less understood. To address this, we examined the function of the IR during zebrafish development. Zebrafish express two isoforms of IR (insra and insrb). Both isoforms were cloned and show high homology to the human insulin receptor and can functionally substitute for the human IR in fibroblasts derived from insr gene-deleted mice. Gene expression studies reveal that these receptors are expressed at moderate levels in the central nervous system during development. Morpholino-mediated selective knockdown of each of the IR isoforms causes growth retardation and profound morphogenetic defects in the brain and eye. These results clearly demonstrate that IR signaling plays essential roles in vertebrate embryogenesis and growth.

Human papillomavirus (HPV) is the main risk factor for cervical cancer; however, some carcinomas occur in the absence of the virus. IGF-IR and an isoform of the insulin receptor, IR-A, play important roles in cancer. In this study we assessed the role of the IGF/insulin receptors in cervical cancer cell lines with different HPV status, SiHa (HPV positive), and C33a (HPV negative). Different patterns of receptor expression were found; while SiHa expressed IGF-IR, IR-A and IR-B, and IR/IGF-IR hybrid receptors, C33a cells expressed the IR-A only. Tyrosine phosphorylation of these receptors in response to their corresponding ligands correlated with the expression level of these receptors in the cell lines. Activation of PI3-K and MAPK pathways was revealed in both cell lines, however, no effects on proliferation, migration, or invasion were observed. Here we show that cervical cancer cell lines--positive and negative for HPV--differ in the type of insulin and IGF-1 receptors expressed. Additional studies are needed for characterization of the role of IR-A in cervical carcinogenesis.

Both obesity and Type 2 diabetes are independently associated with an increased risk of developing cancer and an increased mortality. The etiology is yet to be determined but insulin resistance and hyperinsulinemia maybe important factors. Hyperglycemia, hyperlipidemia and inflammatory cytokines in addition to the insulin-like growth factors are also possible factors involved in the process.