Assessing Structural Racism Measures on Health Outcomes of Asian Americans, Native Hawaiians, and Pacific Islanders: A Scoping Review.

BACKGROUND: Limited literature exists on structural racism measures on health outcomes for Asian Americans, Native Hawaiians, and Pacific Islanders (AAs and NH/PIs). AAs and NH/PIs make up approximately 6.2% of the U.S. population and consist of diverse ethnic subgroups with distinct languages, cultures, religions, socioeconomic statuses, and historical backgrounds. The lack of disaggregated data collection and contextualized measures hinders our understanding of how structural racism affects health outcomes in these populations. METHODS: We conducted a scoping review to assess the extent to which measures of structural racism are used in research with AAs and NH/PIs. Databases, including CINAHL, EBSCO, PsychINFO, PubMed, Scopus, and Social Science Citation Index, were searched for peer-reviewed articles on the measures of and empirical impacts of structural racism on AA and NH/PI health. We identified 23 full-text articles from a pool of 11,660 screened articles. Four articles were included in the final analysis. RESULTS: Among the selected studies, two studies identified an association between racial segregation and mental and behavioral health outcomes within AAs and NH/PIs. The other two studies found redlining on chronic health outcomes in these communities. These studies uncovered associations between government systems and policies and AA and NH/PI health outcomes. DISCUSSION: Existing measures may not adequately capture the complex relationships between structural racism and health outcomes in AAs and NH/PIs. Future research should contextualize and operationalize the multifaceted manifestations of structural racism unique to AAs and NH/PIs to achieve health equity.

Deletion of Smooth Muscle O-GlcNAc Transferase Prevents Development of Atherosclerosis in Western Diet-Fed Hyperglycemic ApoE-/- Mice In Vivo.

Accumulating evidence highlights protein O-GlcNAcylation as a putative pathogenic contributor of diabetic vascular complications. We previously reported that elevated protein O-GlcNAcylation correlates with increased atherosclerotic lesion formation and VSMC proliferation in response to hyperglycemia. However, the role of O-GlcNAc transferase (OGT), regulator of O-GlcNAc signaling, in the evolution of diabetic atherosclerosis remains elusive. The goal of this study was to determine whether smooth muscle OGT (smOGT) plays a direct role in hyperglycemia-induced atherosclerotic lesion formation and SMC de-differentiation. Using tamoxifen-inducible Myh11-CreERT2 and Ogtfl/fl mice, we generated smOGTWT and smOGTKO mice, with and without ApoE-null backgrounds. Following STZ-induced hyperglycemia, smOGTWT and smOGTKO mice were kept on a standard laboratory diet for the study duration. In a parallel study, smOGTWTApoE-/- and smOGTKOApoE-/- were initiated on Western diet at 8-wks-age. Animals harvested at 14-16-wks-age were used for plasma and tissue collection. Loss of smOGT augmented SM contractile marker expression in aortic vessels of STZ-induced hyperglycemic smOGTKO mice. Consistently, smOGT deletion attenuated atherosclerotic lesion lipid burden (Oil red O), plaque area (H&E), leukocyte (CD45) and smooth muscle cell (ACTA2) abundance in Western diet-fed hyperglycemic smOGTKOApoE-/- mice. This was accompanied by increased SM contractile markers and reduced inflammatory and proliferative marker expression. Further, smOGT deletion attenuated YY1 and SRF expression (transcriptional regulators of SM contractile genes) in hyperglycemic smOGTKOApoE-/- and smOGTKO mice. These data uncover an athero-protective outcome of smOGT loss-of-function and suggest a direct regulatory role of OGT-mediated O-GlcNAcylation in VSMC de-differentiation in hyperglycemia.

Cognitive dysfunction and increased phosphorylated tau are associated with reduced O-GlcNAc signaling in an aging mouse model of metabolic syndrome.

Metabolic syndrome (MetS), characterized by hyperglycemia, obesity, and hyperlipidemia, can increase the risk of developing late-onset dementia. Recent studies in patients and mouse models suggest a putative link between hyperphosphorylated tau, a component of Alzheimer's disease-related dementia (ADRD) pathology, and cerebral glucose hypometabolism. Impaired glucose metabolism reduces glucose flux through the hexosamine metabolic pathway triggering attenuated O-linked N-acetylglucosamine (O-GlcNAc) protein modification. The goal of the current study was to investigate the link between cognitive function, tau pathology, and O-GlcNAc signaling in an aging mouse model of MetS, agouti KKAy+/- . Male and female C57BL/6, non-agouti KKAy-/- , and agouti KKAy+/- mice were aged 12-18 months on standard chow diet. Body weight, blood glucose, total cholesterol, and triglyceride were measured to confirm the MetS phenotype. Cognition, sensorimotor function, and emotional reactivity were assessed for each genotype followed by plasma and brain tissue collection for biochemical and molecular analyses. Body weight, blood glucose, total cholesterol, and triglyceride levels were significantly elevated in agouti KKAy+/- mice versus C57BL/6 controls and non-agouti KKAy-/- . Behaviorally, agouti KKAy+/- revealed impairments in sensorimotor and cognitive function versus age-matched C57BL/6 and non-agouti KKAy-/- mice. Immunoblotting demonstrated increased phosphorylated tau accompanied with reduced O-GlcNAc protein expression in hippocampal-associated dorsal midbrain of female agouti KKAy+/- versus C57BL/6 control mice. Together, these data demonstrate that impaired cognitive function and AD-related pathology are associated with reduced O-GlcNAc signaling in aging MetS KKAy+/- mice. Overall, our study suggests that interaction of tau pathology with O-GlcNAc signaling may contribute to MetS-induced cognitive dysfunction in aging.

Sex-specific differences in atherosclerosis, thrombospondin-1, and smooth muscle cell differentiation in metabolic syndrome versus non-metabolic syndrome mice.

Introduction: Metabolic syndrome (MetS) amplifies the risks of atherosclerosis. Despite well-known sexual dimorphism in atherosclerosis, underlying mechanisms are poorly understood. Our previous findings highlight a proatherogenic protein, thrombospondin-1 (TSP-1), in hyperglycemia- or hyperleptinemia (mimicking obesity)-induced atherosclerosis. However, the role of TSP-1 in the development of atherosclerosis prompted by co-existing hyperglycemia and obesity, characteristic of MetS, is unknown. The goal of this study was to examine sex-specific differences in lesion progression in a model of combined MetS and atherosclerosis (KKAyApoE) and interrogate how these differences relate to TSP-1 expression. Methods: Male and female KKAy+/-ApoE-/- (with ectopic agouti gene expression) and age-matched non-agouti KKAy-/-ApoE-/- littermates were placed on a standard laboratory diet from 4 to 24 weeks age followed by blood and tissue harvests for biochemical, molecular, and aortic root morphometric studies. Results: Metabolic profiling confirmed MetS phenotype of KKAy+/-ApoE-/-; however, only male genotypes were glucose intolerant with elevated VLDL-cholesterol and VLDL-triglyceride levels. Aortic root morphometry demonstrated profound lipid-filled lesions, increased plaque area, and augmented inflammatory and SMC abundance in MetS vs non-MetS males. This increase in lesion burden was accompanied with elevated TSP-1 and attenuated LMOD-1 (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in male MetS aortic vessels. In contrast, while lipid burden, plaque area, and TSP-1 expression increased in MetS and non-MetS female mice, there was no significant difference between these genotypes. Increased collagen content was noted in MetS and non-MetS genotypes, specific to female mice. Measurement of plasma testosterone revealed a link between the atherogenic phenotype and abnormally high or low testosterone levels. To interrogate whether TSP-1 plays a direct role in SMC de-differentiation in MetS, we generated KKAy+/- mice with and without global TSP-1 deletion. Immunoblotting showed increased SM contractile markers in male KKAy+/-TSP-1-/- aortic vessels vs male KKAy+/-TSP-1+/ +. In contrast, TSP-1 deletion had no effect on SM contractile marker expression in female genotypes. Conclusion: Together, the current study implicates a role of plasma testosterone in sex-specific differences in atherosclerosis and TSP-1 expression in MetS vs non-MetS mice. Our data suggest a sex-dependent differential role of TSP-1 on SMC de-differentiation in MetS. Collectively, these findings underscore a fundamental link between TSP-1 and VSMC phenotypic transformation in MetS.

Leptin in Atherosclerosis: Focus on Macrophages, Endothelial and Smooth Muscle Cells.

Increasing adipose tissue mass in obesity directly correlates with elevated circulating leptin levels. Leptin is an adipokine known to play a role in numerous biological processes including regulation of energy homeostasis, inflammation, vascular function and angiogenesis. While physiological concentrations of leptin may exhibit multiple beneficial effects, chronically elevated pathophysiological levels or hyperleptinemia, characteristic of obesity and diabetes, is a major risk factor for development of atherosclerosis. Hyperleptinemia results in a state of selective leptin resistance such that while beneficial metabolic effects of leptin are dampened, deleterious vascular effects of leptin are conserved attributing to vascular dysfunction. Leptin exerts potent proatherogenic effects on multiple vascular cell types including macrophages, endothelial cells and smooth muscle cells; these effects are mediated via an interaction of leptin with the long form of leptin receptor, abundantly expressed in atherosclerotic plaques. This review provides a summary of recent in vivo and in vitro studies that highlight a role of leptin in the pathogenesis of atherosclerotic complications associated with obesity and diabetes.

TSP-1 (Thrombospondin-1) Deficiency Protects ApoE-/- Mice Against Leptin-Induced Atherosclerosis.

OBJECTIVE: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE-/- and TSP-1-/-/ApoE-/- double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE-/- mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE-/- mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE-/-; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE-/- on western diet, independent of plasma lipid alterations. CONCLUSIONS: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE-/- mice.

Increasing evidence suggests thrombospondin-1 (TSP-1), a potent proatherogenic matricellular protein, as a putative link between hyperglycemia and atherosclerotic complications in diabetes. We previously reported that the micronutrient chromium picolinate (CrP), with long-standing cardiovascular benefits, inhibits TSP-1 expression in glucose-stimulated human aortic smooth muscle cells in vitro. Here, we investigated the atheroprotective action of orally administered CrP in type 1 diabetic apolipoprotein E-deficient (ApoE-/-) mice and elucidated the role of TSP-1 in this process. CrP decreased lipid burden and neointimal thickness in aortic root lesions of hyperglycemic ApoE-/- mice; also, smooth muscle cell (SMC), macrophage and leukocyte abundance was prevented coupled with reduced cell proliferation. Attenuated lesion progression was accompanied with inhibition of hyperglycemia-induced TSP-1 expression and reduced protein O-glycosylation following CrP treatment; also, PCNA and vimentin (SMC synthetic marker) expression were reduced while SM-MHC (SMC contractile marker) levels were increased. To confirm a direct role of TSP-1 in diabetic atherosclerosis, hyperglycemic TSP-1-/-/ApoE-/- double knockout mice were compared with age-matched hyperglycemic ApoE-/- littermates. Lack of TSP-1 prevented lesion formation in hyperglycemic ApoE-/- mice, mimicking the atheroprotective phenotype of CrP-treated mice. These results suggest that therapeutic TSP-1 inhibition may have important atheroprotective potential in diabetic vascular disease.

Relationship of Anterior Alveolar Dimensions with Mandibular Divergence in Class I Malocclusion - A Cephalometric Study.

INTRODUCTION: One of the major limiting factors in retraction of proclined teeth is the width of the alveolus both in maxilla and mandible. AIM: The objective of this study was to assess the maxillary and mandibular anterior alveolar dimensions and to correlate with mandibular divergence in Class I bi-dento-alveolar protrusion patients. MATERIALS AND METHODS: Pretreatment lateral cephalograms (n=88) were analysed using a composite analysis with cephalometric software. Both maxillary and mandibular anterior alveolar widths and heights were measured and correlated with mandibular divergence. One-way analysis (ANOVA) and Pearson correlation test were used to compare and establish the significance between groups. RESULTS: Segregation of the data based on variation in the bi-cortical widths and heights showed that lesser alveolar widths and greater alveolar heights were associated with the high angled subjects and greater alveolar widths and lesser heights were associated with low angled subjects. CONCLUSION: Patients with hyperdivergent mandible exhibited thin anterior alveolar width and greater alveolar height whereas low angled subjects had wider alveolar width and lesser alveolar height. Orthodontic treatment plan for retraction of anterior teeth must be based on these differences caused by variations in mandibular divergence.

Leptin augments recruitment of IRF-1 and CREB to thrombospondin-1 gene promoter in vascular smooth muscle cells in vitro.

We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation, and coimmunoprecipitation assays revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (-1019 to -1016) and CREB (-1198 to -1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a cooperative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in vascular smooth muscle cells.

Anti-atherogenic effect of trivalent chromium-loaded CPMV nanoparticles in human aortic smooth muscle cells under hyperglycemic conditions in vitro.

Atherosclerosis, a major macrovascular complication associated with diabetes, poses a tremendous burden on national health care expenditure. Despite extensive efforts, cost-effective remedies are unknown. Therapies for atherosclerosis are challenged by a lack of targeted drug delivery approaches. Toward this goal, we turn to a biology-derived drug delivery system utilizing nanoparticles formed by the plant virus, Cowpea mosaic virus (CPMV). The aim herein is to investigate the anti-atherogenic potential of the beneficial mineral nutrient, trivalent chromium, loaded CPMV nanoparticles in human aortic smooth muscle cells (HASMC) under hyperglycemic conditions. A non-covalent loading protocol is established yielding CrCl3-loaded CPMV (CPMV-Cr) carrying 2000 drug molecules per particle. Using immunofluorescence microscopy, we show that CPMV-Cr is readily taken up by HASMC in vitro. In glucose (25 mM)-stimulated cells, 100 nM CPMV-Cr inhibits HASMC proliferation concomitant to attenuated proliferating cell nuclear antigen (PCNA, proliferation marker) expression. This is accompanied by attenuation in high glucose-induced phospho-p38 and pAkt expression. Moreover, CPMV-Cr inhibits the expression of pro-inflammatory cytokines, transforming growth factor-ß (TGF-ß) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), in glucose-stimulated HASMCs. Finally glucose-stimulated lipid uptake is remarkably abrogated by CPMV-Cr, revealed by Oil Red O staining. Together, these data provide key cellular evidence for an atheroprotective effect of CPMV-Cr in vascular smooth muscle cells (VSMC) under hyperglycemic conditions that may promote novel therapeutic ventures for diabetic atherosclerosis.

Trivalent chromium inhibits TSP-1 expression, proliferation, and O-GlcNAc signaling in vascular smooth muscle cells in response to high glucose in vitro.

Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr(3+) (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr(3+) is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase, a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr(3+) attenuates reactive oxygen species formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an antiproliferative effect of Cr(3+) that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr(3+) to modulate aberrant vascular smooth muscle cell function associated with hyperglycemia-induced vascular complications.

Upregulation of thrombospondin-1 expression by leptin in vascular smooth muscle cells via JAK2- and MAPK-dependent pathways.

Hyperleptinemia, characteristic of diabetes and a hallmark feature of human obesity, contributes to the increased risk of atherosclerotic complications. However, molecular mechanisms mediating leptin-induced atherogenesis and gene expression in vascular cells remain incompletely understood. Accumulating evidence documents a critical role of a potent antiangiogenic and proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in atherosclerosis. Although previous studies reported elevated TSP-1 levels in both diabetic and obese patients and rodent models, there is no direct information on TSP-1 expression in vascular cells in response to leptin. In the present study, we show that leptin upregulates TSP-1 expression in cultured human aortic smooth muscle cells (HASMC) in vitro, and this increase occurs at the level of transcription, revealed by mRNA stability and TSP-1 promoter-reporter assays. Utilizing specific pharmacological inhibitors and siRNA approaches, we demonstrate that upregulation of TSP-1 expression by leptin is mediated by JAK2/ERK/JNK-dependent mechanisms. Furthermore, we report that while ERK and JNK are required for both the constitutive and leptin-induced expression of TSP-1, JAK-2 appears to be specifically involved in leptin-mediated TSP-1 upregulation. Finally, we found that increased HASMC migration and proliferation in response to leptin is significantly inhibited by a TSP-1 blocking antibody, thereby revealing the physiological significance of leptin-TSP-1 crosstalk. Taken together, these findings demonstrate, for the first time, that leptin has a direct regulatory effect on TSP-1 expression in HASMCs, underscoring a novel role of TSP-1 in hyperleptinemia-induced atherosclerotic complications.

Endothelin-mediated in vivo pressor responses following TRPV1 activation.

Transient receptor potential vanilliod 1 (TRPV1) channels have recently been postulated to play a role in the vascular complications/consequences associated with diabetes despite the fact that the mechanisms through which TRPV1 regulates vascular function are not fully known. Accordingly, our goal was to define the mechanisms by which TRPV1 channels modulate vascular function and contribute to vascular dysfunction in diabetes. We subjected mice lacking TRPV1 [TRPV1((-/-))], db/db, and control C57BLKS/J mice to in vivo infusion of the TRPV1 agonist capsaicin or the α-adrenergic agonist phenylephrine (PE) to examine the integrated circulatory actions of TRPV1. Capsaicin (1, 10, 20, and 100 µg/kg) dose dependently increased MAP in control mice (5.7 ± 1.6, 11.7 ± 2.1, 25.4 ± 3.4, and 51.6 ± 3.9%), which was attenuated in db/db mice (3.4 ± 2.1, 3.9 ± 2.1, 7.0 ± 3.3, and 17.9 ± 6.2%). TRPV1((-/-)) mice exhibited no changes in MAP in response to capsaicin, suggesting the actions of this agonist are specific to TRPV1 activation. Immunoblot analysis revealed decreased aortic TRPV1 protein expression in db/db compared with control mice. Capsaicin-induced responses were recorded following inhibition of endothelin A and B receptors (ET(A) /ET(B)). Inhibition of ET(A) receptors abolished the capsaicin-mediated increases in MAP. Combined antagonism of ET(A) and ET(B) receptors did not further inhibit the capsaicin response. Cultured endothelial cell exposure to capsaicin increased endothelin production as shown by an endothelin ELISA assay, which was attenuated by inhibition of TRPV1 or endothelin-converting enzyme. TRPV1 channels contribute to the regulation of vascular reactivity and MAP via production of endothelin and subsequent activation of vascular ET(A) receptors. Impairment of TRPV1 channel function may contribute to vascular dysfunction in diabetes.

A novel transcriptional mechanism of cell type-specific regulation of vascular gene expression by glucose.

OBJECTIVE: Vascular diabetic complications are associated with abnormal extracellular matrix and dysfunction of vascular cells, which later result in aberrant angiogenesis and development of atherosclerotic lesions. The tissue and cell specificity of the effects of high glucose are well recognized, but the underlying cell type-specific molecular mechanisms controlled by glucose are still unclear. We sought to identify cell type-specific mechanisms by which high glucose regulates transcription of genes in vascular cells. METHODS AND RESULTS: Thrombospondin-1 is a potent antiangiogenic protein associated with development of several diabetic complications and regulated by high glucose in multiple cell types. We report that distinct cell type-specific mechanisms regulate thrombospondin-1 gene (THBS1) transcription in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in response to high glucose: although a proximal fragment of 280 nucleotides is sufficient to drive transcription in ECs, THBS1 was regulated cooperatively by interaction between proximal (-272 to -275) and distal (-1016 to -1019) promoter elements in VSMCs. Transcription factors activated by high glucose in VSMCs were cell type-specific. The formation of a single complex interacting with both distal and proximal glucose-responsive elements of THBS1 promoter in VSMCs was confirmed using gel-shift assays, binding sequence decoy oligomers, and specific mutant promoter fragments. CONCLUSIONS: Transcriptional response of vascular cells to high glucose is cell type-specific and involves activation of distinct transcription factors, providing a basis for tissue-specific changes of vasculature in diabetics.

Antidiabetogenic effects of chromium mitigate hyperinsulinemia-induced cellular insulin resistance via correction of plasma membrane cholesterol imbalance.

Previously, we found that a loss of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure contributes to insulin-induced insulin resistance. Interestingly, we also demonstrated that chromium picolinate (CrPic), a dietary supplement thought to improve glycemic status in insulin-resistant individuals, augments insulin-regulated glucose transport in insulin-sensitive 3T3-L1 adipocytes by lowering PM cholesterol. Here, to gain mechanistic understanding of these separate observations, we tested the prediction that CrPic would protect against insulin-induced insulin resistance by improving PM features important in cytoskeletal structure and insulin sensitivity. We found that insulin-induced insulin-resistant adipocytes display elevated PM cholesterol with a reciprocal decrease in PM PIP2. This lipid imbalance and insulin resistance was corrected by the cholesterol-lowering action of CrPic. The PM lipid imbalance did not impair insulin signaling, nor did CrPic amplify insulin signal transduction. In contrast, PM analyses corroborated cholesterol and PIP2 interactions influencing cytoskeletal structure. Because extensive in vitro study documents an essential role for cytoskeletal capacity in insulin-regulated glucose transport, we next evaluated intact skeletal muscle from obese, insulin-resistant Zucker (fa/fa) rats. Because insulin resistance in these animals likely involves multiple mechanisms, findings that cholesterol-lowering restored F-actin cytoskeletal structure and insulin sensitivity to that witnessed in lean control muscle were striking. Also, experiments using methyl-beta-cyclodextrin to shuttle cholesterol into or out of membranes respectively recapitulated the insulin-induced insulin-resistance and protective effects of CrPic on membrane/cytoskeletal interactions and insulin sensitivity. These data predict a PM cholesterol basis for hyperinsulinemia-associated insulin resistance and importantly highlight the reversible nature of this abnormality.

Glycosylation mediates up-regulation of a potent antiangiogenic and proatherogenic protein, thrombospondin-1, by glucose in vascular smooth muscle cells.

Accelerated development of atherosclerotic lesions remains the most frequent and dangerous complication of diabetes, accounting for 80% of deaths among diabetics. However, our understanding of the pathways mediating glucose-induced gene expression in vascular cells remains controversial and incomplete. We have identified an intracellular metabolic pathway activated by high glucose in human aortic smooth muscle cells that mediates up-regulation of thrombospondin-1 (TSP-1). TSP-1 is a potent antiangiogenic and proatherogenic protein that may represent an important link between diabetes and vascular complications. Using different glucose analogs and metabolites sharing distinct, limited metabolic steps with glucose, we demonstrated that activation of TSP-1 transcription is mediated by the hexosamine pathway of glucose catabolism, possibly resulting in modulation of the activity of nuclear proteins activity through their glycosylation. Specific inhibitors of glutamine: fructose 6-phosphate amidotransferase (GFAT), an enzyme controlling the hexosamine pathway, as well as direct inhibitors of protein glycosylation efficiently inhibited TSP-1 transcription and the activity of a TSP-1 promoter-reporter construct stimulated by high glucose. Overexpression of recombinant GFAT resulted in increased TSP-1 levels. Pharmacological inhibition of GFAT or protein glycosylation inhibited increased proliferation of human aortic smooth muscle cells caused by glucose. We have demonstrated that the hexosamine metabolic pathway mediates up-regulation of TSP-1 by high glucose. Our results suggest that the hexosamine pathway and intracellular glycosylation may control important steps in initiation and development of atherosclerotic lesions.

Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin.

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (F-actin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nm) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mm) and high (25 mm) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP(2)) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP(2) and found a noticeable loss of this lipid from the plasma membrane. Using a PIP(2) delivery system, we replenished plasma membrane PIP(2) in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulin-induced insulin resistance involving defects in PIP(2)/actin regulation.

Regulation of hepatic glucose metabolism by leptin in pig and rat primary hepatocyte cultures.

Direct effects of leptin on gluconeogenesis in rat hepatocytes are equivocal, and model systems from other species have not been extensively explored in assessing the regulation of glucose metabolism by leptin. Therefore, the goal of the present study was to compare the effects of leptin on gluconeogenesis in pig and rat hepatocyte cultures as well as to investigate an underlying mechanism of action at the level of phosphoenolpyruvate carboxykinase (PEPCK). In rat hepatocytes, leptin exposure (3 h, 50 and 100 nM) attenuated glucagon-stimulated hepatic gluconeogenesis by 35 and 38% (P < 0.05), respectively. However, leptin did not produce any significant acute effect in pig hepatocytes. Leptin exposure for 24 h failed to produce any significant effect on gluconeogenesis in either rat or pig hepatocytes cultured in the presence of glucagon or dexamethasone. Mechanistically, there was a 25-35% decrease (P < 0.05) in glucagon-induced PEPCK mRNA levels in rat but not pig hepatocytes cultured with leptin. This effect on PEPCK mRNA was not due to an alteration in the relative abundance of the leptin receptor or the ability of PEPCK to respond to cAMP. The nonuniformity of the effects of leptin on gluconeogenesis in pig and rat hepatocytes indicates differences in leptin action between species. Furthermore, the unique action of leptin in porcine hepatocytes points to the utility of this model system for biomedical research and also underscores the value of comparative studies.

Hepatic glutamine metabolism.

Expression of high activities of both glutamine synthetase and glutaminase allows the liver to play a major role in the regulation of glutamine homeostasis. The liver shows net glutamine output in metabolic acidosis, in prolonged starvation and animals bearing tumors, net glutamine uptake in the postabsorptive state, on consuming high protein diets, and in uncontrolled diabetes or sepsis. Liver glutamine synthetase is expressed only in a small population of perivenous cells that allows it to salvage any ammonia not incorporated into urea in periportal cells. Hepatic glutaminase is a unique isozyme found only in periportal liver parenchymal cells where it provides glutamate and ammonia for the urea cycle. Control of hepatic glutamine metabolism occurs almost exclusively through changes in the activity of glutaminase, with no change in glutamine synthetase flux.