Michael J. Haas

Obesity-related Changes in High-density Lipoprotein Metabolism

Obesity is associated with a 3‐or‐more‐fold increase in the risk of fatal and nonfatal myocardial infarction ( 1 , 2 , 3 , 4 , 5 , 6 ). The American Heart Association has reclassified obesity as a major, modifiable risk factor for coronary heart disease ( 7 ). The increased prevalence of premature coronary heart disease in obesity is attributed to multiple factors ( 8 , 9 , 10 ). A principal contributor to this serious morbidity is the alterations in plasma lipid and lipoprotein levels. The dyslipidemia of obesity is commonly manifested as high plasma triglyceride levels, low high‐density lipoprotein cholesterol (HDLc), and normal low‐density lipoprotein cholesterol (LDLc) with preponderance of small dense LDL particles ( 7 , 8 , 9 , 10 ). However, there is a considerable heterogeneity of plasma lipid profile in overweight and obese people. The precise cause of this heterogeneity is not entirely clear but has been partly attributed to the degree of visceral adiposity and insulin resistance. The emergence of glucose intolerance or a genetic predisposition to familial combined hyperlipidemia will further modify the plasma lipid phenotype in obese people ( 11 , 12 , 13 , 14 , 15 ).

Diabetes-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1) expression.

The endothelial or epithelial tight junctions create a barrier to diffusion of solutes. Since experimental diabetes mellitus is associated with considerable alterations in the blood-brain barrier (BBB), it is possible that specific tight junction proteins may be altered in diabetes. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and mRNA levels in cerebral tissue of streptozotocin-induced diabetic rats and the results were compared to insulin treated diabetic rats and vehicle injected control rats. The cerebral occludin content in diabetic rats (115.4 ± 18.6 arbitrary units) was significantly reduced compared to insulin-treated diabetic rats (649.1 ± 141.2) or control rats (552.9 ± 82.9), p < 0.001. The ZO-1 content of cerebral tissue from diabetic rats (1240.6 ± 199.7 arbitrary units) was not significantly altered compared to controls (1310.8 ± 256.9). The cerebral occludin mRNA content relative to G3PDH mRNA was 1.35 ± 0.07 and 1.34 ± 0.19 in control and diabetic rats respectively. The cerebral ZO-1 mRNA content relative to G3PDH mRNA in diabetic and control rats was 1.135 ± 0.123 and 0.956 ± 0.038 respectively. These differences did not achieve statistical significance. It is concluded that diabetes alters the molecular anatomy of the tight junctions in cerebral tissue by altering the content of select structural proteins.

Hyperglycemia-induced endoplasmic reticulum stress in endothelial cells

OBJECTIVE Hyperglycemia-induced endothelial cell dysfunction in vascular disease can occur due to increased oxidative stress and a concomitant increase in endoplasmic reticulum (ER) stress. To investigate whether these cellular stresses are independent or causally linked, we determined whether or not specific glycolytic intermediates that induce oxidative stress also induce ER stress. METHODS Human umbilical vein endothelial cells were treated with dextrose, partially metabolizable (e.g., fructose and galactose) and non-metabolizable sugars (e.g., 3-O-methyglucose), and various intermediates of the glycolytic and tricarboxylic acid pathways. Activation of the unfolded protein response and subsequent generation of ER stress was measured by the ER stress-responsive alkaline phosphatase method, and superoxide (SO) generation was measured using the hydro-ethidene-fluorescence method. The mitochondrial origin of the SO and the generation of ER stress by dextrose and the intermediate metabolites were confirmed with experiments using allopurinol and diphenyleneiodonium chloride to block SO generation by xanthine oxidase and nicotinamide adenosine dinucleotide phosphate oxidase, respectively. RESULTS Although ER stress could be induced by glycolytic intermediates up to and including pyruvate, the SO generation occurred in the presence of glycolytic and mitochondrial metabolites. CONCLUSION Although the mitochondria are the site of signals generated by dextrose to initiate oxidative stress, the dextrose-induced ER stress, unlike SO generation, does not require pyruvate oxidation in the mitochondria.

Age-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1)

The endothelial or epithelial tight junctions create a rate-limiting barrier to diffusion of solutes. A major determinant of the barrier function is the density of tight junction proteins. Since aging is associated with significant alterations in the blood-brain barrier (BBB) it is possible that specific tight junction proteins may be altered in the cerebrum of aging rats. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and their mRNA in cerebral tissue of 3-, 12- and 24-month-old rats. The cerebral occludin content in 24-month-old rats (732.5+/-99.9 arbitrary units) was significantly reduced compared to 12-month-old rats (1043.4+/-131.8) or 3-month-old rats (1021.4+/-62.8), P<0.01. The cerebral ZO-1 protein content in 24-month-old rats (161.7+/-8.1 arbitrary units) and 12-month-old rats (144.3+/-35.9) were not significantly reduced compared to 4-month-old rats (189.0+/-27.2). The occludin mRNA content relative to G3PDH mRNA was 1.11+/-0.05, 1.11+/-0.07 and 1.00+/-0.05 in 3-, 12- and 24-month-old rats, respectively. The differences did not achieve statistical significance. The ZO-1 mRNA content of cerebral tissue relative to G3PDH mRNA was significantly increased in 24-month-old rats compared to 3-month-old rats (1.280+/-0.030 vs. 0.956+/-0.038), P<0.001. It is concluded that aging in rats may alter the molecular anatomy of the BBB by altering the content of select structural proteins of tight junctions.

Effects of antioxidants on glucose-induced oxidative stress and endoplasmic reticulum stress in endothelial cells

AIM Hyperglycemia-induced endothelial cell dysfunction can be the result of increased oxidative stress and concomitant increase in endoplasmic reticulum (ER) stress. To test the extent of coupling between these two stresses, the effect of antioxidant vitamins on glucose-induced oxidative stress and ER stress in endothelial cells were studied. METHODS Human umbilical vein endothelial cells (HUVEC) were treated with physiological (5.5mM) or supra-physiological (27.5mM) dextrose concentrations, and ER stress and oxidative stress were measured. Additional experiments were carried out in HUVEC over-expressing exogenous glucose transporter-1 (Glut-1) and treated with 5.5mM dextrose. RESULTS Supra-physiological dextrose concentrations increased both ER stress and oxidative stress. However, while oxidative stress could be effectively inhibited with alpha-tocopherol and ascorbic acid, these antioxidants had no effect on ER stress. Increasing intracellular glucose levels by exogenous expression of Glut-1 in endothelial cells also increased oxidative stress and ER stress. Whereas the oxidative stress in these cells was reduced with alpha-tocopherol and ascorbic acid and dimethylsulfoxide, the ER stress could not be ameliorated with alpha-tocopherol and ascorbic acid. CONCLUSIONS These results indicate that ER stress can be uncoupled from oxidative stress and antioxidants can ameliorate the latter without altering the ER stress induced by hyperglycemia.

Astrocytic and neuronal biochemical markers in the sera of subjects with diabetes mellitus.

To determine if clinical diabetes is associated with disruption of the blood-brain barrier (BBB) and/or brain injury, enzyme-linked immunoassays and Western blots were used to measure serum levels of S100B, NSE and their auto-antibodies in type 1 and type 2 diabetic subjects. Serum S100B concentrations in type 2 diabetic subjects, but not in type 1 diabetic subjects, were significantly lower than those found in healthy controls. There were no significant differences in serum NSE levels of either type 1 or type 2 diabetics compared to healthy controls. However, there was a significant increase in antibodies to NSE in both type 1 and type 2 diabetic subjects compared to controls, whereas diabetics and controls had equally very low levels of anti S100B auto-antibodies. These studies suggest that diabetes in humans may be associated with alterations in the BBB integrity that allow the emergence of antibodies against neuronal antigens.

Glucose-induced endoplasmic reticulum stress is independent of oxidative stress: A mechanistic explanation for the failure of antioxidant therapy in diabetes.

Oxidative stress contributes to the pathogenesis of diabetes and its complications. However, a large number of interventional studies have failed to show any health benefits of antioxidants. The overwhelming failure of antioxidant therapy to prevent disease can be explained by inadequacy of the doses of antioxidants used, short duration of therapy, or poor timing of initiation of the supplementation. A more likely reason for failure of antioxidants to reduce diabetes-related complications is the multiplicity of mechanisms of glucotoxicity that are independent of oxidative stress. Recently, endoplasmic reticulum (ER) stress has emerged as an important contributor to diabetes-related complications. Multiple lines of experimental evidence indicate that ER stress in endothelial cells can be uncoupled from oxidative stress induced by hyperglycemia, and antioxidants can ameliorate the latter without altering the ER stress. These observations provide a novel mechanistic explanation for the failure of antioxidant therapy in interventional clinical trials.

Inflammation, high-density lipoprotein and cardiovascular dysfunction.

Purpose of review This review describes the evidence that supports the hypothesis that high-density lipoprotein (HDL) is atheroprotective due to its antiinflammatory effects and benefits on vascular health. Recent findings Recent investigations have shown that HDL may inhibit atherosclerosis by promoting healthy endothelial function and by limiting or inhibiting the activation of macrophage and other immune cells. Receptors for HDL clearly regulate immune system function as well as cellular stress. Recent studies also suggest that participation of HDL in the process of reverse cholesterol transport may inhibit growth factor and cytokine receptor signaling by depleting cholesterol from lipid rafts. However, inflammation can also be associated with circulating dysfunctional HDL, which often possesses both prooxidative and proinflammatory properties. Summary These studies suggest that HDL-based therapeutics have potential in treating both acute and chronic conditions associated with inflammation. These studies also reveal several other pathways that may be targeted for therapeutic drug development.

Regulation of high‐density lipoprotein by inflammatory cytokines: establishing links between immune dysfunction and cardiovascular disease

Coronary artery disease is a primary co‐morbidity in metabolic diseases such as metabolic syndrome, diabetes and obesity. One contributing risk factor for coronary artery disease is low high‐density lipoprotein‐cholesterol (HDLc). Several factors influence steady‐state HDLc levels, including diet, genetics and environment. Perhaps more important to coronary artery disease is factors that attribute to the dynamics of reverse cholesterol transport, storage, and excretion of excess cholesterol. HDLc biogenesis, clearance and innate ability to serve as a cholesterol acceptor and transporter all contribute to HDLcs function as a negative regulator of cardiovascular disease. With the recent failure of torcetrapid, focus is being placed on HDLc biology and its role in various metabolic diseases. Low HDLc levels are often associated with an increased state of background inflammation. Recently, several syndromes with clear pro‐inflammatory components have been shown to be inversely correlated with low HDLc levels in the absence of obesity, diabetes and metabolic syndrome. Early studies with HDLc during the acute‐phase response suggest that HDLc is substantially physically modified during acute infection and sepsis, and recent studies show that HDLc is physically modified by chronic pro‐inflammatory disease. In this review, several of these connections are described and cytokine signalling related to HDLc is examined. Copyright

Monosaccharide-enriched diets cause hyperleptinemia without hypophagia

To determine the effect of monosaccharide-enriched diets on plasma leptin and food consumption, body weight, food intake, and serum glucose, insulin, and leptin concentrations were measured in rats maintained on a 10-d course of 60% glucose or 60% fructose diet. The serum leptin concentration in rats fed a high-glucose diet (7.60 +/- 0.6 ng/mL) or a high-fructose diet (5.12 +/- 0.8 ng/mL) was significantly increased compared with that in control rats (2.45 +/- 0.10 ng/mL; P < 0.001). To ascertain that the observed effect was related to hyperinsulinemia, a group of rats were infused with exogenous insulin or rendered insulin resistent with a high-fat diet. When hyperinsulinemia was induced with exogenous infusion, the serum leptin was increased (5.56 +/- 0.23 ng/mL; P < 0.001). High-fat feeding was associated with increased serum leptin (12.1 +/- 1.4 ng/mL) and insulin levels. The increased serum leptin concentration was not associated with decreased food intake. We conclude that monosaccharide-enriched diets, probably through hyperinsulinemia or relative or absolute insulin resistance, cause hyperleptinemia, which does not appear to change feeding behavior.