Becky M. Sebastian

Taurine Supplementation Prevents Ethanol-Induced Decrease in Serum Adiponectin and Reduces Hepatic Steatosis in Rats

Chronic ethanol feeding decreases expression of adiponectin by adipocytes and circulating adiponectin. Adiponectin treatment during chronic ethanol feeding prevents liver injury in mice. Chronic ethanol feeding also increases oxidative and endoplasmic reticulum (ER) stress in adipose tissue. Here we tested the hypothesis that supplemental taurine, an amino acid that functions as a chemical chaperone/osmolyte and enhances cellular antioxidant activity, would prevent ethanol‐induced decreases in adiponectin expression and attenuate liver injury. Serum adiponectin concentrations decreased as early as 4 to 7 days after feeding rats a 36% ethanol diet. This rapid decrease was associated with increased oxidative, but not ER, stress in subcutaneous adipose tissue. Taurine prevented ethanol‐induced oxidative stress and increased inflammatory cytokine expression in adipose tissue. Ethanol feeding also rapidly decreased expression of transcription factors regulating adiponectin expression (CCAAT/enhancer binding protein α; peroxisome proliferator‐activated receptor α/γ) in subcutaneous adipose tissue. Taurine prevented the ethanol‐induced decrease in CCAAT/enhancer binding protein α and peroxisome proliferator‐activated receptor α, normalizing adiponectin messenger (m)RNA and serum adiponectin concentrations. In the liver, taurine prevented ethanol‐induced oxidative stress and attenuated tumor necrosis factor α expression and steatosis, at least in part, by increasing expression of genes involved in fatty acid oxidation. Conclusion: In subcutaneous adipose tissue, taurine decreased ethanol‐induced oxidative stress and cytokine expression, as well as normalized expression of adiponectin mRNA. Taurine prevented ethanol‐induced decreases in serum adiponectin; normalized adiponectin was associated with a reduction in hepatic oxidative stress, tumor necrosis factor α expression, and steatosis. Taken together, these data demonstrate that taurine has important protective effects against ethanol‐induced tissue injury in both adipose and liver tissue. (HEPATOLOGY 2009.)

Chronic Ethanol and Triglyceride Turnover in White Adipose Tissue in Rats INHIBITION OF THE ANTI-LIPOLYTIC ACTION OF INSULIN AFTER CHRONIC ETHANOL CONTRIBUTES TO INCREASED TRIGLYCERIDE DEGRADATION

Chronic ethanol consumption disrupts whole-body lipid metabolism. Here we tested the hypothesis that regulation of triglyceride homeostasis in adipose tissue is vulnerable to long-term ethanol exposure. After chronic ethanol feeding, total body fat content as well as the quantity of epididymal adipose tissue of male Wistar rats was decreased compared with pair-fed controls. Integrated rates of in vivo triglyceride turnover in epididymal adipose tissue were measured using 2H2O as a tracer. Triglyceride turnover in adipose tissue was increased due to a 2.3-fold increase in triglyceride degradation in ethanol-fed rats compared with pair-fed controls with no effect of ethanol on triglyceride synthesis. Because increased lipolysis accompanied by the release of free fatty acids into the circulation is associated with insulin resistance and liver injury, we focused on determining the mechanisms for increased lipolysis in adipose tissue after chronic ethanol feeding. Chronic ethanol feeding suppressed β-adrenergic receptor-stimulated lipolysis in both in vivo and ex vivo assays; thus, enhanced triglyceride degradation during ethanol feeding was not due to increased β-adrenergic-mediated lipolysis. Instead, chronic ethanol feeding markedly impaired insulin-mediated suppression of lipolysis in conscious rats during a hyperinsulinemic-euglycemic clamp as well as in adipocytes isolated from epididymal and subcutaneous adipose tissue. These data demonstrate for the first time that chronic ethanol feeding increased the rate of triglyceride degradation in adipose tissue. Furthermore, this enhanced rate of lipolysis was due to a suppression of the anti-lipolytic effects of insulin in adipocytes after chronic ethanol feeding.

Ethanol-induced oxidative stress via the CYP2E1 pathway disrupts adiponectin secretion from adipocytes.

BACKGROUND Adipose tissue is an important target for ethanol action. One important effect of ethanol is to reduce the secretion of adiponectin from adipocytes; this decrease is associated with lowered circulating adiponectin in rodent models of chronic ethanol feeding. Adiponectin is an insulin-sensitizing, anti-inflammatory adipokine; decreased adiponectin activity may contribute to tissue injury in response to chronic ethanol. Here, we investigated the role of cytochrome P450 2E1 (CYP2E1) and oxidative stress in the mechanism for impaired adiponectin secretion from adipocytes in response to ethanol. METHODS Male Wistar rats were fed a liquid diet containing ethanol as 36% of calories or pair-fed a control diet for 4 weeks. 3T3-L1 adipocyte cultures, expressing CYP2E1 or not, were exposed to ethanol or 4-hydroxynonenal (4-HNE). RESULTS Chronic ethanol feeding to rats suppressed the secretion of adiponectin from isolated epididymal adipocytes. Ethanol feeding induced the expression of CYP2E1 in adipocytes and increased markers of oxidative stress, including 4-HNE and protein carbonyls. Because adiponectin is posttranslationally processed in the endoplasmic reticulum and Golgi, we investigated the impact of ethanol on the redox status of high-density microsomes. Chronic ethanol decreased the ratio of reduced glutathione to oxidized glutathione (4.6:1, pair-fed; 2.9:1, ethanol-fed) in high-density microsomes isolated from rat epididymal adipose tissue. We next utilized the 3T3-L1 adipocyte-like cell model to interrogate the mechanisms for impaired adiponectin secretion. Culture of 3T3-L1 adipocytes overexpressing exogenous CYP2E1, but not those overexpressing antisense CYP2E1, with ethanol increased oxidative stress and impaired adiponectin secretion from intracellular pools. Consistent with a role of oxidative stress in impaired adiponectin secretion, challenge of 3T3-L1 adipocytes with 4-HNE also reduced adiponectin mRNA expression and secretion, without affecting intracellular adiponectin concentration. CONCLUSIONS These data demonstrate that CYP2E1-dependent reactive oxygen species production in response to ethanol disrupts adiponectin secretion from adipocytes.

Identification of a cytochrome P4502E1/Bid/C1q-dependent axis mediating inflammation in adipose tissue after chronic ethanol feeding to mice.

Background: Chronic alcohol consumption leads to inflammation in adipose tissue, disrupting normal metabolic activity of adipocytes. Results: Expression of an alcohol metabolizing enzyme, cytochrome P4502E1, initiates inflammation in adipose. Bid-dependent apoptosis and activation of complement then exacerbate this initial response. Conclusion: Adipose inflammation during alcohol feeding develops in response to cytochrome P450 expression. Significance: Preventing adipose inflammation may prevent the pathphysiological effects of ethanol. Chronic, heavy alcohol exposure results in inflammation in adipose tissue, insulin resistance, and liver injury. Here we have identified a CYP2E1/Bid/C1q-dependent pathway that is activated in response to chronic ethanol and is required for the development of inflammation in adipose tissue. Ethanol feeding for 25 days to wild-type (C57BL/6J) mice increased expression of multiple markers of adipose tissue inflammation relative to pair-fed controls independent of increased body weight or adipocyte size. Ethanol feeding increased the expression of CYP2E1 in adipocytes, but not stromal vascular cells, in adipose tissue and Cyp2e1−/− mice were protected from adipose tissue inflammation in response to ethanol. Ethanol feeding also increased the number of TUNEL-positive nuclei in adipose tissue of wild-type mice but not in Cyp2e1−/− or Bid −/− mice. Apoptosis contributed to adipose inflammation, as the expression of multiple inflammatory markers was decreased in mice lacking the Bid-dependent apoptotic pathway. The complement protein C1q binds to apoptotic cells, facilitating their clearance and activating complement. Making use of C1q-deficient mice, we found that activation of complement via C1q provided the critical link between CYP2E1/Bid-dependent apoptosis and onset of adipose tissue inflammation in response to chronic ethanol. In summary, chronic ethanol increases CYP2E1 activity in adipose, leading to Bid-mediated apoptosis and activation of complement via C1q, finally resulting in adipose tissue inflammation. Taken together, these data identify a novel mechanism for the development of adipose tissue inflammation that likely contributes to the pathophysiological effects of ethanol.

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Background: Complement is implicated in obesity and insulin resistance; however, the specific complement activation pathway involved is not known. Results: C1q in the classical pathway is required for activation of complement in response to high fat diets. Conclusion: C1q is an important contributor to high fat diet-induced insulin resistance. Significance: C1q may be an important therapeutic target for treating the derangements in metabolism associated with obesity. Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.

Methods to Investigate the Effects of Chronic Ethanol on Adipocytes

Chronic ethanol consumption dysregulates glucose and lipid homeostasis, is associated with insulin resistance, and alters serum levels of adipokines including adiponectin and tumor necrosis factor-alpha. However, the mechanisms involved in these chronic ethanol-induced pathologies are not fully understood. Adipose tissue has been implicated as an important contributor to chronic ethanol-induced disease states and, therefore, the effects of chronic ethanol feeding in rats on adipocytes has been investigated. Three major functions of the adipocyte include glucose transport, adipokine secretion, and triglyceride breakdown via lipolysis. Included in this chapter are protocols for studying the effect of chronic ethanol feeding on these adipocyte functions.