Hyperglycemia and advanced glycosylation end products suppress adipocyte apoE expression: implications for adipocyte triglyceride metabolism.

Endogenous adipocyte apolipoprotein E (apoE) plays an important role in adipocyte lipoprotein metabolism and lipid flux. A potential role for hyperglycemia in regulating adipocyte apoE expression and triglyceride metabolism was examined. Exposure of adipocytes to high glucose or advanced glycosylation end product-BSA significantly suppressed apoE mRNA and protein levels. This suppression was significantly attenuated by antioxidants or inhibitors of the NF-κB transcription pathway. Hyperglycemia in vivo led to adipose tissue oxidant stress and significant reduction in adipose tissue and adipocyte apoE mRNA level. Incubation with antioxidant in organ culture completely reversed this suppression. Hyperglycemia also reduced adipocyte triglyceride synthesis, and this could be completely reversed by adenoviral-mediated increases in apoE. To more specifically evaluate an in vivo role for adipocyte apoE expression on organismal triglyceride distribution in vivo, WT or apoE knockout (EKO) adipose tissue was transplanted in EKO recipient mice. After 12 wk, WT adipocytes transplanted in EKO mice accumulated more triglyceride compared with transplanted EKO adipocytes. In addition, EKO recipients of WT adipose tissue had reduced hepatic triglyceride content compared with EKO recipients transplanted with EKO adipose tissue. Our results demonstrate that hyperglycemia and advanced glycosylation end products suppress the expression of adipocyte apoE in vitro and in vivo and thereby reduce adipocyte triglyceride synthesis. In vivo results using adipose tissue transplantation suggest that reduction of adipocyte apoE, and subsequent reduction of adipocyte triglyceride accumulation, could influence lipid accumulation in nonadipose tissue.

Oxidative stress regulates adipocyte apolipoprotein e and suppresses its expression in obesity.

OBJECTIVE: Endogenous expression of apolipoprotein E (apoE) has a significant impact on adipocyte lipid metabolism and is markedly suppressed in obesity. Adipose tissue oxidant stress is emerging as an important mediator of adipocyte dysfunction. These studies were undertaken to evaluate the role of oxidant stress for regulation of adipocyte apoE. RESEARCH DESIGN AND METHODS: ApoE gene and protein expression in 3T3-L1 adipocytes or mature adipocytes and adipose tissue from C57/BL6 mice was evaluated after induction of oxidant stress. The response of adipose tissue and adipocytes from obese compared with lean mice to antioxidants was also assessed. RESULTS: Oxidant stress in 3T3-L1 cells or adipocytes and adipose tissue from lean mice significantly reduced apoE mRNA and protein level. Inclusion of an antioxidant eliminated this reduction. Oxidant stress was accompanied by activation of the nuclear factor-kappaB (NF-kappaB) transcription complex, and its effect on apoE was eliminated by an NF-kappaB activation inhibitor. Treatment of freshly isolated adipose tissue or mature adipocytes from obese mice with antioxidant increased apoE expression but had no effect on cells or tissue from lean mice. Incubation of freshly isolated adipocytes from lean mice with stromovascular cells from obese mice significantly suppressed adipocyte apoE compared with incubation with stromovascular cells from lean mice, but this suppression was reversed by inclusion of antioxidant or a neutralizing antibody to tumor necrosis factor-alpha. CONCLUSIONS: Oxidant stress significantly modulates adipose tissue and adipocyte apoE expression. Furthermore, oxidant stress contributes to suppression of adipocyte apoE in obesity. This suppression depends on interaction between adipose tissue stromovascular cells and adipocytes.

The sodium bicarbonate cotransporter: structure, function, and regulation.

The role of the Na(+)-coupled HCO(3)(-) transporter (NBC) family is indispensable in acid-base homeostasis. Almost all tissues express a member of the NBC family. NBC has been studied extensively in the kidney and plays a role in proximal tubule HCO(3)(-) reabsorption. Although the exact function of this transporter family on other tissues is not very clear, the ubiquitous expression of NBC family suggests a role in cell pH regulation. Altered NBC activity caused by mutations of the gene responsible for NBC protein expression results in pathophysiologic conditions. Mutations of NBC resulting in important clinical disorders have been reported extensively on one member of the NBC family, the kidney NBC (NBC1). These mutations have led to several structural studies to understand the mechanism of the abnormal NBC1 activity.

Role of NH(2) and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1.

Sodium bicarbonate cotransporter 1 (NBC1) mediates 80% of bicarbonate reabsorption by the kidney, but the molecular determinants for activity, targeting, and cell membrane stability are poorly understood. We generated truncation mutants involving the entire NH(2) (DeltaN424) or the entire COOH (DeltaC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. DeltaN424 and DeltaC92 targeted to the plasma membrane of HEK293 cells or to the basolateral membrane of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the DeltaN424, DeltaC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total DeltaC92 expression in the cell was constant. We found that decreased DeltaC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both DeltaN424 and DeltaC92 were cotransfected together and more stable expression of DeltaC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH(2) epitope-specific antibody or vice versa. Similar findings were observed with Ni-NTA pull-down assay. Cotransfection of both mutants partially restored NBC1 activity. In summary, NBC1 targets to the basolateral membrane of OK cells by a default mechanism and the COOH terminus plays a role on NBC1 stability in the basolateral membrane.

A central role for Pyk2-Src interaction in coupling diverse stimuli to increased epithelial NBC activity.

Regulation of renal Na-HCO cotransporter (NBC1) activity by cholinergic agonists, ANG II, and acute acidosis (CO(2)) requires both Src family kinase (SFK) and classic MAPK pathway activation. The nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) couples discrete G protein-coupled receptor and growth factor receptor signaling to SFK activation. We examined the role of Pyk2-SFK interaction in coupling these stimuli to increased NBC1 activity in opossum kidney cells. Carbachol increased tyrosine autophosphorylation of endogenous Pyk2 and ectopically expressed wild-type Pyk2 and were abrogated by kinase-dead mutant (Pyk2-KD) overexpression. Pyk2 phosphorylation was calcium/calmodulin dependent, and Pyk2 associated with Src by means of SH2 domain interaction. Pyk2 phosphorylation and Pyk2-Src interaction by carbachol were mimicked by both ANG II and CO(2). To correlate Pyk2 autophosphorylation and Pyk2-Src interaction with NBC activity, cotransporter activity was measured in untransfected cells and in cells overexpressing Pyk2-KD in the presence or absence of carbachol, ANG II, or CO(2). In Pyk2-KD-overexpressing cells, the effect of carbachol, ANG II, and CO(2) was abolished. We conclude that Pyk2 plays a central role in coupling carbachol, ANG II, and CO(2) to increased NBC activity. This coupling is mediated by Pyk2 autophosphorylation and Pyk2-Src interaction.