Gene transfer technology : a tool for studying gene function and role in corneal pathogenesis [abstract]

Transforming growth factor β (TGFb) is associated with many corneal pathologies, diseases and dystrophies. The function of TGFb in adult corneas cannot be studied using conventional transgenic approach because TGFb1 and TGFb2 deficient transgenic animals suffer multiple inflammatory diseases, severe developmental defects, and death by 3-4 weeks of age. This study tested the hypothesis that selective tissue-targeted gene transfer approaches will permit examination of TGFb gene function in the adult cornea without altering TGFb expression in vital organs

Co-administration of vancomycin and piperacillin-tazobactam is associated with increased renal dysfunction in adult and pediatric burn patients

Background: Burn patients are prone to infections which often necessitate broad antibiotic coverage. Vancomycin is a common antibiotic after burn injury and is administered alone (V), or in combination with imipenem-cilastin (V/IC) or piperacillin-tazobactam (V/PT). Sparse reports indicate that the combination V/PT is associated with increased renal dysfunction. The purpose of this study was to evaluate the short-term impact of the three antibiotic administration types on renal dysfunction. Methods: All pediatric and adult patients admitted to our centers between 2004 and 2016 with a burn injury were included in this retrospective review if they met the criteria of exposition to either V, V/IC, or V/PT for at least 48 h, had normal baseline creatinine, and no pre-existing renal dysfunction. Creatinine was monitored for 7 days after initial exposure; the absolute and relative increase was calculated, and patient renal outcomes were classified according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria depending on creatinine increases and estimated creatinine clearance. Secondary endpoints (demographic and clinical data, incidences of septicemia, and renal replacement therapy) were analyzed. Antibiotic doses were modeled in logistic and linear multivariable regression models to predict categorical KDIGO events and relative creatinine increase. Results: Out of 1449 patients who were screened, 718 met the inclusion criteria, 246 were adults, and 472 were children. Between the study cohorts V, V/IC, and V/PT, patient characteristics at admission were comparable. V/PT administration was associated with a statistically higher serum creatinine, and lower creatinine clearance compared to patients receiving V alone or V/IC in adults and children after burn injury. The incidence of KDIGO stages 1, 2, and 3 was higher after V/PT treatment. In children, the incidence of KDIGO stage 3 following administration of V/PT was greater than after V/IC. In adults, the incidence of renal replacement therapy was higher after V/PT compared with V or V/IC. Multivariate modeling demonstrated that V/PT is an independent predictor of renal dysfunction. Conclusion: Co-administration of vancomycin and piperacillin-tazobactam is associated with increased renal dysfunction in pediatric and adult burn patients when compared to vancomycin alone or vancomycin plus imipenem-cilastin. The mechanism of this increased nephrotoxicity remains elusive and warrants further scientific evaluation

Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform. Methods: To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5−/−). Results: Ablation of ACSL5 reduced ACSL activity by ∼80% in jejunal mucosa, ∼50% in liver, and ∼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5−/−, as compared to control ACSL5loxP/loxP, mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5−/− had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5−/− mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (∼16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (∼13-fold) were increased in ACSL5−/− as compared to ACSL5loxP/loxP. Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5−/− mice as compared to control mice. Conclusions: In summary, ACSL5−/− mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies suggest that ACSL5 is an important regulator of whole-body energy metabolism and ablation of ACSL5 may antagonize the development of obesity and insulin resistance. Keywords: Dietary fat absorption, Acyl-CoA, ACSL, Intestine, Liver, FGF2

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction

Objective: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. Methods: We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. Results: Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. Conclusion: These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE). Keywords: Adipocytes, Fatty acid metabolism, Obesity, Arachidonic acid, Polyunsaturated fatty aci

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction.

ObjectiveRegulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction.MethodsWe developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings.ResultsAd-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro.ConclusionThese studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE)