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Alzheimer's disease (AD) is the most common form of neurodegenerative disease worldwide. A large body of work implicates insulin resistance in the development and progression of AD. Moreover, impairment in mitochondrial function, a common symptom of insulin resistance, now represents a fundamental aspect of AD pathobiology. Ceramides are a class of bioactive sphingolipids that have been hypothesized to drive insulin resistance. Here, we describe preliminary work that tests the hypothesis that hyperinsulinemia pathologically alters cerebral mitochondrial function in AD mice via accrual of the ceramides. Homozygous male and female ApoE4 mice, an oft-used model of AD research, were given chronic injections of PBS (control), insulin, myriocin (an inhibitor of ceramide biosynthesis), or insulin and myriocin over four weeks. Cerebral ceramide content was assessed using liquid chromatography-mass spectrometry. Mitochondrial oxygen consumption rates were measured with high-resolution respirometry, and H 2 O 2 emissions were quantified via biochemical assays on brain tissue from the cerebral cortex. Significant increases in brain ceramides and impairments in brain oxygen consumption were observed in the insulin-treated group. These hyperinsulinemia-induced impairments in mitochondrial function were reversed with the administration of myriocin. Altogether, these data demonstrate a causative role for insulin in promoting brain ceramide accrual and subsequent mitochondrial impairments that may be involved in AD expression and progression.

Yerba maté, a herbal tea derived from Ilex paraguariensis , has previously been reported to be protective against obesity-related and other cardiometabolic disorders. Using high-resolution respirometry and reverse-phase high-performance liquid chromatography, the effects of four weeks of yerba maté consumption on mitochondrial efficiency and cellular redox status in skeletal muscle, adipose, and liver, tissues highly relevant to whole-body metabolism, were explored in healthy adult mice. Yerba maté treatment increased the mitochondrial oxygen consumption in adipose but not in the other examined tissues. Yerba maté increased the ATP concentration in skeletal muscle and decreased the ATP concentration in adipose. Combined with the observed changes in oxygen consumption, these data yielded a significantly higher ATP:O 2 , a measure of mitochondrial efficiency, in muscle and a significantly lower ATP:O 2 in adipose, which was consistent with yerba maté-induced weight loss. Yerba maté treatment also altered the hepatic glutathione (GSH)/glutathione disulfide (GSSG) redox potential to a more reduced redox state, suggesting the treatment's potential protective effects against oxidative stress and for the preservation of cellular function. Together, these data indicate the beneficial, tissue-specific effects of yerba maté supplementation on mitochondrial bioenergetics and redox states in healthy mice that are protective against obesity.

Adipocyte mitochondrial respiration may influence metabolic fuel partitioning into oxidation versus storage, with implications for whole-body energy expenditure. Although insulin has been shown to influence mitochondrial respiration, the effects of dietary macronutrient composition have not been well characterized. The aim of this exploratory study was to test the hypothesis that a high-carbohydrate diet lowers the oxygen flux of adipocyte mitochondria ex vivo. Among participants in a randomized-controlled weight-loss maintenance feeding trial, those consuming a high-carbohydrate diet (60% carbohydrate as a proportion of total energy, n = 10) had lower rates of maximal adipose tissue mitochondrial respiration than those consuming a moderate-carbohydrate diet (40%, n = 8, p = 0.039) or a low-carbohydrate diet (20%, n = 9, p = 0.005) after 10 to 15 weeks. This preliminary finding may provide a mechanism for postulated calorie-independent effects of dietary composition on energy expenditure and fat deposition, potentially through the actions of insulin on fuel partitioning., (© 2022. The Author(s).)

Objective: The rampant growth of obesity worldwide has stimulated explosive research into human metabolism. Energy expenditure has been shown to be altered by diets differing in macronutrient composition, with low-carbohydrate, ketogenic diets eliciting a significant increase over other interventions. The central aim of this study was to explore the effects of the ketone β-hydroxybutyrate (βHB) on mitochondrial bioenergetics in adipose tissue., Methods: We employed three distinct systems-namely, cell, rodent, and human models. Following exposure to elevated βHB, we obtained adipose tissue to quantify mitochondrial function., Results: In every model, βHB robustly increased mitochondrial respiration, including an increase of roughly 91% in cultured adipocytes, 113% in rodent subcutaneous adipose tissue (SAT), and 128% in human SAT. However, this occurred without a commensurate increase in adipose ATP production. Furthermore, in cultured adipocytes and rodent adipose, we quantified and observed an increase in the gene expression involved in mitochondrial biogenesis and uncoupling status following βHB exposure., Conclusions: In conclusion, βHB increases mitochondrial respiration, but not ATP production, in mammalian adipocytes, indicating altered mitochondrial coupling. These findings may partly explain the increased metabolic rate evident in states of elevated ketones, and may facilitate the development of novel anti-obesity interventions.

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice. KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling. KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diesel exhaust particles (DEPs) are known pathogenic pollutants that constitute a significant quantity of air pollution. Given the ubiquitous presence of macrophages throughout the body, including the lungs, as well as their critical role in tissue and organismal metabolic function, we sought to determine the effect of DEP exposure on macrophage mitochondrial function. Following daily DEP exposure in mice, pulmonary macrophages were isolated for mitochondrial analyses, revealing reduced respiration rates and dramatically elevated H 2 O 2 levels. Serum ceramides and inflammatory cytokines were increased. To determine the degree to which the changes in mitochondrial function in macrophages were not dependent on any cross-cell communication, primary pulmonary murine macrophages were used to replicate the DEP exposure in a cell culture model. We observed similar changes as seen in pulmonary macrophages, namely diminished mitochondrial respiration, but increased H 2 O 2 production. Interestingly, when treated with myriocin to inhibit ceramide biosynthesis, these DEP-induced mitochondrial changes were mitigated. Altogether, these data suggest that DEP exposure may compromise macrophage mitochondrial and whole-body function via pathologic alterations in macrophage ceramide metabolism.

Because low-carbohydrate diets are effective strategies to improve insulin resistance, the hallmark of type 2 diabetes, the purpose of reporting these clinical cases was to reveal the meaningful changes observed in 90 days of low-carbohydrate (LC) ketogenic dietary intervention in female type 2 diabetics aged 18-45. Eleven women (BMI 36.3 kg/m 2 ) who were recently diagnosed with type 2 diabetes based on HbA1c over 6.5% (8.9%) volunteered to participate in an intensive dietary intervention to limit dietary carbohydrates to under 30 grams daily for 90 days. The main outcome was to determine the degree of change in HbA1c, while secondary outcomes included body weight, blood pressure, and blood lipids. The volunteers lost significant weight (85.7 ± 3.2 kg to 76.7 ± 2.8 kg) and lowered systolic (134.0 ± 1.6 to 123.3 ± 1.1 mmHg) and diastolic (89.9 ± 1.3 to 82.6 ± 1.0 mmHg) blood pressure. HbA1c dropped to 5.6%. Most blood lipids were significantly altered, including HDL cholesterol (43.1 ± 4.4 to 52.3 ± 3.3 mg/dl), triglycerides (177.0 ± 19.8 to 92.1 ± 8.7 mg/dl), and the TG : HDL ratio (4.7 ± 0.8 to 1.9 ± 0.2). LDL cholesterol was not significantly different. AST and ALT, plasma markers of liver health, were reported for eight patients and revealed no significant changes. These findings indicate that a short-term intervention emphasizing protein and fat at the expense of dietary carbohydrate functionally reversed the diabetes diagnosis, as defined by HbA1c. Furthermore, the intervention lowered body weight and blood pressure, while eliciting favorable changes in blood lipids., Competing Interests: BTB, SLB, and RHH are coowners of Insulin IQ. BTB is a scientific advisor for Unicity International.

Aim: To review the evidence for behavioural interventions to reduce drooling in children with neurodisability., Method: A detailed search in eight databases sought studies that: (1) included participants aged 0 to 18 years with neurodisability and drooling; (2) provided behavioural interventions targeting drooling or a drooling-related behaviour; and (3) used experimental designs. Two reviewers extracted data from full-text papers independently. Results were tabulated for comparison. The Risk of Bias assessment in N-of-1 Trials scale for single case experimental designs (SCEDs) and the Cochrane risk of bias assessment tool for randomized controlled trials (RCTs) were applied., Results: Of an initial yield of 763, seven SCEDs and one RCT were included. Behavioural interventions included the use of reinforcement, prompting, self-management, instruction, extinction, overcorrection, and fading. Each assessed body functions or structures' outcomes (drooling frequency and severity); three included activity outcomes (mouth drying, head control, eye contact, and vocalizations) and none assessed participation or quality of life. While each study reported positive effects of intervention, risk of bias was high., Interpretation: Low-level evidence suggests behavioural interventions may be useful for treatment of drooling in children with neurodisability. Well-designed intervention studies are urgently needed to determine effectiveness., What This Paper Adds: Behavioural interventions used to treat drooling included reinforcement, prompting, self-management, extinction, overcorrection, instruction, and fading. Interventions targeted body structures and function-level outcomes and activity-level outcomes. Low-level evidence supports the use of behavioural intervention to treat drooling., (© 2018 Mac Keith Press.)

The clinical benefit of ketosis has historically and almost exclusively centered on neurological conditions, lending insight into how ketones alter mitochondrial function in neurons. However, there is a gap in our understanding of how ketones influence mitochondria within skeletal muscle cells. The purpose of this study was to elucidate the specific effects of β-hydroxybutyrate (β-HB) on muscle cell mitochondrial physiology. In addition to increased cell viability, murine myotubes displayed beneficial mitochondrial changes evident in reduced H₂O₂ emission and less mitochondrial fission, which may be a result of a β-HB-induced reduction in ceramides. Furthermore, muscle from rats in sustained ketosis similarly produced less H₂O₂ despite an increase in mitochondrial respiration and no apparent change in mitochondrial quantity. In sum, these results indicate a general improvement in muscle cell mitochondrial function when β-HB is provided as a fuel.

Background & Aims: Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection., Methods: Fetal rats were tolerized in utero with 10(5) Huh 7 cells. One day after birth, rats were transplanted with 5 x 10(6) Huh 7 cells and, a week later, inoculated with HCV, genotype 1., Results: In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions., Conclusions: HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxynucleotide (ODN) against the 5'-noncoding region (5'-NCR) of the hepatitis C virus. Purity of the conjugates was confirmed by sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a band corresponding to approximately 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5'-NCR and core region and showed HCV sequence-specific cleavage by the appearance of an expected approximately 1000 nt fragment of RNA. Cleavage was not seen by RNase H alone, or ODN alone. Delivery studies using (32)P- and (125)I-labeling showed that while RNAse H failed to enter cells, the conjugate was efficiently taken into the cells. To assess intracellular effects, a cell line, Huh-7/CMV-NCRCDeltaluc, which expresses HCV mRNA (nt 1-585) fused to a marker gene, was transfected with the conjugate. Reporter gene expression was suppressed by 51.2% with the conjugate compared to only 39.7% by ODN alone, 35.8% by a mixture of RNase H plus ODN, and not at all by RNase H alone. In conclusion, the RNase H-ODN conjugate effectively cleaved an HCV transcript in vitro and inhibited expression of an HCV-marker fusion construct in a liver-derived cell line.

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.

Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA-RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA-RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H-oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.

Aim: To determine whether a combination of cell targeting and sequence recognition of nucleic acids can provide specificity for the inhibition of viral gene expression., Methods: Antisense oligonucleotides complexed to a protein-based DNA carrier system were used to target hepatocytes for the inhibition of human hepatitis viral gene expression. The DNA carrier system contained an asialoglycoprotein as a cell-targeting component, which could direct the uptake of complexed DNA specifically to asialoglycoprotein receptors present selectively on the surface of mammalian hepatocytes., Results: HBV and HCV viral gene expression were substantially and specifically inhibited by use of antisense oligonucleotides complexed to a protein-based DNA carrier system., Conclusion: Targeted delivery of nucleic acids by use of receptor-mediated endocytosis can result in inhibition of viral gene expression without host toxicity.

Immune tolerance of human cells without generalized immunosuppression was created in groups of normal fetal rats at 17 days of gestation by inoculation ip with primary human hepatocytes in utero. One day after birth, suspensions of human hepatocytes were transplanted via intrasplenic injection and one week later groups of rats were inoculated with hepatitis B virus (HBV). Tolerized rats that were transplanted with human hepatocytes and subsequently infected with HBV produced hepatitis B surface antigen (HBsAg) in serum beginning on day 3. Levels rose fivefold and remained stable at 0.75 pg/ml through at least 60 days. Of cells that stained positive for human serum albumin, approximately 30% were found to be also positive for HBsAg by immunohistochemistry. Serum HBV DNA was detectable from 1 to 15 weeks postinfection. Finally, covalently closed circular DNA, reflecting HBV replication, was found in liver and serum. Controls that were tolerized and not transplanted, but inoculated with HBV, as well as untreated controls, had no evidence of HBV gene expression or replication under identical conditions. The data support the conclusion that primary human hepatocytes transplanted into genetically immunocompetent rodent hosts, survive and maintain sufficient differentiation to produce human serum albumin and be infected by HBV.

AIM:To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression.METHODS:A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L-lysine(PL), and LLO.Two marker genes, luciferase and beta galactosidase in plasmids were complexed and administered in vitro to Huh7(ASG receptor (+) and SK Hep1(ASG receptor (-) cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated.RESULTS:An LLO containing conjugate retained cell target-ing specificity and membranolytic activity. In ASG receptor (+) cells,luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of betagalactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies.CONCLUSIONS:ASOR-PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

A simple and convenient method for the purification of the hemolytic toxin listeriolysin O (LLO) from Listeria monocytogenes is described. Supernatants from bacteria cultures were purified by application to a CH2 spiral cartridge concentrator (Amicon) and ion exchange chromatography. A critical step is removal of contaminating RNA. The purified proteins had characteristics described for bacterial thiol-activated hemolysins: activation by a reducing agent (DTT) and inactivation by cholesterol. In addition, the molecular weight of 58, 000 and pH-dependent hemolytic activity of this purified protein are consistent with the previously published characteristics of LLO., (Copyright 1999 Academic Press.)

Type I collagen synthesis and deposition is generally indicative of irreversible damage in alcohol-induced cirrhosis in humans. However, in rodents, ethanol alone does not readily cause hepatic fibrosis. To determine whether this is because of a lack of ethanol-responsive elements, an artificial enhancer construct controlling rat type I collagen gene transcription was prepared in transgenic mice. The gene construct, ColCAT3.6, was a chimeric sequence containing the marker chloramphenicol acetyltransferase (CAT) gene linked to 3.5 kb of the rat alpha 1(I) 5'-flanking DNA, and 115 base pairs (bp) of transcribed collagen gene. Groups of transgenic mice were given 4 g/kg ethanol orally, twice daily for 4 weeks. As a positive control for hepatic fibrosis, transgenic mice were given intraperitoneal injections of CCl4, twice weekly for 4 weeks. Livers were assayed for CAT activity. Endogenous mouse collagen alpha 1(I) messenger RNA (mRNA) and transgene CAT mRNA were measured by RNase protection assays. Collagen synthesis in livers from the transgenic mice treated with ethanol were increased over controls, but the levels were not significantly different. Endogenous collagen alpha 1(I) steady-state mRNA levels in ethanol-treated mice were not significantly different compared with saline-treated controls. However, the transgene mRNA levels in ethanol-treated animals increased approximately 21-fold compared with saline-treated controls, as measured by RNase protection assays. Furthermore, the transgene product as measured by CAT activity in ethanol-treated mice was significantly increased threefold over saline-treated controls. We conclude that the 5'-flanking region of the rat alpha 1(I) collagen gene does contain regulatory elements that are strongly responsive to ethanol administration.

We have demonstrated previously that the carboxyl- and amino-terminal propeptides of type I procollagen can inhibit procollagen synthesis by specifically decreasing procollagen mRNA levels. The objective of the present experiments was to determine the mechanism by which propeptides cause these pretranslational effects. IMR-90 fibroblasts were exposed to medium containing carboxyl-terminal propeptide of type I procollagen, and nuclear run-off assays were performed by hybridization to a specific alpha 1 chain type I procollagen cDNA probe. Specific type I procollagen transcription rates were found to be decreased by 50% in the presence of 75 nM carboxyl-terminal propeptide compared with control (untreated) cells. Total cellular transcription rates as well as beta-actin mRNA rates were not affected significantly by any concentration of carboxyl-terminal propeptide. Propeptide radiolabeled with 125I was found to be taken up by cultured cells. Furthermore, exogenous carboxyl-terminal propeptide levels increased in the cytosolic compartment and eventually reached a steady-state level of 18 +/- 2 pmol/g cell protein by 30 min. Of particular interest was the finding that levels of radiolabeled carboxyl-terminal propeptide were also detected in the nuclear fraction and increased with time, reaching a plateau after 60 min of incubation. Incubation of nuclei from IMR-90 cells in medium containing varying concentrations of carboxyl-terminal propeptide resulted in nuclear transcription rates that were decreased by 40% compared with untreated controls. beta-Actin nuclear message levels remained unchanged under identical conditions. We conclude that carboxyl-terminal propeptide of type I procollagen can be internalized and become associated with the nuclear compartment. This suggests a feedback regulatory role on procollagen synthesis by a direct effect on procollagen gene transcription.