Ceramides Mediate Insulin-Induced Impairments in Cerebral Mitochondrial Bioenergetics in ApoE4 Mice.

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 H2O2 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.

CFTR genotype analysis of Asians in international registries highlights disparities in the diagnosis and treatment of Asian patients with cystic fibrosis.

PURPOSE: Cystic fibrosis (CF) is not well-characterized in Asians, potentially resulting in delayed diagnosis and poor prognosis. We characterized CF in Asian subgroups to address these disparities. METHODS: De-identified ethnicity and CFTR variant data were obtained from the United States, United Kingdom, and Canadian CF registries. We measured the prevalence of CF, CFTR variant allele frequencies, effectiveness of screening panels, and eligibility for modulator therapies. RESULTS: The prevalence of CF was 1 in 74,982 people (Canada) to 1 in 13,340 people (United Kingdom) for South Asians and 1 in 256,541 (Canada) to 1 in 52,563 (United Kingdom) for other Asians, suggesting 26,000 to 146,000 patients with CF in South Asia. p.(F508del) variant was markedly less frequent in Asians than in non-Hispanic Whites. Splicing and nonsense variants occurred at high allelic frequencies in Asians, resulting in 41% to 49% of South Asians and 21% to 39% of other Asians being ineligible for CFTR modulator therapies. Hologic/EU2v1 panels failed to identify 37% to 47% of South Asian and 23% to 46% of other Asian patients with CF. CONCLUSIONS: Among Asians, CF appears to be more common in South Asians. A significant CF population may exist in South Asia. CFTR variants in South and other Asians markedly differ from non-Hispanic Whites causing inequities in newborn screening, diagnosis, and treatment. New strategies are necessary to mitigate these health care disparities.

High-Mobility Group Box 1 Disrupts Metabolic Function with Cigarette Smoke Exposure in a Ceramide-Dependent Manner.

We have previously found that cigarette smoke disrupts metabolic function, in part, by increasing muscle ceramide accrual. To further our understanding of this, we sought to determine the role of the cytokine high-mobility group box 1 (HMGB1), which is increased with smoke exposure, in smoke-induced muscle metabolic perturbations. To test this theory, we determined HMGB1 from lungs of human smokers, as well as from lung cells from mice exposed to cigarette smoke. We also treated cells and mice directly with HMGB1, in the presence or absence of myriocin, an inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in ceramide biosynthesis. Outcomes included assessments of insulin resistance and muscle mitochondrial function. HMGB1 was significantly increased in both human lungs and rodent alveolar macrophages. Further testing revealed that HMGB1 treatment elicited a widespread increase in ceramide species and reduction in myotube mitochondrial respiration, an increase in reactive oxygen species, and reduced insulin-stimulated Akt phosphorylation. Inhibition of ceramide biosynthesis with myriocin was protective. In mice, by comparing treatments of HMGB1 injections with or without myriocin, we found that HMGB1 injections resulted in increased muscle ceramides, especially C16 and C24, which were necessary for reduced muscle mitochondrial respiration and compromised insulin and glucose tolerance. In conclusion, HMGB1 may be a necessary intermediate in the ceramide-dependent metabolic consequences of cigarette smoke exposure.

DRA involvement in linaclotide stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Critical role of down-regulated in adenoma bicarbonate transporter in linaclotide stimulated intestinal bicarbonate secretion.

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also alter duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum. Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression or function. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA) inhibition, regardless of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Linaclotide increased apical membrane expression of DRA in non-CF and CF differentiated enteroids. These data provide insights into the action of linaclotide and suggest linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Lipopolysaccharide Disrupts Mitochondrial Physiology in Skeletal Muscle via Disparate Effects on Sphingolipid Metabolism.

Lipopolysaccharides (LPS) are prevalent pathogenic molecules that are found within tissues and blood. Elevated circulating LPS is a feature of obesity and sepsis, both of which are associated with mitochondrial abnormalities that are key pathological features of LPS excess. However, the mechanism of LPS-induced mitochondrial alterations remains poorly understood. Herein we demonstrate the necessity of sphingolipid accrual in mediating altered mitochondrial physiology in skeletal muscle following LPS exposure. In particular, we found LPS elicited disparate effects on the sphingolipids dihydroceramides (DhCer) and ceramides (Cer) in both cultured myotubes and in muscle of LPS-injected mice. Although LPS-treated myotubes had reduced DhCer and increased Cer as well as increased mitochondrial respiration, muscle from LPS-injected mice manifested a reverse trend, namely elevated DhCer, but reduced Cer as well as reduced mitochondrial respiration. In addition, we found that LPS treatment caused mitochondrial fission, likely via dynamin-related protein 1, and increased oxidative stress. However, inhibition of de novo sphingolipid biosynthesis via myriocin protected normal mitochondrial function in spite of LPS, but inhibition of DhCer desaturase 1, which increases DhCer, but not Cer, exacerbated mitochondrial respiration with LPS. In an attempt to reconcile the incongruent effects of LPS in isolated muscle cells and whole muscle tissue, we incubated myotubes with conditioned medium from treated macrophages. In contrast to direct myotube LPS treatment, conditioned medium from LPS-treated macrophages reduced myotube respiration, but this was again mitigated with sphingolipid inhibition. Thus, macrophage sphingolipid production appears to be necessary for LPS-induced mitochondrial alterations in skeletal muscle tissue.