Screening for Novel Inhibitors of Amyloid Beta Aggregation and Toxicity as Potential Drugs for Alzheimer's Disease.

AD is the most common neurodegenerative disorder characterized by progressive memory impairment and cognitive deficits. The pathology of AD is still unclear; however, several studies have shown that the aggregation of the Aß peptide in the CNS is an exclusively pathological process involved in AD. Currently, there is no proven medication to cure or prevent the disease progression. Nevertheless, various therapeutic approaches for AD show only relief of symptoms and mostly work on cognitive recovery. However, one of the promising approaches for therapeutic intervention is to use inhibitors for blocking the Aß peptide aggregation process. Recently, herbal phenolic compounds have been shown to have a therapeutic property for treatment of AD due to their multifaceted action. In this study, we investigated the effectiveness of SA, Gn Rb1, and DMyr on inhibiting the aggregation and toxicity of Aß40 and Aß42 using different biochemical and cell-based assays. Our results showed that SA and DMyr inhibit Aß40 and Aß42 fibrillation, seeded aggregation, and toxicity. Gn Rb1 did not have any effect on the aggregation or toxicity induced by Aß40 and Aß42. Moreover, SA and DMyr were able to disaggregate the preformed fibrils. Overall, these compounds may be used alone or synergistically and could be considered as a lead for designing new compounds that could be used as effective treatment of AD and related disorders.

Structural and Biophysical Characterization of Stable Alpha-Synuclein Oligomers.

The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson's disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain ß-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA.

Inhibition of α-Synuclein Seeding-Dependent Aggregation by ssDNA Aptamers Specific to C-Terminally Truncated α-Synuclein Fibrils.

Neuropathologically, Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of insoluble aggregates of α-synuclein (α-syn) in the Lewy bodies (LBs). In addition to full-length α-syn fibrils, C-terminally truncated α-syn is also abundant in the LBs that acts as seeds and facilitates the aggregation of the full-length α-syn in vitro and in vivo and induces toxicity. Hence, identifying molecules that can inhibit the seeding activity of these truncated forms is of great importance. Here, we report the first in vitro selection of aptamers targeting the fibrillar forms of different C-terminally truncated α-syn using systematic evolution by an exponential enrichment method followed by quantitative high-throughput DNA sequencing. We identify a panel of aptamers that bound with high specificity to different truncated forms of α-syn fibrils with no cross-reactivity toward other amyloid fibrils. Interestingly, two of the aptamers (named Apt11 and Apt15) show higher affinity to most C-terminally truncated forms of α-syn fibrils with an evident inhibition of α-syn-seeded aggregation in vitro by Apt11. This inhibition is further confirmed by circular dichroism, Congo red binding assay, and electronic microscopy. Moreover, Apt11 is also found to reduce the insoluble phosphorylated form of α-syn at Ser-129 (pS129-α-syn) in the cell model and also can inhibit α-syn aggregation using RT-QuIC reactions seeded with brain homogenates extracted from patients affected by PD. The aptamers discovered in this study represent potential useful tools for research and diagnostics or therapy toward PD and DLB.

Disease-Associated α-Synuclein Aggregates as Biomarkers of Parkinson Disease Clinical Stage.

BACKGROUND AND OBJECTIVES: Robust biomarkers that can mirror Parkinson disease (PD) are of great significance. In this study, we present a novel approach to investigate disease-associated α-synuclein (αSyn) aggregates as biomarkers of PD clinical stage. METHODS: We combined both seed amplification assay (SAA) and ELISA to provide a quantitative test readout that reflects the clinical severity of patients with PD. To attain this goal, we initially explored the potential of our test using 2 sets of human brain homogenates (pilot and validation sets) and then verified it with 2 independent human CSF cohorts; discovery (62 patients with PD and 34 controls) and validation (49 patients with PD and 48 controls) cohorts. RESULTS: We showed that oligomers-specific ELISA robustly quantified SAA end product from patients with PD or dementia with Lewy bodies with high sensitivity and specificity scores (100%). Analysis also demonstrated that seeding activity could be detected earlier with oligomeric ELISA as the test readout rather than SAA alone. Of more importance, multiplexing the assays provided robust information about the patients' clinical disease stage. In the discovery cohort, levels of CSF-seeded αSyn oligomers correlated with the severity of the clinical symptoms of PD as measured by the Unified Parkinson Disease Rating Scale (UPDRS) motor (r = 0.58, p < 0.001) and Hoehn and Yahr (H&Y) scores (r = 0.43, p < 0.01). Similar correlations were observed in the validation cohort between the concentrations of CSF-seeded αSyn oligomers and both UPDRS motor (r = 0.50, p < 0.01) and H&Y scores (r = 0.49, p < 0.01). At 20 hours, receiver operating characteristic curves analysis yielded a sensitivity of 91.9% (95% CI 82.4%-96.5%) and a specificity of 85.3% (95% CI 69.8%-93.5%), with an area under the curve of 0.969 for CSF-seeded αSyn oligomers differentiating those with PD from controls in the discovery CSF cohort, whereas, a sensitivity of 80.7% (95% CI 69.1%-88.5%), a specificity of 76.5% (95% CI 60.0%-87.5%), and area under the curve of 0.860 were generated with thioflavin T maximum intensity of fluorescence at the same time point. DISCUSSION: We showed that combining SAA and ELISA assays is a more promising diagnostic tool than SAA alone, providing information about the disease stage by correlating with clinical measures of disease severity. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that CSF-seeded αSyn oligomers can accurately discriminate patients with PD and normal controls and CSF-seeded αSyn oligomers levels correlate with PD severity.

Immune-related biomarkers for Parkinson's disease.

Despite the increasing number of studies on Parkinson's disease and it being the second most common neurodegenerative disorder in the world, no established diagnostic markers or disease modifying therapies are available. Understanding the mechanisms involved in its pathogenesis and identifying markers capable of diagnosing or tracking progression of PD is greatly needed. Among the several factors identified to be involved in Parkinson's disease, the immune system has had increasingly growing evidence that presents a fresh avenue to investigate the pathology of the disease. The involvement of the immune system in the pathology of Parkinson's disease has been linked to an interaction between the peripheral and central nervous system immune response. Whether this involvement is due to an immune response being a cause or consequence of Parkinson's disease pathology is still a matter of debate. Players investigated include cytokines, chemokines, and immune-cells found in both the central and peripheral immune system. Herein, we discuss advances in the current literature on these immune-related markers and their potential use as markers for Parkinson's disease diagnosis and progression.

Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System.

The nuclease activity of the CRISPR-Cas9 system relies on the delivery of a CRISPR-associated protein 9 (Cas9) and a single guide RNA (sgRNA) against the target gene. CRISPR components are typically delivered to cells as either a Cas9/sgRNA ribonucleoprotein (RNP) complex or a plasmid encoding a Cas9 protein along with a sequence-specific sgRNA. Multiple transfection reagents are known to deliver CRISPR-Cas9 components, and delivery vectors are being developed for different purposes by several groups. Here, we repurposed a dual-fluorescence (RFP-GFP-GFP) reporter system to quantify the uptake level of the functional CRISPR-Cas9 components into cells and compare the efficiency of CRISPR delivery vectors. Using this system, we developed a novel and rapid cell-based microplate reader assay that makes possible real-time, rapid, and high throughput quantification of CRISPR nuclease activity. Cells stably expressing this dual-fluorescent reporter construct facilitated a direct quantification of the level of the internalized and functional CRISPR-Cas9 molecules into the cells without the need of co-transfecting fluorescently labeled reporter molecules. Additionally, targeting a reporter gene integrated into the genome recapitulates endogenous gene targeting. Thus, this reporter could be used to optimize various transfection conditions of CRISPR components, to evaluate and compare the efficiency of transfection agents, and to enrich cells containing desired CRISPR-induced mutations.

α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity.

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129­α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129­α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129­α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129­α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129­α-syn (WT­α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129­α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129­α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129­α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129­α-syn as a measure of efficacy in clinical trials.

Prion-like α-synuclein pathology in the brain of infants with Krabbe disease.

Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene that causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (n = 4) compared to infant controls (n = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease.

Novel engineered nanobodies specific for N-terminal region of alpha-synuclein recognize Lewy-body pathology and inhibit in-vitro seeded aggregation and toxicity.

Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (α-syn). Phage display screening of the library allowed the identification of a nanobody, Nbα-syn01, specific for α-syn. Unlike previously developed nanobodies, Nbα-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα-syn01 and the engineered bivalent format BivNbα-syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα-syn01 and its bivalent format recognized preferentially α-syn fibrils compared to the monomeric form. Nbα-syn01 and BivNbα-syn01 were also able to inhibit α-syn-seeded aggregation in vitro and reduced α-syn-seeded aggregation and toxicity in cells showing their potential to reduce α-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα-syn01 binds the N-terminal region of the α-syn aggregated form. Overall, these results highlight the potential of Nbα-syn01 and BivNbα-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders.

Nicotinamide Mononucleotide Prevents Free Fatty Acid-Induced Reduction in Glucose Tolerance by Decreasing Insulin Clearance.

The NAD-dependent deacetylase SIRT1 improves ß cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents ß cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on ß cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced ß cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased ß cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.

Natural Alkaloid Compounds as Inhibitors for Alpha-Synuclein Seeded Fibril Formation and Toxicity.

The accumulation and aggregation of α-synuclein (α-syn) is the main pathologic event in Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-Syn-seeded fibril formation and its induced toxicity occupy a major role in PD pathogenesis. Thus, assessing compounds that inhibit this seeding process is considered a key towards the therapeutics of synucleinopathies. Using biophysical and biochemical techniques and seeding-dependent cell viability assays, we screened a total of nine natural compounds of alkaloid origin extracted from Chinese medicinal herbs. Of these compounds, synephrine, trigonelline, cytisine, harmine, koumine, peimisine, and hupehenine exhibited in vitro inhibition of α-syn-seeded fibril formation. Furthermore, using cell viability assays, six of these compounds inhibited α-syn-seeding-dependent toxicity. These six potent inhibitors of amyloid fibril formation and toxicity caused by the seeding process represent a promising therapeutic strategy for the treatment of PD and other synucleinopathies.

Fibrillar form of α-synuclein-specific scFv antibody inhibits α-synuclein seeds induced aggregation and toxicity.

Synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.

Investigating the presence of doubly phosphorylated α-synuclein at tyrosine 125 and serine 129 in idiopathic Lewy body diseases.

Aggregation of the protein α-synuclein (α-syn) into insoluble intracellular assemblies termed Lewy bodies (LBs) is thought to be a critical pathogenic event in LB diseases such as Parkinson's disease and dementia with LBs. In LB diseases, the majority of α-syn is phosphorylated at serine 129 (pS129), suggesting that this is an important disease-related post-translational modification (PTM). However, PTMs do not typically occur in isolation and phosphorylation at the proximal tyrosine 125 (pY125) residue has received considerable attention and has been inconsistently reported to be present in LBs. Furthermore, the proximity of Y125 to S129 means that some pS129 antibodies may have epitopes that include Y125, in which case phosphorylation of Y125 will impede recognition of α-syn. This would potentially lead to underestimating LB pathology burdens if pY125 occurs alongside pS129. To address the apparent controversy in the literature regarding the detection of pY125, we investigated its presence in the LB pathology. We generated pS129 antibodies whose epitope includes or does not include Y125 and compared the extent of α-syn pathology recognized in mouse models of α-synucleinopathies, human brain tissue lysates and fixed post-mortem brain tissues. Our study demonstrated no difference in α-syn pathology recognized between pS129 antibodies, irrespective of whether Y125 was part of the epitope or not. Furthermore, evaluation with pY125 antibodies whose epitope does not include S129 demonstrated no labeling of LB pathology. This study reconciles disparate results in the literature and demonstrates pY125 is not a key component of LB pathology in murine models or human tissues in idiopathic LB diseases.

Inhibition of alpha-synuclein seeded fibril formation and toxicity by herbal medicinal extracts.

BACKGROUND: Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. METHODS: Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process. RESULTS: Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking ß-sheet content after subjecting them to proteinase K digestion. CONCLUSIONS: Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.

Hyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis.

BACKGROUND: CEACAM1 regulates insulin sensitivity by promoting insulin clearance. Accordingly, global C57BL/6J.Cc1-/- null mice display hyperinsulinemia due to impaired insulin clearance at 2 months of age, followed by insulin resistance, steatohepatitis, visceral obesity and leptin resistance at 6 months. The study aimed at investigating the primary role of hepatic CEACAM1 in insulin and lipid homeostasis independently of its metabolic effect in extra-hepatic tissues. METHODS: Liver-specific C57BL/6J.AlbCre+Cc1fl/fl mice were generated and their metabolic phenotype was characterized by comparison to that of their littermate controls at 2-9 months of age, using hyperinsulinemic-euglycemic clamp analysis and indirect calorimetry. The effect of hyperphagia on insulin resistance was assessed by pair-feeding experiments. RESULTS: Liver-specific AlbCre+Cc1fl/fl mutants exhibited impaired insulin clearance and hyperinsulinemia at 2 months, followed by hepatic insulin resistance (assessed by hyperinsulinemic-euglycemic clamp analysis) and steatohepatitis at ~ 7 months of age, at which point visceral obesity and hyperphagia developed, in parallel to hyperleptinemia and blunted hypothalamic STAT3 phosphorylation in response to an intraperitoneal injection of leptin. Hyperinsulinemia caused hypothalamic insulin resistance, followed by increased fatty acid synthase activity, which together with defective hypothalamic leptin signaling contributed to hyperphagia and reduced physical activity. Pair-feeding experiment showed that hyperphagia caused systemic insulin resistance, including blunted insulin signaling in white adipose tissue and lipolysis, at 8-9 months of age. CONCLUSION: AlbCre+Cc1fl/fl mutants provide an in vivo demonstration of the key role of impaired hepatic insulin clearance and hyperinsulinemia in the pathogenesis of secondary hepatic insulin resistance independently of lipolysis. They also reveal an important role for the liver-hypothalamic axis in the regulation of energy balance and subsequently, systemic insulin sensitivity.

Exenatide induces carcinoembryonic antigen-related cell adhesion molecule 1 expression to prevent hepatic steatosis.

Exenatide, a glucagon-like peptide-1 receptor agonist, induces insulin secretion. Its role in insulin clearance has not been adequately examined. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance to maintain insulin sensitivity. Feeding C57BL/6J mice a high-fat diet down-regulates hepatic Ceacam1 transcription to cause hyperinsulinemia, insulin resistance, and hepatic steatosis, as in Ceacam1 null mice (Cc1-/- ). Thus, we tested whether exenatide regulates Ceacam1 expression in high-fat diet-fed mice and whether this contributes to its insulin sensitizing effect. Exenatide (100 nM) induced the transcriptional activity of wild-type Ceacam1 promoter but not the constructs harboring block mutations of peroxisome proliferator-activated receptor response element and retinoid X receptor alpha, individually or collectively, in HepG2 human hepatoma cells. Chromatin immunoprecipitation analysis demonstrated binding of peroxisome proliferator-activated receptor gamma to Ceacam1 promoter in response to rosiglitazone and exenatide. Consistently, exenatide induced Ceacam1 messenger RNA expression within 12 hours in the absence but not in the presence of the glucagon-like peptide-1 receptor antagonist exendin 9-39. Exenatide (20 ng/g body weight once daily intraperitoneal injection in the last 30 days of feeding) restored hepatic Ceacam1 expression and insulin clearance to curb diet-induced metabolic abnormalities and steatohepatitis in wild-type but not Cc1-/- mice fed a high-fat diet for 2 months. Conclusion: Exenatide promotes insulin clearance in parallel with insulin secretion to prevent chronic hyperinsulinemia and the resulting hepatic steatosis, and this contributes to its insulin sensitizing effect. Our data further highlight the relevance of physiologic insulin metabolism in maintaining insulin sensitivity and normal lipid metabolism. (Hepatology Communications 2018;2:35-47).

Liver-specific reconstitution of CEACAM1 reverses the metabolic abnormalities caused by its global deletion in male mice.

AIMS/HYPOTHESIS: The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Mice with global null mutation (Cc1 -/-) or with liver-specific inactivation (L-SACC1) of Cc1 (also known as Ceacam1) gene display hyperinsulinaemia resulting from impaired insulin clearance, insulin resistance, steatohepatitis and obesity. Because increased lipolysis contributes to the metabolic phenotype caused by transgenic inactivation of CEACAM1 in the liver, we aimed to further investigate the primary role of hepatic CEACAM1-dependent insulin clearance in insulin and lipid homeostasis. To this end, we examined whether transgenic reconstitution of CEACAM1 in the liver of global Cc1 -/- mutant mice reverses their abnormal metabolic phenotype. METHODS: Insulin response was assessed by hyperinsulinaemic-euglycaemic clamp analysis and energy balance was analysed by indirect calorimetry. Mice were overnight-fasted and refed for 7 h to assess fatty acid synthase activity in the liver and the hypothalamus in response to insulin release during refeeding. RESULTS: Liver-based rescuing of CEACAM1 restored insulin clearance, plasma insulin level, insulin sensitivity and steatohepatitis caused by global deletion of Cc1. It also reversed the gain in body weight and total fat mass observed with Cc1 deletion, in parallel to normalising energy balance. Mechanistically, reversal of hyperphagia appeared to result from reducing fatty acid synthase activity and restoring insulin signalling in the hypothalamus. CONCLUSIONS/INTERPRETATION: Despite the potential confounding effects of deleting Cc1 from extrahepatic tissues, liver-based rescuing of CEACAM1 resulted in full normalisation of the metabolic phenotype, underscoring the key role that CEACAM1-dependent hepatic insulin clearance pathways play in regulating systemic insulin sensitivity, lipid homeostasis and energy balance.

Age-dependent insulin resistance in male mice with null deletion of the carcinoembryonic antigen-related cell adhesion molecule 2 gene.

AIMS/HYPOTHESIS: Cc2 -/- mice lacking the gene encoding the carcinoembryonic-antigen-related cell adhesion molecule 2 (Cc2 [also known as Ceacam2]) exhibit hyperphagia that leads to obesity and insulin resistance. This starts at 2 months of age in female mice. Male mutants maintain normal body weight and insulin sensitivity until the last age previously examined (7-8 months), owing to increased sympathetic tone to white adipose tissue and energy expenditure. The current study investigates whether insulin resistance develops in mutant male mice at a later age and whether this is accompanied by changes in insulin homeostasis. METHODS: Insulin response was assessed by insulin and glucose tolerance tests. Energy balance was analysed by indirect calorimetry. RESULTS: Male Cc2 -/- mice developed overt metabolic abnormalities at about 9 months of age. These include elevated global fat mass, hyperinsulinaemia and insulin resistance (as determined by glucose and insulin intolerance, fed hyperglycaemia and decreased insulin signalling pathways). Pair-feeding experiments showed that insulin resistance resulted from hyperphagia. Indirect calorimetry demonstrated that older mutant male mice had compromised energy expenditure. Despite increased insulin secretion caused by Cc2 deletion, chronic hyperinsulinaemia did not develop in mutant male mice until about 9 months of age, at which point insulin clearance began to decline substantially. This was probably mediated by a marked decrease in hepatic CEACAM1 expression. CONCLUSIONS/INTERPRETATION: The data demonstrate that at about 9 months of age, Cc2 -/- male mice develop a reduction in energy expenditure and energy imbalance which, combined with a progressive decrease in CEACAM1-dependent hepatic insulin clearance, causes chronic hyperinsulinaemia and sustained age-dependent insulin resistance. This represents a novel mechanistic underpinning of age-related impairment of hepatic insulin clearance.

Reduced Hepatic Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Level in Obesity.

Impairment of insulin clearance is being increasingly recognized as a critical step in the development of insulin resistance and metabolic disease. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Null deletion or liver-specific inactivation of Ceacam1 in mice causes a defect in insulin clearance, insulin resistance, steatohepatitis, and visceral obesity. Immunohistological analysis revealed reduction of hepatic CEACAM1 in obese subjects with fatty liver disease. Thus, we aimed to determine whether this occurs at the hepatocyte level in response to systemic extrahepatic factors and whether this holds across species. Northern and Western blot analyses demonstrate that CEACAM1 mRNA and protein levels are reduced in liver tissues of obese individuals compared to their lean age-matched counterparts. Furthermore, Western analysis reveals a comparable reduction of CEACAM1 protein in primary hepatocytes derived from the same obese subjects. Similar to humans, Ceacam1 mRNA level, assessed by quantitative RT-PCR analysis, is significantly reduced in the livers of obese Zucker (fa/fa, ZDF) and Koletsky (f/f) rats relative to their age-matched lean counterparts. These studies demonstrate that the reduction of hepatic CEACAM1 in obesity occurs at the level of hepatocytes and identify the reduction of hepatic CEACAM1 as a common denominator of obesity across multiple species.

Loss of Hepatic CEACAM1: A Unifying Mechanism Linking Insulin Resistance to Obesity and Non-Alcoholic Fatty Liver Disease.

The pathogenesis of human non-alcoholic fatty liver disease (NAFLD) remains unclear, in particular in the context of its relationship to insulin resistance and visceral obesity. Work on the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in mice has resolved some of the related questions. CEACAM1 promotes insulin clearance by enhancing the rate of uptake of the insulin-receptor complex. It also mediates a negative acute effect of insulin on fatty acid synthase activity. This positions CEACAM1 to coordinate the regulation of insulin and lipid metabolism. Fed a regular chow diet, global null mutation of Ceacam1 manifest hyperinsulinemia, insulin resistance, obesity, and steatohepatitis. They also develop spontaneous chicken-wire fibrosis, characteristic of non-alcoholic steatohepatitis. Reduction of hepatic CEACAM1 expression plays a significant role in the pathogenesis of diet-induced metabolic abnormalities, as bolstered by the protective effect of hepatic CEACAM1 gain-of-function against the metabolic response to dietary fat. Together, this emphasizes that loss of hepatic CEACAM1 links NAFLD to insulin resistance and obesity.