Development and Implementation of a Medical School Course Integrating Basic, Clinical, and Health Systems Sciences.

OBJECTIVE: In recent years, significant steps have been made in integrating basic science and clinical medicine. There remains a gap in adding the third pillar of education: health systems science (HSS). Core clerkships represent an ideal learning venue to integrate all three. Students can experience the value of integrating basic science as they learn clinical medicine in environments where HSS is occurring all around them. METHODS: We outline the creation of Sciences and Art of Medicine Integrated (SAMI), a course that runs parallel with the clerkship year and integrates basic science and HSS with clinical medicine. A complete description of the planning and implementation of SAMI is provided. We include the participants and educational setting, the goals and objectives, and the structure of each session. To encourage the integration of basic science, HSS, and clinical medicine, students utilize a series of tools, described in detail. Examples of each tool are provided utilizing a case of a patient presenting with obstructive sleep apnea. RESULTS: We successfully implemented this course with positive reception from students. CONCLUSION: This course represents a step not only toward the integration of HSS with basic science and clinical medicine but also an advancement in training future clinicians to provide high-value care. Future curricular development must consider the validation of a measure of clinical reasoning that assesses a student's ability to think in a cognitively integrated fashion about basic science, HSS, and clinical medicine demonstrated by enhanced justification of clinical reasoning and a more holistic approach to planning patient care.

Preparing Students to Deliver Care to Diverse Populations.

The purpose of this study was to increase student exposure to diverse patients using patient ID cards in problem-based learning (PBL) at Case Western Reserve University (CWRU). The pre-clerkship curriculum capitalizes on facilitated small-group, case-based discussions to promote inquiry and learning of the foundational sciences. Quantitative and qualitative results supported a finding of added value to case-discussions and the humanization of case-patients. The inclusion of the patient ID cards resulted in most students indicating that it helped them learn about and prepare to care for their future population of diverse patients. The patient ID cards will allow us to develop specific learning objectives about the demographics to increase learning about diverse patient care. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-022-01648-0.

Teaching the Nutrition Focused Physical Exam (NFPE) To Medical Students Using an Interdisciplinary Approach.

Nutritional status greatly affects the health of patients, yet the time devoted to nutrition curriculum in medical school is minimal. We implemented a novel approach of teaching the Nutrition Focused Physical Exam (NFPE) as a tool to demonstrate the importance of assessing the nutritional status of patients and learning about malnutrition and nutrient deficiencies.

Changing Medical Education, Overnight: The Curricular Response to COVID-19 of Nine Medical Schools.

Issue: Calls to change medical education have been frequent, persistent, and generally limited to alterations in content or structural re-organization. Self-imposed barriers have prevented adoption of more radical pedagogical approaches, so recent predictions of the 'inevitability' of medical education transitioning to online delivery seemed unlikely. Then in March 2020 the COVID-19 pandemic forced medical schools to overcome established barriers overnight and make the most rapid curricular shift in medical education's history. We share the collated reports of nine medical schools and postulate how recent responses may influence future medical education. Evidence: While extraneous pandemic-related factors make it impossible to scientifically distinguish the impact of the curricular changes, some themes emerged. The rapid transition to online delivery was made possible by all schools having learning management systems and key electronic resources already blended into their curricula; we were closer to online delivery than anticipated. Student engagement with online delivery varied with different pedagogies used and the importance of social learning and interaction along with autonomy in learning were apparent. These are factors known to enhance online learning, and the student-centered modalities (e.g. problem-based learning) that included them appeared to be more engaging. Assumptions that the new online environment would be easily adopted and embraced by 'technophilic' students did not always hold true. Achieving true distance medical education will take longer than this 'overnight' response, but adhering to best practices for online education may open a new realm of possibilities. Implications: While this experience did not confirm that online medical education is really 'inevitable,' it revealed that it is possible. Thoughtfully blending more online components into a medical curriculum will allow us to take advantage of this environment's strengths such as efficiency and the ability to support asynchronous and autonomous learning that engage and foster intrinsic learning in our students. While maintaining aspects of social interaction, online learning could enhance pre-clinical medical education by allowing integration and collaboration among classes of medical students, other health professionals, and even between medical schools. What remains to be seen is whether COVID-19 provided the experience, vision and courage for medical education to change, or whether the old barriers will rise again when the pandemic is over.

The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn's Disease-Like Ileitis.

Background: Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn's disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice. Methods: Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation. Results: Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene-carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice. Conclusions: Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal reactivity only in individuals with a pro-inflammatory predisposition, such as CD.

Macrophages with a deletion of the phosphoenolpyruvate carboxykinase 1 (Pck1) gene have a more proinflammatory phenotype.

Phosphoenolpyruvate carboxykinase (Pck1) is a metabolic enzyme that is integral to the gluconeogenic and glyceroneogenic pathways. However, Pck1's role in macrophage metabolism and function is unknown. Using stable isotopomer MS analysis in a mouse model with a myeloid cell-specific Pck1 deletion, we show here that this deletion increases the proinflammatory phenotype in macrophages. Incubation of LPS-stimulated bone marrow-derived macrophages (BMDM) with [U-13C]glucose revealed reduced 13C labeling of citrate and malate and increased 13C labeling of lactate in Pck1-deleted bone marrow-derived macrophages. We also found that the Pck1 deletion in the myeloid cells increases reactive oxygen species (ROS). Of note, this altered macrophage metabolism increased expression of the M1 cytokines TNFα, IL-1ß, and IL-6. We therefore conclude that Pck1 contributes to M1 polarization in macrophages. Our findings provide important insights into the factors determining the macrophage inflammatory response and indicate that Pck1 activity contributes to metabolic reprogramming and polarization in macrophages.

Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism.

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery. The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.

Zinc finger protein 407 overexpression upregulates PPAR target gene expression and improves glucose homeostasis in mice.

The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors is central to the pathophysiology and treatment of metabolic disease through the receptors' ability to regulate the expression of genes involved in glucose homeostasis, adipogenesis, and lipid metabolism. However, the mechanism by which PPAR is regulated remains incompletely understood. We generated a transgenic mouse strain (ZFP-TG) that overexpressed Zfp407 primarily in muscle and heart. Transcriptome analysis by RNA-Seq identified 1,300 differentially expressed genes in the muscle of ZFP-TG mice, among which PPAR target genes were significantly enriched. Among the physiologically important PPARγ target genes, Glucose transporter (Glut)-4 mRNA and protein levels were increased in heart and muscle. The increase in Glut4 and other transcriptional effects of Zfp407 overexpression together decreased body weight and lowered plasma glucose, insulin, and HOMA-IR scores relative to control littermates. When placed on high-fat diet, ZFP-TG mice remained more glucose tolerant than their wild-type counterparts. Cell-based assays demonstrated that Zfp407 synergistically increased the transcriptional activity of all PPAR subtypes, PPARα, PPARγ, and PPARδ. The increased PPAR activity was not associated with increased PPAR mRNA or protein levels, suggesting that Zfp407 posttranslationally regulates PPAR activity. Collectively, these results demonstrate that Zfp407 overexpression improved glucose homeostasis. Thus, Zfp407 represents a new drug target for treating metabolic disease.

Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.

Multiple pathways of programmed cell death are important in liver homeostasis. Hepatocyte death is associated with progression of nonalcoholic fatty liver disease, and inhibition of apoptosis partially protects against liver injury in response to a high-fat diet (HFD). However, the contribution of necroptosis, a caspase-independent pathway of cell death, to HFD-induced liver injury is not known. Wild-type C57BL/6 and receptor interacting protein (RIP) 3-/- mice were randomized to chow or HFD. HFD-fed C57BL/6 mice increased expression of RIP3, the master regulator of necroptosis, as well as phosphorylated mixed lineage kinase domain-like, an effector of necroptotic cell death, in liver. HFD did not increase phosphorylated mixed lineage kinase domain-like in RIP3-/- mice. HFD increased fasting insulin and glucose, as well as glucose intolerance, in C57BL/6 mice. RIP3-/- mice were glucose-intolerant even on the chow diet; HFD further increased fasting glucose and insulin but not glucose intolerance. HFD also increased hepatic steatosis, plasma alanine aminotransferase activity, inflammation, oxidative stress, and hepatocellular apoptosis in wild-type mice; these responses were exacerbated in RIP3-/- mice. Importantly, increased inflammation and injury were associated with early indicators of fibrosis in RIP3-/- compared to C57BL/6 mice. Culture of AML12 hepatocytes with palmitic acid increased cytotoxicity through apoptosis and necrosis. Inhibition of RIP1 with necrostatin-1 or small interfering RNA knockdown of RIP3 reduced palmitic acid-induced cytotoxicity. CONCLUSION: Absence of RIP3, a key mediator of necroptosis, exacerbated HFD-induced liver injury, associated with increased inflammation and hepatocyte apoptosis, as well as early fibrotic responses; these findings indicate that shifts in the mode of hepatocellular death can influence disease progression and have therapeutic implications because manipulation of hepatocyte cell death pathways is being considered as a target for treatment of nonalcoholic fatty liver disease. (Hepatology 2016;64:1518-1533).

Initial evaluation of hepatic T1 relaxation time as an imaging marker of liver disease associated with autosomal recessive polycystic kidney disease (ARPKD).

Autosomal recessive polycystic kidney disease (ARPKD) is a potentially lethal multi-organ disease affecting both the kidneys and the liver. Unfortunately, there are currently no non-invasive methods to monitor liver disease progression in ARPKD patients, limiting the study of potential therapeutic interventions. Herein, we perform an initial investigation of T1 relaxation time as a potential imaging biomarker to quantitatively assess the two primary pathologic hallmarks of ARPKD liver disease: biliary dilatation and periportal fibrosis in the PCK rat model of ARPKD. T1 relaxation time results were obtained for five PCK rats at 3 months of age using a Look-Locker acquisition on a Bruker BioSpec 7.0 T MRI scanner. Six three-month-old Sprague-Dawley (SD) rats were also scanned as controls. All animals were euthanized after the three-month scans for histological and biochemical assessments of bile duct dilatation and hepatic fibrosis for comparison. PCK rats exhibited significantly increased liver T1 values (mean ± standard deviation = 935 ± 39 ms) compared with age-matched SD control rats (847 ± 26 ms, p = 0.01). One PCK rat exhibited severe cholangitis (mean T1 = 1413 ms), which occurs periodically in ARPKD patients. The observed increase in the in vivo liver T1 relaxation time correlated significantly with three histological and biochemical indicators of biliary dilatation and fibrosis: bile duct area percent (R = 0.85, p = 0.002), periportal fibrosis area percent (R = 0.82, p = 0.004), and hydroxyproline content (R = 0.76, p = 0.01). These results suggest that hepatic T1 relaxation time may provide a sensitive and non-invasive imaging biomarker to monitor ARPKD liver disease.

The Signal Transducer and Activator of Transcription 1 (STAT1) Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes.

The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. ß-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.

Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy.

TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1ß secretion. The 17C-6 mice have increased IL-1ß in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis.

Dysregulated intrahepatic CD4+ T-cell activation drives liver inflammation in ileitis-prone SAMP1/YitFc mice.

BACKGROUND AND AIMS: Liver inflammation is a common extraintestinal manifestation of inflammatory bowel disease (IBD); however, whether liver involvement is a consequence of a primary intestinal defect or results from alternative pathogenic processes remains unclear. Therefore, we sought to determine the potential pathogenic mechanism(s) of concomitant liver inflammation in an established murine model of IBD. METHODS: Liver inflammation and immune cell subsets were characterized in ileitis-prone SAMP1/YitFc (SAMP) and AKR/J (AKR) control mice, lymphocyte-depleted SAMP (SAMPxRag-1-/-), and immunodeficient SCID recipient mice receiving SAMP or AKR donor CD4+ T-cells. Proliferation and suppressive capacity of CD4+ T-effector (Teff) and T-regulatory (Treg) cells from gut-associated lymphoid tissue (GALT) and livers of SAMP and AKR mice were measured. RESULTS: Surprisingly, prominent inflammation was detected in 4-wk-old SAMP livers, prior to histologic evidence of ileitis, while both disease phenotypes were absent in age-matched AKRs. SAMP liver disease was characterized by abundant infiltration of lymphocytes, required for hepatic inflammation to occur, a Th1-skewed environment, and phenotypically-activated CD4+ T-cells. SAMP intrahepatic CD4+ T-cells also had the ability to induce liver and ileal inflammation when adoptively transferred into SCID recipients, whereas GALT-derived CD4+ T-cells produced milder ileitis, but not liver inflammation. Interestingly, SAMP intrahepatic CD4+ Teff cells showed increased proliferation compared to both SAMP GALT- and AKR liver-derived CD4+ Teff cells, while SAMP intrahepatic Tregs were decreased among CD4+ T-cells and impaired in in vitro suppressive function compared to AKR. CONCLUSIONS: Activated intrahepatic CD4+ T-cells induce liver inflammation and contribute to experimental ileitis via locally-impaired hepatic immunosuppressive function.

Erlotinib protects against LPS-induced endotoxicity because TLR4 needs EGFR to signal.

Several components of the canonical pathway of response to lipopolysaccharide (LPS) are required for the EGF-dependent activation of NFκB. Conversely, the ability of Toll-like Receptor 4 (TLR4) to activate NFκB in response to LPS is impaired by down regulating EGF receptor (EGFR) expression or by using the EGFR inhibitor erlotinib. The LYN proto-oncogene (LYN) is required for signaling in both directions. LYN binds to the EGFR upon LPS stimulation, and erlotinib impairs this association. In mice, erlotinib blocks the LPS-induced expression of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and ameliorates LPS-induced endotoxity, revealing that EGFR is essential for LPS-induced signaling in vivo.

PTPRT regulates high-fat diet-induced obesity and insulin resistance.

Obesity is a risk factor for many human diseases. However, the underlying molecular causes of obesity are not well understood. Here, we report that protein tyrosine phosphatase receptor T (PTPRT) knockout mice are resistant to high-fat diet-induced obesity. Those mice avoid many deleterious side effects of high-fat diet-induced obesity, displaying improved peripheral insulin sensitivity, lower blood glucose and insulin levels. Compared to wild type littermates, PTPRT knockout mice show reduced food intake. Consistently, STAT3 phosphorylation is up-regulated in the hypothalamus of PTPRT knockout mice. These studies implicate PTPRT-modulated STAT3 signaling in the regulation of high-fat diet-induced obesity.

The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6.

Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.

The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.

Genetic resistance to liver fibrosis on A/J mouse chromosome 17.

BACKGROUND: Because the histological and biochemical progression of liver disease is similar in alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH), we hypothesized that the genetic susceptibility to these liver diseases would be similar. To identify potential candidate genes that regulate the development of liver fibrosis, we studied a chromosome substitution strain (CSS-17) that contains chromosome 17 from the A/J inbred strain substituted for the corresponding chromosome on the C57BL/6J (B6) genetic background. Previously, we identified quantitative trait loci (QTLs) in CSS-17, namely obesity-resistant QTL 13 and QTL 15 (Obrq13 and Obrq15, respectively), that were associated with protection from diet-induced obesity and hepatic steatosis on a high-fat diet. METHODS: To test whether these or other CSS-17 QTLs conferred resistance to alcohol-induced liver injury and fibrosis, B6, A/J, CSS-17, and congenics 17C-1 and 17C-6 were either fed Lieber-DeCarli ethanol (EtOH)-containing diet or had carbon tetrachloride (CCl4 ) administered chronically. RESULTS: The congenic strain carrying Obrq15 showed resistance from alcohol-induced liver injury and liver fibrosis, whereas Obrq13 conferred susceptibility to liver fibrosis. From published deep sequencing data for chromosome 17 in the B6 and A/J strains, we identified candidate genes in Obrq13 and Obrq15 that contained single-nucleotide polymorphisms (SNPs) in the promoter region or within the gene itself. NADPH oxidase organizer 1 (Noxo1) and NLR family, CARD domain containing 4 (Nlrc4) showed altered hepatic gene expression in strains with the A/J allele at the end of the EtOH diet study and after CCl4 treatment. CONCLUSIONS: Aspects of the genetics for the progression of ASH are unique compared to NASH, suggesting that the molecular mechanisms for the progression of disease are at least partially distinct. Using these CSSs, we identified 2 candidate genes, Noxo1 and Nlrc4, which modulate genetic susceptibility in ASH.

A self-defeating anabolic program leads to ß-cell apoptosis in endoplasmic reticulum stress-induced diabetes via regulation of amino acid flux.

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing ß-cells in type 2 diabetes mellitus. ß-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in ß-cells in vivo. Paradoxically, chronic ER stress in ß-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in ß-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Alcohol-induced liver injury is modulated by Nlrp3 and Nlrc4 inflammasomes in mice.

Alcoholic liver disease (ALD) is characterized by increased hepatic lipid accumulation (steatosis) and inflammation with increased expression of proinflammatory cytokines. Two of these cytokines, interleukin-1 ß (IL-1 ß ) and IL-18, require activation of caspase-1 via members of the NOD-like receptor (NLR) family. These NLRs form an inflammasome that is activated by pathogens and signals released through local tissue injury or death. NLR family pyrin domain containing 3 (Nlrp3) and NLR family CARD domain containing protein 4 (Nlrc4) have been studied minimally for their role in the development of ALD. Using mice with gene targeted deletions for Nlrp3 (Nlrp3(-/-)) and Nlrc4 (Nlrc4(-/-)), we analyzed the response to chronic alcohol consumption. We found that Nlrp3(-/-) mice have more severe liver injury with higher plasma alanine aminotransferase (ALT) levels, increased activation of IL-18, and reduced activation of IL-1B. In contrast, the Nlrc4(-/-) mice had similar alcohol-induced liver injury compared to C57BL/6J (B6) mice but had greatly reduced activation of IL-1 ß . This suggests that Nlrp3 and Nlrc4 inflammasomes activate IL-1 ß and IL-18 via caspase-1 in a differential manner. We conclude that the Nlrp3 inflammasome is protective during alcohol-induced liver injury.