Divya P. Kumar

A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.

Background & Aims The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development. Methods A stable isogenic cross between C57BL/6J (B6) and 129S1/SvImJ (S129) mice were fed a high fat diet with ad libitum consumption of glucose and fructose in physiologically relevant concentrations and compared to mice fed a chow diet and also to both parent strains. Results Following initiation of the obesogenic diet, B6/129 mice developed obesity, insulin resistance, hypertriglyceridemia and increased LDL-cholesterol. They sequentially also developed steatosis (4–8 weeks), steatohepatitis (16–24 weeks), progressive fibrosis (16 weeks onwards) and spontaneous hepatocellular cancer (HCC). There was a strong concordance between the pattern of pathway activation at a transcriptomic level between humans and mice with similar histological phenotypes (FDR 0.02 for early and 0.08 for late time points). Lipogenic, inflammatory and apoptotic signaling pathways activated in human NASH were also activated in these mice. The HCC gene signature resembled the S1 and S2 human subclasses of HCC (FDR 0.01 for both). Only the B6/129 mouse but not the parent strains recapitulated all of these aspects of human NAFLD. Conclusions We here describe a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH. Lay summary We have developed a diet-induced mouse model of non-alcoholic steatohepatitis (NASH) and hepatic cancers in a cross between two mouse strains (129S1/SvImJ and C57Bl/6J). This model mimics all the physiological, metabolic, histological, transcriptomic gene signature and clinical endpoints of human NASH and can facilitate preclinical development of therapeutic targets for NASH.

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity.

Background: Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the folding activity seen in Hsp70s. Results: In contrast to Hsp70s, Hsp110s exhibit distinct peptide substrate binding properties. Conclusion: The peptide substrate binding properties determine the chaperone activity differences between Hsp70s and Hsp110s. Significance: Our studies shed light on the molecular mechanism of the chaperone activities of Hsp70s and Hsp110s. The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.

The four hydrophobic residues on the Hsp70 inter-domain linker have two distinct roles.

The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaKs in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.

Release of GLP-1 and PYY in response to the activation of G protein-coupled bile acid receptor TGR5 is mediated by Epac/PLC-ε pathway and modulated by endogenous H2S

Activation of plasma membrane TGR5 receptors in enteroendocrine cells by bile acids is known to regulate gastrointestinal secretion and motility and glucose homeostasis. The endocrine functions of the gut are modulated by microenvironment of the distal gut predominantly by sulfur-reducing bacteria of the microbiota that produce H2S. However, the mechanisms involved in the release of peptide hormones, GLP-1 and PYY in response to TGR5 activation by bile acids and the effect of H2S on bile acid-induced release of GLP-1 and PYY are unclear. In the present study, we have identified the signaling pathways activated by the bile acid receptor TGR5 to mediate GLP-1 and PYY release and the mechanism of inhibition of their release by H2S in enteroendocrine cells. The TGR5 ligand oleanolic acid (OA) stimulated Gαs and cAMP formation, and caused GLP-1 and PYY release. OA-induced cAMP formation and peptide release were blocked by TGR5 siRNA. OA also caused an increase in PI hydrolysis and intracellular Ca2+. Increase in PI hydrolysis was abolished in cells transfected with PLC-ε siRNA. 8-pCPT-2′-O-Me-cAMP, a selective activator of Epac, stimulated PI hydrolysis, and GLP-1 and PYY release. L-Cysteine, which activates endogenous H2S producing enzymes cystathionine-γ-lyase and cystathionine-β-synthase, and NaHS and GYY4137, which generate H2S, inhibited PI hydrolysis and GLP-1 and PYY release in response to OA or 8-pCPT-2′-O-Me-cAMP. Propargylglycine, an inhibitor of CSE, reversed the effect of L-cysteine on PI hydrolysis and GLP-1 and PYY release. We conclude: (i) activation of Gαs-coupled TGR5 receptors causes stimulation of PI hydrolysis, and release of GLP-1 and PYY via a PKA-independent, cAMP-dependent mechanism involving Epac/PLC-ε/Ca2+ pathway, and (ii) H2S has potent inhibitory effects on GLP-1 and PYY release in response to TGR5 activation, and the mechanism involves inhibition of PLC-ε/Ca2+ pathway.

The presence and severity of nonalcoholic steatohepatitis is associated with specific changes in circulating bile acids

The histologic spectrum of nonalcoholic fatty liver disease (NAFLD) includes fatty liver (NAFL) and steatohepatitis (NASH), which can progress to cirrhosis in up to 20% of NASH patients. Bile acids (BA) are linked to the pathogenesis and therapy of NASH. We (1) characterized the plasma BA profile in biopsy‐proven NAFL and NASH and compared to controls and (2) related the plasma BA profile to liver histologic features, disease activity, and fibrosis. Liquid chromatography/mass spectrometry quantified BAs. Descriptive statistics, paired and multiple group comparisons, and regression analyses were performed. Of 86 patients (24 controls, 25 NAFL, and 37 NASH; mean age 51.8 years and body mass index 31.9 kg/m2), 66% were women. Increased total primary BAs and decreased secondary BAs (both P < 0.05) characterized NASH. Total conjugated primary BAs were significantly higher in NASH versus NAFL (P = 0.047) and versus controls (P < 0.0001). NASH had higher conjugated to unconjugated chenodeoxycholate (P = 0.04), cholate (P = 0.0004), and total primary BAs (P < 0.0001). The total cholate to chenodeoxycholate ratio was significantly higher in NAFLD without (P = 0.005) and with (P = 0.02) diabetes. Increased key BAs were associated with higher grades of steatosis (taurocholate), lobular (glycocholate) and portal inflammation (taurolithocholate), and hepatocyte ballooning (taurocholate). Conjugated cholate and taurocholate directly and secondary to primary BA ratio inversely correlated to NAFLD activity score. A higher ratio of total secondary to primary BA decreased (odds ratio, 0.57; P = 0.004) and higher conjugated cholate increased the likelihood of significant fibrosis (F≥2) (P = 0.007). Conclusion: NAFLD is associated with significantly altered circulating BA composition, likely unaffected by type 2 diabetes, and correlated with histological features of NASH; these observations provide the foundation for future hypothesis‐driven studies of specific effects of BAs on specific aspects of NASH. (Hepatology 2018;67:534‐548).

Activation of Transmembrane Bile Acid Receptor TGR5 Modulates Pancreatic Islet α Cells to Promote Glucose Homeostasis

The physiological role of the TGR5 receptor in the pancreas is not fully understood. We previously showed that activation of TGR5 in pancreatic β cells by bile acids induces insulin secretion. Glucagon released from pancreatic α cells and glucagon-like peptide 1 (GLP-1) released from intestinal L cells regulate insulin secretion. Both glucagon and GLP-1 are derived from alternate splicing of a common precursor, proglucagon by PC2 and PC1, respectively. We investigated whether TGR5 activation in pancreatic α cells enhances hyperglycemia-induced PC1 expression thereby releasing GLP-1, which in turn increases β cell mass and function in a paracrine manner. TGR5 activation augmented a hyperglycemia-induced switch from glucagon to GLP-1 synthesis in human and mouse islet α cells by GS/cAMP/PKA/cAMP-response element-binding protein-dependent activation of PC1. Furthermore, TGR5-induced GLP-1 release from α cells was via an Epac-mediated PKA-independent mechanism. Administration of the TGR5 agonist, INT-777, to db/db mice attenuated the increase in body weight and improved glucose tolerance and insulin sensitivity. INT-777 augmented PC1 expression in α cells and stimulated GLP-1 release from islets of db/db mice compared with control. INT-777 also increased pancreatic β cell proliferation and insulin synthesis. The effect of TGR5-mediated GLP-1 from α cells on insulin release from islets could be blocked by GLP-1 receptor antagonist. These results suggest that TGR5 activation mediates cross-talk between α and β cells by switching from glucagon to GLP-1 to restore β cell mass and function under hyperglycemic conditions. Thus, INT-777-mediated TGR5 activation could be leveraged as a novel way to treat type 2 diabetes mellitus.

Preclinical models of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) can manifest as non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). NASH is often associated with progressive fibrosis which can lead to cirrhosis and hepatocellular carcinoma (HCC). NASH is increasing as an aetiology for end-stage liver disease as well as HCC. There are currently no approved therapies for NASH. A major barrier to development of therapeutics for NASH is the lack of preclinical models of disease that are appropriately validated to represent the biology and outcomes of human disease. Many in vitro and animal models have been developed. In vitro models do not fully capture the hepatic and extrahepatic milieu of human NASH and large animal models are expensive and logistically difficult to use. Therefore, there is considerable interest in the development and validation of mouse models for NAFLD, including NASH. Several models based on varying genetic or dietary manipulations have been developed. However, the majority do not recreate steatohepatitis, strictly defined as the presence of hepatocellular ballooning with or without Mallory-Denk bodies, accompanied by inflammation in the presence of macrovesicular steatosis. Others lack validation against human disease. Herein, we describe the best practices in development of mouse models of NASH. We further review existing models and the literature supporting their use as a surrogate for human disease. Finally, data on models to evaluate protective genes are discussed. It is hoped that this review will provide guidance for the interpretation of data derived from mouse models and also for the development and validation of newer models.

Bile acids: emerging role in management of liver diseases.

Bile acids are well known for their effects on cholesterol homeostasis and lipid digestion. Since the discovery of bile acid receptors, of which there are farnesoid X receptor (FXR), a nuclear receptor, and the plasma membrane G-protein receptor, as well as Takeda G-protein coupled receptor clone 5, further roles have been elucidated for bile acids including glucose and lipid metabolism as well as inflammation. Additionally, treatment with bile acid receptor agonists has shown a decrease in the amount of atherosclerosis plaque formation and decreased portal vascular resistance and portal hypotension in animal models. Furthermore, rodent models have demonstrated antifibrotic activity using bile acid receptor agonists. Early human data using a FXR agonist, obeticholic acid, have shown promising results with improvement of histological activity and even a reduction of fibrosis. Human studies are ongoing and will provide further information on bile acid receptor agonist therapies. Thus, bile acids and their derivatives have the potential for management of liver diseases and potentially other disease states including diabetes and the metabolic syndrome.

Hypercontractility of Intestinal Longitudinal Smooth Muscle Induced by Cytokines Is Mediated by the Nuclear Factor-κB/AMP-Activated Kinase/Myosin Light Chain Kinase Pathway

Recent studies have identified AMP-activated kinase (AMPK) as a target of Ca2+/calmodulin-dependent kinase kinase (CaMKKβ) and a negative regulator of myosin light-chain (MLC) kinase (MLCK). The present study examined whether a change in expression or activity of AMPK is responsible for hypercontractility of intestinal longitudinal muscle during inflammation or in response to proinflammatory cytokines. In mouse colonic longitudinal muscle cells, acetylcholine (ACh) stimulated AMPK and MLCK phosphorylation and activity and induced MLC20 phosphorylation and muscle contraction. Blockade of CaMKKβ with STO609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) inhibited AMPK and MLCK phosphorylation and augmented MLCK activity, MLC20 phosphorylation, and smooth muscle cell contraction. In muscle cells isolated from the colon of TNBS (2,4,6-trinitrobenzenesulfonic acid)-treated mice or from strips treated with interleukin-1β or tumor necrosis factor-α, nuclear factor κB was activated as indicated by an increase in p65 phosphorylation and IκBα degradation, and AMPK was phosphorylated at a cAMP-dependent protein kinase (PKA)–specific site (Ser485) that is distinct from the stimulatory CaMKKβ site (Thr172), resulting in attenuation of ACh-stimulated AMPK activity and augmentation of MLCK activity and muscle cell contraction. Inhibition of nuclear factor-κB activity with MG-132 (carbobenzoxy-l-leucyl-l-leucyl-l-leucinal Z-LLL-CHO) or PKA activity with myristoylated PKA inhibitor 14-22 amide blocked phosphorylation of AMPK at Ser485 and restored MLCK activity and muscle cell contraction to control levels. The results imply that PKA released from IκBα complex phosphorylated AMPK at a PKA-specific site and inhibited its activity, thereby relieving the inhibitory effect of AMPK on MLCK and increasing MLCK activity and muscle cell contraction. We conclude that hypercontractility of intestinal longitudinal muscle induced by inflammation or proinflammatory cytokines is mediated by nuclear factor κB/PKA-dependent inhibition of AMPK and activation of MLCK.

Cytokine-Induced S-Nitrosylation of Soluble Guanylyl Cyclase and Expression of Phosphodiesterase 1A Contribute to Dysfunction of Longitudinal Smooth Muscle Relaxation

The effect of proinflammatory cytokines on the expression and activity of soluble guanylyl cyclase (sGC) and cGMP–phosphodiesterases (PDEs) was determined in intestinal longitudinal smooth muscle. In control muscle cells, cGMP levels are regulated via activation of sGC and PDE5; the activity of the latter is regulated via feedback phosphorylation by cGMP-dependent protein kinase. In muscle cells isolated from muscle strips cultured with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) or obtained from the colon of TNBS (2,4,6-trinitrobenzene sulfonic acid)-treated mice, expression of inducible nitric oxide synthase (iNOS) was induced and sGC was S-nitrosylated, resulting in attenuation of nitric oxide (NO)–induced sGC activity and cGMP formation. The effect of cytokines on sGC S-nitrosylation and activity was blocked by the iNOS inhibitor 1400W [N-([3-(aminomethyl)phenyl]methyl)ethanimidamide dihydrochloride]. The effect of cytokines on cGMP levels measured in the absence of IBMX (3-isobutyl-1-methylxanthine), however, was partly reversed by 1400W or PDE1 inhibitor vinpocetine and completely reversed by a combination of 1400W and vinpocetine. Expression of PDE1A was induced and was accompanied by an increase in PDE1A activity in muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice; the effect of cytokines on PDE1 expression and activity was blocked by MG132 (benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), an inhibitor of nuclear factor κB activity. NO-induced muscle relaxation was inhibited in longitudinal muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice, and this inhibition was completely reversed by the combination of both 1400W and vinpocetine. Inhibition of smooth muscle relaxation during inflammation reflects the combined effects of decreased sGC activity via S-nitrosylation and increased cGMP hydrolysis via PDE1 expression.