Translational characterization of the temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut during diet-induced NASH development in Ldlr-/-.Leiden mice.

Background: NAFLD progression, from steatosis to inflammation and fibrosis, results from an interplay of intra- and extrahepatic mechanisms. Disease drivers likely include signals from white adipose tissue (WAT) and gut. However, the temporal dynamics of disease development remain poorly understood. Methods: High-fat-diet (HFD)-fed Ldlr-/-.Leiden mice were compared to chow-fed controls. At t = 0, 8, 16, 28 and 38w mice were euthanized, and liver, WAT depots and gut were analyzed biochemically, histologically and by lipidomics and transcriptomics together with circulating factors to investigate the sequence of pathogenic events and organ cross-talk during NAFLD development. Results: HFD-induced obesity was associated with an increase in visceral fat, plasma lipids and hyperinsulinemia at t = 8w, along with increased liver steatosis and circulating liver damage biomarkers. In parallel, upstream regulator analysis predicted that lipid catabolism regulators were deactivated and lipid synthesis regulators were activated. Subsequently, hepatocyte hypertrophy, oxidative stress and hepatic inflammation developed. Hepatic collagen accumulated from t = 16 w and became pronounced at t = 28-38 w. Epididymal WAT was maximally hypertrophic from t = 8 w, which coincided with inflammation development. Mesenteric and subcutaneous WAT hypertrophy developed slower and did not appear to reach a maximum, with minimal inflammation. In gut, HFD significantly increased permeability, induced a shift in microbiota composition from t = 8 w and changed circulating gut-derived metabolites. Conclusion: HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin resistance, essentially as observed in obese NAFLD patients, underlining their translational value. We demonstrate that marked epididymal-WAT inflammation, and gut permeability and dysbiosis precede the development of NAFLD stressing the importance of a multiple-organ approach in the prevention and treatment of NAFLD.

Serine catabolism generates liver NADPH and supports hepatic lipogenesis.

Carbohydrate can be converted into fat by de novo lipogenesis, a process upregulated in fatty liver disease. Chemically, de novo lipogenesis involves polymerization and reduction of acetyl-CoA, using NADPH as the electron donor. The feedstocks used to generate acetyl-CoA and NADPH in lipogenic tissues remain, however, unclear. Here we show using stable isotope tracing in mice that de novo lipogenesis in adipose is supported by glucose and its catabolism via the pentose phosphate pathway to make NADPH. The liver, in contrast, derives acetyl-CoA for lipogenesis from acetate and lactate, and NADPH from folate-mediated serine catabolism. Such NADPH generation involves the cytosolic serine pathway in liver running in the opposite direction to that observed in most tissues and tumours, with NADPH made by the SHMT1-MTHFD1-ALDH1L1 reaction sequence. SHMT inhibition decreases hepatic lipogenesis. Thus, liver folate metabolism is distinctively wired to support cytosolic NADPH production and lipogenesis. More generally, while the same enzymes are involved in fat synthesis in liver and adipose, different substrates are used, opening the door to tissue-specific pharmacological interventions.

Pharmacologic inhibition of ketohexokinase prevents fructose-induced metabolic dysfunction.

OBJECTIVE: Recent studies suggest that excess dietary fructose contributes to metabolic dysfunction by promoting insulin resistance, de novo lipogenesis (DNL), and hepatic steatosis, thereby increasing the risk of obesity, type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH), and related comorbidities. Whether this metabolic dysfunction is driven by the excess dietary calories contained in fructose or whether fructose catabolism itself is uniquely pathogenic remains controversial. We sought to test whether a small molecule inhibitor of the primary fructose metabolizing enzyme ketohexokinase (KHK) can ameliorate the metabolic effects of fructose. METHODS: The KHK inhibitor PF-06835919 was used to block fructose metabolism in primary hepatocytes and Sprague Dawley rats fed either a high-fructose diet (30% fructose kcal/g) or a diet reflecting the average macronutrient dietary content of an American diet (AD) (7.5% fructose kcal/g). The effects of fructose consumption and KHK inhibition on hepatic steatosis, insulin resistance, and hyperlipidemia were evaluated, along with the activation of DNL and the enzymes that regulate lipid synthesis. A metabolomic analysis was performed to confirm KHK inhibition and understand metabolite changes in response to fructose metabolism in vitro and in vivo. Additionally, the effects of administering a single ascending dose of PF-06835919 on fructose metabolism markers in healthy human study participants were assessed in a randomized placebo-controlled phase 1 study. RESULTS: Inhibition of KHK in rats prevented hyperinsulinemia and hypertriglyceridemia from fructose feeding. Supraphysiologic levels of dietary fructose were not necessary to cause metabolic dysfunction as rats fed the American diet developed hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis, which were all reversed by KHK inhibition. Reversal of the metabolic effects of fructose coincided with reductions in DNL and inactivation of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP). We report that administering single oral doses of PF-06835919 was safe and well tolerated in healthy study participants and dose-dependently increased plasma fructose indicative of KHK inhibition. CONCLUSIONS: Fructose consumption in rats promoted features of metabolic dysfunction seen in metabolic diseases such as T2D and NASH, including insulin resistance, hypertriglyceridemia, and hepatic steatosis, which were reversed by KHK inhibition.

Discovery of PF-06835919: A Potent Inhibitor of Ketohexokinase (KHK) for the Treatment of Metabolic Disorders Driven by the Overconsumption of Fructose.

Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (8), currently in phase 2 clinical trials. The discovery of 8 was built upon our originally reported, fragment-derived lead 1 and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.

Finding the Sweet Spot: Parsing Tissue-Specific Contributions of Fructose Metabolism.

The abundance of dietary sweeteners and overconsumption of fructose are widely thought to promote metabolic disease. In this issue of Cell Metabolism, Andres-Hernando et al. (2020) identify the liver as the major site of fructose metabolism-mediated metabolic dysfunction and identify a surprising role for intestinal fructose metabolism in driving fructose intake.

Human sebum requires de novo lipogenesis, which is increased in acne vulgaris and suppressed by acetyl-CoA carboxylase inhibition.

Sebum plays important physiological roles in human skin. Excess sebum production contributes to the pathogenesis of acne vulgaris, and suppression of sebum production reduces acne incidence and severity. We demonstrate that sebum production in humans depends on local flux through the de novo lipogenesis (DNL) pathway within the sebocyte. About 80 to 85% of sebum palmitate (16:0) and sapienate (16:1n10) were derived from DNL, based on stable isotope labeling, much higher than the contribution of DNL to triglyceride palmitate in circulation (~20%), indicating a minor contribution by nonskin sources to sebum lipids. This dependence on local sebocyte DNL was not recapitulated in two widely used animal models of sebum production, Syrian hamsters and Göttingen minipigs. Confirming the importance of DNL for human sebum production, an acetyl-CoA carboxylase inhibitor, ACCi-1, dose-dependently suppressed DNL and blocked synthesis of fatty acids, triglycerides, and wax esters but not free sterols in human sebocytes in vitro. ACCi-1 dose-dependently suppressed facial sebum excretion by ~50% (placebo adjusted) in human individuals dosed orally for 2 weeks. Sebum triglycerides, wax esters, and free fatty acids were suppressed by ~66%, whereas non-DNL-dependent lipid species, cholesterol, and squalene were not reduced, confirming selective modulation of DNL-dependent lipids. Last, individuals with acne vulgaris exhibited increased sebum production rates relative to individuals with normal skin, with >80% of palmitate and sapienate derived from DNL. These findings highlight the importance of local sebocyte DNL for human skin sebaceous gland biology and illuminate a potentially exploitable therapeutic target for the treatment of acne vulgaris.

The Small Intestine Converts Dietary Fructose into Glucose and Organic Acids.

Excessive consumption of sweets is a risk factor for metabolic syndrome. A major chemical feature of sweets is fructose. Despite strong ties between fructose and disease, the metabolic fate of fructose in mammals remains incompletely understood. Here we use isotope tracing and mass spectrometry to track the fate of glucose and fructose carbons in vivo, finding that dietary fructose is cleared by the small intestine. Clearance requires the fructose-phosphorylating enzyme ketohexokinase. Low doses of fructose are ∼90% cleared by the intestine, with only trace fructose but extensive fructose-derived glucose, lactate, and glycerate found in the portal blood. High doses of fructose (≥1 g/kg) overwhelm intestinal fructose absorption and clearance, resulting in fructose reaching both the liver and colonic microbiota. Intestinal fructose clearance is augmented both by prior exposure to fructose and by feeding. We propose that the small intestine shields the liver from otherwise toxic fructose exposure.

Discovery of Fragment-Derived Small Molecules for in Vivo Inhibition of Ketohexokinase (KHK).

Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.

RNAi-based therapeutic strategies for metabolic disease.

RNA interference (RNAi) is a robust gene silencing mechanism that degrades mRNAs complementary to the antisense strands of double-stranded, short interfering RNAs (siRNAs). As a therapeutic strategy, RNAi has an advantage over small-molecule drugs, as virtually all genes are susceptible to targeting by siRNA molecules. This advantage is, however, counterbalanced by the daunting challenge of achieving safe, effective delivery of oligonucleotides to specific tissues in vivo. Lipid-based carriers of siRNA therapeutics can now target the liver in metabolic diseases and are being assessed in clinical trials for the treatment of hypercholesterolemia. For this indication, a chemically modified oligonucleotide that targets endogenous small RNA modulators of gene expression (microRNAs) is also under investigation in clinical trials. Emerging 'self-delivery' siRNAs that are covalently linked to lipophilic moieties show promise for the future development of therapies. Besides the liver, inflammation of the adipose tissue in patients with obesity and type 2 diabetes mellitus may be an attractive target for siRNA therapeutics. Administration of siRNAs encapsulated within glucan microspheres can silence genes in inflammatory phagocytic cells, as can certain lipid-based carriers of siRNA. New technologies that combine siRNA molecules with antibodies or other targeting molecules also appear encouraging. Although still at an early stage, the emergence of RNAi-based therapeutics has the potential to markedly influence our clinical future.

Glucan particles for selective delivery of siRNA to phagocytic cells in mice.

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 µm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle-safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of beta1,3-D-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 microg kg(-1) siRNA directed against tumour necrosis factor alpha (Tnf-alpha) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-alpha levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-alpha and interleukin-1beta production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.

Tumor necrosis factor-alpha induces caspase-mediated cleavage of peroxisome proliferator-activated receptor gamma in adipocytes.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFalpha) in adipose tissue of obese subjects potently suppresses the expression of PPARgamma and attenuates adipocyte functions. Here we show that PPARgamma is a substrate of caspase-3 and caspase-6 during TNFalpha receptor signaling in adipocytes, and the consequent PPARgamma cleavage disrupts its nuclear localization. TNFalpha treatment of 3T3-L1 adipocytes decreases full-length PPARgamma while increasing the level of a 45-kDa immunoreactive PPARgamma fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARgamma protein in response to TNFalpha in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARgamma cleavage product. Dispersion of nuclear PPARgamma to the cytoplasm in response to TNFalpha treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFalpha down-regulates PPARgamma protein and PPARgamma-mediated metabolic processes in adipose cells.

Tumor necrosis factor alpha (TNFalpha) stimulates Map4k4 expression through TNFalpha receptor 1 signaling to c-Jun and activating transcription factor 2.

Tumor necrosis factor alpha (TNFalpha) is a cytokine secreted by macrophages and adipocytes that contributes to the low grade inflammation and insulin resistance observed in obesity. TNFalpha signaling decreases peroxisome proliferator-activated receptor gamma and glucose transporter isoform 4 (GLUT4) expression in adipocytes, impairing insulin action, and this is mediated in part by the yeast Ste20 protein kinase ortholog Map4k4. Here we show that Map4k4 expression is selectively up-regulated by TNFalpha, whereas the expression of the protein kinases JNK1/2, ERK1/2, p38 stress-activated protein kinase, and mitogen-activated protein kinase kinases 4/7 shows little or no response. Furthermore, the cytokines interleukin 1beta (IL-1beta) and IL-6 as well as lipopolysaccharide fail to increase Map4k4 mRNA levels in cultured adipocytes under conditions where TNFalpha elicits a 3-fold effect. Using agonistic and antagonistic antibodies and small interfering RNA (siRNA) against TNFalpha receptor 1 (TNFR1) and TNFalpha receptor 2 (TNFR2), we show that TNFR1, but not TNFR2, mediates the increase in Map4k4 expression. TNFR1, but not TNFR2, also mediates a potent effect of TNFalpha on the phosphorylation of JNK1/2 and p38 stress-activated protein kinase and their downstream transcription factor substrates c-Jun and activating transcription factor 2 (ATF2). siRNA-based depletion of c-Jun and ATF2 attenuated TNFalpha action on Map4k4 mRNA expression. Consistent with this concept, the phosphorylation of ATF2 along with the expression and phosphorylation of c-Jun by TNFalpha signaling was more robust and prolonged compared with that of IL-1beta, which failed to modulate Map4k4. These data reveal that TNFalpha selectively stimulates the expression of a key component of its own signaling pathway, Map4k4, through a TNFR1-dependent mechanism that targets the transcription factors c-Jun and ATF2.

Dexamethasone suppresses phospholipase C activation and insulin secretion from isolated rat islets.

Dexamethasone inhibits insulin secretion from isolated islets. In the present experiments, possible underlying biochemical mechanisms responsible for defective secretion were explored. Dexamethasone (1 micromol/L) had no immediate deleterious effect on 15 mmol/L glucose-induced insulin release from perifused rat islets. However, a 3-hour preincubation period with 1 micromol/L dexamethasone resulted in parallel reductions in both the first (64%) and second phases (74%) of 15 mmol/L glucose-induced insulin secretion monitored during a dynamic perifusion. When measured after the perifusion, there were no differences in insulin content or in the capacity of control or dexamethasone-treated islets to use glucose. Dexamethasone (1 micromol/L) preexposure also reduced phorbol ester- and potassium-induced secretion. In additional experiments, islets were labeled for 3 hours with 3H-inositol in the presence or absence of 1 micromol/L dexamethasone. The steroid did not affect total 3H-inositol incorporation during the labeling period. However, the capacity of 15 mmol/L glucose, 30 mmol/L KCl, and 100 micromol/L carbachol to activate phospholipase C (PLC), monitored by the accumulation of labeled inositol phosphates, was significantly reduced in dexamethasone-pretreated islets. Inclusion of the nuclear glucocorticoid receptor antagonist RU486 (mifepristone, 10 micromol/L) abolished the adverse effects of dexamethasone on both glucose-induced inositol phosphate accumulation and insulin secretion. Quantitative Western blot analyses revealed that the islet contents of PLCdelta1, PLCbeta1, beta2, beta3, and protein kinase C alpha were unaffected by dexamethasone pretreatment. These findings demonstrate that dexamethasone pretreatment impairs insulin secretion via a genomic action and that impaired activation of the PLC/protein kinase C signaling system is involved in the evolution of its inhibitory effect on secretion.

Effects of prior 5-hydroxytryptamine exposure on rat islet insulin secretory and phospholipase C responses.

Glucose-induced insulin secretion is inhibited by 5-hydroxytryptamine (5HT). In the present studies the specificity of 5HT inhibition of release and the potential biochemical mechanisms involved were investigated. Dose-dependent inhibition of 15 mM glucose-induced secretion was induced by a prior 3 h incubation with 5HT. At the highest 5HT concentration (500 microM) employed, both first and second phase responses to 15 mM glucose were reduced 50-60%. In addition, this level (500 microM) of 5HT virtually abolished 10 mM glucose-induced secretion. In contrast, secretion in response to the protein kinase C activator phorbol 12-myristate 13-acetate (500 nM) was immune to 500 microM 5HT pre-treatment. Glucose usage rates were comparable in both control and 500 microM 5HT-pretreated islets. However, the generation of inositol phosphates and the efflux of 3H-inositol from 3H-inositol-prelabeled islets in response to stimulatory glucose were impaired in parallel with insulin secretion. Based on these observations the following conclusions were reached: (1) 5HT impairs glucose-induced insulin release by altering glucose-induced activation of phospholipase C. (2) Biochemical events distal to phospholipase C remain intact despite this proximal biochemical lesion. (3) Amperometric analysis of 5HT release from 5HT-pretreated islets must take into consideration its profound adverse impact on glucose-induced insulin secretion.

Effects of muscarinic receptor type 3 knockout on mouse islet secretory responses.

The impact of muscarinic type 3 receptor knockout (M3KO) on the cholinergic regulation of insulin secretion and phospholipase C (PLC) activation was determined. Islets isolated from control, wild-type mice or heterozygotes responded with comparable insulin secretory responses to 15 mM glucose. This response was markedly amplified by the inclusion of 10 microM carbachol. While 15 mM glucose-induced release remained similar to wild-type and heterozygote responses in M3KO mice, the stimulatory impact of carbachol was abolished. Stimulation with 15 mM glucose plus 50 microM carbachol increased fractional efflux rates of myo-[2-3H]inositol from control wild-type and heterozygote islets but not from M3KO islets. Fed plasma insulin levels of M3KO mice were reduced 68% when compared to values obtained from combined wild-type and heterozygote animals. These studies support the conclusion that the M3 receptor in islets is coupled to PLC activation and insulin secretion and that cholinergic stimulation of the islets may play an important role in the regulation of plasma insulin levels.