Safety and immunogenicity of Px563L, a recombinant anthrax vaccine candidate, in a two-dose regimen for post-exposure prophylaxis in healthy adults.

Px563L is a next-generation anthrax vaccine candidate consisting of a protein subunit, mutant recombinant protective antigen SNKE167-ΔFF-315-E308D (mrPA), and liposome-embedded monophosphoryl lipid A (MPLA) adjuvant. Px563L has the potential to deliver an improved safety and immunogenicity profile relative to the currently licensed vaccine, which is produced from filtered B. anthracis culture supernatants. We conducted a Phase 1, double-blind, placebo-controlled, dose-escalation study in 54 healthy subjects to evaluate Px563L at 3 dose levels of mrPA (10, 50, and 80 mcg). For each dose level, 18 subjects were randomized in an 8:8:2 ratio to Px563L (mrPA with adjuvant), RPA563 (mrPA only) or placebo (saline). Each subject received an intramuscular (IM) injection on Day 0 and Day 28. Primary safety and immunogenicity analysis was conducted after all subjects completed the Day70 visit, a duration deemed clinically relevant for post-exposure prophylaxis. Long-term safety was assessed through Day 393. Vaccinations with Px563L at all dose levels were well-tolerated. There were no serious adverse events or adverse events (AE) leading to early withdrawal. In all treatment groups, most AEs were due to injection site reactions, and all AEs at the 10 and 50 mcg dose levels were mild. For the primary immunogenicity endpoint (protective toxin neutralizing antibody 50% neutralization factor [TNA NF50]), titers started to increase significantly after the second administration of Px563L, from Day35 through Day70, with the geometric mean and lower bound of the 95% confidence interval exceeding 0.56, a threshold correlating with significant survival in animal models of anthrax exposure. In conclusion, Px563L, administered as two IM doses 28 days apart, was well-tolerated and elicited a protective antibody response starting at seven days after the second vaccination. These findings support the continued development of Px563L in a two-dose regimen for anthrax post-exposure prophylaxis. ClinicalTrials.gov identifier NCT02655549.

Week 4 Liver Fat Reduction on MRI as an Early Predictor of Treatment Response in Participants with Nonalcoholic Steatohepatitis.

Background Pharmacologic treatment of nonalcoholic steatohepatitis (NASH) is long term in nature; thus, early noninvasive treatment response assessment is important for therapeutic decision making. Purpose To investigate potential early predictors of the 12-week treatment response estimated by using the MRI-based proton-density fat fraction (PDFF). Materials and Methods In this secondary analysis of a prospective phase Ib clinical trial evaluating a candidate treatment (MET409, a farnesoid X receptor agonist) for NASH, participants were analyzed at baseline and at 4 and 12 weeks after either active treatment with MET409 or placebo treatment between June 2019 and January 2020. Correlation and multiple linear regression analyses were used to identify clinical, laboratory, and imaging predictors of the relative PDFF change at week 12 (W12). Multivariate logistic regression analysis was used to develop predictive models for an at least 30% relative PDFF reduction at W12, a well-validated indicator of histologic improvement. Model performance was characterized by using area under the receiver operating characteristic curve (AUC) analysis, sensitivity, and specificity. Results A total of 48 participants were analyzed (median age, 57 years; age range, 40-62 years; 32 women), among whom 30 received MET409 and 18 received a placebo. The week 4 (W4) relative changes in PDFF (regression coefficient = 1.24, P < .001) and the serum alkaline phosphatase (ALP) level (regression coefficient = -0.29, P = .03) were predictors of the W12 relative PDFF change. An at least 19.3% relative PDFF reduction at W4 yielded an AUC of 0.98 (sensitivity, 89%; specificity, 95%) for predicting an at least 30% relative PDFF reduction at W12. The addition of ALP to the predictive model did not improve model performance. Conclusion In participants with nonalcoholic steatohepatitis enrolled in a phase Ib treatment trial, the relative change in the MRI-based proton-density fat fraction (PDFF) at week 4 was highly predictive of the treatment response estimated by using the week 12 MRI-based PDFF. © RSNA, 2021 Online supplemental material is available for this article.

A structurally optimized FXR agonist, MET409, reduced liver fat content over 12 weeks in patients with non-alcoholic steatohepatitis.

BACKGROUND & AIMS: The benefits of farnesoid X receptor (FXR) agonists in patients with non-alcoholic steatohepatitis (NASH) have been validated, although improvements in efficacy and/or tolerability remain elusive. Herein, we aimed to assess the performance of a structurally optimized FXR agonist in patients with NASH. METHODS: In this 12-week, randomized, placebo-controlled study, we evaluated MET409 - a non-bile acid agonist with a unique chemical scaffold - in patients with NASH. Patients were randomized to receive either 80 mg (n = 20) or 50 mg (n = 19) of MET409, or placebo (n = 19). RESULTS: At Week 12, MET409 lowered liver fat content (LFC), with mean relative reductions of 55% (80 mg) and 38% (50 mg) vs. 6% in placebo (p <0.001). MET409 achieved ≥30% relative LFC reduction in 93% (80 mg) and 75% (50 mg) of patients vs. 11% in placebo (p <0.001) and normalized LFC (≤5%) in 29% (80 mg) and 31% (50 mg) of patients vs. 0% in placebo (p <0.05). An increase in alanine aminotransferase (ALT) was observed with MET409, confounding Week 12 changes from baseline (-25% for 80 mg, 28% for 50 mg). Nonetheless, MET409 achieved ≥30% relative ALT reduction in 50% (80 mg) and 31% (50 mg) of patients vs. 17% in placebo. MET409 was associated with on-target high-density lipoprotein cholesterol decreases (mean changes of -23.4% for 80 mg and -20.3% for 50 mg vs. 2.6% in placebo) and low-density lipoprotein cholesterol (LDL-C) increases (mean changes of 23.7% for 80 mg and 6.8% for 50 mg vs. -1.5% in placebo). Pruritus (mild-moderate) occurred in 16% (50 mg) and 40% (80 mg) of MET409-treated patients. CONCLUSION: MET409 lowered LFC over 12 weeks in patients with NASH and delivered a differentiated pruritus and LDL-C profile at 50 mg, providing the first clinical evidence that the risk-benefit profile of FXR agonists can be enhanced through structural optimization. LAY SUMMARY: Activation of the farnesoid X receptor (FXR) is a clinically validated approach for treating non-alcoholic steatohepatitis (NASH), although side effects such as itching or increases in low-density lipoprotein cholesterol are frequently dose-limiting. MET409, an FXR agonist with a unique chemical structure, led to significant liver fat reduction and delivered a favorable side effect profile after 12 weeks of treatment in patients with NASH. These results provide the first clinical evidence that the risk-benefit profile of FXR agonists can be enhanced.

Role of islet-, gut-, and adipocyte-derived hormones in the central control of food intake and body weight: implications for an integrated neurohormonal approach to obesity pharmacotherapy.

Contrary to its historical epithet as a lifestyle disorder, obesity is now widely recognized as having a neurobiological basis. This progress is due to our knowledge not only about energy homoeostatic pathways within the central nervous system (CNS), but also about the role of peripheral peptide hormones acting upon the CNS. These hormones include long-term adiposity signals, such as leptin, that inform the CNS primarily of changes in the body's overall fat and energy reserves, and short-term signals such as amylin, peptide YY (PYY) and ghrelin, that primarily reflect changes in the immediate nutritive state (energy intake). The limited weight loss effects achieved with current monotherapy approaches to obesity have been attributed, at least in part, to the redundancies and potent counter-regulatory responses within the neurohormonal feedback loop governing energy balance. Recently, we reported that combinations of amylin, leptin and PYY(3-36) resulted in additive and/or synergistic interactions and caused marked weight loss in the diet-induced obese rat model, which to date has reasonably predicted the clinical effects of several hormones in obese humans. If confirmed in ongoing translational clinical research studies, these findings may provide a physiological rationale for a novel, integrated neurohormonal approach to pharmacotherapy for obesity.

Enhancing energy and glucose metabolism by disrupting triglyceride synthesis: Lessons from mice lacking DGAT1.

Although the ability to make triglycerides is essential for normal physiology, excess accumulation of triglycerides results in obesity and is associated with insulin resistance. Inhibition of triglyceride synthesis, therefore, may represent a feasible strategy for the treatment of obesity and type 2 diabetes. Acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) is one of two DGAT enzymes that catalyze the final reaction in the known pathways of mammalian triglyceride synthesis. Mice lacking DGAT1 have increased energy expenditure and insulin sensitivity and are protected against diet-induced obesity and glucose intolerance. These metabolic effects of DGAT1 deficiency result in part from the altered secretion of adipocyte-derived factors. Studies of DGAT1-deficient mice have helped to provide insights into the mechanisms by which cellular lipid metabolism modulates systemic carbohydrate and insulin metabolism, and a better understanding of how DGAT1 deficiency enhances energy expenditure and insulin sensitivity may identify additional targets or strategies for the treatment of obesity and type 2 diabetes.

Inhibition of triglyceride synthesis as a treatment strategy for obesity: lessons from DGAT1-deficient mice.

Because the ability to make triglycerides is essential for the accumulation of adipose tissue, inhibition of triglyceride synthesis may ameliorate obesity and its related medical consequences. Acyl coenzyme A (CoA):diacylglycerol acyltransferase 1 (DGAT1) is 1 of 2 DGAT enzymes that catalyze the final reaction in the known pathways of mammalian triglyceride synthesis. Mice lacking DGAT1 are resistant to obesity and have increased sensitivity to insulin and leptin. DGAT1-deficient mice are also resistant to diet-induced hepatic steatosis. The effects of DGAT1 deficiency on energy and glucose metabolism result in part from the altered secretion of adipocyte-derived factors. Although complete DGAT1 deficiency causes alopecia and impairs development of the mammary gland, these abnormalities are not observed in mice with partial DGAT1 deficiency. These findings suggest that pharmacological inhibition of DGAT1 may be a feasible therapeutic strategy for human obesity and type 2 diabetes.

Role of adipocyte-derived factors in enhancing insulin signaling in skeletal muscle and white adipose tissue of mice lacking Acyl CoA:diacylglycerol acyltransferase 1.

Mice that lack acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in mammalian triglyceride synthesis, have decreased adiposity and increased insulin sensitivity. Here we show that insulin-stimulated glucose transport is increased in the skeletal muscle and white adipose tissue (WAT) of chow-fed DGAT1-deficient mice. This increase in glucose transport correlated with enhanced insulin-stimulated activities of phosphatidylinositol 3-kinase, protein kinase B (or Akt), and protein kinase Clambda (PKC-lambda), three key molecules in the insulin-signaling pathway, and was associated with decreased levels of serine-phosphorylated insulin receptor substrate 1 (IRS-1), a molecule implicated in insulin resistance. Similar findings in insulin signaling were also observed in DGAT1-deficient mice fed a high-fat diet. Interestingly, the increased PKC-lambda activity and decreased serine phosphorylation of IRS-1 were observed in chow-fed wild-type mice transplanted with DGAT1-deficient WAT, consistent with our previous finding that transplantation of DGAT1-deficient WAT enhances glucose disposal in wild-type recipient mice. Our findings demonstrate that DGAT1 deficiency enhances insulin signaling in the skeletal muscle and WAT, in part through altered expression of adipocyte-derived factors that modulate insulin signaling in peripheral tissues.

Insulin-induced activation of atypical protein kinase C, but not protein kinase B, is maintained in diabetic (ob/ob and Goto-Kakazaki) liver. Contrasting insulin signaling patterns in liver versus muscle define phenotypes of type 2 diabetic and high fat-induced insulin-resistant states.

Insulin resistance in type 2 diabetes is characterized by defects in muscle glucose uptake and hepatic overproduction of both glucose and lipids. These hepatic defects are perplexing because insulin normally suppresses glucose production and increases lipid synthesis in the liver. To understand the mechanisms for these seemingly paradoxical defects, we examined the activation of atypical protein kinase C (aPKC) and protein kinase B (PKB), two key signaling factors that operate downstream of phosphatidylinositol 3-kinase and regulate various insulin-sensitive metabolic processes. Livers and muscles of three insulin-resistant rodent models were studied. In livers of type 2 diabetic non-obese Goto-Kakazaki rats and ob/ob-diabetic mice, the activation of PKB was impaired, whereas activation of aPKC was surprisingly maintained. In livers of non-diabetic high fatfed mice, the activation of both aPKC and PKB was maintained. In contrast to the maintenance of aPKC activation in the liver, insulin activation of aPKC was impaired in muscles of Goto-Kakazaki-diabetic rats and ob/ob-diabetic and non-diabetic high fat-fed mice. These findings suggest that, at least in these rodent models, (a) defects in aPKC activation contribute importantly to skeletal muscle insulin resistance observed in both high fat feeding and type 2 diabetes; (b) insulin signaling defects in muscle are not necessarily accompanied by similar defects in liver; (c) defects in hepatic PKB activation occur in association with, and probably contribute importantly to, the development of overt diabetes; and (d) maintenance of hepatic aPKC activation may explain the continued effectiveness of insulin for stimulating certain metabolic actions in the liver.

Obesity resistance and enhanced glucose metabolism in mice transplanted with white adipose tissue lacking acyl CoA:diacylglycerol acyltransferase 1.

Recent studies have identified the white adipose tissue (WAT) as an important endocrine organ that regulates energy and glucose metabolism via a number of secreted factors. Mice lacking acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in mammalian triglyceride synthesis, are protected against diet-induced obesity and glucose intolerance because of increased energy expenditure and enhanced insulin sensitivity. Because DGAT1 is highly expressed in WAT, we hypothesized that DGAT1 deficiency affects the expression of adipocyte-derived factors that regulate energy and glucose metabolism. Here we show that the transplantation of DGAT1-deficient WAT decreases adiposity and enhances glucose disposal in wild-type mice. Analysis of DGAT1-deficient WAT revealed a twofold increase in the expression of adiponectin, a molecule that enhances fatty acid oxidation and insulin sensitivity, and this increase may account in part for the transplantation-induced metabolic changes. Our results highlight the importance of the endocrine function of WAT and suggest that an alteration in this function contributes to the increased energy expenditure and insulin sensitivity in DGAT1-deficient mice.

Analysis of energy expenditure at different ambient temperatures in mice lacking DGAT1.

Mice lacking acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triglyceride synthesis, have increased energy expenditure and therefore are resistant to obesity. Because ambient temperature can significantly affect energy expenditure in mice, we undertook these studies to determine the effects of different ambient temperatures on energy expenditure, food intake, and thermoregulation in DGAT1-deficient [Dgat1(-/-)] mice. Dgat1(-/-) mice had increased energy expenditure irrespective of changes in the ambient temperature. Although core temperature was normal, surface temperature was increased in Dgat1(-/-) mice, most likely reflecting an active mechanism to dissipate heat from increased thermogenesis. Dgat1(-/-) mice had increased food intake at baseline, and this hyperphagia became more pronounced upon exposure to cold. When fasted in a cold environment, Dgat1(-/-) mice developed hypothermia, which was associated with hypoglycemia. These results suggest that the hyperphagia in Dgat1(-/-) mice is a secondary mechanism that compensates for the increased utilization of fuel substrates. Our findings offer insights into the mechanisms of hyperphagia and increased energy expenditure in a murine model of obesity resistance.

Dissociation of obesity and impaired glucose disposal in mice overexpressing acyl coenzyme a:diacylglycerol acyltransferase 1 in white adipose tissue.

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two DGAT enzymes known to catalyze the final step in mammalian triglyceride synthesis. Mice deficient in DGAT1 are resistant to obesity and have enhanced insulin sensitivity. To understand better the relationship between triglyceride synthesis and energy and glucose metabolism, we generated transgenic (aP2-Dgat1) mice in which expression of murine DGAT1 in the white adipose tissue (WAT) was twofold higher than normal. aP2-Dgat1 mice that were fed a regular diet had larger adipocytes and greater total fat pad weight than wild-type (WT) mice. In response to a high-fat diet, aP2-Dgat1 mice became more obese ( approximately 20% greater body weight after 15 weeks) than WT mice. However, the increase in adiposity in aP2-Dgat1 mice was not associated with impaired glucose disposal, as demonstrated by glucose and insulin tolerance tests. Correlating with this finding, triglyceride deposition in the liver and skeletal muscle, two major target tissues of insulin, was similar in aP2-Dgat1 and WT mice. Thus, DGAT1 overexpression in murine WAT provides a model in which obesity does not impair glucose disposal. Our findings support the lipotoxicity hypothesis that the deposition of triglycerides in insulin-sensitive tissues other than adipocytes causes insulin resistance.

Fatty acids, triglycerides, and glucose metabolism: recent insights from knockout mice.

PURPOSE OF REVIEW: Cellular lipid metabolism plays an important role in modulating glucose metabolism. Recent models of mice with disruptions in genes involved in cellular fatty acid and triglyceride metabolism have provided insight into the long recognized but incompletely understood relationship between fatty acid metabolism and glucose metabolism. RECENT FINDINGS: Here we review findings from mice with deficiency in selected genes involved in the cellular uptake, storage, and hydrolysis of fatty acids. Our review is organized from the perspective of a fatty acid, as it makes its way from the circulation into the anabolic and then catabolic pathways in the cell. Although we focus primarily on the phenotypes of knockout mice, we also point out several transgenic models in which the overexpression phenotype provides complementary information. SUMMARY: The inactivation of enzymes in the anabolic process of fatty acid uptake and storage is more likely to enhance tissue glucose disposal or insulin secretion, whereas disruptions in the catabolic process tend to impair insulin action or secretion. These findings suggest that pharmacological inhibition of fatty acid uptake or storage may be an effective strategy for treating insulin resistance and diabetes.

Deficiency of acyl coenzyme a:diacylglycerol acyltransferase 1 increases leptin sensitivity in murine obesity models.

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known enzymes that catalyze the final step in mammalian triglyceride synthesis. We have reported that DGAT1-deficient mice have increased insulin and leptin sensitivity, likely accounting for their protection against diet-induced obesity and insulin resistance. Here we show that DGAT1 deficiency enhanced the response to peripheral leptin infusion in Agouti yellow and leptin-deficient (ob/ob) mice, two genetic models of obesity and insulin resistance. Interestingly, DGAT1 deficiency did not enhance the response to intracerebroventricular leptin infusion. Moreover, DGAT1 deficiency did not alter the expression of key hypothalamic genes involved in leptin signaling or in the regulation of food intake and energy expenditure. Thus, the leptin-sensitizing effect of DGAT1 deficiency is present in both leptin-resistant and leptin-deficient genetic models of obesity and may occur in part by enhancing the effects of leptin in peripheral tissues.

Determination of adipocyte size by computer image analysis.

Methods that allow rapid and accurate determination of adipocyte size are important to studies of energy and glucose metabolism. The direct measurement of adipocyte size by microscopy is widely used, although the method is tedious and time consuming. Computer-assisted image analysis can overcome most of the disadvantages associated with this technique. We report a new method for determining adipocyte size by measuring the cross-sectional area of adipocytes with computer image analysis. This method allows a large number of adipocytes to be measured rapidly with computer hardware and software that are readily available.

Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1.

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in mammalian triglyceride synthesis. DGAT1-deficient mice are resistant to diet-induced obesity through a mechanism involving increased energy expenditure. Here we show that these mice have decreased levels of tissue triglycerides, as well as increased sensitivity to insulin and to leptin. Importantly, DGAT1 deficiency protects against insulin resistance and obesity in agouti yellow mice, a model of severe leptin resistance. In contrast, DGAT1 deficiency did not affect energy and glucose metabolism in leptin-deficient (ob/ob) mice, possibly due in part to a compensatory upregulation of DGAT2 expression in the absence of leptin. Our results suggest that inhibition of DGAT1 may be useful in treating insulin resistance and leptin resistance in human obesity.

Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport.

Exercise increases glucose transport in muscle by activating 5'-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. In rat extensor digitorum longus (EDL) muscles, (a) AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-d-riboside (AICAR), activated PYK2, ERK and aPKCs; (b) effects of AICAR on ERK and aPKCs were blocked by tyrosine kinase inhibitor, genistein, and MEK1 inhibitor, PD98059; and (c) effects of AICAR on aPKCs and 2-deoxyglucose (2-DOG) uptake were inhibited by genistein, PD98059, and PLD-inhibitor, 1-butanol. Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-zeta pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-zeta. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.

Leptin modulates the effects of acyl CoA:diacylglycerol acyltransferase deficiency on murine fur and sebaceous glands.

Acyl CoA:diacylglycerol acyltransferase (DGAT) is a ubiquitously expressed enzyme that catalyzes the final reaction in the major pathways of triglyceride synthesis. Mice lacking DGAT1 (Dgat(-/-)) demonstrate significant changes in lipid metabolism in several tissues, including the skin. Here we report the effects of DGAT1 deficiency on fur and sebaceous glands. Adult Dgat(-/-) mice had dry fur and hair loss, which were associated with atrophic sebaceous glands and fur lipid abnormalities. As a result, Dgat(-/-) mice had impaired water repulsion and defective thermoregulation after water immersion. These phenotypes were mostly absent in Dgat(-/-) mice with leptin deficiency, indicating an unexpected role for leptin in modulating the skin phenotype. Our findings indicate that DGAT1 plays an important role in normal fur and sebaceous gland physiology and provide evidence that leptin modulates these processes in the skin.