Angiotensin II (Ang II) has been implicated in stimulating myocyte <em>growth</em> in vitro, but the mechanism for such stimulation is still an open question. To understand the role of Ang II, we studied its effect on protein synthesis in rat neonatal and adult myocytes. Ang II (10(-8) mol/L) stimulated protein synthesis in neonatal myocytes by 43+/-3.5% over control. To prevent the proliferation of <em>fibroblasts</em>, bromodeoxyuridine was added, and protein synthesis in neonatal myocytes was reduced to <em>21</em>+/-2.2% over control. In adult myocytes (cultured without bromodeoxyuridine), Ang II stimulated [3H]leucine incorporation by 24+/-2.3% over control; with bromodeoxyuridine, that stimulation was reduced significantly (13+/-0.93% over control). These data suggest that the presence of <em>fibroblasts</em> in the cultures may control myocyte <em>growth</em>. When supernatant from pure <em>fibroblast</em> culture was added to myocyte preparations, a significant increase (49.8+/-3.5% over control) in protein synthesis occurred. Pretreatment of these <em>fibroblasts</em> with Ang II (10(-3) mol/L) further stimulated protein synthesis, suggesting that Ang II directly stimulates the production of a <em>factor</em> from <em>fibroblasts</em>. The stimulatory effect of Ang II on the release of the <em>factor</em> can be completely blocked by pretreatment with losartan, an Ang II receptor (AT1) blocker. Our data are the first to demonstrate a paracrine effect of a <em>fibroblast</em>-derived <em>factor</em> that modulates myocyte <em>growth</em>. <em>Fibroblast</em>-derived <em>factor</em> loses its biological activity by (1) tryptic digestion, (2) exposure to pH below 4.0 and above 9.0, and (3) heating to 95 degrees C. The molecular weight of the <em>factor</em> is approximately 65 kD. The antibodies against <em>fibroblast</em> <em>growth</em> <em>factor</em> (both acidic and basic) could not inhibit this <em>factor</em>'s stimulatory effect. Furthermore, this <em>factor</em> is heart specific and is produced at least up to the 16th passage of neonatal rat heart <em>fibroblasts</em>. Skin <em>fibroblasts</em>, aortic endothelial cells, and aortic smooth muscle cells do not produce this protein. Our data suggest that the observed myocyte <em>growth</em> by Ang II comes about via <em>fibroblast</em>-derived <em>factor</em>, which is increased by Ang II. Cross talk between <em>fibroblasts</em> and myocytes is an important <em>factor</em> in stimulating myocyte <em>growth</em> by Ang II.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a novel metabolic regulator of glucose and lipid metabolism; however, the exact mechanism of action and regulation of FGF<em>21</em> is not fully understood. Metabolic status plays an important role in the regulation of FGF<em>21</em>, and we therefore examined whether metformin, an indirect AMPK-activator, regulates FGF<em>21</em> expression in hepatocytes. FGF<em>21</em> mRNA and protein expression were determined after incubation of primary cultured rat and human hepatocytes with metformin for 24 hours. To study the role of AMPK in the putative regulation of FGF<em>21</em>, hepatocytes were incubated with Compound C (an AMPK inhibitor) in the presence of metformin. A strong dose-dependent increase in FGF<em>21</em> expression was observed in both rat and human hepatocytes treated with metformin. This effect was blocked by addition of the AMPK-inhibitor Compound C. The study shows that metformin is a potent inducer of hepatic FGF<em>21</em> expression and that the effect of metformin seems to be mediated through AMPK activation. As FGF<em>21</em> therapy normalizes blood glucose in animal models of type 2 diabetes, the induction of hepatic FGF<em>21</em> by metformin might play an important role in metformin's antidiabetic effect.

OBJECTIVE

Traditional therapies for arteriosclerotic disease often fail as a result of an exaggerated fibroproliferative response (recurrent stenosis) at the site of the intervention. Lysyl oxidase, secreted by activated vascular smooth muscle cells and fibroblasts, catalyzes a key step in the cross-linking and stabilization of collagen and elastin in the vascular wall. We hypothesized that lysyl oxidase messenger RNA (mRNA) and protein expression are time-dependent and precede collagen accumulation and luminal narrowing after arterial balloon injury in the rat.

METHODS

A 2F balloon-tipped catheter was used to injure the right common carotid artery in male Sprague-Dawley rats. Injured right and control (uninjured) left common carotid arteries were harvested at 0, 0.25, 1, 3, 7, 14, 21, 28, and 60 days for mRNA quantitation and immunohistochemical analysis. Steady-state lysyl oxidase mRNA levels were quantitated with real-time reverse transcription polymerase chain reaction (TaqMan). Immunohistochemical staining with antibodies to alpha-smooth muscle cell actin and lysyl oxidase, and Movat pentachrome staining were performed for qualitative assessment of changes in the cellular and extracellular matrix components of the vessel wall. Post-injury intimal area was measured from hematoxylin and eosin-stained specimens at each time point.

RESULTS

When compared with sham-operated control arteries, lysyl oxidase expression in balloon-injured arteries increased significantly to 212% by day 3 after injury, and remained elevated through day 21, with a decrease toward baseline levels by day 28. Lysyl oxidase protein expression did not peak until day 14, and persisted through day 28. Collagen accumulation peaked at day 28, corresponding to the maximal increase in intimal area, with later accumulation of proteoglycans and ground substance in the intimal lesion.

CONCLUSIONS

Our results indicate that lysyl oxidase mRNA and protein expression is time-dependent after balloon injury of the rat carotid artery and that expression appears to precede maximal collagen accumulation and corresponding increases in intimal area. This suggests that lysyl oxidase may have an important role in stabilization of collagen and elastin at sites of vascular injury and that modulation of lysyl oxidase activity may be a viable method to prevent or reduce recurrent stenosis.

CONCLUSIONS

Failure of traditional therapies for ischemic arteriosclerotic disease is often due to an exaggerated fibroproliferative response (recurrent stenosis) at the site of intervention. Recurrent stenosis can be viewed as an injury-repair process, with an initial stage characterized by cellular proliferation followed by deposition of extracellular matrix. This study focuses on lysyl oxidase, a key enzyme involved in stabilization of collagen and elastin. This study demonstrates that lysyl oxidase messenger RNA and protein expression are time-dependent, preceding collagen accumulation and corresponding increases in intimal area. Accumulation of extracellular matrix is a major factor in growth of the restenotic lesion, and modulation of lysyl oxidase activity may offer a therapeutic method for decreasing or preventing recurrent stenosis.

Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on -12, 10, 24, and 136 DIM for gene expression analysis. <em>Growth</em> hormone receptor (GHR), insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear <em>factor</em> 4alpha (HNF4alpha), <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>), and peroxisome proliferator-activated receptor alpha (PPARalpha) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41 x 10(4) message copies for -12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4alpha mRNA was not altered by DIM or any of the treatments. Abundance of PPARalpha mRNA was unchanged through 24 DIM but increased by 136 DIM. There was a trend for an interaction of bST, diet, and DIM on PPARalpha mRNA abundance from 24 to 136 DIM because the amount of PPARalpha mRNA increased in SSN, SSY, and AFN cows but was not altered in AFY cows. The amount of FGF-<em>21</em> mRNA increased markedly in early lactation but, like HNF4alpha mRNA, was not affected by bST, diet, or their interactions. These results indicate 1) that bST induced increases in hepatic expression of GHR, IGF-I, and IGFBP3 when cows were in negative energy balance in early lactation, 2) there was no effect of reduced dietary n-6/n-3 content on hepatic gene expression, and 3) there was support for a potential homeorhetic role of hepatic FGF-<em>21</em> via uncoupling the somatotropin-IGF-axis in early lactation.

OBJECTIVE

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand-receptor pair frequently overexpressed in solid tumors.

UNASSIGNED

Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFκB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14.

METHODS

Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of

UNASSIGNED

Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics.

RESULTS

In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment.

CONCLUSIONS

RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced

UNASSIGNED

Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258-66. ©2014 AACR.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> is a novel regulator of glucose and lipid metabolism. Recently, increased FGF-<em>21</em> mRNA expression in muscle was found in patients with type 2 diabetes, but the role for FGF-<em>21</em> in muscle is not well understood. Patients with HIV-infection and lipodystrophy are characterised by various degree of lipid-driven insulin resistance. We hypothesized that muscle FGF-<em>21</em> mRNA would be altered in HIV patients with lipodystrophy.

METHODS

Twenty-five HIV-infected men with lipodystrophy (LD) and 15 age-matched healthy controls, received an oral glucose tolerance test and a euglycemic-hyperinsulinemic clamp (50 mU/m2/min) combined with 6,6-H2 glucose infusion. Muscle biopsies were obtained and FGF-<em>21</em> mRNA and glycogen synthase (GS) activity were measured.

RESULTS

Subjects with HIV were insulin resistant compared with non-HIV subjects. Compared to controls, HIV subjects demonstrated a twofold increase of plasma FGF-<em>21</em> from 70.4±56.8 pg/ml vs 109.1±71.8 pg/ml, respectively (p = 0.04) and an eight-fold increase in muscular FGF-<em>21</em> mRNA expression (p = 0.001). Muscle FGF-<em>21</em> mRNA correlated inversely with the rate of disappearance of glucose during insulin clamp (r = -0.54, p = 0.0009), and the GS fractional velocity in muscle (r = -0.39, p = 0.03), and directly with fasting insulin (r = 0.50, p = 0.0022), HOMA-IR (r = 0.47, p = 0.004), triglycerides (r = 0.60. P = 0.0001), waist-to-hip ratio (r = 0.51, p = 0.0001) and limb fat mass (-0.46, p = 0.004), but not to plasma FGF-<em>21</em>.

CONCLUSIONS

FGF-<em>21</em> mRNA is increased in skeletal muscle in HIV patients and correlates to whole-body (primarily reflecting muscle) insulin resistance, but not to plasma FGF-<em>21</em>. Those findings add to the evidence that FGF-<em>21</em> is a myokine and may suggest that muscle FGF-<em>21</em> is working in a local manner.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has evolved as a major metabolic regulator, the pharmacological administration of which causes weight loss, insulin sensitivity and glucose control in rodents and humans. To understand the molecular mechanisms by which FGF<em>21</em> exerts its metabolic effects, we developed a human in vitro model of adipocytes to examine crosstalk between FGF<em>21</em> and insulin signaling. Human adipose stem cell-derived (hASC) adipocytes were acutely treated with FGF<em>21</em> alone, insulin alone, or in combination. Insulin signaling under these conditions was assessed by measuring tyrosine phosphorylation of insulin receptor (InsR), insulin receptor substrate-1 (IRS-1), and serine 473 phosphorylation of Akt, followed by a functional assay using 14C-2-deoxyglucose [14C]-2DG to measure glucose uptake in these cells. FGF<em>21</em> alone caused a modest increase of glucose uptake, but treatment with FGF<em>21</em> in combination with insulin had a synergistic effect on glucose uptake in these cells. The presence of FGF<em>21</em> also effectively lowered the insulin concentration required to achieve the same level of glucose uptake compared to the absence of FGF<em>21</em> by 10-fold. This acute effect of FGF<em>21</em> on insulin signaling was not due to IR, IGF-1R, or IRS-1 activation. Moreover, we observed a substantial increase in basal S473-Akt phosphorylation by FGF<em>21</em> alone, in contrast to the minimal shift in basal glucose uptake. Taken together, our data demonstrate that acute co-treatment of hASC-adipocytes with FGF<em>21</em> and insulin can result in a synergistic improvement in glucose uptake. These effects were shown to occur at or downstream of Akt, or separate from the canonical insulin signaling pathway.

Mitochondria are highly adaptable organelles that can facilitate communication between tissues to meet the energetic demands of the organism. However, the mechanisms by which mitochondria can nonautonomously relay stress signals remain poorly understood. Here we report that mitochondrial mutations in the young, preprogeroid polymerase gamma mutator (POLG) mouse produce a metabolic state of starvation. As a result, these mice exhibit signs of metabolic imbalance including thermogenic defects in brown adipose tissue (BAT). An unexpected benefit of this adaptive response is the complete resistance to diet-induced obesity when POLG mice are placed on a high-fat diet (HFD). Paradoxically, HFD further increases oxygen consumption in part by inducing thermogenesis and mitochondrial biogenesis in BAT along with enhanced expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). Collectively, these findings identify a mechanistic link between FGF<em>21</em>, a long-known marker of mitochondrial disease, and systemic metabolic adaptation in response to mitochondrial stress.

OBJECTIVE

This phase I trial assessed the safety, dose limiting toxicity (DLT) and pharmacodynamics of PX-12 in adult patients with advanced refractory cancers.

METHODS

PX-12 was administered to sequential cohorts as a 72-h infusion utilizing a portable infusion pump on days 1, 2, and 3 of a <em>21</em>-day cycle at a starting dose level of 300 mg/m(2)/day and escalating dose levels till DLT was observed. Plasma thioredoxin (Trx-1), vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and FGF-2 (<em>fibroblast</em> <em>growth</em> <em>factor</em>) levels were measured predose and during infusion of PX-12.

RESULTS

Patients (n = 14) were enrolled to the following dose cohorts, 300 mg/m(2) (n = 3), 400 mg/m(2) (n = 10) and 500 mg/m(2) (n = 1). Common grade 1/2 toxicities included fatigue, taste alteration and odor caused by expired drug metabolite. DLTs were one episode each of grade 3 hypoxia at the 400 mg/m(2) and grade 3 reversible pneumonitis at the 500 mg/m(2) dose levels. Best response was stable disease in a patient with rectal cancer. Predose Trx-1 levels (n = 12) ranged from 5.1 to 30.0 ng/mL (median 12.6 ng/mL).

CONCLUSIONS

PX-12 administered at 400 mg/m(2)/day by 72-hour infusion appears safe and tolerable. Inhibition of thioredoxin is a strategy worth evaluation with next generation of inhibitors.

BACKGROUND

The diagnosis of mitochondrial disease requires a complex synthesis of clinical, biochemical, histological, and genetic investigations. An expanding number of mitochondrial diseases are being recognized, despite their phenotypic diversity, largely due to improvements in methods to detect mutations in affected individuals and the discovery of genes contributing to mitochondrial function. Improved understanding of the investigational pitfalls and the development of new laboratory methodologies that lead to a molecular diagnosis have necessitated the field to rapidly adopt changes to its diagnostic approach.

METHODS

We review the clinical, investigational and genetic challenges that have resulted in shifts to the way we define and diagnose mitochondrial disease. Incorporation of changes, including the use of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) and next generation sequencing techniques, may allow affected patients access to earlier molecular diagnosis and management.

CONCLUSIONS

There have been important shifts in the diagnostic paradigm for mitochondrial disease. Diagnosis of mitochondrial disease is no longer reliant on muscle biopsy alone, but should include clinical assessment accompanied by the use of serological biomarkers and genetic analysis. Because affected patients will be defined on a molecular basis, oligosymptomatic mutation carriers should be included in the spectrum of mitochondrial disease. Use of new techniques such as the measurement of serum FGF-<em>21</em> levels and next-generation-sequencing protocols should simplify the diagnosis of mitochondrial disease.

CONCLUSIONS

Improvements in the diagnostic pathway for mitochondrial disease will result in earlier, cheaper and more accurate methods to identify patients with mitochondrial disease. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

The potential mechanisms of action of polyphenols in nonalcoholic fatty liver disease (NAFLD) are overlooked. Here, we evaluate the beneficial therapeutic effects of hydroxytyrosol (HT), the major metabolite of the oleuropein, in a nutritional model of insulin resistance (IR) and NAFLD by high-fat diet. Young male rats were divided into three groups receiving (1) standard diet (STD; 10.5% fat), (2) high-fat diet (HFD; 58.0% fat) and (3) HFD+HT (10 mg/kg/day by gavage). After 5 weeks, the oral glucose tolerance test was performed, and at 6th week, blood sample and tissues (liver and duodenum) were collected for following determinations. The HT-treated rats showed a marked reduction in serum AST, ALT and cholesterol and improved glucose tolerance and insulin sensitivity, reducing homeostasis model assessment index. HT significantly corrected the metabolic impairment induced by HFD, increasing hepatic peroxisome proliferator activated receptor PPAR-α and its downstream-regulated gene <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, the phosphorylation of acetyl-CoA carboxylase and the mRNA carnitine palmitoyltransferase 1a. HT also reduced liver inflammation and nitrosative/oxidative stress decreasing the nitrosylation of proteins, reactive oxygen species production and lipid peroxidation. Moreover, HT restored intestinal barrier integrity and functions (fluorescein isothiocyanate-dextran permeability and mRNA zona occludens ZO-1). Our data demonstrate the beneficial effect of HT in the prevention of early inflammatory events responsible for the onset of IR and steatosis, reducing hepatic inflammation and nitrosative/oxidative stress and restoring glucose homeostasis and intestinal barrier integrity.

We previously reported that molecular hydrogen (H2) acts as a novel antioxidant to exhibit multiple functions. Moreover, long-term drinking of H2-water (water infused with H2) enhanced energy expenditure to improve obesity and diabetes in db/db mice accompanied by the increased expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) by an unknown mechanism. H2 was ingested by drinking of H2-water or by oral administration of an H2-producing material, MgH2. The comprehensive gene expression profile in the liver of db/db mice was analyzed by DNA microarray. The molecular mechanisms underlying the gene expression profile was investigated using cultured HepG2 cells. Moreover, the effects on lifespan of drinking H2-water were examined using wild-type mice that were fed a fatty diet. Pathway analyses based on comprehensive gene expression revealed the increased expression of various genes involved in fatty acid and steroid metabolism. As a transcription pathway, the PPARα signaling pathway was identified to upregulate their genes by ingesting H2. As an early event, the gene expression of PGC-1α was transiently increased, followed by increased expression of FGF<em>21</em>. The expression of PGC-1α might be regulated indirectly through sequential regulation by H2, 4-hydroxy-2-nonenal, and Akt/FoxO1 signaling, as suggested in cultured cell experiments. In wild-type mice fed the fatty diet, H2-water improved the level of plasma triglycerides and extended their average of lifespan. H2 induces expression of the PGC-1α gene, followed by stimulation of the PPARα pathway that regulates FGF<em>21</em>, and the fatty acid and steroid metabolism.

Neuroblasts exist within the human subependymal zone (SEZ); however, it is debated to what extent neurogenesis changes during normal aging. It is also unknown how precursor proliferation may correlate with the generation of neuronal and glial cells or how expression of <em>growth</em> <em>factors</em> and receptors may change throughout the adult lifespan. We found evidence of dividing cells in the human SEZ (n D 50) in conjunction with a dramatic age-related decline (<em>21</em>-103 years) of mRNAs indicative of proliferating cells (Ki67) and immature neurons (doublecortin). Microglia mRNA (ionized calcium-binding adapter molecule 1) increased during aging, whereas transcript levels of stem/precursor cells (glial fibrillary acidic protein delta and achaete-scute homolog 1), astrocytes (vimentin and pan-glial fibrillary acidic protein), and oligodendrocytes (oligodendrocyte lineage transcription <em>factor</em> 2) remained stable. Epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) mRNAs increased throughout adulthood, while transforming <em>growth</em> <em>factor</em> alpha (TGFα), EGF, Erb-B2 receptor tyrosine kinase 4 (ErbB4) and FGF receptor 1 (FGFR1) mRNAs were unchanged across adulthood. Cell proliferation mRNA positively correlated with FGFR1 transcripts. Immature neuron and oligodendrocyte marker expression positively correlated with TGFα and ErbB4 mRNAs, whilst astrocyte transcripts positively correlated with EGF, FGF2, and FGFR1 mRNAs. Microglia mRNA positively correlated with EGF and FGF2 expression. Our findings indicate that neurogenesis in the human SEZ continues well into adulthood, although proliferation and neuronal differentiation may decline across adulthood. We suggest that mRNA expression of EGF- and FGF-related family members do not become limited during aging and may modulate neuronal and glial fate determination in the SEZ throughout human life.

Malnutrition is considered a leading cause of <em>growth</em> attenuation in children. When food is replenished, spontaneous catch-up (CU) <em>growth</em> usually occurs, bringing the child back to its original <em>growth</em> trajectory. However, in some cases, the CU <em>growth</em> is not complete, leading to a permanent <em>growth</em> deficit. This review summarizes our current knowledge regarding the mechanism regulating nutrition and <em>growth</em>, including systemic <em>factors</em>, such as insulin, <em>growth</em> hormone, insulin- like <em>growth</em> <em>factor</em>-1, vitamin D, <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>, etc., and local mechanisms, including autophagy, as well as regulators of transcription, protein synthesis, miRNAs and epigenetics. Studying the molecular mechanisms regulating CU <em>growth</em> may lead to the establishment of better nutritional and therapeutic regimens for more effective CU <em>growth</em> in children with malnutrition and <em>growth</em> abnormalities. It will be fascinating to follow this research in the coming years and to translate the knowledge gained to clinical benefit.

Adipocytes play important roles in regulating cardiovascular health and disease. However, the molecular mechanism underlying the endocrine role of brown adipose tissue (BAT) in pathological cardiac remodeling remains unknown. Herein we show that adenosine A2A receptor (A2AR) knockout (A2ARKO) causes interscapular BAT (iBAT) dysfunction, leading to accelerated cardiac remodeling in hypertension compared with wild-type (WT) mice. Surgical iBAT depletion induces dramatic cardiac remodeling in WT but not in A2ARKO hypertensive mice. AMPK/PGC1α signaling-induced <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) in brown adipocytes is required for A2AR-mediated inhibition of hypertensive cardiac remodeling. Recombinant FGF<em>21</em> administration improves cardiac remodeling in iBAT-depleted hypertensive mice. More importantly, brown adipocyte-specific A2ARKO inhibits FGF<em>21</em> production and accelerates cardiac damage in hypertension. Consistently, brown adipocyte-specific FGF<em>21</em> knockout abolishes the effects of A2AR agonism in attenuating hypertensive cardiac remodeling. Our findings reveal a distinctive endocrine role of BAT in hypertensive cardiac remodeling via activating A2AR/FGF<em>21</em> pathway.

1. Currently, there is no satis<em>factor</em>y treatment for pulmonary fibrosis. Emodin, a component in Chinese herbs, has been shown to have an antifibrotic effect on pancreatic fibrosis and liver fibrosis. In the present study, we tested the hypothesis that emodin may attenuate the development of pulmonary fibrosis. 2. Mice were randomly divided into five groups (n = 16 in each). One group was a control group; the remaining four groups were treated with intratracheal instillation of 3 mg/kg bleomycin (BLM). The following day, emodin (5, 10 or 20 mg/kg per day, p.o.) treatment was started for three of the BLM-treated groups and was continued for <em>21</em> days. The fourth BLM-treated group (and the control group) received daily 0.5% sodium carboxymethyl cellulose (placebo) by gavage over the same period. 3. Bleomycin challenge provoked severe pulmonary fibrosis, with marked increases in fibrosis fraction, hydroxyproline content and myeloperoxidase activity in lung tissue. Emodin treatment (10 and 20 mg/kg per day, p.o.) attenuated all these biochemical indices, as well as histopathological alterations induced by BLM. Furthermore, in mice injected with BLM, elevated levels of transforming <em>growth</em> <em>factor</em>-beta1, interleukin (IL)-4 and IL-13 were found in bronchoalveolar lavage fluid. These increases were significantly inhibited by 10 and 20 mg/kg per day emodin. 4. In cell culture, exposure of cells to 6.25, 12.5, 25 or 50 micromol/L emodin for 24 h decreased <em>fibroblast</em> proliferation. Treatment of cells with the same concentrations of emodin for 72 h decreased collagen production by <em>fibroblasts</em>. In addition, emodin (6.25, 12.5, 25 or 50 micromol/L) inhibited the steady state expression of alpha1 (I) procollagen and alpha2 (I) procollagen mRNA in a dose-dependent manner. 5. The results of the present study suggest that emodin may be effective in the treatment of pulmonary fibrosis.

The endocrine hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is induced in the adaptive response to nutrient deprivation, where it serves to regulate the integrated response to fasting via its primary receptor complex, FGF receptor 1 coupled with the co<em>factor</em> β-klotho (KLB) in target tissues. Curiously, endogenous FGF<em>21</em> levels are also elevated in preclinical models of obesity and in obese/diabetic individuals. In addition to higher FGF<em>21</em> levels, reduced KLB expression in liver and adipose tissue has been noted in these same individuals, suggesting that obesity may represent an FGF<em>21</em> resistant state. To explore the contribution of tissue-specific KLB levels to endogenous FGF<em>21</em> activity, in both fasting and high-fat diet feeding conditions, we generated animals overexpressing KLB in liver (LKLBOE) or adipose (ATKLBOE). Supportive of tissue-specific partitioning of FGF<em>21</em> action, after chronic high-fat feeding, ATKLBOE mice gained significantly less weight than WT. Reduced weight gain was associated with elevated caloric expenditure, accompanied by a reduced respiratory exchange ratio and lower plasma free fatty acids levels, suggestive of augmented lipid metabolism. In contrast, LKLBOE had no effect on body weight but did reduce plasma cholesterol. The metabolic response to fasting was enhanced in LKLBOE mice, evidenced by increased ketone production, whereas no changes in this were noted in ATKLBOE mice. Taken together, these data provide further support that specific effects of FGF<em>21</em> are mediated via engagement of distinct target organs. Furthermore, enhancing KLB expression in adipose may sensitize to endogenous FGF<em>21</em>, thus representing a novel strategy to combat metabolic disease.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an important endogenous regulator involved in the regulation of glucose and lipid metabolism. FGF<em>21</em> expression is strongly induced in animal and human subjects with metabolic diseases, but little is known about the molecular mechanism. Endoplasmic reticulum (ER) stress plays an essential role in metabolic homeostasis and is observed in numerous pathological processes, including type 2 diabetes, overweight, nonalcoholic fatty liver disease (NAFLD). In this study, we investigate the correlation between the expression of FGF<em>21</em> and ER stress. We demonstrated that TG-induced ER stress directly regulated the expression and secretion of FGF<em>21</em> in a dose- and time-dependent manner. FGF<em>21</em> is the target gene for activating transcription <em>factor</em> 4 (ATF4) and CCAAT enhancer binding protein homologous protein (CHOP). Suppression of CHOP impaired the transcriptional activation of FGF<em>21</em> by TG-induced ER stress in CHOP-/- mouse primary hepatocytes (MPH), and overexpression of ATF4 and CHOP resulted in FGF<em>21</em> promoter activation to initiate the transcriptional programme. In mRNA stability assay, we indicated that ER stress increased the half-life of mRNA of FGF<em>21</em> significantly. In conclusion, FGF<em>21</em> expression is regulated by ER stress via ATF- and CHOP-dependent transcriptional mechanism and posttranscriptional mechanism, respectively.

The eukaryotic translation initiation <em>factor</em> 2α (eIF2α) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2α protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF<em>21</em> expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf<em>21</em> expression in cell-based screens and by implication of the ISR-inducible activating transcription <em>factor</em> 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non-cell-autonomous metabolic regulation by induced expression of a potent myokine.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), an endocrine <em>factor</em> predominantly secreted from liver, possesses multiple beneficial effect on energy metabolism and insulin sensitivity in animals. This study aimed to investigate the acute change of serum FGF<em>21</em> in response to glucose challenge in humans.

METHODS

A 75-g oral glucose tolerance test was performed among 20 healthy subjects, 18 with impaired glucose tolerance (IGT) and <em>21</em> with type 2 diabetes mellitus (T2DM). Blood samples were collected for measurement of FGF<em>21</em> and other biochemical parameters. The associations of FGF<em>21</em> with insulin and other metabolic parameters were analyzed.

RESULTS

Fasting serum FGF<em>21</em> levels increased progressively from healthy, IGT to T2DM subjects (P < 0.05 for global trend). After oral glucose administration, the serum FGF<em>21</em> level showed a similar biphasic change in all three groups. It declined to a nadir level at 60 min and then increased gradually to its peak level at 180 min. FGF<em>21</em> levels at different time points of oral glucose tolerance test negatively correlated with glucose levels in all subjects, and the fold change of serum FGF<em>21</em> at different time points (compared with the basal level) were inversely associated with fold changes of insulin (P = 0.012) and C-peptide (P = 0.043) levels in healthy subjects but not in IGT and T2DM patients.

CONCLUSIONS

The dynamic change of circulating FGF<em>21</em> was associated with alterations in insulin levels in response to glucose challenge in humans. These findings support the role of FGF<em>21</em> as a potential regulator of insulin secretion and glucose metabolism in humans.

Obesity and diabetes are major challenges to global health, and there is an urgent need for interventions that promote weight loss. Dietary restriction of methionine promotes leanness and improves metabolic health in mice and humans. However, poor long-term adherence to this diet limits its translational potential. In this study, we develop a short-term methionine deprivation (MD) regimen that preferentially reduces fat mass, restoring normal body weight and glycemic control to diet-induced obese mice of both sexes. The benefits of MD do not accrue from calorie restriction, but instead result from increased energy expenditure. MD promotes increased energy expenditure in a sex-specific manner, inducing the <em>fibroblast</em> <em>growth</em> <em>factor</em> (Fgf)-<em>21</em>-uncoupling protein (Ucp)-1 axis only in males. Methionine is an agonist of the protein kinase mechanistic target of rapamycin complex (mTORC)-1, which has been proposed to play a key role in the metabolic response to amino acid-restricted diets. In our study, we used a mouse model of constitutive hepatic mTORC1 activity and demonstrate that suppression of hepatic mTORC1 signaling is not required for the metabolic effects of MD. Our study sheds new light on the mechanisms by which dietary methionine regulates metabolic health and demonstrates the translational potential of MD for the treatment of obesity and type 2 diabetes.-Yu, D., Yang, S. E., Miller, B. R., Wisinski, J. A., Sherman, D. S., Brinkman, J. A., Tomasiewicz, J. L., Cummings, N. E., Kimple, M. E., Cryns, V. L., Lamming, D. W. Short-term methionine deprivation improves metabolic health via sexually dimorphic, mTORC1-independent mechanisms.

Interest has been focused on differentiating anatomical, molecular, and physiological characteristics of the types of mammalian adipose tissues. White adipose tissue (WAT) and brown adipose tissue (BAT) are the two main forms of adipose tissue in humans. WAT functions as an endocrine organ and serves as a reservoir of energy in the form of triglycerides. The hormones released by WAT are called adipokines. BAT consists of a group of specialized cells with abundant uncoupling protein 1 (UCP1) in the inner mitochondrial membrane and also fulfills endocrine functions. Following the identification of functional (BAT) in human adults, there has been a great deal of interest in finding out how it is induced, its localization, and the mechanisms by which it regulates thermogenesis. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a key regulator of the differentiation to brown adipocytes. The main mechanisms occur through enhancing UCP1 expression. In addition, following exposure to cold or exercise, FGF<em>21</em> induces upregulation of local peroxisome proliferator-activated receptor gamma co-activator (PGC)-1-alfa and thus promotes thermogenesis in adipose tissue and skeletal muscle. FGF<em>21</em> integrates several pathways allowing the regulation of human energy balance, glucose levels, and lipid metabolism. Such mechanisms and their clinical relevance are summarized in this review.

BACKGROUND

Activated fibroblasts (myofibroblasts) play a critical role in cardiac fibrosis; however, their origin in the diseased heart remains unclear, warranting further investigation. Recent studies suggest the contribution of bone marrow fibroblast progenitor cells (BM-FPCs) in pressure overload-induced cardiac fibrosis. We have previously shown that interleukin-10 (IL10) suppresses pressure overload-induced cardiac fibrosis; however, the role of IL10 in inhibition of BM-FPC-mediated cardiac fibrosis is not known. We hypothesized that IL10 inhibits pressure overload-induced homing of BM-FPCs to the heart and their transdifferentiation to myofibroblasts and thus attenuates cardiac fibrosis.

METHODS

Pressure overload was induced in wild-type (WT) and IL10 knockout (IL10KO) mice by transverse aortic constriction. To determine the bone marrow origin, chimeric mice were created with enhanced green fluorescent protein WT mice marrow to the IL10KO mice. For mechanistic studies, FPCs were isolated from mouse bone marrow.

RESULTS

Pressure overload enhanced BM-FPC mobilization and homing in IL10KO mice compared with WT mice. Furthermore, WT bone marrow (from enhanced green fluorescent protein mice) transplantation in bone marrow-depleted IL10KO mice (IL10KO chimeric mice) reduced transverse aortic constriction-induced BM-FPC mobilization compared with IL10KO mice. Green fluorescent protein costaining with α-smooth muscle actin or collagen 1α in left ventricular tissue sections of IL10KO chimeric mice suggests that myofibroblasts were derived from bone marrow after transverse aortic constriction. Finally, WT bone marrow transplantation in IL10KO mice inhibited transverse aortic constriction-induced cardiac fibrosis and improved heart function. At the molecular level, IL10 treatment significantly inhibited transforming growth factor-β-induced transdifferentiation and fibrotic signaling in WT BM-FPCs in vitro. Furthermore, fibrosis-associated microRNA (miRNA) expression was highly upregulated in IL10KO-FPCs compared with WT-FPCs. Polymerase chain reaction-based selective miRNA analysis revealed that transforming growth factor-β-induced enhanced expression of fibrosis-associated miRNAs (miRNA-21, -145, and -208) was significantly inhibited by IL10. Restoration of miRNA-21 levels suppressed the IL10 effects on transforming growth factor-β-induced fibrotic signaling in BM-FPCs.

CONCLUSIONS

Our findings suggest that IL10 inhibits BM-FPC homing and transdifferentiation to myofibroblasts in pressure-overloaded myocardium. Mechanistically, we show for the first time that IL10 suppresses Smad-miRNA-21-mediated activation of BM-FPCs and thus modulates cardiac fibrosis.

<AbstractText>Epidemiological studies have shown that increased circulating branched-chain amino acids (BCAAs) are associated with insulin resistance and type 2 diabetes (T2D). This may result from altered energy metabolism or dietary habits.</AbstractText><AbstractText>We hypothesized that a lower intake of BCAAs improves tissue-specific insulin sensitivity.</AbstractText><AbstractText>This randomized, placebo-controlled, double-blinded, crossover trial examined well-controlled T2D patients receiving isocaloric diets (protein: 1 g/kg body weight) for 4 wk. Protein requirements were covered by commercially available food supplemented ≤60% by an AA mixture either containing all AAs or lacking BCAAs. The dietary intervention ensured sufficient BCAA supply above the recommended minimum daily intake. The patients underwent the mixed meal tolerance test (MMT), hyperinsulinemic-euglycemic clamps (HECs), and skeletal muscle and white adipose tissue biopsies to assess insulin signaling.</AbstractText><AbstractText>After the BCAA- diet, BCAAs were reduced by 17% during fasting (P < 0.001), by 13% during HEC (P < 0.01), and by 62% during the MMT (P < 0.001). Under clamp conditions, whole-body and hepatic insulin sensitivity did not differ between diets. After the BCAA- diet, however, the oral glucose sensitivity index was 24% (P < 0.01) and circulating <em>fibroblast</em>-<em>growth</em> <em>factor</em> <em>21</em> was <em>21</em>% higher (P < 0.05), whereas meal-derived insulin secretion was 28% lower (P < 0.05). Adipose tissue expression of the mechanistic target of rapamycin was 13% lower, whereas the mitochondrial respiratory control ratio was 1.7-fold higher (both P < 0.05). The fecal microbiome was enriched in Bacteroidetes but depleted of Firmicutes.</AbstractText><AbstractText>Short-term dietary reduction of BCAAs decreases postprandial insulin secretion and improves white adipose tissue metabolism and gut microbiome composition. Longer-term studies will be needed to evaluate the safety and metabolic efficacy in diabetes patients.This trial was registered at clinicaltrials.gov as NCT03261362.</AbstractText>