Systemic proteome adaptions to 7-day complete caloric restriction in humans.

Surviving long periods without food has shaped human evolution. In ancient and modern societies, prolonged fasting was/is practiced by billions of people globally for religious purposes, used to treat diseases such as epilepsy, and recently gained popularity as weight loss intervention, but we still have a very limited understanding of the systemic adaptions in humans to extreme caloric restriction of different durations. Here we show that a 7-day water-only fast leads to an average weight loss of 5.7 kg (±0.8 kg) among 12 volunteers (5 women, 7 men). We demonstrate nine distinct proteomic response profiles, with systemic changes evident only after 3 days of complete calorie restriction based on in-depth characterization of the temporal trajectories of ~3,000 plasma proteins measured before, daily during, and after fasting. The multi-organ response to complete caloric restriction shows distinct effects of fasting duration and weight loss and is remarkably conserved across volunteers with >1,000 significantly responding proteins. The fasting signature is strongly enriched for extracellular matrix proteins from various body sites, demonstrating profound non-metabolic adaptions, including extreme changes in the brain-specific extracellular matrix protein tenascin-R. Using proteogenomic approaches, we estimate the health consequences for 212 proteins that change during fasting across ~500 outcomes and identified putative beneficial (SWAP70 and rheumatoid arthritis or HYOU1 and heart disease), as well as adverse effects. Our results advance our understanding of prolonged fasting in humans beyond a merely energy-centric adaptions towards a systemic response that can inform targeted therapeutic modulation.

Differential Effects of One Meal per Day in the Evening on Metabolic Health and Physical Performance in Lean Individuals.

Background: Generally, food intake occurs in a three-meal per 24 h fashion with in-between meal snacking. As such, most humans spend more than ∼ 12-16 h per day in the postprandial state. It may be reasoned from an evolutionary point of view, that the human body is physiologically habituated to less frequent meals. Metabolic flexibility (i.e., reciprocal changes in carbohydrate and fatty acid oxidation) is a characteristic of metabolic health and is reduced by semi-continuous feeding. The effects of time-restricted feeding (TRF) on metabolic parameters and physical performance in humans are equivocal. Methods: To investigate the effect of TRF on metabolism and physical performance in free-living healthy lean individuals, we compared the effects of eucaloric feeding provided by a single meal (22/2) vs. three meals per day in a randomized crossover study. We included 13 participants of which 11 (5 males/6 females) completed the study: age 31.0 ± 1.7 years, BMI 24.0 ± 0.6 kg/m2 and fat mass (%) 24.0 ± 0.6 (mean ± SEM). Participants consumed all the calories needed for a stable weight in either three meals (breakfast, lunch and dinner) or one meal per day between 17:00 and 19:00 for 11 days per study period. Results: Eucaloric meal reduction to a single meal per day lowered total body mass (3 meals/day -0.5 ± 0.3 vs. 1 meal/day -1.4 ± 0.3 kg, p = 0.03), fat mass (3 meals/day -0.1 ± 0.2 vs. 1 meal/day -0.7 ± 0.2, p = 0.049) and increased exercise fatty acid oxidation (p < 0.001) without impairment of aerobic capacity or strength (p > 0.05). Furthermore, we found lower plasma glucose concentrations during the second half of the day during the one meal per day intervention (p < 0.05). Conclusion: A single meal per day in the evening lowers body weight and adapts metabolic flexibility during exercise via increased fat oxidation whereas physical performance was not affected.

FSH Levels Are Related to E-cadherin Expression and Subcellular Location in Nonfunctioning Pituitary Tumors.

CONTEXT: Gonadotroph pituitary neuroendocrine tumors (PitNETs) can express follicle-stimulating hormone (FSH) and luteinizing hormone (LH) or be hormone negative, but they rarely secrete hormones. During tumor development, epithelial cells develop a mesenchymal phenotype. This process is characterized by decreased membranous E-cadherin and translocation of E-cadherin to the nucleus. Estrogen receptors (ERs) regulate both E-cadherin and FSH expression and secretion. Whether the hormone status of patients with gonadotroph PitNETs is regulated by epithelial-to-mesenchymal transition (EMT) and ERs is unknown. OBJECTIVES: To study the effect of EMT on hormone expression in gonadotroph nonfunctioning (NF)-PitNETs. DESIGN: Molecular and clinical analyses of 105 gonadotroph PitNETs. Immunohistochemical studies and real-time quantitative polymerase chain reaction were performed for FSH, LH, E-cadherin, and ERα. Further analyses included blood samples, clinical data, and radiological images. SETTING: All patients were operated on in the same tertiary referral center. RESULTS: NF-PitNET with high FSH expression had decreased immunohistochemical staining for membranous E-cadherin (P < .0001) and increased staining for nuclear E-cadherin (P < .0001). Furthermore, high FSH expression was associated with increased ERα staining (P = .0002) and ERα mRNA (P = .0039). Circulating levels of plasma-FSH (P-FSH) correlated with FSH staining in gonadotroph NF-PitNET (P = .0025). Tumor size and invasiveness was not related to FSH staining, E-cadherin, or ERα. LH expression was not associated with E-cadherin or ERα. CONCLUSION: In gonadotroph PitNETs, FSH staining is related to E-cadherin, ERα expression, and circulating levels of P-FSH. There was no association between FSH staining and invasiveness. The clinical significance of these findings will be investigated in ongoing prospective studies.

TGFBR3L-An Uncharacterised Pituitary Specific Membrane Protein Detected in the Gonadotroph Cells in Non-Neoplastic and Tumour Tissue.

Here, we report the investigation of transforming growth factor beta-receptor 3 like (TGFBR3L), an uncharacterised pituitary specific membrane protein, in non-neoplastic anterior pituitary gland and pituitary neuroendocrine tumours. A polyclonal antibody produced within the Human Protein Atlas project (HPA074356) was used for TGFBR3L staining and combined with SF1 and FSH for a 3-plex fluorescent protocol, providing more details about the cell lineage specificity of TGFBR3L expression. A cohort of 230 pituitary neuroendocrine tumours were analysed. In a subgroup of previously characterised gonadotroph tumours, correlation with expression of FSH/LH, E-cadherin, oestrogen (ER) and somatostatin receptors (SSTR) was explored. TGFBR3L showed membranous immunolabeling and was found to be gonadotroph cell lineage-specific, verified by co-expression with SF1 and FSH/LH staining in both tumour and non-neoplastic anterior pituitary tissues. TGFBR3L immunoreactivity was observed in gonadotroph tumours only and demonstrated intra-tumour heterogeneity with a perivascular location. TGFBR3L immunostaining correlated positively to both FSH (R = 0.290) and LH (R = 0.390) immunostaining, and SSTR3 (R = 0.315). TGFBR3L correlated inversely to membranous E-cadherin staining (R = -0.351) and oestrogen receptor ß mRNA (R = -0.274). In conclusion, TGFBR3L is a novel pituitary gland specific protein, located in the membrane of gonadotroph cells in non-neoplastic anterior pituitary gland and in a subset of gonadotroph pituitary tumours.

GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress.

Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles.

Epinephrine increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown, and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated in condition with decreased GS activation. Saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously in Wistar rats (∼130 g) with low (24-h-fasted), normal (normal diet), and high glycogen content (fasted-refed), and epitrochlearis muscles were removed after 3 h and incubated ex vivo, eliminating epinephrine action. Epinephrine injection reduced glycogen content in epitrochlearis muscles with high (120.7 ± 17.8 vs. 204.6 ± 14.5 mmol/kg, P < 0.01) and normal glycogen (89.5 ± 7.6 vs. 152 ± 8.1 mmol/kg, P < 0.01), but not significantly in muscles with low glycogen (90.0 ± 5.0 vs. 102.8 ± 7.8 mmol/kg, P = 0.17). In saline-injected rats, GS phosphorylation at sites 2+2a, 3a+3b, and 1b was higher and GS activity lower in muscles with high compared with low glycogen. GS sites 2+2a and 3a+3b phosphorylation decreased and GS activity increased in muscles where epinephrine decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where epinephrine injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a+3b phosphorylation independently of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where epinephrine injection decreased glycogen content. In conclusion, epinephrine stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low, glycogen content. Epinephrine-stimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content.

The myokine decorin is regulated by contraction and involved in muscle hypertrophy.

The health-promoting effects of regular exercise are well known, and myokines may mediate some of these effects. The small leucine-rich proteoglycan decorin has been described as a myokine for some time. However, its regulation and impact on skeletal muscle has not been investigated in detail. In this study, we report decorin to be differentially expressed and released in response to muscle contraction using different approaches. Decorin is released from contracting human myotubes, and circulating decorin levels are increased in response to acute resistance exercise in humans. Moreover, decorin expression in skeletal muscle is increased in humans and mice after chronic training. Because decorin directly binds myostatin, a potent inhibitor of muscle growth, we investigated a potential function of decorin in the regulation of skeletal muscle growth. In vivo overexpression of decorin in murine skeletal muscle promoted expression of the pro-myogenic factor Mighty, which is negatively regulated by myostatin. We also found Myod1 and follistatin to be increased in response to decorin overexpression. Moreover, muscle-specific ubiquitin ligases atrogin1 and MuRF1, which are involved in atrophic pathways, were reduced by decorin overexpression. In summary, our findings suggest that decorin secreted from myotubes in response to exercise is involved in the regulation of muscle hypertrophy and hence could play a role in exercise-related restructuring processes of skeletal muscle.