Plasma MANF is increased in fasting humans

The study population consisted of 40 adults with an average age of 54.5 ± 12.5 years (range 22–77 years) who volunteered to fast for therapeutic effects on various health conditions (Table 1). The average concentration of MANF in human plasma samples before fasting was 6.1 ± 2.3 ng/ml (median: 5.8 ng/ml; range: 2.6–14.0 ng/ml, Fig. 1a). In samples collected after fasting, the average MANF concentration was 7.5 ± 3.0 ng/ml (median: 7.1 ng/ml; range: 3.2–16.8 ng/ml). The mean MANF plasma concentration increased by 1.4 ng/ml (+23%) during fasting (p < 0.001) (Fig. 1a,b). The MANF concentration in plasma increased in 72.5% (n = 29/40) of the study subjects with an average increase of 2.3 ± 2.0 ng/ml (range: 0.2–11.0 ng/ml, +44%) from pre-fasting MANF concentration (Fig. 1b). In cases of decreased plasma MANF levels (n = 9/40, 22.5%), the average decrease was −1.2 ± 1.0 ng/ml (range: −0.1 to −3.6 ng/ml, −17%). MANF concentration was unchanged in two samples (5%).

Table 1 Selected background information of the human study group. Full size table

Figure 1 Plasma MANF levels increase with therapeutic fasting in humans. (a) MANF concentrations in human plasma samples by ELISA. (b) MANF concentration increased in 72.5% (n = 29/40) of cases after fasting (red lines). In 22.5% (n = 9/40) of cases, MANF decreased (blue lines) and in two cases (5%) remained unchanged (green lines). (c) Circulating concentrations of MANF (ng/ml) and adiponectin (μg/ml) correlated inversely before fasting (r s = −0.36, p = 0.04, n = 32). (d) Percentage change in plasma MANF and adiponectin levels between pre- and post-fasting samples correlated inversely (r s = −0.49, p = 0.01, n = 32). (e) Plasma adiponectin concentrations before and after fasting by ELISA. (f) Adiponectin concentration increased in 59.4% (n = 19/32) and decreased in 40.6% (n = 13/32) of cases after fasting (red and blue lines, respectively). Statistical significance was analysed by Wilcoxon signed ranks test in (b,f). Correlations were analysed by Spearman’s rank correlation in (c,d). n.s. = not significant. Full size image

The average duration of fasting was 15.3 ± 6.3 days (range 9–49 days, Table 1). We found no correlation between time of fasting and MANF concentration (ng/ml) after fasting (r s = −0.16, p = 0.34, n = 40), or the percentage change in MANF during of fasting (r s = 0.07, p = 0.66, n = 40; data not shown).

Correlation analysis of circulating MANF with BMI, glucose, and insulin

MANF serum levels did not differ between lean (BMI < 25 kg/m2), overweight (BMI 25–29.9 kg/m2), or obese (BMI ≥ 30 kg/m2) subjects in pre- or post-fasting situations. The percentage change in MANF during fasting also did not differ between the different BMI groups (p > 0.05; data not shown). Circulating MANF concentrations showed no correlation with weight, BMI, waist circumference, or with blood lipids (Table 2). Furthermore, no correlations were observed between MANF and blood glucose or insulin levels, or with the levels measured after oral glucose tolerance test (OGTT). No correlation was found between MANF levels and homeostatic model assessment (HOMA) indexes for the estimation of insulin resistance (IR) and beta cell function (%β) (HOMA2 IR or HOMA2%β, respectively).

Table 2 Correlation analysis between circulating MANF levels and clinical variables. Full size table

Pre-fasting MANF and adiponectin show an inverse correlation

We found an inverse correlation between MANF and adiponectin concentrations measured in pre-fasting plasma samples (r s = −0.36, p = 0.04, n = 32, Fig. 1c). Furthermore, the percentage change in MANF concentrations during fasting correlated inversely with the percentage change in adiponectin levels (r s = −0.49, p = 0.01, n = 32, Fig. 1d). In contrast to plasma MANF, average adiponectin levels measured before and after fasting (9.4 ± 5.7 μg/ml and 10.5 ± 4.9 μg/ml, respectively) did not significantly differ. Plasma adiponectin concentration was increased in 59.4% (n = 19/32) and decreased in 40.6% (n = 13/32) of the study subjects (p = 0.15; Fig. 1e,f). No correlations were found between the plasma levels of MANF and leptin or insulin like-growth factor-1 (IGF-1) (Table 2).

Generation and validation of mouse MANF ELISA

To quantify endogenous MANF protein levels in mouse serum and tissues, we developed a mouse (m)MANF ELISA and tested its specificity by MANF KO mouse samples15. The sensitivity of the mouse MANF ELISA was 29 pg/ml. The assay detected both mouse and human MANF but did not detect homologous human cerebral dopamine neurotrophic factor (CDNF) with 59% amino acid identity31 and did not respond to MANF KO mouse serum or tissue lysates in contrast to samples from wild-type mice (Supplementary Table S1). The mMANF ELISA yielded a slightly different concentration-response curve for recombinant mouse (rm) and human (rh) MANF proteins (Supplementary Table S1). The endogenous MANF concentration in mouse serum samples was calculated by the standard curve of rmMANF protein whereas endogenous mouse MANF in tissue lysates was calculated by the standard curve of rhMANF. This was because mouse tissue samples only gave acceptable dilutional linearity when compared to the rhMANF standard curve (Supplementary Table S2).

Recombinant mMANF could be measured with acceptable accuracy and precision (total error within 15%) at concentrations of 31.25 to 1000 pg/ml. The average intra- and interassay variations were 5.1% coefficient of variation (CV) (range: 3.7–7.5%) and 9.7% CV (range: 5.4–16.5%), respectively (Supplementary Table S3). The dynamic range of the mMANF ELISA for rhMANF was 62.5 to 1,000 pg/ml and the average intra- and interassay variations were 9.8% CV (range: 7.5–13.0%) and 8.6% CV (range: 4.7–13.4%), respectively (Supplementary Table S3). The linearity of dilution was within 80% to 106% in mouse serum samples (n = 7) and 90% to 112% in mouse tissue lysates (n = 5) (Supplementary Table S4). The average recovery of 50 to 250 pg/ml spikes of recombinant mMANF and hMANF to study samples was 96 ± 8% in mouse sera (n = 18) and 99 ± 5% in tissue lysates, respectively (n = 6, Supplementary Table S5).

We found that mouse MANF serum concentrations measured using mouse MANF ELISA correlated positively with the absorbance values at 414 nm (Abs 414nm ), signifying the extent of haemolysis in the serum samples (r s = 0.86, p < 0.001, n = 90, Supplementary Fig. S1a). By excluding mouse serum samples with Abs 414nm ≥ 0.3, the positive correlation between MANF concentrations and Abs 414nm was abolished (r s = 0.23, p = 0.16, n = 39, Supplementary Fig. S1b), indicating that low levels of haemolysis do not dictate serum MANF concentration values. Therefore, only the MANF values that were quantified from mouse cardiac serum samples with a cut-off limit of 0.3 for Abs 414nm we included in the analysis.

MANF protein levels are increased in the liver after change from high-fat to normal diet and weight loss in mice

To further investigate the changes of MANF levels in relation to diet change in a mammalian model system, we analysed MANF concentrations in serum samples from mice on diets with different fat content. A group of DIO mice was fed a HFD until 13 weeks-of-age and then switched to a ND for 2 weeks (HFD/ND), while a parallel group remained on the HFD. A control group of mice was fed a ND throughout the experiment (n = 12/group).

The average weight of the ND, HFD, and HFD/ND groups differed over time as analysed using repeated measures ANOVA (F(2,33) = 19.7, p < 0.001). Follow-up pairwise comparisons of the interaction with Bonferroni correction revealed that the mean weight in the HFD and HFD/ND groups fed a HFD was higher than in the ND group at the ages of 10 to 13 weeks (p < 0.001, Fig. 2a). After the change from HFD to ND, the mean weight of HFD/ND group was significantly lower than that of the HFD group (p < 0.001) and similar to that of the ND group (p = 1.0) as measured at the age of 14 and 15 weeks (Fig. 2a).

Figure 2 MANF concentration increases in mouse liver after change from high-fat to normal diet. (a) Average body weight was higher in mice on high fat diet (HFD and HDF/ND groups) than controls on a normal diet (p < 0.001, weeks 10–13). After change from a HFD to a ND, the HFD/ND group lost weight when compared with the HFD group (p < 0.001, weeks 14 and 15). The average weight of the HFD/ND group decreased similarly as the ND group (p = 1.0, weeks 14 and 15). (b) Serum MANF levels did not differ between the study groups as measured by ELISA. (c) MANF concentration increased in the liver of the HFD/ND group compared to the HFD or ND groups (p = 0.001 for both). (d) Serum MANF (ng/ml) and liver MANF (ng/mg total protein) correlated positively (r = 0.61, p = 0.037, n = 12) in the HFD/ND group. Statistical significance was analysed by repeated measures ANOVA followed by pairwise comparisons with Bonferroni correction in (a). Differences between group means were analysed by one-way ANOVA followed by Tukey HSD test in (b,c). Correlation was analysed by Pearson’s correlation in (d). n.s. = not significant; HFD, high-fat diet; ND, normal diet. Full size image

The MANF serum concentration in the ND group at 15 weeks-of-age was 3.86 ± 1.09 ng/ml (range: 2.32–5.85 ng/ml, n = 10). In the HFD group, the MANF concentration was 2.84 ± 0.57 ng/ml (range: 2.11–3.82 ng/ml, n = 6), while in the HDF/ND group the corresponding concentration was 4.44 ± 1.76 ng/ml (range: 1.36–6.82 ng/ml, n = 12, Fig. 2b). The average concentration of serum MANF was 1.62 ng/ml (i.e., 58%) higher in the HFD/ND group than the HFD group. The differences in MANF concentrations between the ND, HFD, and HFD/ND groups did not reach statistical significance (p = 0.076). Serum MANF concentration in the HFD/ND group did not correlate with weight (r = 0.24, p = 0.46, n = 12, data not shown) as measured at 15 weeks-of-age.

To study the effects of diet change on MANF levels in tissues which are important for systemic energy homeostasis4, we quantified MANF concentrations in the liver, skeletal muscle, and pancreas of the mice. MANF levels did not differ between the ND, HFD, or HFD/ND study groups in the pancreas or skeletal muscle (data not shown). However, the mean MANF concentration increased by 72% in the livers of mice from the HFD/ND group (p = 0.001, Fig. 2c). The liver MANF concentration in the ND and HFD group was 78.7 ± 7.7 and 79.5 ± 34.5 ng/mg total protein, respectively, while in the HFD/ND group the corresponding concentration was 136.4 ± 46.8 ng/mg total protein (p = 0.001, n = 12 in all, Fig. 2c). We observed that liver and serum MANF levels correlated positively in the HFD/ND group (r = 0.61, p = 0.037, n = 12, Fig. 2d) but not in the ND (r = 0.28, p = 0.43, n = 10, data not shown) or HFD group (r = 0.29, p = 0.57, n = 6, data not shown).

Inverse correlation between liver MANF and serum adiponectin in mice

Serum MANF and adiponectin levels did not correlate statistically significantly in the mice of the HFD/ND group (r = −0.53, p = 0.09, n = 11, Fig. 3a). However, we found a strong inverse correlation between circulating adiponectin and liver MANF protein concentrations (r = −0.84, p = 0.001, n = 11, Fig. 3b).

Figure 3 Concentrations of circulating adiponectin and liver MANF correlate inversely in mice after diet change. (a) Trend of inverse correlation between serum MANF and adiponectin concentrations (r = −0.53, p = 0.09, n = 11) in the HFD/ND group. (b) Inverse correlation between liver MANF and circulating adiponectin levels (r = −0.84, p = 0.001, n = 11) in the HFD/ND group. Correlation was analysed by Pearson’s correlation in (a,b). HFD, high-fat diet; ND, normal diet. Full size image

Liver MANF concentrations are associated with changes in UPR markers

We next analysed the transcript levels of UPR-related genes in liver samples from the ND, HFD, and HFD/ND groups to determine if the observed increase in liver MANF levels of the HFD/ND group was related to ongoing ER stress. In the liver samples of the obese HFD group, transcript levels of Atf6a, spliced X-box-binding protein 1 (sXbp1), activating transcription factor 4 (Atf4), DNA damage-inducible transcript 3 (Ddit3, also known as Chop), and DnaJ homolog subfamily B member 9 (Dnajb9, also known as Erdj4) were increased compared to the ND group, suggesting that the three UPR sensors ATF6α, IRE1α, and PERK were activated. Activated ATF6α translocates to the Golgi where it is cleaved to produce a transcription factor that induces expression of UPR target genes, including chaperones and a pro-apoptotic transcription factor Chop32,33. sXbp1, generated downstream of IRE1α, induces expression chaperones and components of ER associated degradation (e.g. Erdj419). ATF4, downstream of PERK, regulates induction of Chop34.

The diet change from HFD to ND led to a decrease in Atf6α, Chop, and Erdj4 levels in the liver, implying attenuation of UPR during the 2 weeks of fat withdrawal. In contrast, the mean transcript levels of Manf (p = 0.003), Grp78 (p = 0.039), and total Xbp1 (tXBP1) (p < 0.001) were increased in the HFD/ND group compared to the ND group (Fig. 4a). The levels of sXbp1 (p = 0.006) remained high after the diet change in the HFD/ND group when compared with the ND group.

Figure 4 Expression of Manf and UPR genes in livers of mice after diet change as measured by qRT-PCR. (a) Expression of Erdj4, Atf6α, and Chop was downregulated by diet fat removal and weight loss in the HFD/ND group compared to the HFD group. (b) MANF protein and mRNA levels correlated positively in the HFD/ND group (r = 0.79, p = 0.02, n = 12). (c) Manf and Grp78 mRNA levels correlated positively (r = 0.94, p < 0.001, n = 12) in the HFD/ND group. Statistical significance was analysed by one-way ANOVA followed by Tukey HSD test in (a). Correlation was analysed by Pearson’s correlation in (b,c). HFD, high-fat diet; ND, normal diet. Full size image

Manf transcript and protein levels correlated positively in the livers of the HFD/ND group (r = 0.79, p = 0.002, n = 12, Fig. 4b). Furthermore, Manf levels correlated positively with those of Grp78 (r = 0.94, p < 0.001, n = 12, Fig. 4c) in the HFD/ND group. Similarly, a positive correlation between liver Manf and Grp78 was found in the ND (r = 0.72, p = 0.009, n = 12, data not shown) and HFD (r = 0.83, p = 0.001, n = 12, data not shown) groups.