Effect of lenti-adiponectin-GFP-NaKtide on adiposity, metabolic balance, and locomotion in C57BL6 mice fed a WD

The effectiveness and specificity of the NaKtide lentiviral-construct were evaluated by immunofluorescence studies in C57BL6 mice fed a WD. Since adiponectin is expressed specifically in adipocytes, the lentiviral construct with NaKtide driven by an adiponectin promoter was used to achieve NaKtide expression specifically in adipocytes (Fig. S1). Results showed that NaKtide expression was exclusively present in the adipose tissue and not visibly expressed in liver and brain tissues (Fig. S2). Further, our results showed that mice fed a WD exhibited a significant increase in body weight over a period of 12 weeks compared to the mice on normal chow (Fig. 1A). The increase in the body weight was significantly attenuated by the transduction of lenti-adiponectin-GFP-NaKtide (Fig. 1A). Our results also demonstrated a significant increase in the visceral and subcutaneous fat, liver weight, and heart weight in the mice fed a WD, which was markedly reduced with lenti-adiponectin-GFP-NaKtide treatment (Table 1). Food intake and energy intake did not differ amongst the different experimental groups (Fig. 1B,C). Energy expenditure was determined as heat production rate in units of kcal/kg/day3. The results showed significant reduction in the energy expenditure of mice fed a WD compared to control group; this energy expenditure was significantly increased with lenti-adiponectin-GFP-NaKtide treatment (Fig. 1D). Further, our results showed that oxygen consumption in mice fed a WD alone (2491 ± 149 mL/kg/hr) was significantly (p < 0.01) decreased as compared to control (3104 ± 88 mL/kg/hr). Treatment with lenti-adiponectin-GFP-NaKtide (3146 ± 232 mL/kg/hr) significantly (p < 0.01) improved the oxygen consumption as compared to WD alone. Since obesity has been associated with decreased locomotion3,28, we further looked at the effect of lenti-adiponectin-GFP-NaKtide on movement. Locomotor activity was measured as previously described3. Mice fed a WD showed decreased locomotion, and this was normalized by treatment with the lenti-adiponectin-GFP-NaKtide (Fig. 1E).

Figure 1 Effect of lenti-adiponectin-GFP-NaKtide on adiposity, metabolic balance, and locomotion in C57BL6 mice fed a WD. Body weight (A). Food intake (B) Energy intake (C) Energy expenditure (D), and Locomotion (E) determined via CLAMS cages after 48 hours. Data are displayed as”scatter plots” showing data points and “box plots” showing the distribution of a continuous variable as described in the Methods section. N = 12–14/group; *p < 0.05 vs. Control (CTR), **p < 0.01 vs. CTR, #p < 0.05 vs. Western Diet (WD), ##p < 0.01 vs. WD. Full size image

Table 1 Effect of lenti-adipo-NaKtide on weights and mitochondrial and inflammatory markers in adipose tissue. Full size table

Effect of lenti-adiponectin-GFP-NaKtide on adipocyte phenotype and systemic inflammatory profile in C57BL6 mice fed a WD

The WD altered the morphological phenotype of visceral adipocytes with dramatic increases in the size and fat content of these cells. Mice treated with the lenti-adiponectin-GFP-NaKtide showed an improved adipocyte phenotype as evidenced by significantly increased numbers of adipocyte, but significant decreases in adipocyte cell area, when compared to mice fed a WD alone (Fig. 2A–C). Mice injected with lenti-adiponectin-GFP-NaKtide and fed the normal chow diet showed no significant differences in any of the aforementioned measurements from the control mice. There was also no significant difference noted between mice injected with the empty vector control, the lenti-adiponectin-GFP lacking the NaKtide sequence that were fed a WD when compared to mice fed a WD alone. We next evaluated the markers directly tied to the altered adipocyte phenotype that plays a causal role in the aggravation of systemic oxidative stress. Our Western blot analysis showed the upregulated expression of lipogenic marker, fatty acid synthase (FAS) and adipogenic markers, peroxisome proliferator-activated receptor gamma (PPARγ) and mesoderm specific transcript gene (MEST) in WD fed mice. This upregulation was significantly attenuated in lenti-adiponectin-GFP-NaKtide transduced mice (Fig. 2D–F). Since adipose mitochondria participate in energy expenditure, we measured genes of mitochondrial biogenesis in adipose tissue. Our results showed a significant down regulated expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a major regulator of mitochondrial biogenesis, in mice fed a WD, as compared to control. This down regulation was prevented by lenti-adiponectin-GFP-NaKtide treatment (Fig. 2G). Similarly, the expression of genes related to mitochondrial biogenesis and the “browning” phenomenon of adipose tissue, including expression of mitofusin (MFN) 1 and 2, and sirtuin 1 (Sirt1) were significantly down regulated in mice fed a WD. Importantly, the expression of the aforementioned genes were all significantly increased upon treatment with lenti-adiponectin-GFP-NaKtide (Table 1). Further, our results showed that the expression level of the inflammatory cytokine, tumor necrosis factor alpha (TNFα), was significantly attenuated by treatment with lenti-adiponectin-GFP-NaKtide (Table 1). Leptin, a hormone secreted by adipocytes, is a potent inducer of ROS generation by promoting inflammation and oxidative stress29,30. Mice fed a WD exhibited upregulated levels of leptin, as compared to control. This was significantly attenuated by lenti-adiponectin-GFP-NaKtide treatment (Table 1).

Figure 2 Effect of lenti-adiponectin-GFP-NaKtide on adipocyte phenotype, mitochondrial biogenesis, inflammatory markers, and Na/K-ATPase signaling cascade in C57BL6 mice fed a WD. Representative H&E staining in visceral adipose tissue. Images taken with 20x objective lens; scale bar represents 100 µm (A). Quantitative analysis of the adipocyte number (B) and adipocyte area (C) in visceral adipose tissue. Western blot analysis of visceral adipose tissue homogenates, with data shown as mean band density normalized to GAPDH, for fatty acid synthase (FAS) (D), peroxisome proliferator-activated receptor gamma (PPARγ) (E), mesoderm specific transcript (MEST) (F) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) (G). Quantitative analysis of protein carbonylation levels (shown as 2, 4-dinitrophenylhydrazine (DNP) expression) with Coomassie staining as a loading control (H). Immunoblot analysis of α-1 subunit (I) and α-2 subunit (J) with data shown as mean band density normalized to GAPDH. pSrc immunoblot analysis with data shown as mean band density normalized to total Src (K). All gels have been cropped above and below the band, and the full blots have been included in Supplementary Fig. S5D–K. N = 12–14/group; *p < 0.05 vs. CTR, **p < 0.01 vs. CTR, #p < 0.05 vs. WD, ##p < 0.01 vs. WD. Full size image

The insulin resistance in mice fed a WD was reversed by treatment with lenti-adiponectin-GFP-NaKtide (Fig. 3A). Inflammatory cytokines are both indicative of high levels of oxidative stress as well as contribute to the production of oxidants2. Mice fed a WD showed significantly upregulated plasma levels of the inflammatory cytokines TNFα, monocyte chemoattractant protein-1 (MCP1), and interleukin-6 (IL-6) (Fig. 3B–D). Treatment with the lenti-adiponectin-GFP-NaKtide ameliorated the increases in the plasma concentrations of these inflammatory cytokines. Our results showed increases in plasma leptin concentrations in mice fed a WD. This was attenuated by treatment with lenti-adiponectin-GFP-NaKtide (Fig. 3E).

Figure 3 Effect of lenti-adiponectin-GFP-NaKtide on systemic inflammatory cytokines, and metabolic profile in C57BL6 mice fed a WD. Glucose tolerance test (A), Plasma levels of TNFα (B), monocyte chemoattractant protein-1 (MCP1) (C) interleukin-6 (IL-6) (D) and leptin (E) assessed by ELISA assay. N = 12–14/group; *p < 0.05 vs. CTR, **p < 0.01 vs. CTR, #p < 0.05 vs. WD, ##p < 0.01 vs. WD. Full size image

Effect of lenti-adiponectin-GFP-NaKtide on Na/K-ATPase/Src signaling cascade in C57BL6 mice fed a WD

Protein carbonylation is an established method for assessing oxidative stress31,32. Our results showed a significant increase in protein carbonylation, measured by 2,4-dinitrophenylhydrazine (DNP), in mice fed a WD which was attenuated by treatment with the lenti-adiponectin-GFP-NaKtide (Fig. 2H). Expression of the α1 subunit of the Na/K-ATPase was significantly down regulated in visceral fat of mice fed a WD. This was prevented by lenti-adiponectin-GFP-NaKtide treatment (Fig. 2I). Conversely, the up-regulated expression of the α2 subunit in mice fed a WD was significantly attenuated by lenti-adiponectin-GFP-NaKtide (Fig. 2J). Treatment with lenti-adiponectin-GFP-NaKtide also prevented Src activation in mice fed a WD (Fig. 2K).

Effect of lenti-adiponectin-GFP-NaKtide on neurodegeneration in C57BL6 mice fed a WD

Obesity and systemic oxidative stress have been implicated with neurodegenerative disorders, so we next examined the effect of lenti-adiponectin-GFP-NaKtide on these parameters. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the production of dopamine; the expression of both TH and the type 2 dopamine receptor (D2) correlate with locomotor activity33. TH staining showed decreased expression of TH in the prefrontal cortex of the brain in mice fed a WD; western blot analysis of TH showed significant decrease in brains of mice fed WD (Fig. 4A,B). Treatment with lenti-adiponectin-GFP-NaKtide negated these changes. Furthermore, expression of D2 receptor in the brain was significantly down regulated in mice fed a WD. This was also improved by the treatment with the lenti-adiponectin-GFP-NaKtide (Fig. 4B). Further, our results demonstrated an increase in protein carbonylation in the brain tissues of mice fed a WD; this was attenuated by the lenti-adiponectin-GFP-NaKtide (Fig. 4C). Our results also showed decreased expression of postsynaptic density protein 95 (PSD95), a marker of synaptic plasticity34 in the brain tissues of WD mice. Treatment with lenti-adiponectin-GFP-NaKtide improved this PSD95 expression (Fig. 4D). Tau, a marker of tangled neuron tracks and a hallmark of neurodegenerative disease34, was up regulated in the brains of mice fed WD and markedly reduced by the lenti-adiponectin-GFP-NaKtide (Fig. 4E). The TUNEL assay demonstrated that apoptosis was increased in the brain tissue of mice fed WD. Apoptosis was also attenuated by the lenti-adiponectin-GFP-NaKtide treatment (4F, G).

Figure 4 Effect of lenti-adiponectin-GFP-NaKtide on neurodegeneration in brain tissue in C57BL6 mice fed a WD. Representative and quantitative analysis based on tyrosine hydroxylase staining (A), images taken with 20X objective lens; scale represents 100 µm. Immunoblot analysis for tyrosine hydroxylase (TH) (B). Protein carbonylation levels with Coomassie staining as a loading control (C). Immunoblot analysis of D2 receptor with data shown as mean band density normalized to GAPDH (D). Immunoblot analysis for marker of synaptic plasticity, PSD95 (E), and marker of tangles in neuron tracks, Tau (F). Representative images and quantification of the TUNEL assay in brain tissue (G–H). All gels have been cropped above and below the band, and the full blots have been included in Supplementary Fig. S6 B-F. N = 12–14/group; *p < 0.05 vs. CTR, **p < 0.01 vs. CTR, #p < 0.05 vs. WD, ##p < 0.01 vs. WD. Full size image

Effect of lenti-adiponectin-GFP-NaKtide on hepatic histology, inflammation, and fibrosis in C57BL6 mice fed a WD

Next, we aimed to determine whether NaKtide targeted specifically to adipocytes was able to attenuate the development of nonalcoholic steatohepatitis (NASH). H&E staining of liver sections from C57BL6 mice fed a WD showed inflammation and increased lipid accumulation in the liver as compared to the control group (Fig. S3A). Treatment with lenti-adiponectin-GFP-NaKtide exhibited decreased lipid and inflammatory cell infiltration. We note that the viral transduction did not lead to demonstrable hepatic NaKtide expression (Fig. S1) implying an indirect effect. Mice fed a WD had significantly increased lipid accumulation in the liver compared to mice fed a normal chow diet as demonstrated by Oil Red O staining. Administration of lenti-adiponectin-GFP-NaKtide decreased lipid accumulation in mice fed a WD (Fig. S3B). CD36, a fatty acid transport protein, contributes to the progression of NASH3. Our results showed that CD36 mRNA expression was decreased by lenti-adiponectin-GFP-NaKtide treatment as compared to the mice fed a WD (Fig. S3C). Similarly, TNFα and F4/80 mRNA expression, markers of inflammation and macrophage/kupffer cells infiltration, were also increased in mice fed a WD as compared to control mice (Fig. S3D,E). These changes were attenuated by the lenti-adiponectin-GFP-NaKtide treatment. Furthermore, mRNA expression of hepatic matrix metalloproteinases (MMP) 2 and 9, genes related to fibrogenesis, were also elevated in mice fed a WD, and these increases were also attenuated with lenti-adiponectin-GFP-NaKtide treatment (Fig. S2F,G). These findings indicate that the lenti-adiponectin-GFP-NaKtide not only improves the metabolic profile in adipocytes, but also has profound effects on the liver through indirect mechanisms.

Correlational analysis of markers associated with reprogramming of adipocyte phenotype, NASH and neurodegeneration in C57BL6 mice fed a WD

First, we examined the degree of correlation between the various measurements performed in our study. These data are summarized in Fig. 5A. Our results showed a number of strong correlations. In particular, it was clear that the plasma levels of inflammatory cytokines correlated with locomotor activity (Fig. 5A). When we performed multiple linear regression analysis, it was clear that a model consisting of plasma TNFα and IL-6 predicted the locomotor activity with excellent accuracy (R2 = 0.84) (Fig. 5B). We have also included a webpage illustrating heat map and corresponding box plates as Fig. S4.