PSTi8 with metformin ameliorates perimenopause induced steatohepatitis associated ER stress by regulating SIRT-1/SREBP-1c axis

Abstract

Aims: Hepatic steatosis in women confronting menopause is the manifestation of substantial fructose consumption and forms a positive feedback loop to develop endoplasmic reticulum (ER) stress. Previously pancreastatin inhibitor peptide-8 (PSTi8) and Metformin (Met) combination effectively ameliorated hepatic lipid accumulation in high fructose diet (HFrD) fed diabetic mice models at reduced doses. Moreover, SIRT-1 plays a crucial role in the regulation of SREBP-1c. Hence we hypothesized that Met and PSTi8 in combination (at therapeutic lower doses) could mitigate hepatic steatosis linked ER stress by activating SIRT-1 and precluding SREBP-1c in HFrD fed 4-Vinylcyclohexenediepoxide (HVCD) induced perimenopausal rats.

Main methods: HVCD rats were fed HFrD for 12 weeks, accompanied by 14 days of treatment with Met, PSTi8, and combination. We confirmed model establishment by estrus cycle study, estradiol level, and intraperitoneal glucose tolerance test. Plasma lipid profile and liver function were determined. Also, mRNA and protein expressions were examined. Moreover, distribution of SIRT-1 and SREBP-1c was detected in HepG2 cells by immunofluorescence staining.

Key findings: HVCD group displayed augmented insulin resistance (IR), lipogenesis, and ER stress in the liver. Combination therapy improved the estrus cyclicity, estradiol, and lipid profile of HVCD rats. Met and PSTi8 combination reduced hepatic SREBP-1c and triggered SIRT-1 expression in high fructose-induced insulin-resistant HepG2 cells; consequently, combination therapy attenuated ER stress.

Significance: Succinctly, present research promotes impetus concerning the remedial impact of Met with PSTi8 at lower therapeutic doses to ameliorate hepatic IR, steatosis, and associated ER stress by revamping the SIRT-1/SREBP-1c axis in perimenopausal rats.

Keywords: Combination therapy (Met + PSTi8); ER stress; Hepatic steatosis; Metformin; PSTi8; Perimenopausal rat; SIRT-1; SREBP-1c.

Figure 1

Estrus cycle study and examination of plasma estradiol level in HFrD/VCD-induced perimenopausal rat model. (a) Experimental design and treatment intervention (b) Estrus cycle evaluation for 10 days and stages of the estrus cycle, (c) representing different stages of estrus cycle including (d) serum estradiol level. P: proestrus phase characterized by the presence of nucleated epithelial cells (indicated by black arrow); E: estrus phase, characterized by the presence of cornified cells (indicated by white arrow); M: metestrus phase, characterized by the presence of nucleated epithelial cells, cornified cells, and leukocyte infiltration (indicated by the red arrow); D: diestrus, characterized by the presence of leukocyte infiltration. Results are shown as means ± SEM (n = 6). Significance among different groups presented as β, Control vs HVCD, δ, HVCD vs HVCD + Met; ε, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance represented as ^βp<0.05; ^φφp<0.01; ^εεεp<0.001.

Figure 2

HFrD fed perimenopausal rat model development and effect of combination therapy on glucose intolerance. (a) body weight, (b) complete fat mass %, (c) complete lean mass %, (d) IPGTT and its relevant (e) AUC with acute treatment of Met 150 mg/kg, PSTi8 5 mg/kg and combination Met 75 mg/kg + PSTi8 2.5 mg/kg, including glucose 2 g/kg, (f) IPGTT and its relevant (g) AUC with chronic treatment of Met 150 mg/kg, PSTi8 1 mg/kg, and combination Met 150 mg/kg + PSTi8 0.5 mg/kg and glucose 2 g/kg. Results are shown as means ± SEM (n = 6). Significance among different groups presented as β, Control vs HVCD; δ, HVCD vs HVCD + Met; ε, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance demonstrated as ^δp<0.05; ^ββp<0.01; ^{βββ,δδδ,εεε,φφφ}p<0.001.

Figure 3

Effect of combination therapy on important elements of liver function. (a) SGOT, (b) SGPT, and (C) serum ALP. Comparative significance results are shown as means ± SEM (n = 6). Significance among groups presented as β, Control vs HVCD, δ, HVCD vs HVCD + Met; δ, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance presentation as ^εεp<0.01; ^{βββ,δδδ,εεε,φφφ}p<0.001.

Figure 4

Combination therapy ameliorated plasma lipid markers and reduced hepatic lipid deposition. Plasma lipid markers (a) LDL, (b) HDL, (c) VLDL, (D) triglyceride, and (e) total cholesterol. (f) Liver histopathological analysis was performed by staining with H & E stain (scale bar 100 μm, 20x magnification). (g) Liver steatosis score analysis and (h) hepatic ballooning score analysis. Data represented as means ± SEM (n = 6). Significance among different groups presented as β, Control vs HVCD, δ, HVCD vs HVCD + Met; ε, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance illustrated as ^β,δ,εp<0.05; ^{ββ,δδ,φφ}p<0.01; ^{βββ,δδδ,εεε,φφφ}p<0.001.

Figure 5

Combination therapy attenuated lipid accumulation in high fructose-induced insulin-resistant HepG2 cells. (a) Nile Red staining to detect lipid accumulation in HFr-induced HepG2 cells and (b) Oil Red O staining (c) Nile Red fluorescence intensity quantification. (d) Quantification of lipid accumulation detected by Oil Red O staining in HFr-induced HepG2 cells. Results were represented as means ± SEM (n = 6). Significance among different groups presented as β, Control vs HFr; δ, HFr vs HFr + Met; ε, HFr vs HFr + PSTi8; φ, HFr vs HFr + Comb. Significance represented as ^εp<0.05; ^{βββ,δδδ,εεε,φφφ}p<0.001.

Figure 6

Combination therapy activates SIRT1 in HepG2 cells and liver of perimenopausal rats. (a) Immunofluorescence imaging of SIRT1 in HFr-induced HepG2 cells, (b) SIRT1 fluorescence intensity quantification (n = 6). (c) SIRT1 relative mRNA expression (n = 6) and (d) protein content (n = 3) in the liver of HVCD rats. SIRT1 mRNA and protein expression normalized with 18s and GAPDH expression, respectively (densitometry by Image J software). Results were represented as means ± SEM (n = 3). For (a and b), Significance among different groups presented as β, Control vs HFr; δ, HFr vs HFr + Met; ε, HFr vs HFr + PSTi8; φ, HFr vs HFr + Comb. For (c and d), significance values represented as β, Control vs HVCD; δ, HVCD vs HVCD + Met; ε, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance showed as ^εp<0.05;^δδ,φφp<0.01;^{βββ,δδδ,εεε, φφφ}p<0.001.

Figure 7

Combination therapy improved SREBP-1c expression in HFr induced HepG2 cells and hepatic SREBP-1c expression in perimenopausal rats. (a) Immunofluorescence imaging of HFr-induced HepG2 cells, (b) SREBP-1c fluorescence intensity quantification (n = 6). (c) Relative mRNA expression of SREBP-1c in the liver, studied by qRT-PCR (n = 6). (d) Total form of SREBP-1c (molecular weight 122kDa) presented by western blotting indicates protein expression levels (n = 3). We investigated relative mRNA expression of downstream markers of SREBP-1c. These are represented as (e) FAS (f) ACC-1 (g) SCD-1. Further, we determined the relative mRNA expression of fat oxidation related genes (h) CPT-1a (i) UCP-2. The mRNA and protein expressions were normalized with corresponding 18s and GAPDH expression, respectively. Results were represented as means ± SEM (n = 6). For (a and b), significance among different groups displayed as β, Control vs HFr; δ, HFr vs HFr + Met; ε, HFr vs HFr + PSTi8; φ, HFr vs HFr + Comb. For (c to i), Significance among groups showed as β, Control vs HVCD; δ, HVCD vs HVCD + Met; ε, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance represented as ^φp<0.05;^δδ,εεp<0.01;^{βββ,δδδ,εεεφφφ}p<0.001.

Figure 8

Combination therapy ameliorated the ER stress-induced PERK-eIF2a pathway and improved insulin signaling. We detected protein expression levels of hepatic ER stress pathway (a) GRP78, (b) p-PERK, and (c) p-eIF2a (d) ATF4. Concomitantly combination of Met and PSTi8 improved insulin sensitivity in liver as identified by improved insulin signaling. We detected protein phosphorylation of (e) p-(Ser-473) AKT (f) p-(Ser-307) IRS-1. Results are presented as means ± SEM (n = 3). Significance among groups presented as β, Control vs HVCD, δ, HVCD vs HVCD + Met; ε, HVCD vs HVCD + PSTi8; φ, HVCD vs HVCD + Comb. Significance represented as ^φp<0.05; ^{ββ,εε,φφp<0.01;βββ,δδδ,εεε,φφφ}p<0.001.