Immortalized Human Hepatocytes-hTERT

Overexpression of Centenarian Variants of SIRT6 does not Affect Hepatocyte Insulin Sensitivity

NAFLD, impacting 30% of the global adult population, escalates into more severe conditions like cirrhosis and hepatocellular carcinoma with no approved specific treatments. The discovery of genetic variants of SIRT6, a protein regulating aging and cellular stress, offers insights into novel therapeutic avenues. Frohlich et al. employed in vitro hepatic models using immortalized human hepatocytes (IHH) and hepatic stellate cells to investigate the metabolic and secretomic profiles affected by centenarian-associated SIRT6 mutations.

Figure 1A shows the far-red fluorescence protein Katushka2S signal in the LV cassette, either alone or with SIRT6 versions (WT, N308K, or N308K/A313S), demonstrating successful infection. No signal was detected in IHH control cells. Western blot analysis (Fig. 1B and C) showed a significant increase in SIRT6 levels in cells transfected with LV-SIRT6 compared to empty or CTL cells. SIRT6 represses gene transcription by deacetylating H3K9 and H3K56. Thus, in SIRT6 overexpression (OE) groups, acetylated histone H3K9 levels were significantly reduced, and H3K56Ac levels showed a decreasing trend (Fig. 1B and C), indicating increased SIRT6 expression and deacetylase activity. Given the close association of insulin receptor substrate PI3K (phosphoinositide 3-kinase) and AKT signaling pathways with metabolic disorders and insulin resistance, they tested by immunoblotting whether SIRT6 overexpression and longevity variants affected insulin sensitivity/PI3K/AKT pathway activation in IHH cells. After serum and glucose starvation, cells were stimulated with human insulin solution (100 nM) for 30 min. pAKT (Ser473) levels were measured, revealing no significant differences among conditions, with or without insulin (Fig. 2A and B).

Fig. 1. Model of stable transfected IHH cells overexpressing SIRT6 allele variants (Frohlich J, Raffaele M, et al., 2022).

Fig. 2. SIRT6 overexpression did not affect pAKT/AKT ratio in IHH (Frohlich J, Raffaele M, et al., 2022).

Prolonged Exposure of Human Hepatocytes to Hyperinsulinemia Enhances Cell Senescence

NAFLD affects the liver through multiple conditions ranging from simple steatosis to advanced hepatocellular carcinoma without available drug treatments. The aging process features cellular senescence which shows connections to various metabolic diseases. Cells experience senescence when exposed to hyperinsulinemia which is related to obesity and insulin resistance. Baboota et al. explored chronic hyperinsulinemia's direct role in inducing senescence in human hepatocytes.

To examine prolonged hyperinsulinemia's effect on hepatic senescence, we treated immortalized human hepatocytes (IHH) with insulin (20 and 100 nM) for 6, 24, or 72 hours. Insulin consistently increased protein senescence markers MDM2, p53, and p21 (Fig. 3A). Similarly, IGF1, activating a related pathway, elevated these markers' levels (Fig. 3A). Immunostaining confirmed the p53 increase from insulin and IGF1 (Fig. 3B). Insulin and IGF1 led to γH2AX puncta, indicating senescence-related DNA damage (Fig. 3B), and raised p16 and SASP markers like IL18, MMP3, IL32, and IL8 mRNA levels. These results show that extended high insulin and IGF1 exposure boosts senescence marker expression in human hepatocytes.

Fig. 3. Senescence markers are increased in human hepatocytes following insulin or insulin-like growth factor 1 (IGF1) exposure (Baboota R K, Spinelli R, et al., 2022).