br Results br Discussion secretase mediated proteolysis of i

Results
Discussion γ-secretase-mediated proteolysis of integral membrane proteins is required for diverse biological processes (Lal and Caplan, 2011). One γ-secretase target of particular interest is Notch, as recent work has shown Notch activity to be increased in obesity and associated metabolic conditions, such as T2D and NAFLD/NASH (Fukuda et al., 2012, Lee et al., 2016, Valenti et al., 2013), perhaps in an ill-fated attempt to regenerate hepatic functional capacity (Geisler and Strazzabosco, 2015). Our data show that treatment with GSIs, or more specific approaches to blocking liver γ-secretase activity (Ncst ASO; L-Ncst or iL-Ncst mice), recapitulates the insulin-sensitizing effects of loss of hepatocyte Notch signaling but simultaneously reduces plasma TGs and non-HDL cholesterol in a Notch-independent manner. These data support the dual role of hepatic γ-secretase to regulate cdc42 homeostasis and lipid homeostasis by diverse mechanisms, but why this proteolytic complex, functionally conserved from flies and worms to humans (Tomita et al., 2001), has evolved to play such a critical role in metabolic processes disrupted by the obese state is unclear. In addition, further study is required to test whether hepatic γ-secretase activity is constitutive or may be regulated by increased nutrient availability and/or lipid flux to the liver. For example, Hif-1α, known to regulate glucose metabolism in neoplastic and obese tissue (Denko, 2008, Jiang et al., 2011), has been shown to increase γ-secretase activity in breast cancer (Villa et al., 2014). Our findings suggest that other hormonal and nutrient stimuli may have similar effects. Our data show that Nicastrin directly targets LDLR for γ-secretase-mediated proteolysis as predicted by a prior proteomics screen (Hemming et al., 2008), which in turn renders LDLR susceptible to lysosomal degradation, and decreases uptake of TG-rich VLDL/LDL particles (Gordts et al., 2016), but we cannot rule out indirect mechanisms that may contribute to TRL clearance. The very short (∼50 aa) LDLR CTD released is then rapidly degraded by the proteasome, consistent with other γ-secretase substrates (Lal and Caplan, 2011). Of note, we find additional LDLR C-terminal fragments that suggest that LDLR may be shed by other secretases prior to γ-secretase cleavage, but the necessity for this processing for Nicastrin recognition and γ-secretase activity warrants further investigation. Nonetheless, our results show that LDLR lacking its short CTD, which represents the end product of γ-secretase activity, is inherently unstable and undergoes lysosomal degradation. This experiment unintentionally recapitulates data from Brown and Goldstein from decades earlier, where C-terminal premature stop mutations of LDLR decreased receptor stability (van Driel et al., 1987). We hypothesize that these artificial mutations actually reveal inherent biology and that γ-secretase prevents normal LDLR recycling and hepatic TRL removal (Figure 6G), perhaps to ensure sufficient lipid delivery to other tissues. Our data further suggest a parallel path for LDLR degradation, above and beyond PCSK9-mediated LDLR lysosomal degradation, with different effects on plasma lipids. PCSK9 loss of function or pharmacologic inhibition lowers LDL cholesterol, but effects on plasma TGs in treated patients are less robust and reproducible (Everett et al., 2015). Consistent with these data, we found that administration of a PCSK9 mAb, alirocumab, did not further reduce plasma TGs in Ncst ASO-treated mice. We postulate that increased “baseline” hepatocyte plasma membrane LDLR, by dint of γ-secretase inhibition, allows rapid binding and uptake of TRLs, which may distinguish this approach from PCSK9 antagonism that preferentially increases LDLR recycling. Alternatively, LDLR post-translational modifications or association with other receptors may determine cargo preference, or it may simply reflect competition for LDLR pools. Notably, simultaneous inhibition of γ-secretase and PCSK9 appeared to lower both TGs and cholesterol, but further work is necessary to test whether PCSK9 and γ-secretase have synergistic and additive effects on lipoprotein metabolism in animal models with elevated non-HDL cholesterol (Lagace, 2014). Nevertheless, these data indicate that γ-secretase inhibition attenuates multiple metabolic comorbidities of the obese state.