Fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance that results from an impaired collagen turnover. Peptidase D (PEPD) plays a vital role in collagen turnover by degrading proline-containing dipeptides. Nevertheless, its specific function and importance in AT is unknown. GWAS identified the rs731839 variant in the locus near PEPD that uncouples obesity from insulin resistance and dyslipidaemia, thus indicating that defective PEPD might impair AT remodelling and exacerbate metabolic complications. Here we show that in human and murine obesity, PEPD expression and activity decrease in AT, coupled to the release of PEPD systemically. Both events, in turn, are associated with the accumulation of fibrosis in AT and insulin resistance. Using pharmacologic and genetic animal models of PEPD down-regulation, we show that whereas dysfunctional PEPD activity provokes AT fibrosis, it is the PEPD secreted by AT the main contributor to inflammation, insulin resistance and metabolic dysfunction. Also, PEPD originated in inflammatory macrophages (Mɸ), plays an essential role promoting fibro-inflammatory responses via activation of EGFR in Mɸ and preadipocytes. Using genetic ablation of pepd in Mɸ that prevents obesity-induced PEPD release, also averts AT fibro-inflammation and obesity-associated metabolic dysfunctions. Taking advantage of factor analysis, we have identified the coupling of prolidase decreased activity and increased systemic levels of PEPD as the essential pathogenic triggers of AT fibrosis and insulin resistance. Thus, PEPD produced by Mɸ qualifies as a biomarker of AT fibro-inflammation and a therapeutic target for AT fibrosis and obesity-associated insulin resistance and type 2 diabetes.

A reduction in GH, as well as IGF1, is associated with non-alcoholic fatty liver disease (NAFLD). However, the relative contribution of changes in circulating GH and IGF1, to hepatic triglyceride accumulation (steatosis), remains to be clearly defined. To study the direct actions of GH on hepatocyte metabolism, we have utilized a mouse model of adult-onset, hepatocyte-specific, GHR knockdown (aHepGHRkd; 10–12 week-old, GHRfl/fl male mice, treated with AAV8-TBGp-Cre). In this and previous reports, we have observed that aHepGHRkd male mice rapidly develop steatosis (after 7 days) associated with enhanced de novo lipogenesis (DNL; measured by deuterated H2O labeling, 10h after 0800h food removal), and low ketone levels, suggestive of reduced hepatic β-oxidation. Of note, aHepGHRkd also reduces plasma IGF1 levels to >80% of GHR-intact controls (GHRfl/fl mice treated with AAV8-TBGp-Null), leading to a rise in GH, due to loss of IGF1 negative feedback to the pituitary/hypothalamus. This reciprocal shift in IGF1/GH is associated with an increase in insulin levels. Therefore, it is possible that the steatosis that develops in aHepGHRkd mice is the consequence of systemic insulin resistance supplying excess substrates (glucose and NEFA) for hepatic lipogenesis. However, inconsistent with this theory is the fact that glucose and NEFA levels are not altered after aHepGHRkd. To tease out the indirect (perhaps driven by high insulin levels) vs. direct effects of GH on hepatocyte lipid accumulation, male aHepGHRkd mice were injected with a vector expressing rat IGF1 (AAV8-TBGp-rIGF1). Reconstitution of hepatocyte IGF1 in aHepGHRkd mice, raised plasma IGF1 and normalized GH, insulin and ketone levels, but hepatic steatosis and DNL remained greater than that of GHR-intact controls, indicating GH directly suppresses hepatic fat accumulation. RNAseq analysis of livers from aHepGHRkd mice showed expression of genes related to carbohydrate metabolism (Gck, Khk) and fatty acid synthesis (Fasn, Srebf1, Usf1), processing (Scd1) and uptake (Cd36) were increased, while genes related to gluconeogenesis (Pck1, Fbp1, G6pc) were reduced. Remarkably, IGF1 reconstitution had no major impact on the hepatic transcriptome of aHepGHRkd mice, with the exception of reducing the expression of Srebf1, consistent with the reduction in circulating insulin levels. Interestingly, carbohydrate-responsive element-binding protein (CHREBP) levels, but not mRNA levels, were greater in aHepGHRkd mice with or without IGF1 reconstitution, consistent with upregulation of CHREBP target genes (Khk and Fasn among others). Taken together, these results suggest GH directly regulates steatosis, at least in part, by suppressing carbohydrate-driven DNL, where additional studies are underway to test this hypothesis.