Neurotensin (NTS), a 13-amino acid peptide discovered by Carraway and Leeman, is mainly synthesized by N cells of the small intestine and released into the bloodstream acting as a circulating hormone or in the brain as a peptidergic neurotransmitter or neuromodulator. The physiological effects of NTS are through its receptors (NTSRs), including NTSR1, NTSR2 and NTSR3 (also called sortilin or SORT1). In a landmark study published in 2012, Melander et al. showed that increased fasting levels of pro-NTS (the final stable 117-amino-acid polypeptide after the NTS precursor is cleaved by convertases) were associated with the development of diabetes and an increased risk of cardiovascular disease and mortality, thus providing the first clinical evidence that excessive NTS secretion results in metabolic disorders and increased morbidity and mortality. More recently, pro-NTS has been shown to be a novel diagnostic biomarker for detection of fatty liver disease and type 2 diabetes mellitus. Previously, we reported that NTS deficiency protected against body weight (BW) gain, insulin resistance and hepatic steatosis associated with the consumption of a high-fat diet (HFD). We further showed that NTS stimulates fatty acid (FA) absorption in the small intestine by attenuating the activation of AMP-activated protein kinase (AMPK) and involving both NTSR1 and NTSR3. In complementary studies, we demonstrated increased lipid accumulation in transgenic Drosophila strains expressing NTS specifically in gut enteroendocrine (EE) cells. Remarkably, in adults, we showed that increased levels of pro-NTS strongly predict new-onset obesity in a graded manner, which is independent of body mass index and insulin resistance.
AMPK exists as a complex consisting of an α catalytic subunit (α1 or α2) and regulatory subunits β (β1 or β2) and γ (γ1 or γ2 or γ3). Reznick et al. found that stimulation of AMPKα2 by 5′-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an AMPK activator, was blunted in the skeletal muscle of aged versus young rats. AMPK activation in the muscle or fat can extend lifespan in Drosophila. Furthermore, AMPK upregulation in Drosophila demonstrates both autonomous effects on intestinal epithelium and nonautonomous effects on distant tissues, such as the brain, linked to reduced insulin-like peptide levels and increased 4E-BP expression, which collectively slow systemic aging, thus demonstrating that localized activation of AMPK in key tissues can slow aging in a non-cell-autonomous manner.
With obesity, AMPK activity is generally reduced in tissues such as skeletal muscle, liver and adipose tissue. Basal AMPK activity is generally higher in young muscle versus aged muscle. Young cells can efficiently activate AMPK in response to metabolic stress such as exercise, fasting or caloric restriction. However, basal AMPK activity is lower in aged skeletal muscle. The ability of cells to activate AMPK in response to stressors is diminished with age. This can lead to a decline in mitochondrial function, decreased energy production and increased susceptibility to metabolic disorders. The importance of AMPK in the intestine has been recognized only recently. Both AICAR and metformin (Met), another AMPK activator, control glucose metabolism in the intestine and maintain whole-body glucose homeostasis. Intraduodenal infusion of Met activated duodenal mucosal AMPK and lowered hepatic glucose production in a HFD-induced rat model with insulin resistance. Mice with knockout (KO) of intestinal epithelium-specific AMPKα1 gained weight, and glucose tolerance was impaired compared with wild-type (WT) mice after 6 weeks of HFD feeding. Intestinal epithelium-specific AMPKα1 deletion impaired intestinal long-chain FA absorption and protected mice from HFD-induced obesity, suggesting the involvement of AMPK in intestinal FA absorption.
FA binding protein 1 (FABP1, also called liver FABP or L-FABP) and FABP2 (also called intestinal FABP or I-FABP) are expressed in enterocytes, but not in crypt cells. Both proteins are highly expressed in the jejunum, the major site of absorption of dietary lipids, compared with the duodenum, ileum and colon. FABP1 and FABP2, both of which bind long-chain FAs, have different functional effects in the intestine. FABP1 regulates genes involved in FA oxidation and the formation of prechylomicron transport vesicles. By contrast, apical cytoplasmic FABP2 binds FAs from the mucosa of fasting rats and transports the FAs into the interior of the cell, suggesting that FABP2 is more likely to be involved in FA uptake from the lumen of the intestine and with the distribution of FAs to metabolic compartments. Utilizing FABP1- and FABP2-deficient mice, Lagakos et al. found that FABP1 directs FAs toward oxidative pathways, while FAPB2 targets dietary FAs toward triglyceride synthesis.
In the present study, FABP1 was upregulated by HFD feeding, which correlates with the HFD-induced inhibition of AMPK activity. Consistently, Met or AICAR activated AMPK and concurrently decreased FABP1 protein expression, further supporting the role of AMPK in controlling intestinal lipid absorption. NTS deficiency restores AMPK signaling as well as FABP1 impaired by HFD feeding and aging in IECs intestinal epithelial cells.
Phospho-AMPKα (Thr172) (2535), AMPKα (2532), phospho-ERK1/2 (Thr202/Tyr204) (4370), ERK1/2 (9102), phospho-Akt (Ser-473) (4058) and pan-Akt (4691) antibodies were from Cell Signaling Technology. AMPKα1 (sc19128), AMPKα2 (sc19129), FABP1 (sc-374537) and FABP2 (sc-374482) antibodies were from Santa Cruz Biotechnology. GFP (ab290) and RFP (ab62341) antibodies were from Abcam. AICAR, Met and PD 98059 were from Cayman. Fetal bovine serum, NTS 1–13, human insulin, palmitic acid (PA), N-acetyl cysteine and β-actin antibody were from Sigma-Aldrich. SYBR green primers for real-time or quantitative PCR (qPCR) were from Integrated DNA Technologies. Noggin-conditioned medium (CM) was purchased from U-Protein Express BV. Dulbecco’s modified Eagle medium (DMEM), advanced DMEM/F12 medium, OptiMEM reduced serum medium, growth factor-reduced Matrigel, B-27 supplement, N-2 supplement, HEPES, GlutaMAX and Zeocin were from ThermoFisher. Mouse EGF was from PeproTech. ChromoTek GFP-Trap agarose was from Proteintech.
The normal rat small intestinal epithelial cell line IEC-6 and the human embryonic kidney cell line HEK-293, purchased from ATCC, were maintained in DMEM, supplemented with 10% fetal bovine serum. pEGFP-N1 was a gift from Antony K. Chen (Addgene plasmid #172281). mRFP1-N1 was a gift from Robert Campbell, Michael Davidson and Roger Tsien (Addgene plasmid #54635). Human NTSR1 and NTSR3 cDNAs were cloned into pEGFP-N1 and mRFP1-N1, respectively. HEK-293 cells were transfected with constructs by LipoFectamine 3000 (ThermoFisher). Stable cell lines (293/GFP), 293/GFP-NTSR1 and 293/RFP-NTSR3) were selected with G418 (800 μg/ml), sorted by flow cytometry and maintained in culture medium containing 500 μg/ml G418.
Gr36C-Gal4 and the NTS transgenic lines have been previously described. The EE-cell-specific Gr36C-Gal4 was used to drive NTS expression. Gr36C-NTS flies were back crossed with w1118 for eight generations to synchronize the genetic backgrounds. Flies were housed in vials at 25
