Background & Aims Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na⁺/H⁺ exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na⁺/H⁺ exchanger activity and Na⁺-dependent fluid absorption in the intestine. Methods We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2; Nherf2^−/−) or LPA₂ (Lpa₂^−/−). Roles of LPA₅ and NHERF2 were determined by analysis of heterologous expression. Results Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA₅ was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA₅ signaling required coexpression of NHERF2, which interacted with LPA₅. LPA-mediated intestinal fluid absorption was impaired in Nherf2^−/− mice, demonstrating the requirement for NHERF2 in LPA₅ activity. However, fluid absorption was unaltered in Lpa₂^−/− mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. Conclusions LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA₅. Regulation by LPA₅ depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

Calcium (Ca²⁺) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca²⁺ regulates the activity of Na⁺/H⁺ exchanger 3 (NHE3). However, the effect of Ca²⁺-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP₃ receptor-binding protein released with IP₃ (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca²⁺-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca²⁺-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca²⁺-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca²⁺ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.

Iron is a mineral essential for blood production and a variety of critical cellular functions. Altered iron metabolism has been increasingly observed in many diseases and disorders, but a comprehensive and mechanistic understanding of the cellular impact of impaired iron metabolism is still lacking. We examined the effects of iron overload or iron deficiency on cellular stress responses and autophagy which collectively regulate cell homeostasis and survival. Acute iron loading led to increased mitochondrial ROS (mtROS) production and damage, lipid peroxidation, impaired autophagic flux, and ferroptosis. Iron-induced mtROS overproduction is the mechanism of increased lipid peroxidation, impaired autophagy, and the induction of ferroptosis. Iron excess-induced ferroptosis was cell-type dependent and regulated by activating transcription factor 4 (ATF4). Upregulation of ATF4 mitigated iron-induced autophagic dysfunction and ferroptosis, whereas silencing of ATF4 expression impaired autophagy and resulted in increased mtROS production and ferroptosis. Employing autophagy-deficient hepatocytes and different autophagy inhibitors, we further showed that autophagic impairment sensitized cells to iron-induced ferroptosis. In contrast, iron deficiency activated the endoplasmic reticulum (ER) stress response, decreased autophagy, and induced apoptosis. Decreased autophagy associated with iron deficiency was due to ER stress, as reduction of ER stress by 4-phenylbutyric acid (4-PBA) improved autophagic flux. The mechanism of decreased autophagy in iron deficiency is a disruption in lysosomal biogenesis due to impaired posttranslational maturation of lysosomal membrane proteins. In conclusion, iron excess and iron deficiency cause different forms of cell stress and death in part through the common mechanism of impaired autophagic function.

Enteric neuronal degeneration, as seen in inflammatory bowel disease, obesity, and diabetes, can lead to gastrointestinal dysmotility. Pyroptosis is a novel form of programmed cell death but little is known about its role in enteric neuronal degeneration. We observed higher levels of cleaved caspase-1, a marker of pyroptosis, in myenteric ganglia of overweight and obese human subjects compared with normal-weight subjects. Western diet–fed (WD-fed) mice exhibited increased myenteric neuronal pyroptosis, delayed colonic transit, and impaired electric field stimulation–induced colonic relaxation responses. WD increased TLR4 expression and cleaved caspase-1 in myenteric nitrergic neurons. Overactivation of nitrergic neuronal NF-κB signaling resulted in increased pyroptosis and delayed colonic motility. In caspase-11–deficient mice, WD did not induce nitrergic myenteric neuronal pyroptosis and colonic dysmotility. To understand the contributions of saturated fatty acids and bacterial products to the steps leading to enteric neurodegeneration, we performed in vitro experiments using mouse enteric neurons. Palmitate and lipopolysaccharide (LPS) increased nitrergic, but not cholinergic, enteric neuronal pyroptosis. LPS gained entry to the cytosol in the presence of palmitate, activating caspase-11 and gasdermin D, leading to pyroptosis. These results support a role of the caspase-11–mediated pyroptotic pathway in WD-induced myenteric nitrergic neuronal degeneration and colonic dysmotility, providing important therapeutic targets for enteric neuropathy.

Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na⁺/H⁺ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP₃) receptor-binding protein released with IP₃ (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2–dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.