Lactotrehalose, an Analog of Trehalose, Increases Energy Metabolism Without Promoting Clostridioides difficile Infection in Mice

BACKGROUND & AIMS: Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clos-tridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lacto-trehalose) has the same metabolic effects of trehalose without expanding C difficile. METHODS: We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lacto-trehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses. RESULTS: Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection. CONCLUSIONS: Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.