Ceramide metabolism associated with chronic dietary nutrient surplus and diminished insulin sensitivity in the liver, muscle, and adipose tissue of cattle

Abstract

High dietary energy and protein supply is common practice in livestock nutrition, aiming to maximize growth and production performance. However, a chronic nutritional surplus induces obesity, promotes insulin insensitivity, and triggers low-grade inflammation. Thirty Holstein bulls were randomly assigned to two groups, low energy and protein (LEP), and high energy and protein (HEP) intake, provided from the 13th to the 20th month of life. Body weight, carcass composition, laminitis score, and circulating insulin and glucose concentrations were assessed. The expression and extent of phosphorylation of insulin signaling proteins were measured in the liver, muscle, and adipose tissue. The sphingolipid metabolome was quantified by a targeted liquid chromatography-mass spectrometry based metabolomics approach. The HEP bulls were obese, had hyperinsulinemia with euglycemia, and expressed clinical signs of chronic laminitis. In the liver, protein kinase B (PKB) phosphorylation was decreased and this was associated with a higher tissue concentration of ceramide 16:0, a sphingolipid that diminishes insulin action by dephosphorylating PKB. In the adipose tissue, insulin receptor expression was lower in HEP bulls, associated with higher concentration of hexosylceramide, which reduces the abundance of functional insulin receptors. Our findings confirm that diet-induced metabolic inflammation triggers ceramide accumulation and disturbs insulin signaling. As insulin insensitivity exacerbates metabolic inflammation, this self-reinforcing cycle could explain the deterioration of metabolic health apparent as chronic laminitis. By demonstrating molecular relationships between insulin signaling and sphingolipid metabolism in three major tissues, our data extend our mechanistic understanding of the role of ceramides in diet-induced metabolic inflammation.

Keywords: ceramide; insulin resistance; metabolic inflammation; obesity; sphingolipids; tissue metabolomics.

FIGURE 1

(A) Body weight, carcass classes and laminitis score in HEP and LEP fed bulls. (B) Serum insulin and plasma glucose of HEP and LEP fed bulls, as published previously (Bäßler et al., 2021). Means ± SD; n = 15.

FIGURE 2

Protein expression and phosphorylation of key components of the insulin signaling pathway in liver, muscle, and retroperitoneal adipose tissue. Insulin receptor β (InsR), mechanistic target of rapamycin (mTOR), protein kinase B (PKB), 5′ adenosine monophosphate-activated protein kinase α (AMPK), and their phosphorylated forms were detected by Western blotting. Means ± SD; n = 15.

FIGURE 3

Partial least squares-discriminant analysis (PLS-DA) scores plots with variable importance in projection (VIP) scores of sphingolipid metabolome profiles of (A) liver, (B) muscle, and (C) subcutaneous adipose tissue of bulls on HEP and LEP dietary regimens (n = 15). Spha, sphinganines; dhCer, dihydroceramides; dhCer-P, dihydroceramide-1-phosphates; Cer, ceramides; Cer-P, ceramide-1-phosphates; GalCer, galactosyl-ceramides; LacCer, lactosyl-ceramides; SM, sphingomyelins; dhSM, dihydrosphingomyelins; Spho: sphingosines.

FIGURE 4

Principal component analysis loading plots for (A) liver, (B) muscle, and (C) subcutaneous and retroperitoneal adipose tissues performed based on insulin signaling pathway proteins, and Cer 16:0, Cer 18:0, Cer 24:0, and sphingolipids subclasses’ abundance. Green and pink points indicate insulin signaling proteins and sphingolipid subclasses, respectively.

FIGURE 5

Schematic overview of the proposed interactions between inflammation, ceramide biosynthesis, and insulin resistance. Boxes highlighted in red indicate the scientific findings of this study. LPS, lipopolysaccharide; Ser, serine; CDase, ceramidase; CerS, ceramide synthase; SMS, sphingomyelin synthase; SMase, sphingomyelinase; PKB, protein kinase B.