Dysfunctional ß-cell longevity in diabetes relies on energy conservation and positive epistasis.

Long-lived PFKFB3-expressing ß-cells are dysfunctional partly because of prevailing glycolysis that compromises metabolic coupling of insulin secretion. Their accumulation in type 2 diabetes (T2D) appears to be related to the loss of apoptotic competency of cell fitness competition that maintains islet function by favoring constant selection of healthy "winner" cells. To investigate how PFKFB3 can disguise the competitive traits of dysfunctional "loser" ß-cells, we analyzed the overlap between human ß-cells with bona fide "loser signature" across diabetes pathologies using the HPAP scRNA-seq and spatial transcriptomics of PFKFB3-positive ß-cells from nPOD T2D pancreata. The overlapping transcriptional profile of "loser" ß-cells was represented by down-regulated ribosomal biosynthesis and genes encoding for mitochondrial respiration. PFKFB3-positive "loser" ß-cells had the reduced expression of HLA class I and II genes. Gene-gene interaction analysis revealed that PFKFB3 rs1983890 can interact with the anti-apoptotic gene MAIP1 implicating positive epistasis as a mechanism for prolonged survival of "loser" ß-cells in T2D. Inhibition of PFKFB3 resulted in the clearance of dysfunctional "loser" ß-cells leading to restored glucose tolerance in the mouse model of T2D.

ß-cell-specific deletion of PFKFB3 restores cell fitness competition and physiological replication under diabetogenic stress.

HIF1α and PFKFB3 play a critical role in the survival of damaged ß-cells in type-2 diabetes while rendering ß-cells non-responsive to glucose stimulation. To discriminate the role of PFKFB3 from HIF1α in vivo, we generated mice with conditional ß-cell specific disruption of the Pfkfb3 gene on a human islet pancreatic polypeptide (hIAPP+/-) background and a high-fat diet (HFD) [PFKFB3ßKO + diabetogenic stress (DS)]. PFKFB3 disruption in ß-cells under DS led to selective purging of hIAPP-damaged ß-cells and the disappearance of insulin- and glucagon positive bihormonal cells. PFKFB3 disruption induced a three-fold increase in ß-cell replication as evidenced by minichromosome maintenance 2 protein (MCM2) expression. Unlike high-, lower DS or switch to restricted chow diet abolished HIF1α levels and reversed glucose intolerance of PFKFB3ßKO DS mice. Our data suggest that replication and functional recovery of ß-cells under DS depend on ß-cell competitive and selective purification of HIF1α and PFKFB3-positive ß-cells.

E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis.

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.

E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity.

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.