Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca²⁺ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca²⁺ release and thereby deplete ER Ca²⁺ stores. The resulting ER Ca²⁺ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca²⁺ release, cytosolic and mitochondrial matrix Ca²⁺ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca²⁺ depletion mediate persistent Ca²⁺ influx, further impairing cytosolic and mitochondrial Ca²⁺ homeostasis. Mitochondrial Ca²⁺ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

Sulforaphane, a natural compound identified by drug repurposing, reduces hepatic glucose production and improves glucose control in type 2 diabetes.

Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca²⁺ increase due to depletion of luminal ER Ca²⁺. Palmitate-induced ER Ca²⁺ depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca²⁺ pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca²⁺ depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca²⁺ loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca²⁺ depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca²⁺ depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.

Aims/hypothesis Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). Methods Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. Results Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca²⁺-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. Conclusions/interpretation These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Type 2 diabetes (T2D) develops after years of prediabetes during which high glucose (glucotoxicity) impairs insulin secretion. We report that the ATP-conducting mitochondrial outer membrane voltage-dependent anion channel-1 (VDAC1) is upregulated in islets from T2D and non-diabetic organ donors under glucotoxic conditions. This is caused by a glucotoxicity-induced transcriptional program, triggered during years of prediabetes with suboptimal blood glucose control. Metformin counteracts VDAC1 induction. VDAC1 overexpression causes its mistargeting to the plasma membrane of the insulin-secreting β cells with loss of the crucial metabolic coupling factor ATP. VDAC1 antibodies and inhibitors prevent ATP loss. Through direct inhibition of VDAC1 conductance, metformin, like specific VDAC1 inhibitors and antibodies, restores the impaired generation of ATP and glucose-stimulated insulin secretion in T2D islets. Treatment of db/db mice with VDAC1 inhibitor prevents hyperglycemia, and maintains normal glucose tolerance and physiological regulation of insulin secretion. Thus, β cell function is preserved by targeting the novel diabetes executer protein VDAC1.

Insulin secretion was studied using adult rat intact islets, dissociated isolated islet cells, and isolated islet cells that had been allowed to reaggregate. The three preparations were maintained for 2 h in tissue culture medium at 37 C in order to allow for equilibration after the isolation procedures and to permit restoration of contacts between the reaggregated cells. The isolated cells appeared well preserved at the ultrastructural level. Evidence for intimate contacts between the reaggregated cells was demonstrated by the reappearance of gap junctions between adjacent cells. Basal insulin release (2.8 mM glucose) during 30 min was elevated in the isolated cell suspension but was restored to the level found in intact islets when isolated cells from the same population were reaggregated. The elevated basal release did not appear to be due to cell damage since there was a commensurate increase in the rate of insulin biosynthesis relative to intact islets. Although there was a marked stimulation of insulin release from the intact islets at 16.7 mM glucose, the response was smaller in the reaggregated cells and in the isolated cell suspension. All three preparations responded to elevated cAMP levels evoked by glucagon in the presence of 16.7 mM glucose. Similar results were obtained when insulin release was studied in various preparations derived from newborn rat pancreatic endocrine cells. Thus, basal insulin release was elevated in isolated cells, whereas cultures with cell contacts displayed lower basal insulin release. Taken together, the restoration of lower basal insulin release and the parallel appearance of contacts between the reaggregated cells suggest that these two phenomena are interrelated. Thus, cell contacts may be important in regulating basal insulin release. Whether such regulation is a consequence merely of cell association or of direct communication between cells remains to be established.