Rebecca L. Pongratz

SGLT2 Deletion Improves Glucose Homeostasis and Preserves Pancreatic β-Cell Function

OBJECTIVE Inhibition of the Na+-glucose cotransporter type 2 (SGLT2) is currently being pursued as an insulin-independent treatment for diabetes; however, the behavioral and metabolic consequences of SGLT2 deletion are unknown. Here, we used a SGLT2 knockout mouse to investigate the effect of increased renal glucose excretion on glucose homeostasis, insulin sensitivity, and pancreatic β-cell function. RESEARCH DESIGN AND METHODS SGLT2 knockout mice were fed regular chow or a high-fat diet (HFD) for 4 weeks, or backcrossed onto the db/db background. The analysis used metabolic cages, glucose tolerance tests, euglycemic and hyperglycemic clamps, as well as isolated islet and perifusion studies. RESULTS SGLT2 deletion resulted in a threefold increase in urine output and a 500-fold increase in glucosuria, as well as compensatory increases in feeding, drinking, and activity. SGLT2 knockout mice were protected from HFD-induced hyperglycemia and glucose intolerance and had reduced plasma insulin concentrations compared with controls. On the db/db background, SGLT2 deletion prevented fasting hyperglycemia, and plasma insulin levels were also dramatically improved. Strikingly, prevention of hyperglycemia by SGLT2 knockout in db/db mice preserved pancreatic β-cell function in vivo, which was associated with a 60% increase in β-cell mass and reduced incidence of β-cell death. CONCLUSIONS Prevention of renal glucose reabsorption by SGLT2 deletion reduced HFD- and obesity-associated hyperglycemia, improved glucose intolerance, and increased glucose-stimulated insulin secretion in vivo. Taken together, these data support SGLT2 inhibition as a viable insulin-independent treatment of type 2 diabetes.

Cytosolic and Mitochondrial Malic Enzyme Isoforms Differentially Control Insulin Secretion

In islet β-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP+-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD+-preferred (ME2) expressed at ∼50%, and the NADP+-specific (ME3) at ∼10% compared with ME1. Selective reduction (89 ± 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mm:41 ± 6%, p < 0.01) and amino acid (4 mm glutamine ± 10 mm leucine: 39 ± 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 ± 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 ± 4% (p < 0.01). Modeling of the metabolism of [U-13C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.

Phosphoenolpyruvate Cycling via Mitochondrial Phosphoenolpyruvate Carboxykinase Links Anaplerosis and Mitochondrial GTP with Insulin Secretion

Pancreatic β-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical KATP-dependent glucose-stimulated insulin secretion (GSIS), there are important KATP-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel 13C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.

Role for malic enzyme, pyruvate carboxylation, and mitochondrial malate import in glucose-stimulated insulin secretion

Pyruvate cycling has been implicated in glucose-stimulated insulin secretion (GSIS) from pancreatic beta-cells. The operation of some pyruvate cycling pathways is proposed to necessitate malate export from the mitochondria and NADP(+)-dependent decarboxylation of malate to pyruvate by cytosolic malic enzyme (ME1). Evidence in favor of and against a role of ME1 in GSIS has been presented by others using small interfering RNA-mediated suppression of ME1. ME1 was also proposed to account for methyl succinate-stimulated insulin secretion (MSSIS), which has been hypothesized to occur via succinate entry into the mitochondria in exchange for malate and subsequent malate conversion to pyruvate. In contrast to rat, mouse beta-cells lack ME1 activity, which was suggested to explain their lack of MSSIS. However, this hypothesis was not tested. In this report, we demonstrate that although adenoviral-mediated overexpression of ME1 greatly augments GSIS in rat insulinoma INS-1 832/13 cells, it does not restore MSSIS, nor does it significantly affect GSIS in mouse islets. The increase in GSIS following ME1 overexpression in INS-1 832/13 cells did not alter the ATP-to-ADP ratio but was accompanied by increases in malate and citrate levels. Increased malate and citrate levels were also observed after INS-1 832/13 cells were treated with the malate-permeable analog dimethyl malate. These data suggest that although ME1 overexpression augments anaplerosis and GSIS in INS-1 832/13 cells, it is not likely involved in MSSIS and GSIS in pancreatic islets.

A Role for Mitochondrial Phosphoenolpyruvate Carboxykinase (PEPCK-M) in the Regulation of Hepatic Gluconeogenesis

Background: PEPCK-M is generally considered irrelevant for glucose production, although gluconeogenesis has never been characterized in its absence. Results: PEPCK-M loss impaired gluconeogenesis from lactate, lowered plasma glucose, insulin, and triglycerides, reduced hepatic glycogen, and increased glycerol turnover. Conclusion: Approximately a third of gluconeogenesis comes from PEPCK-M. Significance: The nutrient-sensitive PEPCK-M has been overlooked and is potentially important for metabolic diseases such as diabetes. Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

Porcine islet xenotransplantation is considered a potential cell‐based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose‐stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3‐fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig‐to‐NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

A β-peptide agonist of the GLP-1 receptor, a class B GPCR

Previous work has shown that certain β(3)-peptides can effectively mimic the side chain display of an α-helix and inhibit interactions between proteins, both in vitro and in cultured cells. Here we describe a β(3)-peptide analog of GLP-1, CC-3(Act), that interacts with the GLP-1R extracellular domain (nGLP-1R) in vitro in a manner that competes with exendin-4 and induces GLP-1R-dependent cAMP signaling in cultured CHO-K1 cells expressing GLP-1R.

Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle

Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell.

Mitochondrial Dysfunction Contributes to Impaired Insulin Secretion in INS-1 Cells with Dominant-negative Mutations of HNF-1α and in HNF-1α-deficient Islets

Maturity Onset Diabetes of the Young-type 3 (MODY-3) has been linked to mutations in the transcription factor hepatic nuclear factor (HNF)-1α, resulting in deficiency in glucose-stimulated insulin secretion. In INS-1 cells overexpressing doxycycline-inducible HNF-1α dominant-negative (DN-) gene mutations, and islets from Hnf-1α knock-out mice, insulin secretion was impaired in response to glucose (15 mm) and other nutrient secretagogues. Decreased rates of insulin secretion in response to glutamine plus leucine and to methyl pyruvate, but not potassium depolarization, indicate defects specific to mitochondrial metabolism. To identify the biochemical mechanisms responsible for impaired insulin secretion, we used 31P NMR measured mitochondrial ATP synthesis (distinct from glycolytic ATP synthesis) together with oxygen consumption measurements to determine the efficiency of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling was significantly higher in DN-HNF-1α cells, such that rates of ATP synthesis were decreased by approximately one-half in response to the secretagogues glucose, glutamine plus leucine, or pyruvate. In addition to closure of the ATP-sensitive K+ channels with mitochondrial ATP synthesis, mitochondrial production of second messengers through increased anaplerotic flux has been shown to be critical for coupling metabolism to insulin secretion. 13C-Isotopomer analysis and tandem mass spectrometry measurement of Krebs cycle intermediates revealed a negative impact of DN-HNF-1α and Hnf-1α knock-out on mitochondrial second messenger production with glucose but not amino acids. Taken together, these results indicate that, in addition to reduced glycolytic flux, uncoupling of mitochondrial oxidative phosphorylation contributes to impaired nutrient-stimulated insulin secretion with either mutations or loss of HNF-1α.

Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb’s cycle flux independent of ATP synthesis

Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with (31)P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by (13)C NMR isotopomer analysis of the fate of [U-(13)C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media.