Endogenous GIP ameliorates impairment of insulin secretion in proglucagon-deficient mice under moderate beta cell damage induced by streptozotocin.

AIMS/HYPOTHESIS: The action of incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is potentiated in animal models defective in glucagon action. It has been reported that such animal models maintain normoglycaemia under streptozotocin (STZ)-induced beta cell damage. However, the role of GIP in regulation of glucose metabolism under a combination of glucagon deficiency and STZ-induced beta cell damage has not been fully explored. METHODS: In this study, we investigated glucose metabolism in mice deficient in proglucagon-derived peptides (PGDPs)-namely glucagon gene knockout (GcgKO) mice-administered with STZ. Single high-dose STZ (200 mg/kg, hSTZ) or moderate-dose STZ for five consecutive days (50 mg/kg × 5, mSTZ) was administered to GcgKO mice. The contribution of GIP to glucose metabolism in GcgKO mice was also investigated by experiments employing dipeptidyl peptidase IV (DPP4) inhibitor (DPP4i) or Gcg-Gipr double knockout (DKO) mice. RESULTS: GcgKO mice developed severe diabetes by hSTZ administration despite the absence of glucagon. Administration of mSTZ decreased pancreatic insulin content to 18.8 ± 3.4 (%) in GcgKO mice, but ad libitum-fed blood glucose levels did not significantly increase. Glucose-induced insulin secretion was marginally impaired in mSTZ-treated GcgKO mice but was abolished in mSTZ-treated DKO mice. Although GcgKO mice lack GLP-1, treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-treated GcgKO mice, but did not increase beta cell area or significantly reduce apoptotic cells in islets. CONCLUSIONS/INTERPRETATION: These results indicate that GIP has the potential to ameliorate glucose intolerance even under STZ-induced beta cell damage by increasing insulin secretion rather than by promoting beta cell survival.

Mesenchymal stem cells ameliorate impaired wound healing through enhancing keratinocyte functions in diabetic foot ulcerations on the plantar skin of rats.

AIMS/HYPOTHESIS: Although the initial healing stage involves a re-epithelialization in humans, diabetic foot ulceration (DFU) has been investigated using rodent models with wounds on the thigh skin, in which a wound contraction is initiated. In this study, we established a rodent model of DFU on the plantar skin and evaluated the therapeutic efficacy of bone-marrow-derived mesenchymal stem cells (BM-MSCs) in this model. METHODS: The wounds made on the hind paws or thighs of streptozotocin induced diabetic or control rats were treated with BM-MSCs. Expression levels of phosphorylated focal adhesion kinase (pFAK), matrix metaroprotease (MMP)-2, EGF, and IGF-1, were evaluated in human keratinocytes, which were cultured in conditioned media of BM-MSCs (MSC-CM) with high glucose levels. RESULTS: Re-epithelialization initiated the healing process on the plantar, but not on the thigh, skin. The therapy utilizing BM-MSCs ameliorated the delayed healing in diabetic rats. In the keratinocytes cultured with MSC-CM, the decreased pFAK levels in the high glucose condition were restored, and the MMP2, EGF, and IGF-1 levels increased. CONCLUSIONS/INTERPRETATION: Our study established a novel rat DFU model. The impaired healing process in diabetic rats was ameliorated by transplantation of BM-MSCs. This amelioration might be accounted for by the modification of keratinocyte functions.

The role of S100B in the interaction between adipocytes and macrophages.

OBJECTIVE: The S100 calcium binding protein B (S100B) implicated in brain inflammation acts via the receptor of advanced glycation end products (RAGE) and is also secreted from adipocytes. We investigated the role of S100B in the interaction between adipocytes and macrophages using a cell-culture model. DESIGN AND METHODS: RAW264.7 macrophages (RAW) were stimulated by recombinant S100B to observe alterations in TNF-α and M1 markers; 3T3-L1 adipocytes (L1) were stimulated by TNF-α to examine S100B secretion. RAW and L1 were then mutually stimulated with conditioned media of each other, or co-cultured. The effects of S100B silencing or a RAGE-neutralizing antibody were also investigated. RESULTS: S100B upregulated TNF-α and M1 markers in RAW, and TNF-α augmented S100B secretion from L1. L1 conditioned media stimulated TNF-α secretion from RAW, and RAW conditioned media increased S100B secretion from L1. The co-culture of RAW and L1 increased TNF-α, S100B, and the expression of M1 markers and the MCP-1 receptor CCR2. The silencing of S100B or RAGE neutralization significantly ameliorated TNF-α hypersecretion from RAW that were stimulated with L1 conditioned media. CONCLUSIONS: Thus, S100B as an adipokine may play a role in the interaction between adipocytes and macrophages to establish a vicious paracrine loop.

Ectopic expression of GIP in pancreatic ß-cells maintains enhanced insulin secretion in mice with complete absence of proglucagon-derived peptides.

Glucagon and glucagon-like peptide-1 (GLP-1) are produced in pancreatic α-cells and enteroendocrine L-cells, respectively, in a tissue-specific manner from the same precursor, proglucagon, that is encoded by glucagon gene (Gcg), and play critical roles in glucose homeostasis. Here, we studied glucose homeostasis and ß-cell function of Gcg-deficient mice that are homozygous for a Gcg-GFP knock-in allele (Gcg(gfp/gfp)). The Gcg(gfp/gfp) mice displayed improved glucose tolerance and enhanced insulin secretion, as assessed by both oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT). Responses of glucose-dependent insulinotropic polypeptide (GIP) to both oral and intraperitoneal glucose loads were unexpectedly enhanced in Gcg(gfp/gfp) mice, and immunohistochemistry localized GIP to pancreatic ß-cells of Gcg(gfp/gfp) mice. Furthermore, secretion of GIP in response to glucose was detected in isolated islets of Gcg(gfp/gfp) mice. Blockade of GIP action in vitro and in vivo by cAMP antagonism and genetic deletion of the GIP receptor, respectively, almost completely abrogated enhanced insulin secretion in Gcg(gfp/gfp) mice. These results indicate that ectopic GIP expression in ß-cells maintains insulin secretion in the absence of proglucagon-derived peptides (PGDPs), revealing a novel compensatory mechanism for sustaining incretin hormone action in islets.

Remodeling of hepatic metabolism and hyperaminoacidemia in mice deficient in proglucagon-derived peptides.

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcg(gfp/gfp): GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action.

Ingestion of a moderate high-sucrose diet results in glucose intolerance with reduced liver glucokinase activity and impaired glucagon-like peptide-1 secretion.

UNLABELLED: Aims/Introduction: Excessive intake of sucrose can cause severe health issues, such as diabetes mellitus. In animal studies, consumption of a high-sucrose diet (SUC) has been shown to cause obesity, insulin resistance and glucose intolerance. However, several in vivo experiments have been carried out using diets with much higher sucrose contents (50-70% of the total calories) than are typically ingested by humans. In the present study, we examined the effects of a moderate SUC on glucose metabolism and the underlying mechanism. MATERIALS AND METHODS: C57BL/6J mice received a SUC (38.5% sucrose), a high-starch diet (ST) or a control diet for 5 weeks. We assessed glucose tolerance, incretin secretion and liver glucose metabolism. RESULTS: An oral glucose tolerance test (OGTT) showed that plasma glucose levels in the early phase were significantly higher in SUC-fed mice than in ST-fed or control mice, with no change in plasma insulin levels at any stage. SUC-fed mice showed a significant improvement in insulin sensitivity. Glucagon-like peptide-1 (GLP-1) secretion 15 min after oral glucose administration was significantly lower in SUC-fed mice than in ST-fed or control mice. Hepatic glucokinase (GCK) activity was significantly reduced in SUC-fed mice. During the OGTT, the accumulation of glycogen in the liver was suppressed in SUC-fed mice in a time-dependent manner. CONCLUSIONS: These results indicate that mice that consume a moderate SUC show glucose intolerance with a reduction in hepatic GCK activity and impairment in GLP-1 secretion. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00208.x, 2012).

Beneficial effects of exendin-4 on experimental polyneuropathy in diabetic mice.

OBJECTIVE: The therapeutic potential of exendin-4, an agonist of the glucagon-like peptide-1 receptor (GLP-1R), on diabetic polyneuropathy (DPN) in streptozotocin (STZ)-induced diabetic mice was investigated. RESEARCH DESIGN AND METHODS: The presence of the GLP-1R in lumbar dorsal root ganglion (DRG) was evaluated by immunohistochemical analyses. DRG neurons were dissected from C57BL6/J mice and cultured with or without Schwann cell-conditioned media in the presence or absence of GLP-1 (7-37) or exendin-4. Then neurite outgrowth was determined. In animal-model experiments, mice were made diabetic by STZ administration, and after 12 weeks of diabetes, exendin-4 (10 nmol/kg) was intraperitoneally administered once daily for 4 weeks. Peripheral nerve function was determined by the current perception threshold and motor and sensory nerve conduction velocity (MNCV and SNCV, respectively). Sciatic nerve blood flow (SNBF) and intraepidermal nerve fiber densities (IENFDs) also were evaluated. RESULTS: The expression of the GLP-1R in DRG neurons was confirmed. GLP-1 (7-37) and exendin-4 significantly promoted neurite outgrowth of DRG neurons. Both GLP-1R agonists accelerated the impaired neurite outgrowth of DRG neurons cultured with Schwann cell-conditioned media that mimicked the diabetic condition. At the doses used, exendin-4 had no effect on blood glucose or HbA(1c) levels. Hypoalgesia and delayed MNCV and SNCV in diabetic mice were improved by exendin-4 without affecting the reduced SNBF. The decreased IENFDs in sole skins of diabetic mice were ameliorated by exendin-4. CONCLUSIONS: Our findings indicate that exendin-4 ameliorates the severity of DPN, which may be achieved by its direct actions on DRG neurons and their axons.