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Nudix hydrolase NUDT19 regulates mitochondrial function and ATP production in murine hepatocytes
(2022)
Changes in intracellular CoA levels are known to contribute to the development of non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes (T2D) in human and rodents. However, the underlying genetic basis is still poorly understood.
Due to their diverse susceptibility towards metabolic diseases, mouse inbred strains have been proven to serve as powerful tools for the identification of novel genetic factors that underlie the patho-physiology of NAFLD and diabetes. Transcriptome analysis of mouse liver samples revealed the nucleoside diphosphate linked moiety X-type motif Nudt19 as novel candidate gene responsible for NAFLD and T2D development. Knockdown (KD) of Nudt19 increased mitochondrial and glycolytic ATP production rates in Hepa 1-6 cells by 41% and 10%, respectively.
The enforced utilization of glutamine or fatty acids as energy substrate reduced uncoupled respiration by 41% and 47%, respectively, in non-target (NT) siRNA transfected cells.
This reduction was prevented upon Nudt19 KD. Furthermore, incubation with palmitate or oleate respectively increased mitochondrial ATP production by 31% and 20%, and uncoupled respiration by 23% and 30% in Nudt19 KD cells, but not in NT cells.
The enhanced fatty acid oxidation in Nudt19 KD cells was accompanied by a 1.3-fold increased abundance of Pdk4.
This study is the first to describe Nudt19 as regulator of hepatic lipid metabolism and potential mediator of NAFLD and T2D development.
Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in the liver.
Various mechanisms such as an increased uptake in fatty acids or de novo synthesis contribute to the development of steatosis and progression to more severe stages. Furthermore, it has been shown that impaired lipophagy, the degradation of lipids by autophagic processes, contributes to NAFLD.
Through an unbiased lipidome analysis of mouse livers in a genetic model of impaired lipophagy, we aimed to determine the resulting alterations in the lipidome. Observed changes overlap with those of the human disease.
Overall, the entire lipid content and in particular the triacylglycerol concentration increased under conditions of impaired lipophagy.
In addition, we detected a reduction in long-chain polyunsaturated fatty acids (PUFAs) and an increased ratio of n-6 PUFAs to n-3 PUFAs, which was due to the depletion of n-3 PUFAs.
Although the abundance of major phospholipid classes was reduced, the ratio of phosphatidylcholines to phosphatidylethanolamines was not affected. In conclusion, this study demonstrates that impaired lipophagy contributes to the pathology of NAFLD and is associated with an altered lipid profile.
However, the lipid pattern does not appear to be specific for lipophagic alterations, as it resembles mainly that described in relation to fatty liver disease.
Dysfunctional islets of Langerhans are a hallmark of type 2 diabetes (T2D). We hypothesize that differences in islet gene expression alternative splicing which can contribute to altered protein function also participate in islet dysfunction. RNA sequencing (RNAseq) data from islets of obese diabetes-resistant and diabetes-susceptible mice were analyzed for alternative splicing and its putative genetic and epigenetic modulators. We focused on the expression levels of chromatin modifiers and SNPs in regulatory sequences. We identified alternative splicing events in islets of diabetes-susceptible mice amongst others in genes linked to insulin secretion, endocytosis or ubiquitin-mediated proteolysis pathways. The expression pattern of 54 histones and chromatin modifiers, which may modulate splicing, were markedly downregulated in islets of diabetic animals. Furthermore, diabetes-susceptible mice carry SNPs in RNA-binding protein motifs and in splice sites potentially responsible for alternative splicing events. They also exhibit a larger exon skipping rate, e.g., in the diabetes gene Abcc8, which might affect protein function. Expression of the neuronal splicing factor Srrm4 which mediates inclusion of microexons in mRNA transcripts was markedly lower in islets of diabetes-prone compared to diabetes-resistant mice, correlating with a preferential skipping of SRRM4 target exons. The repression of Srrm4 expression is presumably mediated via a higher expression of miR-326-3p and miR-3547-3p in islets of diabetic mice. Thus, our study suggests that an altered splicing pattern in islets of diabetes-susceptible mice may contribute to an elevated T2D risk.
Background The Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and the metabolic syndrome. This study aimed to identify genetic variants associated with impaired glucose metabolism using the obese lines BFMI861-S1 and BFMI861-S2, which are genetically closely related, but differ in several traits. BFMI861-S1 is insulin resistant and stores ectopic fat in the liver, whereas BFMI861-S2 is insulin sensitive. Methods In generation 10, 397 males of an advanced intercross line (AIL) BFMI861-S1 x BFMI861-S2 were challenged with a high-fat, high-carbohydrate diet and phenotyped over 25 weeks. QTL-analysis was performed after selective genotyping of 200 mice using the GigaMUGA Genotyping Array. Additional 197 males were genotyped for 7 top SNPs in QTL regions. For the prioritization of positional candidate genes whole genome sequencing and gene expression data of the parental lines were used. Results Overlapping QTL for gonadal adipose tissue weight and blood glucose concentration were detected on chromosome (Chr) 3 (95.8-100.1 Mb), and for gonadal adipose tissue weight, liver weight, and blood glucose concentration on Chr 17 (9.5-26.1 Mb). Causal modeling suggested for Chr 3-QTL direct effects on adipose tissue weight, but indirect effects on blood glucose concentration. Direct effects on adipose tissue weight, liver weight, and blood glucose concentration were suggested for Chr 17-QTL. Prioritized positional candidate genes for the identified QTL were Notch2 and Fmo5 (Chr 3) and Plg and Acat2 (Chr 17). Two additional QTL were detected for gonadal adipose tissue weight on Chr 15 (67.9-74.6 Mb) and for body weight on Chr 16 (3.9-21.4 Mb). Conclusions QTL mapping together with a detailed prioritization approach allowed us to identify candidate genes associated with traits of the metabolic syndrome. In addition, we provided evidence for direct and indirect genetic effects on blood glucose concentration in the insulin-resistant mouse line BFMI861-S1.
Current attempts to prevent and manage type 2 diabetes have been moderately effective, and a better understanding of the molecular roots of this complex disease is important to develop more successful and precise treatment options.
Recently, we initiated the collective diabetes cross, where four mouse inbred strains differing in their diabetes susceptibility were crossed with the obese and diabetes-prone NZO strain and identified the quantitative trait loci (QTL) Nidd13/NZO, a genomic region on chromosome 13 that correlates with hyperglycemia in NZO allele carriers compared to B6 controls.
Subsequent analysis of the critical region, harboring 644 genes, included expression studies in pancreatic islets of congenic Nidd13/NZO mice, integration of single-cell data from parental NZO and B6 islets as well as haplotype analysis.
Finally, of the five genes (Acot12, S100z, Ankrd55, Rnf180, and Iqgap2) within the polymorphic haplotype block that are differently expressed in islets of B6 compared to NZO mice, we identified the calcium-binding protein S100z gene to affect islet cell proliferation as well as apoptosis when overexpressed in MINE cells. In summary, we define S100z as the most striking gene to be causal for the diabetes QTL Nidd13/NZO by affecting beta-cell proliferation and apoptosis. Thus, S100z is an entirely novel diabetes gene regulating islet cell function.
Background: Dietary protein restriction is emerging as an alternative approach to treat obesity and glucose intolerance because it markedly increases plasma fibroblast growth factor 21 (FGF21) concentrations. Similarly, dietary restriction of methionine is known to mimic metabolic effects of energy and protein restriction with FGF21 as a required mechanism. However, dietary protein has been shown to be required for normal bone growth, though there is conflicting evidence as to the influence of dietary protein restriction on bone remodeling. The purpose of the current study was to evaluate the effect of dietary protein and methionine restriction on bone in lean and obese mice, and clarify whether FGF21 and general control nonderepressible 2 (GCN2) kinase, that are part of a novel endocrine pathway implicated in the detection of protein restriction, influence the effect of dietary protein restriction on bone.
Methods: Adult wild-type (WT) or Fgf21 KO mice were fed a normal protein (18 kcal%; CON) or low protein (4 kcal%; LP) diet for 2 or 27 weeks. In addition, adult WT or Gcn2 KO mice were fed a CON or LP diet for 27 weeks. Young New Zealand obese (NZO) mice were placed on high-fat diets that provided protein at control (16 kcal%; CON), low levels (4 kcal%) in a high-carbohydrate (LP/HC) or high-fat (LP/HF) regimen, or on high-fat diets (protein, 16 kcal%) that provided methionine at control (0.86%; CON-MR) or low levels (0.17%; MR) for up to 9 weeks. Long bones from the hind limbs of these mice were collected and evaluated with micro-computed tomography (mu CT) for changes in trabecular and cortical architecture and mass.
Results: In WT mice the 27-week LP diet significantly reduced cortical bone, and this effect was enhanced by deletion of Fgf21 but not Gcn2. This decrease in bone did not appear after 2 weeks on the LP diet. In addition, Fgf21 KO mice had significantly less bone than their WT counterparts. In obese NZO mice dietary protein and methionine restriction altered bone architecture. The changes were mediated by FGF21 due to methionine restriction in the presence of cystine, which did not increase plasma FGF21 levels and did not affect bone architecture.
Conclusions: This study provides direct evidence of a reduction in bone following long-term dietary protein restriction in a mouse model, effects that appear to be mediated by FGF21.
Background: Dietary protein restriction is emerging as an alternative approach to treat obesity and glucose intolerance because it markedly increases plasma fibroblast growth factor 21 (FGF21) concentrations. Similarly, dietary restriction of methionine is known to mimic metabolic effects of energy and protein restriction with FGF21 as a required mechanism. However, dietary protein has been shown to be required for normal bone growth, though there is conflicting evidence as to the influence of dietary protein restriction on bone remodeling. The purpose of the current study was to evaluate the effect of dietary protein and methionine restriction on bone in lean and obese mice, and clarify whether FGF21 and general control nonderepressible 2 (GCN2) kinase, that are part of a novel endocrine pathway implicated in the detection of protein restriction, influence the effect of dietary protein restriction on bone.
Methods: Adult wild-type (WT) or Fgf21 KO mice were fed a normal protein (18 kcal%; CON) or low protein (4 kcal%; LP) diet for 2 or 27 weeks. In addition, adult WT or Gcn2 KO mice were fed a CON or LP diet for 27 weeks. Young New Zealand obese (NZO) mice were placed on high-fat diets that provided protein at control (16 kcal%; CON), low levels (4 kcal%) in a high-carbohydrate (LP/HC) or high-fat (LP/HF) regimen, or on high-fat diets (protein, 16 kcal%) that provided methionine at control (0.86%; CON-MR) or low levels (0.17%; MR) for up to 9 weeks. Long bones from the hind limbs of these mice were collected and evaluated with micro-computed tomography (mu CT) for changes in trabecular and cortical architecture and mass.
Results: In WT mice the 27-week LP diet significantly reduced cortical bone, and this effect was enhanced by deletion of Fgf21 but not Gcn2. This decrease in bone did not appear after 2 weeks on the LP diet. In addition, Fgf21 KO mice had significantly less bone than their WT counterparts. In obese NZO mice dietary protein and methionine restriction altered bone architecture. The changes were mediated by FGF21 due to methionine restriction in the presence of cystine, which did not increase plasma FGF21 levels and did not affect bone architecture.
Conclusions: This study provides direct evidence of a reduction in bone following long-term dietary protein restriction in a mouse model, effects that appear to be mediated by FGF21.
Pancreatic steatosis associates with beta-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals.
Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetesprone NZO mice.
Intermittierendes Fasten
(2020)
Übergewicht und Adipositas erhöhen die Risiken für Stoffwechselstörungen und können zu einem Typ-2-Diabetes führen. Deshalb stellen die Behandlung und Prävention von Fettleibigkeit eine große medizinische Herausforderung dar. Häufig werden eine erhöhte körperliche Aktivität und die Reduktion der täglichen Kalorienaufnahme um 25–30 % angeraten. Eine andere Möglichkeit bietet intermittierendes Fasten, also eine Kalorieneinschränkung über bestimmte Zeiten, d. h. an einem oder mehreren Tagen pro Woche oder über mehr als 14 h pro Tag. Tier- und Humanstudien lieferten Hinweise darauf, dass intermittierendes Fasten bei Adipositas zu einer Verringerung der Körperfettmasse sowie zu Verbesserungen der Stoffwechselparameter und der Insulinsensitivität führt. Diese positiven Effekte werden nicht nur allein durch die Abnahme der Körpermasse, sondern auch durch die Aktivierung von Stoffwechselwegen und zellulären Prozessen ausgelöst, die für Fastenbedingungen spezifisch sind. In diesem Artikel beschreiben wir die derzeit bekannten Mechanismen, die durch intermittierendes Fasten induziert werden, und stellen Ergebnisse aus randomisierten kontrollierten Studien am Menschen vor.
Intermittierendes Fasten
(2020)
A long-term positive energy balance leads to overweight and obesity. Adiposity is the main risk factor for cardiovascular diseases, type 2 diabetes and cancer and is often accompanied by depression. The increasing prevalence creates a major problem for the healthcare system. The conservative management of obesity strives for weight loss by reducing the daily caloric intake and increasing physical activity as well as an improvement in the quality of life supported by psychological interventions. For reducing body weight, intermittent fasting represents an alternative to continuous calorie restriction as it can be easily integrated into daily life. In this form of diet calorie intake is limited in time, i.e. on 2 days in the week or 6-10 h per day. Animal and human studies provide evidence that intermittent fasting over a longer time period is a suitable method to decrease body fat and to improve many metabolic parameters. Fasting alters metabolism and activates specific cellular pathways. These have not only cardioprotective effects but also neuroprotective and antidepressive effects. In this article the currently discussed mechanisms induced by intermittent fasting are highlighted and the essential observations from randomized controlled human trials are presented.
Obesity is a worldwide epidemic and contributes to global morbidity and mortality mediated via the development of nonalcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), cardiovascular (CVD) and other diseases. It is a consequence of an elevated caloric intake, a sedentary lifestyle and a genetic as well as an epigenetic predisposition. This review summarizes changes in DNA methylation and microRNAs identified in blood cells and different tissues in obese human and rodent models. It includes information on epigenetic alterations which occur in response to fat-enriched diets, exercise and metabolic surgery and discusses the potential of interventions to reverse epigenetic modifications.
Background: Hepatic steatosis is a common chronic liver disease that can progress into more severe stages of NAFLD or promote the development of life-threatening secondary diseases for some of those affected. These include the liver itself (nonalcoholic steatohepatitis or NASH; fibrosis and cirrhosis, and hepatocellular carcinoma) or other organs such as the vessels and the heart (cardiovascular disease) or the islets of Langerhans (type 2 diabetes). In addition to elevated caloric intake and a sedentary lifestyle, genetic and epigenetic predisposition contribute to the development of NAFLD and the secondary diseases. Scope of review: We present data from genome-wide association studies (GWAS) and functional studies in rodents which describe polymorphisms identified in genes relevant for the disease as well as changes caused by altered DNA methylation and gene regulation via specific miRNAs. The review also provides information on the current status of the use of genetic and epigenetic factors as risk markers. Major conclusion: With our overview we provide an insight into the genetic and epigenetic landscape of NAFLD and argue about the applicability of currently defined risk scores for risk stratification and conclude that further efforts are needed to make the scores more usable and meaningful.
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g., high-fat diets) or overweight and insulin resistance (e.g., methionine-choline-deficient diets), or they are based on monogenetic defects (e.g., ob/ob mice). In the current study, a Western-type diet containing soybean oil with high n-6-PUFA and 0.75% cholesterol (SOD + Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice, which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast, a soybean oil-containing Western-type diet without cholesterol (SOD) induced only mild steatosis but not hepatic inflammation, fibrosis, weight gain or insulin resistance. Another high-fat diet, mainly consisting of lard and supplemented with fructose in drinking water (LAD + Fru), resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD + Cho, but livers were devoid of inflammation and fibrosis. Although both LAD + Fru-and SOD + Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD + Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. In summary, dietary cholesterol in the SOD + Cho diet may trigger hepatic inflammation and fibrosis. SOD + Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.
Objective: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic 13-cells. <br /> Methods: A 13-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. <br /> Results: The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from 13-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from 13-cell-specific Arfrp1 knockout mice. <br /> Conclusion: Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting. (c) 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Type 2 diabetes (T2D) is a complex metabolic disease regulated by an interaction of genetic predisposition and environmental factors. To understand the genetic contribution in the development of diabetes, mice varying in their disease susceptibility were crossed with the obese and diabetes-prone New Zealand obese (NZO) mouse. Subsequent whole-genome sequence scans revealed one major quantitative trait loci (QTL),Nidd/DBAon chromosome 4, linked to elevated blood glucose and reduced plasma insulin and low levels of pancreatic insulin. Phenotypical characterization of congenic mice carrying 13.6 Mbp of the critical fragment of DBA mice displayed severe hyperglycemia and impaired glucose clearance at week 10, decreased glucose response in week 13, and loss of beta-cells and pancreatic insulin in week 16. To identify the responsible gene variant(s), further congenic mice were generated and phenotyped, which resulted in a fragment of 3.3 Mbp that was sufficient to induce hyperglycemia. By combining transcriptome analysis and haplotype mapping, the number of putative responsible variant(s) was narrowed from initial 284 to 18 genes, including gene models and non-coding RNAs. Consideration of haplotype blocks reduced the number of candidate genes to four (Kti12,Osbpl9,Ttc39a, andCalr4) as potential T2D candidates as they display a differential expression in pancreatic islets and/or sequence variation. In conclusion, the integration of comparative analysis of multiple inbred populations such as haplotype mapping, transcriptomics, and sequence data substantially improved the mapping resolution of the diabetes QTLNidd/DBA. Future studies are necessary to understand the exact role of the different candidates in beta-cell function and their contribution in maintaining glycemic control.
Objective
Insulin regulates mitochondrial function, thereby propagating an efficient metabolism. Conversely, diabetes and insulin resistance are linked to mitochondrial dysfunction with a decreased expression of the mitochondrial chaperone HSP60. The aim of this investigation was to determine the effect of a reduced HSP60 expression on the development of obesity and insulin resistance.
Methods
Control and heterozygous whole-body HSP60 knockout (Hsp60+/−) mice were fed a high-fat diet (HFD, 60% calories from fat) for 16 weeks and subjected to extensive metabolic phenotyping. To understand the effect of HSP60 on white adipose tissue, microarray analysis of gonadal WAT was performed, ex vivo experiments were performed, and a lentiviral knockdown of HSP60 in 3T3-L1 cells was conducted to gain detailed insights into the effect of reduced HSP60 levels on adipocyte homeostasis.
Results
Male Hsp60+/− mice exhibited lower body weight with lower fat mass. These mice exhibited improved insulin sensitivity compared to control, as assessed by Matsuda Index and HOMA-IR. Accordingly, insulin levels were significantly reduced in Hsp60+/− mice in a glucose tolerance test. However, Hsp60+/− mice exhibited an altered adipose tissue metabolism with elevated insulin-independent glucose uptake, adipocyte hyperplasia in the presence of mitochondrial dysfunction, altered autophagy, and local insulin resistance.
Conclusions
We discovered that the reduction of HSP60 in mice predominantly affects adipose tissue homeostasis, leading to beneficial alterations in body weight, body composition, and adipocyte morphology, albeit exhibiting local insulin resistance.
Objective
Insulin regulates mitochondrial function, thereby propagating an efficient metabolism. Conversely, diabetes and insulin resistance are linked to mitochondrial dysfunction with a decreased expression of the mitochondrial chaperone HSP60. The aim of this investigation was to determine the effect of a reduced HSP60 expression on the development of obesity and insulin resistance.
Methods
Control and heterozygous whole-body HSP60 knockout (Hsp60+/−) mice were fed a high-fat diet (HFD, 60% calories from fat) for 16 weeks and subjected to extensive metabolic phenotyping. To understand the effect of HSP60 on white adipose tissue, microarray analysis of gonadal WAT was performed, ex vivo experiments were performed, and a lentiviral knockdown of HSP60 in 3T3-L1 cells was conducted to gain detailed insights into the effect of reduced HSP60 levels on adipocyte homeostasis.
Results
Male Hsp60+/− mice exhibited lower body weight with lower fat mass. These mice exhibited improved insulin sensitivity compared to control, as assessed by Matsuda Index and HOMA-IR. Accordingly, insulin levels were significantly reduced in Hsp60+/− mice in a glucose tolerance test. However, Hsp60+/− mice exhibited an altered adipose tissue metabolism with elevated insulin-independent glucose uptake, adipocyte hyperplasia in the presence of mitochondrial dysfunction, altered autophagy, and local insulin resistance.
Conclusions
We discovered that the reduction of HSP60 in mice predominantly affects adipose tissue homeostasis, leading to beneficial alterations in body weight, body composition, and adipocyte morphology, albeit exhibiting local insulin resistance.
Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy expenditure. Increases in circulating fibroblast growth factor 21 (FGF21) have been implicated as a potential underlying mechanism. Methods We aimed to test whether low-protein diets in the context of a high-carbohydrate or high-fat regimen would also protect against type 2 diabetes in New Zealand Obese (NZO) mice used as a model of polygenetic obesity and type 2 diabetes. Mice were placed on high-fat diets that provided protein at control (16 kJ%; CON) or low (4 kJ%; low-protein/high-carbohydrate [LP/HC] or low-protein/high-fat [LP/HF]) levels. Results Protein restriction prevented the onset of hyperglycaemia and beta cell loss despite increased food intake and fat mass. The effect was seen only under conditions of a lower carbohydrate/fat ratio (LP/HF). When the carbohydrate/fat ratio was high (LP/HC), mice developed type 2 diabetes despite the robustly elevated hepatic FGF21 secretion and increased energy expenditure. Conclusion/interpretation Prevention of type 2 diabetes through protein restriction, without lowering food intake and body fat mass, is compromised by high dietary carbohydrates. Increased FGF21 levels and elevated energy expenditure do not protect against hyperglycaemia and type 2 diabetes per se.
Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.